Veterinaria Italiana, Volume 49 (4), October-December 2013

RIVISTA DI SANITÀ PUBBLICA VETERINARIA

VOLUME 49 (4) - OTTOBRE-DICEMBRE / OCTOBER-DECEMBER 2013

ISSN 0505-401X

Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”

Campo Boario, 64100 TERAMO, Italiatelefono +39 0861 3321fax +39 0861 332251www.izs.it

Questa rivistaè nata nel 1950 con il

nome di Croce Azzurra.Dal 1954 si chiamerà

Veterinaria Italiana.

Rivista trimestrale di Sanità Pubblica Veterinaria,edita dall’Istituto Zooprofilattico Sperimentale

dell’Abruzzo e del Molise “G. Caporale”

A quarterly journal devoted to veterinary public health,veterinary science and medicine,

published by the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ in Teramo, Italy

Volume 49 (4), 2013

Segreteria di redazione Associate EditorsMonica Bucciarelli, Guido Mosca,Mariarosaria Taddeo, Carlo Turilli

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Victor E. Saraiva – BrazilAristarhos M. Seimenis – GreeceArnon Shimshony – IsraelSamba Sidibé – MaliGavin R. Thomson – South AfricaCarlo Turilli – ItalyNorman G. Willis – Canada

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Comitato direttivo Managing Scientific Board

Romano MarabelliFernando Arnolfo

Direttore Editor-in-Chief

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Si ringrazia Fabrizio Piccari per la gentile concessione dell’immagine di copertina.

Publications of the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ (IZSAM) are protected by international copyright law. Users are permitted to read, download, copy, distribute, print, search abstracts; besides they can link to Veterinaria Italiana full pdf files. Should information be required for commercial purposes, prior written permission must be sought from the IZSAM. Published translations and adaptations also require prior written approval from the IZSAM.The views expressed in signed articles are solely the responsibility of the authors. The mention of specific companies or products of manufacturers, whether or not patented, does not imply that these have been endorsed or recommended by the IZSAM in preference to others of a similar nature that are not mentioned.

Acknowledgement is made to Fabrizio Piccari for the cover image.

Volume 49 (4), 2013

LETTER TO THE EDITORValentina Chisu, Rosaura Porcu, Antonio Tanda & Giovanna MasalaFirst isolation and characterization of Chlamydophila abortusfrom abortion tissues of sheep in Sardinia, Italy .....................................331Primo isolamento e caratterizzazione di Chlamydophila abortusda aborti ovini in Sardegna, Italia (riassunto)............................................................................... 331

Federica Giorda, Lisa Guardone, Marialetizia Mancini,Annalisa Accorsi, Fabio Macchioni & Walter MignoneCases of bed bug (Cimex lectularius) infestationsin Northwest Italy .......................................................................................335Casi di infestazione da cimici dei letti (Cimex lectularius)in Italia Nord-occidentale (riassunto) ............................................................................................. 335

Daniela Salvatore, Antonietta Di Francesco, Maria Parigi,Giovanni Poglayen, Maurizio Battistini & Raffaella BaldelliCanine leishmaniasis surveillance programin a San Marino Republic kennel ..............................................................341Programma di sorveglianza della leishmaniosi canina in un caniledella Repubblica di San Marino (riassunto) ................................................................................... 341

Anna Toffan, Michela Bonci, Luca Bano, Viviana Valastro,Marta Vascellari, Ilaria Capua & Calogero TerreginoDiagnostic and clinical observationon the infectious bronchitis virus strain Q1 in Italy ................................347Descrizione di focolai di bronchite infettivasostenuta dal ceppo Q1 recentemente segnalato in Italia (riassunto) ................................... 347

Anjan Mondal, Soumendu Chakravarti, Sharmila B. Majee& Anilkumar Sadashivrao BannalikarDetection of picobirnavirus and rotavirus indiarrhoeic faecal samples of cattle and buffalo calvesin Mumbai metropolis, Western India .......................................................357Presenza di picobirnavirus e rotavirus in campioni fecali diarroicidi vitelli bovini e bufalini nella città di Mumbai, India occidentale (riassunto) ..................... 357

Asim Jana & Anjan MondalSerotyping, pathogenicity and antibiogram of Escherichia coliisolated from raw poultry meat in West Bengal, India ............................361Caratterizzazione di Escherichia coli isolata in campionidi carne cruda di pollo nel Bengala occidentale, India (riassunto) .......................................... 361

Volume 49 (4), 2013

Nadia B. Barile, Sabatino Cappabianca, Luigi Antonetti,Mariaspina Scopa, Eliana Nerone, Giuseppina Mascilongo,Sara Recchi & Antonio D’AloiseNew protocols to improve the deposition and hatchingof Sepia officinalis’ eggs .............................................................................367Nuove procedure per migliorare la deposizione e schiusadi uova di Sepia officinalis (riassunto) ............................................................................................ 367

SHORT COMMUNICATIONBimalendu Mondal, Bhoopendra Bisht, Sanchay K. Biswas & Karam ChandBluetongue virus serotype-1 in goatsin the Pithoragarh area of Uttarakahand, India ......................................375Virus della bluetongue sierotipo 1 in capre nell’area di Pithoragarh,Stato di Uttarakahand, India (riassunto) ........................................................................................ 375

LIBRI/Book reviews(a cura di) Giorgio Battelli, Raffaella Baldelli,Fabio Ostanello, Santino ProsperiGli animali, l'uomo e l'ambiente ................................................................................381Raffaele MastrocolaStorie e geografie di un veterinario di campagna ............................................383

331

1 Istituto Zooprofilattico Sperimentale della Sardegna, Via Duca degli Abruzzi 8, 07100 Sassari, Italy

* Corresponding author at: Istituto Zooprofilattico Sperimentale della Sardegna, Via Duca degli Abruzzi 8, 07100 Sassari, Italy.Tel.: +39 079 289200, e-mail: [email protected].

Parole chiaveAborti ovini,Chlamydophila abortus,Isolamento in colture cellulari,PCR.

RiassuntoChlamydophila abortus (C. abortus) è l’agente responsabile dell’aborto da chlamydia [comunemente conosciuto come Enzootic Abortion of Ewes (EAE)] e provoca gravi perdite economiche per il comparto ovino in tutto il mondo. L’isolamento del patogeno è considerato il “gold standard” e il metodo più sensibile di rilevamento per la diagnosi di infezione da Chlamydia. Nessun dato è attualmente disponibile riguardo l’isolamento di C. abortus da ovini in Sardegna, Italia. Lo scopo di questo studio è stato quello di determinare la presenza di C. abortus durante la stagione degli aborti ovini nel 2010. Un totale di 89 campioni d’aborto (40 feti e 49 placente) provenienti dal Nord della Sardegna sono stati analizzati; 3 placente sono risultate positive alla ricerca del DNA di C. abortus in PCR utilizzando primers specifici per ‘putative outer membrane protein’ (pmp), portando all’identificazione ed al successivo isolamento in coltura cellulare di C. abortus. Questo lavoro documenta il primo isolamento di C. abortus da placenta ovina e integra la conoscenza riguardo uno degli agenti abortigeni circolanti in Sardegna e, più in generale, nel bacino del Mediterraneo.

Primo isolamento e caratterizzazione di Chlamydophila abortusda aborti ovini in Sardegna, Italia

KeywordsCell culture isolation,Chlamydophila abortus,Ovine abortion,PCR.

SummaryChlamydophila abortus (C. abortus) is the responsible agent for chlamydial abortion [commonly known as Enzootic Abortion of Ewes (EAE)] and, as such, it causes major financial losses to the sheep industry worldwide. Isolation of the pathogen is considered the ‘gold standard’ and most sensitive method of detection for diagnosing chlamydial infection. So far, there has been no isolation of C. abortus from ovines in Sardinia, Italy. This letter describes the results of a study conducted on a total of 89 aborted samples (40 foetuses and 49 placentae) collected in 2010 in Northern Sardinia, Italy. Three placentae resulted PCR-positive when analyzed using the putative outer membrane protein (pmp) specific primers, the test lead to the identification and first isolation in cell culture of C. abortus. This letter to the editor describes the first isolation of C. abortus from ovine placentae and increases the knowledge of one of the agents that causes ovine abortion in Sardinia and, more generally, in the Mediterranean basin.

Valentina Chisu, Rosaura Porcu, Antonio Tanda & Giovanna Masala*

First isolation and characterization ofChlamydophila abortus from abortion tissues

of sheep in Sardinia, Italy

LETTER TO THE EDITOR

Veterinaria Italiana 2013, 49 (4), 331-334. doi: 10.12834/VetIt.1303.10 Accepted: 28.10.2013 | Available on line: 18.12.2013

332 Veterinaria Italiana 2013, 49 (4), 331-334. doi: 10.12834/VetIt.1303.10

conjugated monoclonal antibody that detect elementary bodies of C. abortus.

One hundred microliters of genomic DNA from 1000 μl of total homogenated organs were extracted using QIAgen columns (QIAamp tissue kit, Qiagen, Hilden, Germany), according to the manufacturer’s instructions.

Chlamydial DNA was amplified by using oligonucleotide primers PMP-1 [5’-ATGAAACATTCCAGTCTACTGG-3’] and PMP-2 [3’-TTGTGTAGTAATATTATCAAA-5’] targeted to the outer membrane protein (POMP 90-91B) gene which amplify a product of 320 bp (2). A negative and a positive control of C. abortus DNA were included in each test. Polymerase chain reaction products were visualized by electrophoresis in 1.5% agarose gel, stained with ethidium bromide and examined under UV transillumination.

Polymerase chain reaction products were then purified using a QIAquick Spin PCR purification Kit (QUIAGEN, Hilden, Germany) and sequenced using a DNA sequencing kit (dRhodamine Terminator cycle sequencing ready reaction; Applied Biosystems, Courtaboeuf, France), according to the manufacturer’s instructions. All sequences were compared with those of the Chlamydiae present in the GenBank database using the BLAST search tool. Attempts to cultivate and isolate the strain were made from each positive sample. An aliquot of one microliter of purified homogenates was inoculated in flasks of McCoy cells (ECACC 90010305), a heteroploid mouse fibroblast line. Infected McCoy cells were grown in MEM (Minimum Essential modified Medium, GibcoBRL, Grand Island, NY, USA) containing 10% heat-inactivated foetal bovine serum (FBS, GibcoBRL, Grand Island, NY, USA) and cycloheximide (2μg/ml) at 37°C with 5% CO2. When a successful isolation was identified on the chamber slide, the infected cells were passaged by trypsinization into a 25-cm2 tissue culture flask. Bacteria were harvested after direct immunofluorescence test was heavily positive, and total genomic DNA was extracted from the culture using a QIAamp tissue kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Two hundred microliters of elution buffer were used to re-suspend each extracted DNA sample. The genomic DNA was then stored at -20 °C until further processing.

Three placental tissues (3/40), resulted positive for chlamydial DNA by PCR. Sequence analyses of the positive samples showed 100% similarity with 320-bp putative outer membrane protein region of C. abortus (GenBank accession number: EU326104). After 5 days, 2 of the 3 cultures that were infected with placental tissue suspensions, showed the presence of chlamydial-like organisms when tested

Chlamydophila abortus is a zoonotic Gram negative bacterium, it infects epithelial cells and monocyte/macrophages of a wide host range, resulting in a broad spectrum of diseases in humans, as well as in other mammals and birds (3). C. abortus is also one of the most common causes of infectious abortion in sheep and goats worldwide, the so called enzootic abortion of ewes (EAE). The disease has a major economic impact as it represents the most important cause of lamb loss in sheep in several parts of Europe, North America and Africa (6).

In Sardinia, the second largest island of Italy in the middle of the Mediterranean Sea, sheep farming is a very important sector of primary animal production both economically and socially. In this reagion, the primary sector is still of outstanding importance, especially sheep rearing; in the island there are 32,209 farms, of which 14,854 are sheep farms with a total of 3,140,442 sheep corresponding to nearly half of the total Italian sheep population. These farms are registered in the Italian National database (BDN data 2012, Reg. CE 1760/2000).

The incidence of ovine abortion is very high because of the high animal density (149/km2 against 23/km2 on the mainland), poor breeding conditions (traditional herding practices and transhumance) and inadequate prophylaxis. The economic losses due to lamb mortality and reduced milk production are estimated at about 10 million euro per year (6).

Chlamydial studies based on clinical and serological features were conducted in Sardinia using indirect immunofluorescence antibody tests to detect antibodies against C. abortus. Moreover, PCR assay was then used to identify C. abortus in samples from adult ovine abortions (6). Historically, the isolation of the pathogen has been considered the ‘gold standard’ and most sensitive method of detection for diagnosing chlamydial infection. Isolation involves the cultivation of organisms from clinical samples in either embryonated hen’s eggs or cell culture. So far, there has been no direct detection or isolation of chlamydiae in Sardinia. The aim of this study was to detect and isolate C. abortus from ovine placental tissues in Sardinia.

A total of 89 ovine aborted samples (40 foetuses and 49 placentae) were brought to the Istituto Zooprofilattico Sperimentale della Sardegna. The aborted-sheep came from semi-wild flocks and it was not possible to distinguish primiparae from pluriparae sheep. The 49 placentae and the 40 foetuses (40 brains, 3 livers and 6 spleens) were then analyzed for detection and identification of C. abortus antigens using acetone-fixed tissue sample smears. Slides were subsequently analyzed by a direct immunofluorescence (IFD) test (IMAGEN Chlamydia test, Oxoid, Ely, United Kingdom) containing fluorescein isothiocyanate (FITC)

Isolation and characterization of C. abortus in Sardinia, Italy Chisu et al.

333Veterinaria Italiana 2013, 49 (4), 331-334. doi: 10.12834/VetIt.1303.10

of significant economic importance and represents principally a pathogen of ruminants. Sheep can become infected at any age and during any season, but the highest period of risk is undoubtedly at lambing time. Infectious ovine abortion is a major health problem in sheep worldwide, having significant financial and welfare implications. For this reason, an early and accurate diagnosis of the cause of abortion is important, so that appropriate control measures can be adopted to limit or prevent the spread of infection. DNA detection is more rapid and economic than isolation and it can be considered a useful technique for the diagnosis of the previously mentioned pathogens (1). Nevertheless, the isolation of these pathogens from aborted samples represents the gold standard for definitive diagnosis and for the charaterizating of isolated strains. However, isolation requires obtaining samples in optimal conditions (they must be fresh, with little or no contamination, and free of toxic factors), containing a threshold number of live and viable microorganisms. In fact, contamination with other bacteria, inadequate transport conditions, autolysis, and other factors may all adversely affect isolation. Samples should be placed in chlamydia transport medium (SPG) before being despatched to laboratory in order to prevent contaminations.

If the diagnosis is confirmed rapidly and reliably, then control measures can be implemented to reduce the impact of infection in current and future years, thus protecting the economic viability of the flocks. As the disease is commonly transmitted within flocks by infected stock, controls performed at farms level may be beneficial. Farmers will implement control measures where the cost of action is lower than the benefits. Therefore knowledge of the costs and benefits is required in order to support the uptake of disease control strategies.

by IFD (Figure 1). Moreover, chlamydial DNA was confirmed by PCR analyses. Two Chlamydia isolates have been stably maintained in McCoy cells at 37°C as a persistent infection by changing the cell culture medium every 10 days to 2 weeks.

This study documents the first isolation of C. abortus from ovine placental tissues in Sardinia. In the authors’ previous studies the prevalence of abortion and the role of different infectious agent in abortion were investigated. From 1999 to 2005, 12/213 placentae from sheep resulted positive to C. abortus after PCR (4, 5). C. abortus is a pathogen

Chisu et al. Isolation and characterization of C. abortus in Sardinia, Italy

Figure 1. Presence of chlamydial-like organisms after IFD analysis in McCoy cultures that were infected with 1ml of suspensions of placental tissues.

334

1. Hartley J.C., Kaye S., Stevenson S., Bennett J. & Ridgway G. 2001. PCR detection and molecular identification of Chlamydiaceae species. J Clin Microbiol, 39(9), 3072-3079.

2. Longbottom D., Russell M., Dunbar S.M., Jones G.E. & Herring A.J. 1998. Molecular cloning and characterization of the genes coding for the highly immunogenic cluster of 90-kilodalton envelope proteins from the Chlamydia psittaci subtype that causes abortion in sheep. Infect Immun, 66(4), 1317-1324.

3. Longbottom D. & Coulter L.J. 2003. Animal chlamydioses and zoonotic implications. J Comp Pathol, 128(4), 217-244.

4. Masala G., Porcu R., Sanna G., Tanda A. & Tola S. 2005. Role of Chlamydophila abortus in ovine and caprine abortion in Sardinia, Italy. Vet Res Commun, 29 (suppl 1), 117-123.

References

5. Masala G., Porcu R., Daga C., Denti S., Canu G., Patta C. & Tola S. 2007. Dection of pathogens in ovine and caprine abortion samples from Sardinia, Italy, by PCR. J Vet Diagn Invest,19(1), 96-98.

6. Sachse K., Vretou E., Livingstone M., Borel N., Pospischilet A. & Longbottom D. 2009. Recent developments in the laboratory diagnosis of chlamydial infections. Vet Microbiol, 135(1-2), 2-21.

7. Thomson N.R., Yeats C., Bell K., Holden M.T., Bentley S.D., Livingstone M., Cerdeño-Tárraga A.M., Harris B., Doggett J., Ormond D., Mungall K., Clarke K., Feltwell T., Hance Z., Sanders M., Quail M.A., Price C., Barrell B.G., Parkhill J. & Longbottom D. 2005. The Chlamydophila abortus genome sequence reveals an array of variable proteins that contribute to interspecies variation. Genome Res, 15(5), 629-640.

Veterinaria Italiana 2013, 49 (4), 331-334. doi: 10.12834/VetIt.1303.10

Isolation and characterization of C. abortus in Sardinia, Italy Chisu et al.

335

Veterinaria Italiana 2013, 49 (4), 335-340. doi: 10.12834/VetIt.1306.03Accepted: 15.11.2013 | Available on line: 18.12.2013

SummaryBed bugs (Cimex lectularius) have been a common problem for humans for at least 3,500 years and in Europe their presence was endemic until the end of World War II, when infestations began to decrease. However, since the beginning of the 21st century new cases of infestations have been reported in developed countries. Many theories have been put forward to explain this change of direction, but none has been scientifically proven. The aim of this study is to provide some reports of bed bug infestations in Northern Italy (Liguria, Piedmont and Aosta valley regions) and a brief summary about their identification, clinical significance, bioecology and control. From 2008 to date, 17 bed bug infestations were identified in Northwest Italy. Knowledge about the presence and distribution of bed bugs in Italy is scanty, prior to this work only 2 studies reported the comeback of these arthropods in the Italian territory; further investigations would be necessary to better understand the current situation.

RiassuntoLe cimici dei letti (Cimex lectularius) sono un problema comune per l’umanità da almeno 3.500 anni. In Europa la loro presenza è stata endemica fino al termine della II Guerra Mondiale, quando è stato osservata una diminuzione del numero di infestazioni. Tuttavia, dai primi anni 2000, nei paesi sviluppati è stata riscontrata una nuova recrudescenza. Sono state prese in considerazione numerose teorie per fornire una spiegazione a tale inversione di tendenza, ma nessuna è stata scientificamente provata. L’obiettivo di questo lavoro è di riportare diversi casi d’infestazione da cimici dei letti in Italia Nord-occidentale (Liguria, Piemonte e Valle d’Aosta) e offrire informazioni riguardo alla loro identificazione, al loro significato clinico, alla bioecologia e al controllo. Dal 2008 sono stati identificati 17 casi d’infestazione nell’area di studio. Ad oggi, i dati sulla reale presenza e distribuzione delle cimici dei letti in Italia sono limitati; prima del presente lavoro 2 soli studi avevano documentato il ritorno di questi insetti sul territorio italiano. Ulteriori indagini sarebbero necessarie per comprendere meglio l’epidemiologia attuale.

Casi di infestazione da cimici dei letti (Cimex lectularius)in Italia Nord-occidentale

Parole chiaveCimici dei letti,Cimex lectularius,Controllo degli infestanti,Epidemiologia,Identificazione,Infestazione,Italia Nord-occidentale.

KeywordsBed bugs,Cimex lectularius,Epidemiology,Identification,Infestation,Northwestern Italy,Pest management.

1 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Sezione di Imperia,via Nizza 4, 18100 Imperia, Italy

2 Department of Veterinary Science, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy

* Corresponding author at: Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta,Sezione di Imperia, via Nizza 4, 18100 Imperia, Italy.

Tel.: +39 018 3660185, e-mail: [email protected].

Federica Giorda1*, Lisa Guardone2, Marialetizia Mancini1,Annalisa Accorsi2, Fabio Macchioni2 & Walter Mignone1

Cases of bed bug (Cimex lectularius) infestationsin Northwest Italy


From 2008 to date the Institute received 17 samples of ‘potential’ bed bugs, delivered directly by private citizens who had found them in their own homes. The specific identification of arthropods was conducted through direct observation with a stereoscope (Nikon model SMZ800, Tokio, Japan), following the identification keys of Usinger (46) and Tremblay (45). The order Hemiptera or Rhynchota includes species of insects that range in size from 1 mm to around 1 cm and are characterized by a stiliform mouthpart in wich mandibles and maxillae are sheathed in a modified labium forming a ‘beak’ or ‘rostrum’ [from the greek ρύνχός = (rhynchos)], which is capable to penetrate plant or animal tissues to suck out the liquids. The suborder Heteroptera is characterized by a ‘rostrum’ in the anterior region of the head. Wings, when present, are generally constituted by a pair of forewings, or hemelytra, generally more sclerificate, and a pair of membranous hind wings. Insects of the family Cimicidae are apterous and brown, while the genus Cimex can be identified by anatomical details, as a second antennal segment subequal to the interocular space and the pronotum 1 ½ or more times as wide as head. The bat bug L. boueti is smaller than C. lectularius and C. hemipterus and differs from C. lectularius in having a narrower pronotum, only slightly wider than the head, and very long legs. C. lectularius and C. hemipterus are morphologically similar; the distinction is based on an upturned lateral flange on the margin of the pronotum which makes this structure wider in the common bed bug compared to that of the second species (46).

In order to understand the methods of control used in Italy, in addition to a scientific bibliographic research (6, 13, 16, 11, 25, 28, 29, 31, 41, 49) the following sources were examined:

• catalogs and guidelines of the companies for the production of equipment and insecticides for pest control (4, 9, 10, 30);

• technical manuals and seminars organized by the National Association of Pest Control Companies (2, 3, 6, 7, 8, 20, 27, 34, 42);

• pest control journals (16, 20).

ResultsResults are given in details in Table I, of the 17 samples received, all specimens were identified as belonging to the species C. lectularius (Figure 1): 3 infestations occurred in 2008 (Liguria), 5 in 2009 (Liguria), 2 in 2010 (Liguria), 3 in 2011 (2 in Piemonte and 1 in Liguria), 3 in 2012 (of which 2 in Liguria and 1 in Piemonte), and 1 in 2013 (Piemonte). Interestingly, no cases had been registered between 2001 and 2008. All infestations involved adult specimens, with the exception of 3

IntroductionBed bugs are nocturnal haematophagous arthropods belonging to the family Cimicidae, order Hemiptera. The family includes 6 subfamilies, 23 genera and 91 species. Three species are considered ectoparasites of humans: Cimex lectularius, the common bed bug, is a cosmopolitan species associated with humans, bats and chickens; Cimex hemipterus, the tropical bed bug, is a parasite of humans and chickens (15), and Leptocimex boueti belongs to the Cacodminae subfamily and affects bats and people in West Africa (46).

Recent findings of bed bugs specimens in tombs in Egypt prove the relationship between humans and cimicids for at least 3,500 years (33) and both the ancient Greek and Latin literature testifies the existence of C. lectularius (43). The parasite has been recorded through history in Greece in 400 BC, in Italy in AD 77, in Germany in the 11th century, in France in the 13th century and in England in 1583, although the wide dissemination of this insect throughout the world probably did not begin until the 16th century (22).

Until 1945 bed bugs presence was endemic worldwide; documents report that in 1930 one third of the London population was infested (24). After the World War II a decrease of bed bugs incidence in developed nations was observed (24), due to social and economic progress and possibly to the development of new insecticides (e.g. diclodifeniltricloetano-DDT), while their presence remained widespread in poor countries (19, 21). Since the late 90’s there has been a global resurgence of the number of infestations (15, 37). In Europe cases have been reported in the UK (5, 17, 48), Germany (26) and Spain (18). The distribution of bed bugs in Italy is scarcely known; to the authors’ knowledge, prior to this study only reports from North-Eastern and Central Italy were available (28, 29).

The aim of this article is to provide a documented evidence of the presence of bed bug infestations in Northwest Italy and a brief summary about their identification, clinical significance, biology and control. Furthermore we want to direct the reader to a policy of non-chemical control.

Materials and methodsSince 2001, the section of Imperia of Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta (IZS) has conducted a national entomological surveillance plan and has established a provincial office of Veterinary Public Health Services accredited for the identification of arthropods. The study area covers 34,085 km² (corresponding to the regions of Piemonte, Liguria and Valle d’Aosta).

Cases of bed bug (Cimex lectularius) infestations in Northwest Italy Giorda et al.

337

been recently bought from a shop and an antique market, respectively.

DiscussionOur results are a contribution to the epidemiology of bed bugs in Italy and suggest a diffusion of such infestations, in agreement with data from other countries (15). Bed bugs’ epidemiology in Italy is poorly characterized: 2 human infestations have been reported in Pisa in 2003, other 2 in Italian trains in 2005 (28) and more recently a study identified 23 cases of C. lectularius infestations between 2006 and 2011 in the North-East of the country (29). However, the presence of C. lectularius is probably underestimated due to several reasons among which the scarce public awareness, the reticence of people to inform physicians (15), the reluctance of accommodation industry’s to declare the pest presence and a certain lack of exchange of epidemiological data between pest control companies and scientific research institutes. Also in the case of our study a possible underestimation of the actual number of cases should be taken into account, due to the fact that data from pest control companies were not included. Interestingly, the information gathered from the examined technical sources reveal that bed bugs start to become a relevant topic in the industry of pest management in Italy since 2002 (2, 9, 10).

Furthermore, it has to be mentioned that the identification of bed bugs is not straightforward. C. lectularius is 4/5 x 3 mm, dorso-ventrally flattened and females are larger than males. Adult bed bugs are reddish-brown to dark-brown depending on climatic conditions and feeding status. The nymphal stages are translucent and cream in color during the first instar and their internal structure turns reddish

cases, which also included nymphs (in 2011 and 2012). The samples were collected mainly during spring and summer: 14 infestations occurred from April to September and only 3 in the remaining months (1 in November, 1 in February and 1 in March). All those who brought the specimens for identification were private citizens and reported bites and dermatological reactions. Bed bugs were found in most of the cases in the bedroom; in 1 case the sofa was infested. In 2 cases the source of infestation was second hand furniture, in particular some furniture for the bedroom and a sofa that had


Giorda et al. Cases of bed bug (Cimex lectularius) infestations in Northwest Italy

Figure 1. Adults of Cimex lectularius. (A) Adult specimens observed by stereomicroscopy. (B) Optical microscopy of an adult female.

A B5 mm 1 mm

Table I. Bed bugs specimens identified in the study area, with the area of origin and collection dates.

Year Collection date

Geographical origin (region,

province)

Stage of identified specimens

1 2008 10/07 Liguria, Imperia Adults

2 2008 14/07 Liguria, Savona Adults

3 2008 05/08 Liguria, Genoa Adults




7 2009 07/09 Liguria, La Spezia Adults



10 2010 22/09 Liguria, Savona Adults


12 2011 07/09 Piedmont, Cuneo Adults

13 2011 13/09 Piedmont, Asti Juveniles


15 2012 21/08 Liguria, Genoa Juveniles

16 2012 28/09 Piedmont, Cuneo Adults and juveniles

17 2013 07/03 Piedmont, Verbania Adults

338

after a blood meal. Instars and adults are both haematofagous. The life cycle includes 5 nymphal stages, lasting 2.5-10 days each. The nymphs become progressively dark as they move along the 5 stages. Nymphs are morphologically very similar to adults but smaller (1-4 mm vs 5-6 mm) and sexually immature. Feeding generally occurs within 24 hrs after hatching or molting (15). The duration of the life cycle depends on the temperature, and varies from 24 days at 30°C to 128 days at 18°C. In cold conditions insects can live up to 1.5-2 years without feeding (37). In average temperature conditions of 23°C, it takes approximately 9 days for an egg to hatch (14). Eggs have an elongated aspect, pearly white colour and are about 1mm long. They are laid singly and are coated with transparent adhesive cement (22). Bed bugs favourite hosts are humans, but they can feed also on pets, bats and birds; in all cases the only moment of contact with the host is during the blood meal (14). The carbon dioxide and the body heat attract bed bugs to their hosts (37). Bed bugs are active in the dark and hide under mattresses, curtains, carpets, up-holstered furniture and crevices of walls during the day. Other common sites of infestations include public facilities such as theatres, office waiting rooms, upholstery seats in buses and trains. Heavily infested areas by this insect may be identified by a characteristic sweet smell (22).

In case of suspicion of bed bug presence, monitoring and accurate searches over a full day are required to discover and identify the insects (28).

In case of a proven infestation, eradication is a challenge (15). Nowadays, bed bugs can be eradicated both with the use of insecticides and with physical methods such as thermic disinfestation and cryogenic treatment with liquid nitrogen.

The worldwide most commonly used insecticides against C. lectularius are pyrethroids, carbamates, silicates and insect growth regulators (IGRs) (38). In Italy, while there is a fair number of registered pyrethroids, there is only 1 authorized carbamate (bendiocarb) (11). Among pyrethroids, delthametrin is the most widely used molecule; however, a general resistance of bed bugs to this principle is well documented (38) and pyrethroids are extremely toxic for bees and many aquatic organism (35).

The use of silicates and IGRs is generally not practiced because they are slow to react (2, 3, 9, 10, 40, 41). In this respect, the study conducted by Romero et al. is noteworthy (39), as it showed that up to 6 days are necessary to achieve 100% of mortality in adult bed bugs with silicates. The class of IGRs presents the same drawback as silicates: after being dosed they lead bed bugs to die just after numerous molts. Organophosphates are the most effective group of insecticide, but their use to contrast bed bugs is forbidden in all Europe. To be effective all insecticides



should be applied directly on the bed bug’s body: for this reason the formulation of the insecticide can seriously interfere with its success (15).

In Italy, as well as in other countries (27), non-toxic techniques and monitoring of post-treatment actions are preferred to the use of insecticides (7, 20, 31).

Scientific publications (25, 31, 41, 49), together with seminars, brochures and catalogues edited by the Associazione Nazionale delle Imprese di Disinfestazione (ANID) (2, 3, 4, 6, 7, 8, 9, 10, 13, 16, 11, 20, 27, 30, 32, 34, 40, 42) reveal that the topic 'bed bugs and control strategies' is of fundamental interest to the pest control sector.

Bed bugs can cause unpleasant bite reactions and significant blood loss in chronic infestations (36, 47). The feeding in itself does not produce any pain, while sensitivity reactions are the results of substances injected during feeding (28). Approximately 20 human pathogens have been shown to survive for varying lengths of time in C. lectularius. However, there is little or no evidence that the insect is a vector of these agents (12). People are most often bitten on the limbs, trunk and face and individual reactions vary from no response to anaphylactic reaction. The cutaneous reaction can be localized or systemic. The sign of the bite can often be visible in the middle of the lesion. The local reaction can be similar to an uncomplicated mosquito bite or, in some individuals, can cause large fluid-filled blisters. Erythema is uncommon, but may occur as a result of multiple feedings that cause extensive subcutaneous hemorrhaging (43). Severe infestations are usually accompanied by typical sweet and sickly smell. The severity of the cutaneous reaction depends on the immunologic status of the host, however the exact immune basis of the cutaneous reaction is not known. According to some authors (1), antigens in bed bugs’ saliva stimulate the production of IgG antibodies, while another study (23) showed the presence of IgE antibodies. During the first exposure bed bugs’ bites can be totally asymptomatic; cutaneous reactions can develop up to 11 days after the bite. In most of the cases, however, the reaction occurs within 24 or 48 hrs, and within minutes in hypersensitive individuals. Secondary infections, as folliculitis, cellulitis and impetigo, that can result from a bite are also well documented (15).

Several theories have been provided to explain the recent rise in bed bug infection rate worldwide, although none has been scientifically demonstrated (15). The increase of international travels is considered as the primary reason, since bed bugs can be passively transported over long distances by clothing and luggage (12). Some authors (38) also report that the trade of second-hand furniture can be an easy way to transfer bed bugs, as it has also been shown by the results of this study. The widespread use of air

339

Bed bugs resurgence should be considered as an emerging public health problem and direct cooperation between specialists in the National Health Service is necessary both for diagnosis and management. Further investigations would be necessary to better understand the epidemiology in Italy and Europe and to assess the real public health risk.

AcknowledgmentsThe authors are grateful to Dr Marco Ballardini for Figure 1b.


Giorda et al. Cases of bed bug (Cimex lectularius) infestations in Northwest Italy

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2. Aventis CropScience Italia 2002. Non lasciate alle cimici dei letti il tempo di reagire! Igiene alimenti - disinfestazione & igiene ambientale, 19, 41.

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SummaryThe Republic of San Marino is an autonomous State that, in view of its geographical and environmental features, can be considered a part of the Northern Italian territory, where the canine leishmaniasis (CanL) is endemic. In the past, a CanL focus in the Republic’s kennel was described. As a consequence of this epidemiological situation, a surveillance program was carried-out covering a 6-year period (2006-2012). A total of 1,094 sera were collected from 420 kennel dogs and examined for antibodies to Leishmania infantum by the indirect fluorescent antibody test (IFAT). Eighty-eight (21%) dogs resulted IFAT positive (antibody titre ≥1/40). The overall seroprevalence increased in the first 4 years (2006-2010), going from 5.5% to 26.8% and then decreased in the 2 following years going to 17.9% (2011) and 3.9% (2012). The cumulative incidence constantly increased from 0.6% to 2.6%. This trend could be attributed to a changed infection pressure due to the dog turnover in the kennels. According to the observed incidence values, the CanL focus seems to be stable, supported by autochthonous transmission, new case introduction and Leishmania spp. circulation in owned dogs in the same area.

RiassuntoLa Repubblica di San Marino è uno Stato autonomo confinante con l’Emilia Romagna, dove la leishmaniosi canina (LCan) è endemica. Nel presente studio sono descritti i risultati di un programma di sorveglianza della durata di 6 anni attivato nel canile della Repubblica di San Marino, a seguito di un precedente focolaio di LCan all’interno di tale struttura. Complessivamente sono stati analizzati, mediante immunofluorescenza indiretta (IFI), 1.094 sieri prelevati da 420 cani, 88 dei quali sono risultati positivi (IFI ≥ 1/40). Nel periodo 2006-2010 la sieroprevalenza ha presentato un incremento dal 5,5% al 26,8%, mentre negli anni 2011 e 2012 è stata registrata una diminuzione al 17,9% e 3,9%, rispettivamente. L’incidenza cumulativa è aumentata costantemente da 0,6% a 2,6%. Tale andamento può essere imputato a modifiche della pressione di infezione conseguenti al turnover degli animali. Sulla base dei dati di incidenza osservati, il focolaio di LCan nel canile della Repubblica di San Marino sembra essere attualmente stabile, supportato dalla trasmissione autoctona dell’infezione, dall’introduzione di nuovi casi e dalla circolazione di Leishmania spp. in cani di proprietà presenti nella stessa area geografica.

Programma di sorveglianza della leishmaniosi caninain un canile della Repubblica di San Marino

Parole chiaveCane,Canile,Immunofluorescenza indiretta,IFI,Leishmania infantum,Repubblica di San Marino,Sorveglianza.

KeywordsDog,Kennel,Indirect fluorescent antibody test (IFAT),Leishmania infantum,Republic of San Marino,Surveillance.

1 Unit Department of Veterinary Medical Sciences, Alma Mater Studiorum-University of Bologna,Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy

2 Department of Prevention U.O.S. Public Veterinary Health and Food Hygiene,Via La Toscana 3, 47893 San Marino Republic

* Corresponding author at: Department of Veterinary Medical Sciences, Alma Mater Studiorum-University of Bologna,Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy.

Tel.: +39 051 2097061, e-mail: [email protected]

Daniela Salvatore1, Antonietta Di Francesco1, Maria Parigi1, Giovanni Poglayen1,Maurizio Battistini2 & Raffaella Baldelli1*

Canine leishmaniasis surveillance programin a San Marino Republic kennel


Both roaming or abandoned dogs are sheltered in the kennel: the animals are either re-housed or remain there permanently (31). The canine population consists of about 160 dogs. A mean of 38 dogs enters into the kennel each year.

The dog’s health conditions are checked by the public Veterinary Service of the Republic of San Marino. After the first detection of the CanL focus, each year, from June to September, all dogs are provided with deltamethrin-impregned collars against sand fly bites. Once a year, before the transmission season, serological tests are performed on all housed animals as well as on the new entry dogs.

From 2006 to 2012, a total of 1094 sera from 420 kennel dogs were collected and analysed (Table I). The number of animals tested every year changed, according to the turn over of the animals in the kennel. Only 40 dogs, which never moved from the kennel, were examined throughout the study period.

Anamnestic data (sex, estimated age, breed, origin, date of entry in the kennel, clinical signs and therapeutic treatment referred to leishmaniasis) were recorded for each dog. Two hundred and fifty six out of 420 (61%) animals were male and 159 (37.9%) were female, while sex data were not available about 5 dogs (1.1%). Two hundred and twenty two out of 420 (52.9%) dogs were crossbreed dogs, 70 (16.7%) hunting dogs, 37 (8.8%) belonged to other breeds, 91 (21.7%) were breed-undetermined. Referring to the age, 194 out of 420 (46.2%) were young (< 3 years old), 76 (18.1%) adult (3-7 years old), 57 (13.6%) old (≥ 7 years old), while the age was not established for 93 (22.1%) animals. One hundred and forty two out of 420 dogs originated from the Republic of San

IntroductionCanine leishmaniasis (CanL) due to Leishmania infantum is endemic in the whole Mediterranean basin, representing an important public health problem, since dog is considered the main reservoir of the infection (13).

In Italy, until the 1970s, CanL was endemic in Central and Southern regions, including islands, while Northern Italy was considered free from CanL, with the exception of Liguria and few areas in the Emilia-Romagna region. Since the mid-1980s, a progressive spread of the infection has been observed both in the old foci of Central and Southern regions and in the previously free Northern ones (8, 10, 11, 19, 20, 26, 28, 32, 35).

In the Emilia-Romagna region, in the early 1970s, a severe human visceral leishmaniasis outbreak occurred without evidence of a canine reservoir, although seropositive dogs were identified (18, 24). Several years later, new foci of CanL and the re-emergence of the old one have been reported (3, 20, 22). Starting with 2007, the Emilia-Romagna region promoted a regional surveillance program focusing on vector-borne diseases, including leishmaniasis (34).

The Republic of San Marino is a small autonomous State within the Italian territory, neighbouring CanL endemic areas of the Emilia-Romagna region (3, 6). In 2003-2004, a CanL autochthonous focus in the Republic kennel was observed, showing seroprevalence and incidence values of 7.1% and 6.6%, respectively (4). Further investigations allowed for isolating a strain of L. infantum zymodeme MON-1. Moreover, Phlebotomus perfiliewi, the proved L. infantum vector and typical sand fly in the Emilia-Romagna foci (2, 3, 15), was detected in the kennel. In the present study we report the results of a 6-year surveillance program carried out to evaluate the dynamics of the infection in the Republic’s kennel.

Materials and methodsThe Republic of San Marino (43° 46’ N, 12° 25’ E) is located in the middle of Italy and borders with Rimini and Pesaro-Urbino provinces, respectively in the Emilia-Romagna and Marche regions (Figure 1). The land covers an area of 61,196 km2, mostly hilly and clayey. The climate is continental, with hot summers and cold winters and heavy snowfalls. The summer mean temperatures range between 20°C and 30°C, with peaks of 35°C, the winter temperatures range between -5°C and 10°C. The kennel is sited in a hilly area used for the intensive cultivations of wheat, grapevine and orchard, surrounded by rich vegetation.

Canine leishmaniasis in a San Marino Republic kennel Salvatore et al.

Republic ofSan Marino

Figure 1. Localization of the Republic of San Marino within the Italian territory.

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substituted with 2 mg/kg/SID miltefosine per os for 28 days (33), because of its easier administration.

Data were collected into Microsoft Excel®, and then analysed by Statistical Package for Social Science version 14 (SPSS, Inc., Chicago, USA). The seroprevalence (all seropositive dogs per year/ dogs sheltered per year) and the cumulative incidence (new cases with IFAT titre ≥ 1/160 per year/ new cases with IFAT titre ≥ 1/160 per year + population time at risk) were calculated from 2006 to 2012 and from 2007 to 2011, respectively.

The chi-square test was used to evaluate the relationships between IFAT results and the others variables and to compare the incidence value with the number of new entries per year (p ≤ 0.05). The prevalence values per year were compared using the Fisher’s exact test (p ≤ 0.05).

ResultsSerological results are reported in Table II and III.

Out of 420 dogs tested, 88 (21%) were IFAT positive (≥ 1/40) in at least 1 test during the entire surveillance period. Fourteen dogs showed an active infection (IFAT titres ≥1/160): 5 of them were tested only once and no information about therapy has been reported. The remaining 9 dogs were treated and after the therapy, 6 showed IFAT titres fluctuating from doubtful to negative, 2 dogs confirmed the previous IFAT titres and one dog was tested and treated only once.

Marino and 5 from Emilia-Romagna and Abruzzo regions; the geographical origin was not reported for 254 dogs. Clinical signs of leishmaniasis were observed in 21 dogs in at least one control.

The samples were tested by the indirect fluorescent antibody test (IFAT), the ‘gold standard’ test for the diagnosis of CanL, according to the laboratory procedures described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (36). An in-house antigen consisting of promastigotes of L. infantum strain MHOM/TN/80/IPT1 was used. The cut-off was set at 1/40. Animals with IFAT titre of 1/40 and 1/80 were considered exposed to the parasite. Dogs showing antibody titres ≥ 1/160 were considered infected and treated (14) by 100 mg/kg/SID (semel in die; once a day) s.c. for 28 days in association with 10 mg/kg/BID (bis in die, twice a day) allopurinol per os for 6 months; then meglumine antimoniate was


Salvatore et al. Canine leishmaniasis in a San Marino Republic kennel

Table I. Dogs tested for canine leishmaniasis from 2006 to 2012 in San Marino Republic.

Year Total dogs New Entry2006 165 28

2007 176 51

2008 173 55

2009 166 39

2010 153 45

2011 134 26

2012 127 28

Table II. Serological results on canine leshmaniasis from 2006 to 2012 in San Marino Republic.

Year2006 2007 2008 2009 2010 2011 2012

IFAT results<1/40 156 159 151 141 112 110 106

1/40-1/80 6 13 21 23 37 18 4

≥1/160 3 4 1 2 4 6 1IFAT = indirect fluorescent antibody test

Table III. Serological results on canine leshmaniasis from 2006 to 2012 divided by resident and new entry dogs in a kennel of San Marino Republic.

YearResident dogs New entry dogs

<1/40 1/40-1/80 ≥1/160 <1/40 1/40-1/80 ≥1/1602006 129 5 3 27 1 /

2007 117 6 2 42 7 2

2008 106 12 / 45 9 1

2009 108 18 1 33 5 1

2010 78 29 1 34 8 3

2011 88 15 5 22 3 1

2012 95 3 1 27 1 /

344

The overall seroprevalence increased from 5.5% (2006) to 26.8% (2010) and then decreased to 17.9% (2011) and 3.9% (2012) (Figure 2). Seroprevalence calculated on resident dogs modified significantly (p ≤ 0.05), increasing from 5.8% (2006) to 27.7% (2010) and then decreasing to 4.0% (2012) (Figure 3). The cumulative incidence value constantly increased from 0.6% (2007) to 2.6% (2011) (Figure 2). It is noteworthy that no new cases have been recorded in 2012.

Forty out of 420 dogs have been controlled throughout the monitoring period, 27 of them proved constantly seronegative, while 11 animals fluctuated from a negative to exposed status and 2 seroconverted to IFAT titres ≥1/160 and were treated.

In frequency order, lymphadenopathy, dermatitis, onychogryphosis, alopecia, and poor condition were observed. The comparison of the overall seroprevalence with sex, age, breed and clinical signs of the animals did not show a statistically significant difference (p > 0.05), as well as the comparison of the incidence value with the number of new entries. With reference exclusively to 2009-2011 period, the rate of the old (≥7 years old) seropositive dogs with clinical signs increased significantly from 4.2% (2009) to 6.7% (2011) (p ≤ 0.05).

DiscussionIn the first year of the surveillance program, a seroprevalence value (5.5%) lower than the one (7.1%) previously reported in the same focus (4) was observed. The decreasing of the value could be attributed to the euthanasia applied in 2004 to all seropositive dogs and to the antivectorial measures provided for those seronegative.

During the survey the seroprevalence and the cumulative incidence increased from 5.5% (2006) to 26.8% (2010) and from 0.6% (2007) to

2.6% (2010), respectively; then the seroprevalence decreased to 17.9% (2011) and 3.9% (2012), while the incidence value remained constant. This trend might be explained by the turnover of the animals in the structure, which could contribute to increase/decrease the infection pressure. A higher number of dogs entered the kennel from 2007 to 2010 than it did in 2011-2012 period. It is noteworthy that the greatest number of seropositive animals was introduced in 2010, when the highest seroprevalence was reported. Nevertheless, no significant association has been showed in comparing the prevalence of new entry and resident dogs. These results are different from those observed in a ‘close system’ in which no new dogs were admitted: a significant decreasing of seroprevalence and incidence was reported from the beginning to the end of the study period, related to the therapeutic and prophylactic measures applied (27).

Forty dogs permanently housed in the kennel have been monitored throughout the surveillance period, Although more than the half of them resulted constantly negative, 11 showed fluctuating antibody titres and 2 seroconverted to IFAT titres ≥1/160, suggesting a poor efficacy of the antivectorial measures.

Nine out of 14 dogs showing IFAT titres ≥ 1/160 in at least one control were subjected to recommended therapeutic protocols (14, 34). In most of the dogs, IFAT titres decreased confirming that an appropriate therapy can reduce the parasitic load. Although treated animals continue to harbour the parasite, the therapy represents a key factor to control the infection spread (14, 21), by inducing a significant reduction of the infectious load to sand flies.

No significant association between serological results and sex, breed and clinical signs was detected. Similar results have been reported in studies carried out in Spain and Greece (1, 12, 16), while significant



%

Year

0

0.05

0.1

0.15

0.2

0.25

0.3

2006 2007 2008 2009 2010 2011 2012

PrevalenceIncidence

Figure 2. Prevalence of canine leshmaniasis in dogs of San Marino Republic from 2006 to 2012; incidence from 2007 to 2011.

%

Year

0

0.05

0.1

0.15

0.2

0.25

0.3

2006 2007 2008 2009 2010 2011 2012

Prevalence residentPrevalence new entry

Figure 3. Prevalence of canine leshmaniasis calculated on resident and new entry dogs in a kennel of San Marino Republic from 2006 to 2012.


Salvatore et al. Canine leishmaniasis in a San Marino Republic kennel

period, which was probably insufficient to observe improvements related to control measures.

In the present study, the CanL prevalence is quite similar to the one reported in the kennels of the Emilia-Romagna region (17), mainly in the bordering Rimini province (9), an historical endemic area for about 10 years (2, 3, 6).

In general, the kennel is not a good epidemiological observatory, because the dog population is characterized by a turnover of free roaming or abandoned animals and data about the geographical origin often lack or are incomplete and incorrect. This was the case of the canine population described in this article, which was dynamic, variable and not foreseeable. Nevertheless, several data about the CanL spread in Italy are collected in kennels (5, 9, 23, 29, 30). Despite the limits related to the kennel features, the control of these facilities provides a useful tool to avoid the spread of the infection to free areas, which could easily escalate given the increased number online kennel dog adoptions.

differences with respect to the sex and the breed have been observed in Croatia (37).

Starting with 2009 until 2011, an increased frequency of infected symptomatic old dogs was observed, showing how the risk of exposure to infection raises with the age of the animal, which also prompts a decreasing in the immune competence of the host (7, 25, 29).

ConclusionsAccording to the incidence values observed in the present study, the CanL focus in the Republic kennel seems to be stable, supported by autochthonous transmission, new case introduction and Leishmania spp. circulation in owned dogs in the same area (data not shown).

Incidence values reported are not very high if compared with those registered in a previous study conducted in another kennel (5) for a shorter

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SummaryThis paper describes the diagnostic and clinical observations of an infectious bronchitis virus (IBV) variant, referred to as Q1, in clinically ill chickens in Italy. This IBV variant was described for the first time in 1998 in China. In the autumn of 2011 it caused a small-scale epidemic in non-vaccinated meat chickens in farms located in Northern Italy. The disease was characterized by increased mortality, kidney lesions and proventriculitis. Histopatological observations confirmed the nephritis and described an unusual erosive/necrotic proventriculitis with infiltration of lymphocytes, plasma cells and heterophils, as well as fibroplasia in the lamina propria. Despite these findings and the isolation of the Q1 IB virus directly from proventricular tissue, further studies are necessary to confirm the role of this IBV strain in the development of proventricular lesions. Phylogenetic analysis revealed that all the IBV isolates were very similar and probably had a common origin. The IBV Q1 variant appears to be now endemic in the North of Italy and at times it is detected in vaccinated backyard and commercial broiler farms. The importance of continuous monitoring in controlling the spread of known or emerging IBV variants is underlined.

RiassuntoL’articolo concerne la descrizione clinica e la diagnosi dei focolai sostenuti dalla variante Q1 del virus della bronchite infettiva aviare (IBV). Tale variante, descritta per la prima volta in Cina nel 1998, non era mai stata segnalata al di fuori del continente asiatico. Tuttavia, tra luglio e settembre 2011, in alcuni allevamenti di polli da carne (Hubbard naked neck) destinati al mercato dell’Italia settentrionale è stato rilevato un insolito aumento della mortalità (4,1-9,8%), preceduto da sintomatologia respiratoria caratterizzata da rantoli e scolo nasale. All’esame anatomopatologico sono stati rilevati congestione, edema polmonare, tracheite catarrale e aerosacculite fibrinosa. I reni degli animali deceduti sono risultati pallidi e globosi con depositi di urati intraparenchimatosi e, nei casi più gravi, sulle sierose viscerali. Anche l’apparato gastroenterico è risultato coinvolto con un ispessimento del proventricolo e congestione dello sbocco ghiandolare. L’esame istologico ha confermato la nefrite e ha evidenziato una grave proventricolite di tipo necrotico-erosiva con infiltrazione di linfociti, plasmacellule ed eterofili. L’isolamento di IBV Q1 direttamente dai proventricoli degli animali deceduti fa attribuire all’infezione virale un ruolo primario nella genesi della proventricolite. Ulteriori studi sull’argomento si rendono necessari visto che i dati presenti in letteratura risultano contrastanti. L’analisi filogenetica ha caratterizzato i virus isolati dai diversi allevamenti come appartenenti ad un unico cluster. L’elevata omologia genetica dei ceppi isolati e le connessioni epidemiologiche tra i vari allevamenti colpiti fanno ipotizzare un’origine comune dei focolai. La variante Q1, rilevata negli allevamenti del Nord Italia vaccinati contro la bronchite infettiva, suggerisce che IBV sia diventata endemica nel nostro Paese.

Descrizione di focolai di bronchite infettivasostenuta dal ceppo Q1 recentemente segnalato in Italia

Parole chiaveBronchite infettiva,CK/CH/LDL97I/97,Pollo da carne,Proventricolite,Variante Q1.

KeywordsBroiler,CK/CH/LDL97I/97,Infectious bronchitis,Proventricolitis,Q1 variant.

1 OIE/FAO and National Reference Laboratory for Avian Influenza and Newcastle disease, OIE Collaborating Centre for Diseases at the Human Animal Interface, Istituto Zooprofilattico Sperimentale delle Venezie,

Viale dell’Università 10, 35020 Legnaro (Padova), Italy.2 Veterinary Diagnostic Laboratory, Istituto Zooprofilattico Sperimentale delle Venezie,

Vicolo Mazzini 4, 31020 Villorba (Treviso), Italy.3 Histopathology Laboratory, Istituto Zooprofilattico Sperimentale delle Venezie,

Viale dell’Università 10, 35020 Legnaro (Padova), Italy.

* Corresponding author at: Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università 10, 35020 Legnaro (Padova), Italy.Tel.: +39 049 8084333, e-mail: [email protected].

Anna Toffan1*, Michela Bonci2, Luca Bano2, Viviana Valastro1,Marta Vascellari3, Ilaria Capua1 & Calogero Terregino1

Diagnostic and clinical observationon the infectious bronchitis virus strain Q1 in Italy


and poor growth, sometimes associated with nasal discharge and rales. Total mortality was high, ranging from 4.1 to 9.8%. The affected subjects were 4 to 39 day old Hubbard naked neck chickens supplied by a hatchery located in the North-West of Italy and destined for sale to backyard flocks. Birds had been vaccinated at 1 day of age against Newcastle disease. At the same age, chicks were routinely vaccinated with IBV H120 strain, but vaccination was suspended from July the 4th to August the 13th, as the results of an independent decision taken by the hatchery.

Bacteriology and histopathologyCarcasses (n = 83) belonging to 12 flocks from 7 farms were submitted for necropsy. Samples from the pericardium, liver, brain, spleen, and air sacs of birds showing lesions indicating colisepticaemia (such as pericarditis, perihepatitis or airsacculitis) were plated on both Blood and Eosin-Methylene Blue agar plates. The plates were incubated at 37±1°C for 24-48 hrs under aerobic conditions. Swabs collected from air sacs and pericardium were plated on Columbia sheep blood agar in the presence of a Staphylococcus hycus helper culture and plates were incubated in 5% to 10% carbon dioxide for 18 to 48 hrs at 37°C. Identification of isolated strains was achieved by biochemical tests.

On the basis of the reported clinical signs and observed lesions, the presence of Mycoplasma gallisepticum and Mycoplasma synoviae was also investigated in each farm by polymerase chain reaction (PCR) according to the protocol developed by Garcia et al. (10), from different organs namely: lungs, heart, larynx and joint (n = 15).

Samples of organs that were apparently more affected, such as the proventriculus and kidneys, were immediately fixed in 10% neutral buffered formalin for histological examination. After 24 hrs of fixation, samples were processed, paraffin embedded, stained with haematoxylin and eosin (HE) and observed by standard light microscopy.

VirologyOrgans (kidneys, lungs, trachea, proventriculus, cecal tonsils) were homogenized with sterile quartz and inoculated in 9-11 day old Specific Pathogen-Free (SPF) embryonated fowl eggs for virus isolation according to standard procedures (11). Both 3 and 7-day-long passages were performed and allantoic fluids were analyzed by Real Time Reverse Transcriptase PCR (RT-PCR) following the protocol described below.

Viral RNA was extracted from target organs (kidney, lungs, trachea, proventriculus and cecal

IntroductionInfectious bronchitis (IB) is an acute and highly contagious disease of affecting chickens. The viral agent of IB, a group 3 Coronavirus, is a single-stranded RNA virus characterised by rapid spread and a notable capacity to modify its genome both by spontaneous mutation and genetic recombination (6). The continuous emergence of new variants can threaten intensive poultry production despite the global availability and application of vaccination. The existence of undetected reservoirs for these viruses, which may include wild birds and/or domestic poultry in developing countries, has been suggested by Cavanagh and Gelb (7). The rapidly increasing intensive poultry production occurring in these days in the developing countries, associated with little knowledge of local infectious bronchitis virus (IBV) circulation and, consequently, the inadequate application of vaccines could act as a source of new IB variants (8). For example, an IB variant virus, firstly described in China and called QX, spread rapidly through Europe becoming one of the most economically damaging viruses of poultry of the last decade (8, 29). Indeed several new IBV variants have been emerging in developing country (9, 17, 18). At the same time, other viruses seemed to have disappeared for long time following their first description and only recently re-emerged in different Asian countries. This is the case of the Q1 IBV, also known as CK/CH/LDL97I/97 and described in this paper. The virus was initially detected in China and only after several years its presence has been reported in North Italy (1, 31). The reason why some strains spread readily at a global level while others remain more local is unknown. Indeed, little is known even on how these viruses spread between neighbouring countries.

In the Summer of 2011, an unexpected occurrence of the Q1 IB virus was reported in a diseased poultry flock in the North-East of Italy (26). Subsequently a small-scale IB epidemic caused by this IB variant was observed in the same region.

This paper aims to follow up the clinical and pathological features of first naturally occurring infection with IBV Q1 in Italian meat chickens.

Materials and methods

Case historyBetween mid-July and the beginning of September 2011, 7 commercial broiler farms located in the North-East of Italy noticed the onset of non-specific clinical signs, such as depression, ruffled feathers

Diagnostic and clinical observation on the IBV in Italy Toffan et al.

349

Results

Post-mortem examinationLesions of the respiratory, urinary and digestive apparatus were observed during the necropsy procedure. Lesions of the respiratory organs included congestion and oedema of the lungs and airsacculitis with deposition of fibrinous exudates on air sacs, while lesions of the urinary apparatus ranged from pale and swollen kidneys to urate crystal deposition in the kidney parenchyma and in the ureters and visceral uricosis (Figure 1). Fibrinous exudates were also seen on the heart and liver. Lesions of the digestive tract were confined to the proventriculus and consisted of the thickening of the wall, in some cases associated with proventricular congestion at the point of emergence of the glandular ducts (papilla) (Figure 2). The simultaneous involvement of all 3 apparati was not observed in all chickens. Nonetheless, at least 2 apparati per each group of chickens submitted to our laboratories for post-mortem investigations were affected.

Bacteriology and histopathology: the bacteriological examination of swabs collected from birds showing

tonsils) and RT-PCR targeting the conserved 5’- untranslated region of the viral genome was carried out to screen for the presence of the IBV genome. Four positive samples were processed by RT nested-PCR using primers annealing to a conserved region of the spike protein 1 (S1), as described by Jones et al. (14). The spike protein 1 (S1) gene fragment (approximately 320 nucleotides) of 6 out of 7 isolates (namely: farms 2, 3, 4, 5, 6 and 7) was sequenced. The virus isolated from farm 1 was selected as representative of the study group and processed by one-step RT-PCR following an in-house protocol (primers sequence available upon request), which made the generation of the complete S1 protein sequence (1493 nucleotides) possible. The generated PCR products were processed and sequenced in a 16 capillary ABI 3130 XL Genetic Analyzer (Applied Biosystems, Padova, Italy) genetic analyzed. Virus sequences of the S1 gene fragment were aligned and compared with representative sequences of different IBV variants available in GenBank. The phylogenetic tree was generated using Bayesian methods available in the MrBayes v.3.1.2 software (MrBayes, opensource software, available at http://mrbayes.sourceforge.net/index.php); nucleotide diversity was calculated using the Maximum Composite Likelihood method implemented in the MEGA4 software package (23, 25).

Gross lesions were occasionally observed on birds carcasses, which suggested the presence of viral diseases other than IBV. The organs were subsequently tested by real-time RT-PCR for Newcastle Disease (n = 12), Avian influenza (n = 12) and Infectious Bursal disease (n = 5) according to published protocols (5, 19, 22).

SerologySera collected from infected farms were analyzed by the haemagglutination inhibition (HI) test according to standard protocols (11). Homologous antigens were produced using the Q1 isolate obtained from farm 1. In a nutshell, virus was inoculated in 9-11 day old SPF embryonated fowl eggs and incubated for 72 hrs. Eggs were chilled and allantoic fluid harvested. This was then centrifuged for 92 min at 20,000g and the supernatant discarded; the remaining antigen was re-diluted with a ratio of 1:100 in phospholipase and incubated for 3 hrs at room temperature.

Haemagglutination inhibition was performed against classical IBV variants: M41; 793/B; 624/I; IT02, D274, QX and with the new Q1 antigen. In addition, sera were collected also from the parents of the affected chicks and analyzed in order to assess Q1 IBV antibody level.


Toffan et al. Diagnostic and clinical observation on the IBV in Italy

Figure 1. Kidney, chicken. Swollen kidneys with massive urate crystal deposition in the kidney parenchyma and in the ureters.

Figure 2. Proventriculus, chicken. Thickening of proventricular wall with prominence of papilla.

350

airsacculitis, pericarditis and perihepatitis yielded pure cultures of Escherichia coli. All the samples analysed for M. gallisepticum and M. synoviae were negative.

By histological examination, mild multifocal infiltration of plasma cells and lymphocytes was observed in the interstitium of the renal cortex (Figure 3). The proventriculus was affected by mild to severe proventriculitis, characterized by multifocal erosion and necrosis of the tunica mucosa and glandular epithelium, associated with infiltration of lymphocytes, plasma cells and heterophils, as well as fibroplasia in the lamina propria (Figure 4 and Figure 5).

VirologyInfectious bronchitis virus’ RNA was detected by real-time RT-PCR in the majority of the organs tested, while no positive results were observed for the other

viral diseases tested: Avian Influenza, Newcastle Disease and Infectious Bursal Disease.

Six out of the 7 viral sequences (those from farm 1, 2, 3, 4, 6 and 7) were identical and showed a percentage of identity of 99.2% when compared with the IBV virus of farm 5. For this reason, only the S1 sequences of viruses from farm 1 and farm 5 were included in the phylogenetic tree (Genbank accession number JQ290229 and JQ419754, respectively). The virus from farm 1 showed a nucleotide identity of 100% with the Q1 strain (Genbank accession number AF286302) and 99.2% with the strain CK/CH/LDL97I/97 (Genbank accession number DQ068701). The S1 sequence of the virus from farm 5 showed an identity of 98.3% with the CK/CH/LDL97I/97 strain (Figure 6). Furthermore, close similarity was observed between the viruses under study and the 624/I IBV variant (97.5% and 96.6% with farm 1 and 5, respectively).

Virus isolation was achieved in all organs (lungs, kidneys, trachea, proventriculus) and in most cases after only one passage in SPF embryonated chicken eggs. Typical lesions including dwarfed, haemorrhagic and curled embryos in positive fowl eggs were observed. Virus isolation was achieved from 5 out of 7 real-time RT-PCR positive periventricular tissue coming from 3 different infected farms. The IBV isolate from farm 1 was used as reference antigen for the HI test.

SerologyThe HI test performed on serum samples collected from sick birds confirmed the presence of specific antibodies against Q1 IB variant. Specific HI titres against Q1 were generally 2 to 4 logs greater than those against classical IB virus variants (i.e. M41 and 793B). Cross reactions were observed with other IB variants, mainly with 624/I and D274 (Figure 7).



Figure 3. Kidney, chicken. Interstitial infiltration of lymphocytes and plasma cells in the renal cortex is evident. Hematoxylin and eosin stain. Bar = 50 µm.

Figure 4. Proventriculus, chicken. Diffuse necrosis of superficial and glandular epithelium, associated with infiltration of heterophils lymphocytes and plasmacells in the lamina propria. Hematoxylin and eosin stain. Bar = 100 µm.

Figure 5. Proventriculus, chicken. Diffuse epithelial necrosis, associated with infiltration of heterophils lymphocytes and plasmacells in the lamina propria. Hematoxylin and eosin stain. Bar = 50 µm.



Figure 6. Phylogenetic tree constructed by Bayesian analysis of the S1 gene fragment of IB viruses. The viruses under examination are highlighted with a black circle in the tree. Posterior probabilities of the clades are indicated above the nodes. The scale bar indicates the number of nucleotide substitutions per site.

0

2

4

6

8

10

12

farm 1 .1 (4) farm 1.2 (5) farm 3 (10) farm 4 (20) farm 5.1 (4) farm 5.2 (5) farm 5.3 (6) breeder farm (10)

GM

T lo

g2 H

I tit

re

ID farms

Q1 QX 624/I M41 793B D274 ITA02

Figure 7. GMT (geometric mean titer) of the log2 haemagglutination inhibition test titers of Q1 infected farms and breeder farm tested. Vertical bars refer to standard deviations. Number of sera tested per farm is reported in brackets.



Middle East showing tracheitis, nephritis but no proventriculitis (1, 17). Another IBV variant (QX) of worldwide significance, originating from China, has been primarily reported in association with proventriculitis (29). However, in all the relevant reports, included this one, it is almost impossible to unequivocally demonstrate the role of this IBV strain in the genesis of the proventriculitis, as well, factors other than IBV infection which may be the cause e of proventricular lesions have not been ruled out emphatically. Furthermore, the pathogenicity and tissue tropism of the IBV Q1 strain may change under different field conditions and therefore requires further characterization (12, 16, 31).

From a phylogenetic point of view, the Italian Q1 clustered separately from the classical IBV variants, appearing to have the closest relationship with the 624/I strain. All the viruses isolated during the outbreaks described herein showed a very high homology rate, suggesting a common origin due to a single introduction. A field study showed that all the affected farms were served by the same hatchery located in the North-West of Italy. This hatchery normally buys eggs from a single broiler breeder. However in July 2011 the hatchery purchased an additional batch of eggs from France. At the same time, for commercial reasons, the vaccination against IBV routinely performed (H120 strain by eye drop at one day) was interrupted for nearly 40 days. Additional investigations performed on the broiler parent flock showed no IB virus circulation (data not shown), and high levels of antibodies against IBV induced by multiple vaccinations. Considering all these data, it seems that the virus probably originated from the hatchery or was carried by the lorry used to move one day old chicks to the farms and, due to the lack of protection given by IBV vaccination, it was able to spread.

Very little is known on the protection against this variant induced by the available commercial vaccines. A consistent decrease in Q1 positive farms (Table I) after the re-establishment of vaccination with the H120 strain in the hatchery was observed. Since no other IB vaccines were used by the farmer, we may assume a certain level of cross-protection between H120 and Q1 variant. Nevertheless, at present the IBV Q1 variant appears to be established in the Northen Italy (data not shown), as it is detected, although not frequently, from vaccinated broilers. Further monitoring of the spread of this IB strain in Italy and of the efficacy of vaccination in controlling this variant is therefore highly recommended.

The way in which the Q1 virus may possibly have reached Italy remains unclear. As shown by a recent viral survey performed by Tosi et al. (28) between 2007-2010, in which 368 IB viruses were characterized by RT-PCR or sequencing, it emerged

In serum samples collected from the broiler’s breeder flock, the HI titres were high against all the IB antigens tested without great differences among the different IBV strains. This was expected, since this flock had undergone standard breeder vaccination for IB.

Discussion Infectious bronchitis virus generally causes significant economic losses, primarily due to a reduced productivity rather than to an increased mortality rate. Nevertheless, the outcome of infection can vary depending on the breed of chickens, the presence and adequacy of vaccination, and the concomitant infection with other pathogens (6, 8, 20). In this case report a combination of factors such as the E. coli co-infection, the lack of protection given by a temporary suspension of IBV vaccination and, perhaps, the high susceptibility of the naked neck broiler chicken breed may have led to the onset of a small epidemic of IBV of the Q1 variant, characterized by a significant increase in mortality (ranging from 4 to nearly 10%). It is known that IB infection predisposes birds to E. coli infection, resulting in increased mortality (6). This was also our experience, in that E. coli was detected in samples from the majority of the tested organs, including lungs, air sacs, brain, spleen and liver of the birds submitted to the laboratory. The high mortality rate reported was probably due to the combined action of the two pathogens, IBV Q1 and E. coli.

In the reported outbreak, the main lesions did not differ from those caused by classical IBV strains, such as tracheitis, lung congestion, nephritis, except for those of the proventricular tissue which were frequently observed here, barring a few exceptions (26). Marked thickening of the proventricular wall and congestion of glandular ducts have always been associated with positive real time RT-PCR for IBV RNA. Notably, virus isolation analysis performed directly on this organ confirmed the presence of Q1 IBV in the proventricular tissue. The IBV Q1 variant, together with 2 other similar IBV strains (J2 and T3), was first identified in the proventricular tissue of 25-to 70-day-old layers suffering from the so called ‘avian disease associated with proventriculus’. Samples were collected between 1996 and 1998 in China (31); Yu et al. isolated the newly detected Q1 variant and the disease was experimentally reproduced in SPF chickens. During the experiment, infected SPF chickens showed severe proventriculitis associated with respiratory and enteric signs, whereas no kidney lesions were reported either in the field or in experimentally infected birds. Very recently, this IBV variant, also called CK/CH/LDL97I/97, has re-emerged in vaccinated broiler flocks in China and in the



Table I. Summary of field data and laboratory results of Q1 positive farms.

ID farm Flock number

Age of chickens at submmission

(days)

Total mortality

(%)Gross pathology

Main histopathological

reports

Other patohgens

found*

farm 1

1 16 4.5

Fibrinous exudates on heart, liver and air sacsSevere lung congestionThickening of proventricular wall and congestion at the point of emergence of the glandular ductsMultiple necrotic foci in pancreasCatarrhal enteritis with petechial haemorrhages on the intestinal wallPale and enlarged kidneys

Erosive proventriculitisUrate crystal deposition in kidneys

reovirus† and coccidia

2 28 4.3

Thickening of proventricular wallCongestion of kidneysCatarrhal enteritis with petechial haemorrhages on the intestinal wallBursa slightly enlarged

n.d. reovirus†

farm 2 1 16 9.8

Lung congestionFibrinous exudates on heart, liver and air sacsThickening of proventricular wall and congestion at the point of emergence of the glandular ductsPale and enlarged liverPale and enlarged kidneys

Necrotic proventriculitisNecrotic hepatitisUrate crystal deposition in kidney

no

farm 3 1 19 4.5Lung congestionFibrinous exudates on air sacsSevere urate deposition in kidneys

n.d. no

farm 4

1 12 and 28 5.6

Lung congestionMassive fibrinous exudates on heart and air sacs Thickening of proventricular wallSevere enlarged and congested kidney

Erosive proventriculitisSevere nephritis with urate crystal deposition

no

2 25 4.1Massive fibrinous exudates on heart and air sacsEnlarged hocks with accumulation of fibrinous exudates

n.d. no

3 4 4.5Thickening of proventricular wallSevere urate deposition in kidneys with visceral uricosi

Erosive and necrotic proventiculitis no

farm 5

1 21 and 25 4.9

Fibrinous exudates on air sacs Thickening of proventricular wall and congestion at the point of emergence of the glandular ductsCatarrhal enteritisBursa slightly enlarged

n.d. no

2 39 4.8

Thickening of proventricular wall Enlarged liver Catarrhal enteritis Enlarged and congested bursa Swollen kidneys

Severe necrotic proventriculitis no

farm 6

1 21 5.0

Congestion of lung and tracheaFibrinous exudates on air sacs and liverPale pancreasKidney congestion and urate depositions

NephritisErosive proventiculitis

adenovirus‡

2 27 4.1

Congestion of trachea Thickening of proventricular wall Pale pancreas Enlarged and pale kidneys

Erosive proventriculitis coccidia

farm 7 1 20 4.5Fibrinous exudates on heart, liver and air sacsEnlarged and pale kidneys

n.d. coccidia

n.d.= not done; *Escherichia coli was detected in all tested farms; †Reovirus was isolated in cell cultures from pancreas; ‡Adenovirus was isolated in cell culture from lungs.



1. Ababneh M., Dalab A.E., Alsaad S. & Al-Zghoul M. 2012. Presence of Infectious Bronchitis Virus Strain CK/CH/LDL/97I in the Middle East. ISRN Vet Science, Article ID 201721, doi:10.5402/2012/201721.

2. Abro S.H., Renstrom L.H., Ullman K., Isaksson M., Zohari S., Jansson D.S, Belák S. & Baule C. 2011. Emergence of novel strains of avian infectious bronchitis virus in Sweden. Vet Microbiol, 155(2-4), 237-246.

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4. Callison S.A., Hilt D.A., Boynton T.O, Sample B.F., Robison R., Swayne D.E & Jackwood M.W. 2006. Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens. J Virol Methods, 138(1-2), 60-65.

5. Cattoli G., Susta L., Terregino C. & Brown C. 2011. Newcastle disease: a review of field recognition and current methods of laboratory detection. J Vet Diag Invest, 23(4), 637-656.

6. Cavanagh D. 2007. Coronavirus avian infectious bronchitis virus. Vet Res, 38(2), 281-297.

7. Cavanagh D. & Gelb J. 2008. Infectious bronchitis. In Disease of Poultry (Y.M. Saif, A.M. Fadly, J.R. Glisson, L.R. McDougald, L.K. Nolan & De Swayne eds.), 12th ed. Ames, Iowa, USA, Blackwell Publishing Professional, 117-135 p.

8. De Wit J.J., Cook J.K. & van der Heijden H.M. 2011. Infectious bronchitis virus variants: a review of the history, current situation and control measures. Avian Pathol, 40(3), 223-235.

9. Ducatez M.F., Martin A.M., Owoade A.A., Olatoye I.O., Alkali B.R., Maikano I., Snoeck C.J., Sausy A., Cordioli P. & Muller C.P. 2009. Characterization of a new genotype and serotype of infectious bronchitis virus in Western Africa. J Gen Virol, 90(Pt 11), 1679-1685.

10. Garcia M., Ikuta N., Levisohn S. & Kleven S.H. 2005. Evaluation and comparison of various PCR methods

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14. Jones R.C., Worthington K.J., Capua I. & Naylor C.J. 2005. Efficacy of live infectious bronchitis vaccines against a novel European genotype, Italy 02. Vet Rec, 156(20), 646-647.

15. Krapez U., Slavec B. & Rojs O.Z. 2011. Circulation of infectious bronchitis virus strains from Italy 02 and QX genotypes in Slovenia between 2007 and 2009. Avian Dis, 55(1), 155-161.

16. Liu S., Zhang X., Wang Y., Li C., Liu Q., Han Z., Zhang Q., Kong X. & Tong G. 2009. Evaluation of the protection conferred by commercial vaccines and attenuated heterologous isolates in China against the CK/CH/LDL/97I strain of infectious bronchitis coronavirus. Vet J, 179(1), 130-136.

17. Ma H., Shao Y., Sun C., Han Z., Liu X., Guo H., Liu X., Kong X. & Liu S. 2012. Genetic diversity of avian infectious bronchitis coronavirus in recent years in China. Avian Dis, 56(1),15-28.

18. Mahmood Z.H., Sleman R.R. & Uthman A.U. 2010, Isolation and molecular characterization of Sul/01/09 avian infectious bronchitis virus, indicates the emergence of a new genotype in the Middle East. Vet Microbiol, 150(1-2), 21-27.

Diffusion via commercial routes or by wild birds has been suggested, since increasing evidence proves that IBV has a host range not limited to poultry (6, 8, 30). Whatever its origin, the case report herein described highlights the importance of monitoring programs to control the spread of known or emerging IBV variants.

AcknowledgmentThe authors thank Dr William Dundon and Dr Francesca Ellero for critical editing of the manuscript.

that the detected IB variants belonged mostly to the 793/B, M41, QX and IT02 variants with sporadic detection of D274, 624/I and B1648 (28). It is noteworthy that thre are no records of the Q1 variant in Italy prior 2011. Apparently, this IBV variant had disappeared from the field for a long time, despite several subsequent field surveillances performed in China and in neighbouring countries it had never been detected until recently, when it was detected again in China, Iran, Jordan, Saudi Arabia and Europe (1, 12, 13, 17, 21, 26). Apart from 793/B and, more recently, QX, IBV variants are rarely reported to spread from one continent to another (6, 27, 30).

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25. Tamura K., Dudley J., Nei M. & Kumar S. 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol, 24(8),1596-1599.

26. Toffan A., Terregino C., Mazzacan E., Castaldello I., Capua I. & Bonci M. 2011. Detection of Chinese Q1 strain of infectious bronchitis virus in Europe. Vet Rec, 169(8), 212-213.

27. Toffan A., Monne I., Terregino C., Cattoli G., Hodobo C.T., Gadaga B., Makaya P.V., Mdlongwa E. & Swiswa S. 2011. QX-like infectious bronchitis virus in Africa. Vet Rec, 169(22), 589.

28. Tosi G., Taddei R., Barbieri I., Fiorentini L. & Massi P. 2010. Caratterizzazione molecolare dei ceppi di virus della bronchite infettiva aviare isolati in Italia nel periodo 2007-2009 e nel primo bimestre del 2010. In Atti della Società Italiana di Patologia Aviare, L Convegno Annuale, Forlì, Italy, 217 p.

29. Wang Y.D., Wang Y.L., Zhang Fan G.C., Jiang Y.H., Liu X.E., Ding J. & Wang S.S. 1998. Isolation and identification of glandular stomach type IBV (QXIBV) in chickens. Chinese J Anim Quarantine, 15(1), 1-3.

30. Worthington K.J., Currie R.J. & Jones R.C. 2008. A reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in Western Europe from 2002 to 2006. Avian Pathol, 37(3), 247-257.

31. Yu L., Jiang Y., Low S., Nam S.J., Liu W. & Kwangac J. 2001. Characterization of three infectious bronchitis virus isolates from China associated with proventriculus in vaccinated chickens. Avian Dis, 45(2), 416-424.

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21. Ovchinnikova E.V., Bochkov Y.A., Shcherbakova L.O., Nikonova Z.B, Zinyakov N.G, Elatkin N.P, Mudrak N.S, Borisov A.V. & Drygin V.V. 2011. Molecular characterization of infectious bronchitis virus isolates from Russia and neighbouring countries: identification of intertypic recombination in the S1 gene. Avian Pathol, 40(5), 507-514.

22. Peters M.A., Lin T.L. & Wu CC. 2005. Real-time RT-PCR differentiation and quantitation of infectious bursal disease virus strains using dual-labeled fluorescent probes. J Virol Methods, 127(1), 87-95.

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24. Sumi V., Singh S.D., Dhama K., Gowthaman V., Barathidasan R. & Sukumar K. 2012, Isolation and molecular characterization of infectious bronchitis virus from recent outbreaks in broiler flocks reveals emergence of novel strain in India. Trop Anim Health Pro, 44(7),1791-1795.



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SummaryIn this study 113 diarrhoeic faecal samples obtained from buffalo (n = 68) and cattle (n = 45) calves under 1 years of age were analysed in order to determine the presence of rotavirus infection and the frequency of picobirnavirus excretion. Eleven (9.73%) samples positive for group A rotavirus were identified through RNA-polyacrylamide gel electrophoresis (RNA-PAGE), while 4 (3.53%) samples showed a bisegmented genome with a typical picobirnavirus pattern. This is the first report of picobirnavirus in cattle and buffalo calves from Western India.

RiassuntoQuesto articolo descrive i risultati di uno studio condotto su 113 campioni di feci prelevati da bufali (n = 68) e bovini (n = 45) di età inferiore ad 1 anno e analizzati per rotavirus e picobirnavirus. Dei 113 campioni testati mediante RNA-polyacrylamide gel electrophoresis (RNA-PAGE), 11 (9.73%) sono risultati positivi per il ceppo A del rotavirus mentre 4 (3.53%) hanno mostrato un genoma bisegmentato tipico del picobirnavirus. È la prima volta che picobirnavirus è stato rilevato in bufali e bovini in India occidentale.

Presenza di picobirnavirus e rotavirusin campioni fecali diarroici di vitelli bovini e bufalini

nella città di Mumbai, India occidentale Parole chiaveIndia,Mumbai,Picobirnavirus,RNA- polyacrylamide gel electrophoresis,Rotavirus.

KeywordsIndia,Mumbai,Picobirnavirus,RNA- polyacrylamide gel electrophoresis,Rotavirus.

1 Department of Veterinary Microbiology, West Bengal University of Animal and Fishery Sciences,Belgachia, Kolkata 700037, West Bengal, India.

2 Division of Biological Sciences, Indian Veterinary Research Institute, Bareilly 243122, Uttar Pradesh, India.3 Department of Veterinary Microbiology, Bombay Veterinary College, Parel, Mumbai 400012, Maharashtra, India.

* Corresponding author at: West Bengal University of Animal and Fishery Sciences,Belgachia, Kolkata 700037, West Bengal, India.

Tel: +91 7278770611, e-mail: [email protected]

Anjan Mondal1*, Soumendu Chakravarti2, Sharmila B. Majee3 &Anilkumar Sadashivrao Bannalikar3

Detection of picobirnavirus and rotavirus in diarrhoeic faecal samples of cattle and buffalo

calves in Mumbai metropolis, Western India


Viral RNA was isolated with the guanidinium isothiocyanate using the lysis method (6). In brief, 500 µl of the clarified 10% faecal suspension was vortex-mixed with an equal volume of GIT lysis buffer and 0.1 volume of 2 M sodium acetate, pH 4.6 and kept on ice for 15 min. A mixture containing an equal volume of phenol, chloroform and isoamyl (PCI) alcohol (25:24:1) was added, vortexed, kept on ice for 15 min and centrifuged at 12,000 g for 10 min at 4°C. The supernatant was transferred into sterile tubes (Ratiolab GmbH, Dreieich, Germany). The PCI extraction was repeated twice. The aqueous phase was transferred into fresh tubes, with a mixture containing an equal volume of chloroform and isoamyl alcohol, vortexed and centrifuged at 12,000 g for 10 min at 4°C. Again, the aqueous phase was placed in fresh tubes and 0.1 volume of 3 M sodium acetate, pH 5.2 was added and vortexed. To this, an equal volume of isopropanol was added; tubes were inverted 4-5 times and kept at -20°C overnight for precipitation. RNA was pelleted by centrifugation at 12,000 g for 10 min at 4°C and the pellet was washed with pre-chilled 70% ethanol. The pellet was air dried, dissolved in 30 µl of nuclease free water and stored at -20°C until further use.

RNA-polyacrylamide gel electrophoresisThe genome of PBV was detected and analysed by RNA-polyacrylamide gel electrophoresis (RNA-PAGE) using discontinuous buffer system without SDS as described by Laemmli (14). The PAGE was performed at a constant voltage of 120 V (8-10 V cm) for 3 hrs using 8% resolving and 5% stacking gel. Subsequently, the gel was stained with silver nitrate according to the method described by Svensson et al. (22) with some minor modifications. The segment lengths of PBV were estimated by comparison with the length of genome segments of faecal sample positive for rotavirus.

Results

Molecular analysisRNA-polyacrylamide gel electrophoresis analysis of these samples revealed that 9.73% (11/113) was positive for group A rotavirus, whilst 4 (3.53%) presented a bisegmented genome, the bands of which ranged between segments 3 and 5 compared to the rotavirus segments (Figure 1). Out of 68 faecal samples of buffalo calves tested for viral gastroenteritis, 3 (4.41%) were found positive for picobirnavirus and 8 (11.76%) for rotavirus. Similarly, of 45 faecal samples of cattle calves tested for viral gastroenteritis, 3 (6.66%) were found positive for rotavirus, whereas 1 (2.22%) tested positive for PBV.

IntroductionPicobirnaviruses (PBV) are a group of small viruses whose genome is composed of 2 segments of double-stranded RNA (dsRNA), ranging in size from 2.69 kbp to 2.36 kbp for the larger segment and from 2.36 to1.58 kbp for the smaller segment (7). The virion is non-enveloped, with a diameter of 33 to 35 nm and has icosahedral symmetry. Picobirnavirus has large and small genome profile depending on the size of 2 segments of double-stranded RNA. It was first described by Pereira et al. (17), who detected two bands of bisegmented double-stranded RNA genome by polyacrylamide gel electrophoresis (PAGE) in faecal samples from children. Since then, PBV has been detected in faecal samples from different animals including rats (17), avian (1, 13), guinea-pigs (18), pigs (9), rabbits (8), cattle (3, 12, 24) and giant anteaters (Myrmecophaga tridactyla) (10). Picobirnavirus has been identified in both normal and diarrheic faeces so its pathogenicity is still unclear. Rotaviruses are the major causal agents of acute viral gastroenteritis in young animals as well as in children, as such they are responsible for significant economic losses throughout the world. The group A is the most frequently isolated in the case of rotaviral diarrhoea. The rotavirus belongs to the genus Rotavirus under the family Reoviridae and its genome can be separated into 11 discrete segments by RNA-PAGE. This study reports the detection of PBV and rotavirus in diarrhoeic faecal samples of cattle and buffalo calves in Mumbai metropolis, Western India.


SamplesA total of 113 faecal samples including 68 from buffalo and 45 from cattle were collected from different farms in Mumbai. All sampled animals were under 1 year of age and with diarrhoea. The diarrhoeic faecal samples were collected between October 2008 and October 2009.

A 10% suspension of each faecal sample was prepared in lysis buffer [3 M sodium acetate, pH 5.2, 10% sodium dodecyl sulphate (SDS)]. The suspension was vortexed for 10 min, followed by centrifugation at 10,000 × g (10,381 rpm) for 15 min at 4°C to remove coarse particles and cellular debris. The clarified supernatant was transferred into sterilised vials and stored at -20ºC or processed for RNA extraction.

Viral RNA extractionPicobirnavirus-positive samples were detected using the protocol used for rotavirus genome extraction.

Picobirnavirus in Western India Mondal et al.

359

was 4.67%, whereas earlier studies have reported presence of PBV infections among diarrhoeic and non-diarrhoeic calves as 0.70% (3). Bhattacharya et al. (2) reported that the prevalence of the small genome profile PBV amongst cases of diarrhoea in children in Kolkata was 2.47%. Pereira et al. (19) reported large genome profile PBV infections among diarrhoeic and non-diarrhoeic human cases at prevalence rate of approximately 0.45%.

Rotaviruses are the most commonly identified viral causes of diarrhoea of neonatal food animals (11). In our study the Group A rotavirus was detected in 9.73% of the diarrhoeic calves, this finding is in agreement with Chitambar et al. (5), suggesting that the rotavirus is one of the more important causative agents in neonatal diarrhoea.

Although other studies have also demonstrated the presence of PBV in various hosts with a certain frequency, conclusive data regarding the pathogenicity of this virus are lacking. Some articles tried to associate the presence of PBV with manifestations of gastroenteritis (15, 17). However, since PBV has also been detected in animals without clinical signs, its true role in this clinical manifestation remains to be defined (4, 9, 15). Finally, we would like to highlight that, to the best of our knowledge, this is the first report of a PBV identified in cattle and buffalo calves in Western India.

AcknowledgmentsThe authors extend thanks to the Associate Dean of Bombay Veterinary College, Parel, Mumbai and to Dr Soumendu Chakravarti, Scientist at the Indian Veterinary Research Institute.

Grant supportBombay Veterinary College, Parel, Mumbai, is gratefully acknowledged for providing funds.

The size of the segments was estimated to be approximately 2.36 kb and 1.58 kb for the larger and smaller segments, respectively (Figure 1).

DiscussionThe detection of PBV has been done by RNA-PAGE, because this method allows for sufficiently fractionating the viral RNA from other nucleic acids in the stool samples showing distinct electropherotypic profiles (21). In Brazil, PBV has been detected using PAGE in faeces of diarrhoeic and non-diarrhoeic calves (3). Malik et al. (16) reported 3.67% (5/136) positivity for PBV, showing a typical genomic migration pattern with 2 discrete bands with size of approximately 2.4 and 1.7 kb for the larger and smaller segments, respectively. The overall prevalence of the large PBV genome profile amongst diarrhoeic buffalo calves in Mumbai


Mondal et al. Picobirnavirus in Western India

Figure 1. Electrophoretic analysis of RNA genome segments of rotavirus and picobirnavirus isolated from bovine and buffalo calves.Lane 1: cattle rotavirus.Lane 10: buffalo rotavirus.Lane 3: cattle picobirnavirus.Lanes 6, 7, and 9: buffalo picobirnavirus.Lanes 2, 4, 5, and 8: negative samples.

1 2 3 4 5 6 7 8 9 10

2.36 kbp

1.58 kbp


Picobirnavirus in Western India Mondal et al.

1. Alfieri A.F., Alfieri A.A., Resende J.S. & Resende M. 1988. A new bisegmented double stranded RNA virus in avian feces. Arq Bras Med Vet Zootec, 40, 437-440.

2. Bhattacharya R., Sahoo G.C., Nayak M.K., Rajendran K., Dutta P., Mitra U., Bhattacharya M.K., Naik T.N., Bhattacharya S.K. & Krishnan T. 2007. Detection of genogroup I and II human picobirnaviruses showing small genomic RNA profile causing acute watery diarrhea among children in Kolkata, India. Infect Genet Evol, 7(2), 229-238.

3. Buzinaro M.G., Freitas P.P.S., Kisiellius J.J., Ueda M. & Jerez J.A. 2003. Identification of a bisegmented double-stranded RNA virus (picobirnavirus) in calf faeces. Vet J, 166(2), 185-187.

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5. Chitambar S.D., Arora R., Kolpe A.B., Yadav M.M. & Raut C.G. 2011. Molecular characterization of unusual bovine group A rotavirus G8P14 strains identified in Western India: Emergence of P14 genotype. Vet Microbiol, 148(2-4), 384-388.

6. Chomczynski C.C. & Sacchi N. 1987. Single step method of RNA isolation by acid Guanidinium thiocynate-phenol-chloroform extraction. Anal Biochem, 162(1), 156-159.

7. Costa A.P., Cubel Garcia R.C.N., Labarthe N.V. & Leite J.P.G. 2004. Detection of double-stranded RNA viruses in fecal samples of dogs with gastroenteritis in Rio de Janeiro, Brazil. Arq Bras Med Vet Zootec, 56(4), 554-557.

8. Gallimore C., Lewis D. & Brown D. 1993. Detection and characterization of a novel bisegmented double-stranded RNA virus (picobirnavirus) from rabbit faeces. Arch Virol, 133(1-2), 63-73.

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SummaryThis study has been undertaken to isolate and characterise Escherichia coli strains from raw poultry meat in West Bengal, determine their pathogenicity and identify the prevalent serotypes and their antibiogram. A total of 83 raw poultry meat samples were collected from February to July 2004. Thirty-three samples (39.76%) were positive for E. coli. The majority of highly pathogenic E. coli belonged to O3, O6, O25, O73, O120 whereas the highly enteropathogenic E. coli belonged to O6, O25, and O158. Most isolates (84% - 100%) were sensitive to chloramphenicol, amikacin and gentamicin, they were (92% - 100%) also resistant to novobiocin, cefixime, sulphafurazole, vancomycin. Considering the frequency of E. coli serogroups O6, O25, O158 which are important zoonotic pathogens, special attention needs to be paid in order to maintain strict hygienic measures in the retail meat shops, so to avoid serious health risks for the retailers and for the consumers.

RiassuntoLo studio presentato in questo articolo è stato condotto al fine di isolare e individuare le caratteristiche distintive di ceppi di Escherichia coli rilevati in campioni di carne cruda di pollo prelevati nello stato del Bengala occidentale. Lo studio ha avuto, inoltre, anche lo scopo di determinare la patogenicità dei microrganismi, i sierotipi prevalenti e il loro antibiogramma. Nel periodo febbraio-luglio 2004, sono stati prelevati 83 campioni di carne cruda di pollo di cui 33 campioni (39,7%) sono risultati positivi per E. coli. La maggior parte dei sierotipi altamente patogeni è risultata appartenere ai gruppi O3, O6, O25, O73, O120, quelli altamente eteropatogeni ai gruppi O6, O25 e O158. La maggior parte degli isolati (84% - 100%) ha mostrato sensibilità nei confronti di cloramfenicolo, amikacina e gentamicina, mentre si è rilevata resistenza (92% - 100%) a novobiocina, cefixime, sulfafurazolo e vancomicina. L’articolo evidenzia la necessità di dedicare particolare attenzione al rispetto delle condizioni igenico-sanitarie negli esercizi che trattano carne cruda di pollo al fine di evitare seri rischi per la salute dei consumatori e degli addetti alla vendita. Tale necessità si fa ancor più pressante se si considera la frequenza di E. coli O6, O25, O158 e la loro importanza come agenti di zoonosi.

Caratterizzazione di Escherichia coli isolata in campionidi carne cruda di pollo nel Bengala occidentale, India

Parole chiaveAntibiogramma,Carne di pollo,Escherichia coli,India,Sierogruppo.

KeywordsAntibiogram,Escherichia coli,India,Meat,Poultry,Serogroup.

1 Department of Microbiology, West Bengal University of Animal and Fishery Sciences,Belgachia, Kolkata 700037, West Bengal, India.

2 Department of Veterinary Microbiology, West Bengal University of Animal and Fishery Sciences,Belgachia, Kolkata 700037, West Bengal, India.

* Corresponding author at: Department of Microbiology, West Bengal University of Animal and Fishery Sciences,Belgachia, Kolkata 700037, West Bengal, India.

Tel.: +91 7278770611, e-mail: [email protected]

Asim Jana1* & Anjan Mondal2

Serotyping, pathogenicity and antibiogram ofEscherichia coli isolated from raw poultry meat

in West Bengal, India

362

Pathogenicity testingIn miceEscherichia coli isolates were tested for pathogenicity in white Swiss albino mice as described by Mukherjee et al. (8). Inoculated mice were observed at 6 hrs intervals during the first 24 hrs post inoculation and, subsequently, at 12 hrs intervals for 4 days. Mortality was recorded at each interval observation. Those mice who died due to probable infection were immediately necropsied under strict aseptic condition and the gross pathological changes in the internal organs were recorded and re-isolation of E. coli was attempted from heart blood, pericardial and peritoneal fluids, liver, lungs, and kidney. All survivors were sacrificed on the 5th day post inoculation and all gross pathological lesions, if any, were recorded.

Ligated rabbit gut loop techniqueThe ligated rabbit gut loop technique was performed to determine the enteropathogenicity of E. coli in rabbits. The test was performed following a modified version of the method of Joshi and Kahlon (5). The animals were observed for 24 hrs and were then sacrificed after 24 hrs of operation. Strains which caused full distension with tightening of the test loops were designated as highly enteropathogenic (EP) and assigned grade 4+. Those which caused full distension with loosened loops were noted as moderately enteropathogenic and graded 3+, those with mild EP were graded as 1+, those which caused no distension were recorded as non-EP (–). After grading, each loop was opened lengthwise and fluid volume was measured. The volume (ml)/length (cm)

IntroductionMicrobes in meat, especially those causing food borne diseases, have recently become a matter of great public health concern (11). India could earn a considerable profit though meat export, however its poor quality and high level of microbial load deprive the country of such a possibility. As a concequence of the growing demand for animal proteins, slaughterhouses have to face an increasing load of meat to process. Such an increased load came at the cost of the hygienic aspects of meat production, to which is paid less attention that it is requited. Meat, being it a nutrient-rich substrate, can support the growth of a wide range of micro-organisms, which also include Escherichia coli. The latter has received much attention as a potential public health threat due to the morbidity and mortality rates associated with outbreaks and sporadic cases of human illness (10). This study has been undertaken to isolate and characterise E. coli from raw poultry meat in West Bengal; to determine the pathogenicity; and to identify the prevalent serotypes and their antibiogram.


Collection of samplesA total of 83 raw poultry meat samples was collected between February and July 2004 from various retail shops situated in different local markets of Nadia and Calcutta districts of West Bengal: namely, Mohanpur (N=30), Jaguli (N=20), Kalibazar (N=17) and Belgachia (N=16) (Figure 1). About 10 to 15 grams of meat samples were randomly collected including neck, breast, back, thigh and abdomen of poultry carcasses.

Isolation and identificationSamples were plated on MacConkey agar (HIMEDIA Laboratories, Mumbai, India) and incubated at 37ºC for 24 hrs. The lactose fermenting colonies were reinoculated to Eosin Methylene Blue (HIMEDIA) agar and colonies producing metallic sheen were transferred to Nutrient agar slants and incubated at 37ºC for 24 hrs and stored at 4ºC for further identification. Identification of isolates was done according to the Cruickshank et al. (3) based on staining and biochemical tests.

SerotypingThe isolates were sent to the National Salmonella and Escherichia Centre, Kasauli, Himachal Pradesh, India for further confirmation and serotyping.


Escherichia coli in raw poultry meat in West Bengal, India Jana et al.

Figure 1. Map of India showing the study area.

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pathogens causing food poisoning needs to be emphasised. In the present study, meat samples from poultry birds, collected from different retail shops have been found to be contaminated with different strains of E. coli, which may cause infection to the consumers as well as to those involved in the meat processing chain.

Out of 83 poultry meat samples, 33 (39.76%) samples were positive for E. coli. This finding is consistent with those of Stanescu et al. (16) who reported that 37.1% of broiler carcass samples were contaminated with E. coli. Of the 30 samples of poultry meat collected from the local Mohanpur market, 13 (43.33%) samples yielded E. coli strains, while 20 samples collected from Jaguli, 17 samples from Kalibazar and 16 samples from Belgachia yielded 7 (35.00%), 8 (47.06%) and 5 (43.75%) E. coli strains, respectively. Thus, the present investigation shows the contamination of poultry meat with E. coli strains and highlights the possibility that this may be due to the breach of proper sanitary precautions before and during slaughtering.

All the 33 isolates of E. coli were serotyped in the National Salmonella and Escherichia Centre, in Kasauli, Himachal Pradesh. Out of 33 isolates of E. coli, 18 could be typed, 9 could not be typed and 6 were classified as rough strains (Table II). Table II shows that out of 13 different ‘O’ serogroups of E. coli, O120 serogroup comprising of 3 (9.09%) isolates was the most prevalent in the present study. O6, O81 and O100 serogrpups follow, (N=2; 6.06%) as well as serogroups O3, O25, O32, O73, O101, O104, O107, O109 and O158 (one strain each; 3.03%).

The 13 different serogroups of E. coli were tested for their pathogenicity in Swiss albino mice. Among these, 5 (38.46%) serogroups (O3, O6, O25, O73, O120) were highly pathogenic, 4 (30.77%)

ratio was called mean index of loop. The total viable count of E. coli content in each loop was measured by pour plate methods and finally the lesion or inflammation present on intestinal surface of loops were also recorded.

In chicksOne day old chicks were procured from a poultry farm, in Mohanpur, West Bengal, India and the pathogenicity test to detect the virulence of E. coli strains on the chicks was performed following the method proposed by Savov (14), Dho and Lafont (4). The chicks were randomly divided into groups, each group being comprised of 6 chicks. Representative E. coli strains from each selected serogroup were cultured in nutrient broth and 0.2 ml of fresh broth culture containing 107 viable organisms in the case of E. coli were inoculated intraperitoneally into each group. The control group was inoculated with 0.2 ml of sterile nutrient broth by intraperitoneal route. All the chicks were observed for 3 days. The post-mortem examination was done in freshly dead chicks and all the gross pathological changes in internal organs were recorded.

AntibiogramThe drug sensitivity of different serotypes to different antibiotics was carried out as recommended in Bauer et al. (1) and has been presented in Table I.

Results and Discussion A number of different microorganisms are recognised to cause foodborne illness (15). Among these, the importance of E. coli organisms as potential


Jana et al. Escherichia coli in raw poultry meat in West Bengal, India

Table I. Antimicrobial sensitivity test of Escherichia coli serogroups obtained from raw poultry meat samples collected in West Bengal (India).

Serial No. Antimicrobial agents

No. of strains

Resistant Intermediate SensitiveNo. % No. % No. %

1. Amikacin 13 0 0 1 7.69 12 92.31

2. Cefixime 13 12 92.31 0 0 1 7.69

3. Chloramphenicol 13 0 0 0 0 13 100

4. Ciprofloxacin 13 2 15.38 2 15.38 9 69.23

5. Erythromycin 13 8 61.54 3 23.08 2 15.38

6. Gentamicin 13 2 15.38 0 0 11 84.62

7. Kanamycin 13 1 7.69 8 61.54 4 30.77

8. Methicillin 13 8 61.54 5 38.46 0 0

9. Novobiocin 13 13 100 0 0 0 0

10. Oxytetracycline 13 11 84.62 0 0 2 15.38

11. Sulphafurazole 13 12 92.31 0 0 1 7.69

12. Vancomycin 13 12 92.31 0 0 1 7.69


Escherichia coli in raw poultry meat in West Bengal, India Jana et al.

The 13 ‘O’ serogroups of E. coli were tested for their pathogenicity in 1 day old chicks. The results showed that 4 ‘O’ serogroups (30.77%) (O6, O25, O73, O120) were highly pathogenic and 4 serogroups (30.77%) (O32, O101, O109, O158) were moderately pathogenic, while 3 serogroups (23.08%) (O3, O81, O107) were least pathogenic. Finally, 2 serogroups (15.38%) (O100, O104) were non-pathogenic. These results are consistent to some extent with previous findings described in Mukherjee et al. (8) and Orden et al. (9).

Thirteen ‘O’ serogroups of E. coli isolates were tested with 12 different antimicrobial agents. The results showed that E. coli isolates were 100% sensitive to chloramphenicol, followed by amikacin (92.31%), gentamicin (84.62%), ciprofloxacin (69.23%), erythromycin (15.38%), oxytetracycline (15.38%), cefixime (7.69%), sulphafurazole (7.69%) and vancomycin (7.69%). Intermediate sensitivity showed to kanamycin (61.54%) followed by methecillin (38.46%), erythromycin (23.08%), ciprofloxacin (15.38%) and amikacin (7.69%). For novobiocin, 100% of the isolates showed resistant followed by cefixime (92.31%), sulphafurazole (92.31%), vancomycin (92.31%), oxytetracycline (84.62%), erythromycin (61.54%), methicillin (61.54%), ciprofloxacin (15.38%), gentamicin (15.38%) and kanamycin (7.69%). Antimicrobial sensitivity test of E. coli strains has been presented in Table I. It is noteworthy that these findings were partially correlated to those reported in Sackey et al. (13) and Mishra et al. (7). The possible explanation for this resistance of E. coli against the antibiotics as found in the study could be attributed to the indiscriminate use of these antibiotics in poultry. This high resistance might be due to transmissible drug resistance and resistance might also develop due to mutational changes.

The present investigation sheds some light on high degree of prevalence of pathogenic E. coli strains in poultry meat. Considering the frequency of E. coli serogroups O6, O25, O158 which are important zoonotic pathogens, special attention need to be paid so to maintain strict hygienic measures in slaughterhouses and retail meat shops, in order to avoid the serious health risks for the meat handlers working in the meat shops and for the consumers.

AcknowledgmentsThe authors thank to the Vice-Chancellor and Dean, West Bengal University of Animal and Fishery Sciences, Belgachia, Kolkata, India.

Grant supportThe West Bengal University of Animal and Fishery Sciences is gratefully acknowledged for providing funds.

serogroups (O32, O81, O109, O158) were moderately pathogenic, 2 (15.38%) serogroups (O101, O107) were least pathogenic and 2 (15.38%) serogroups (O100, O104) were non-pathogenic to mice. Most of the serogroups obtained in this investigation were reported in other studies, like Chachra and Katoch (2), Mukherjee et al. (8). Serogroups O100, O101, O104 were not reported from poultry in the relevant literature. It may be the case that such groups come from other livestock or other environmental source distant from poultry origin. E. coli serogroup O158 was found to be associated with gastroenteritis and diarrhoea in infants as well as adults (12). E. coli serogroups, O6 and O25, were found to be associated with infantile and travellers’ diarrhoea (6). So, for what it may concern transmissibility, it is evident that there is every chance for meat handlers and consumers to be infected by the E. coli, particularly in those cases in which scanty higenic measures are maintained.

As for the enteropathogenicity, 3 of the 13 isolated E. coli strains were highly enteropathogenic, 4 were moderately enteropathogenic and 6 were non enteropathogenic. Serogroups O6, O25, O158 were highly enteropathogenic. Serogroups O3, O32, O73, O120 were moderately enteropathogenic strains. Serogroups O101, O109 were least entropathogenic and rated as 1+ whereas serogroups O81, O100, O104, O107 were non enteropathogenic with no distension. The gross lesion in the intestine varied from inflammation, oedema to haemorrhage. In the control loops inoculated with sterilised normal saline, lesions were absent.

Table II. Distribution and frequency of serogroups of Escherichia coli isolates obtained from raw poultry meat in West Bengal (India).

Sl No. Serogroup Frequency Percentage

1. O3 1 3.03

2. O6 2 6.06

3. O25 1 3.03

4. O32 1 3.03

5. O73 1 3.03

6. O81 2 6.06

7. O100 2 6.06

8. O101 1 3.03

9. O104 1 3.03

10. O107 1 3.03

11. O109 1 3.03

12. O120 3 9.09

13. O158 1 3.03

15. Untypeable 9 27.27


Jana et al. Escherichia coli in raw poultry meat in West Bengal, India

1. Bauer A.W., Kirby W.M.M., Sherris J.C. & Truck M. 1966. Antibiotic sensitivity test by a standardized disc method. Am J Clin Path, 45(4), 493-496.

2. Chachra D. & Katoch R.C. 1996. Prevalence of Escherichia coli and Salmonella among domestic poultry in Himachal Pradesh. Indian J Poult Sci, 31(1), 38-44.

3. Cruickshank R., Duguid J.P., Marmion B.P. & Swain R.H.A. 1975. Medical microbiology, 12th Ed, Vol. 1, Churchil Livingstone, London, 236 pp.

4. Dho M. & Lafont J.P. 1984. Adhesive poperties and iron uptake in Escherichia coli lethal and non-lethal for chicks. Avian Dis, 28(4), 1016-1025.

5. Joshi V.K. & Kahlon S.S. 1984. Prevalence of enteropathogenic E. coli in market milk of Ludhiana city. Indian J Dairy Sci, 37(1), 5-10.

6. Levine M.M. 1987. Escherichia coli that cause diarrhoea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohaemorrhagic and enteroadherent. J Infect Dis, 155(3), 377-389.

7. Mishra A., Sharda R., Chhabra D. & Tanwani S.K. 2002. Antibiogram of Escherichia coli isolates from domestic poultry. Indian Vet J, 79(8), 863-864.

8. Mukherjee B.N., Mondal D. & Mishra S.K. 1997. Pathogenicity of E. coli isolated from chicks in laboratory animals. Indian J Anim Health, 36(2), 151-156.

9. Orden J.A., Ruiz-Santa-Quiteria J.A., Cid D., García S. & de la Fuente R. 1999. Prevalence and characteristics of

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necrotoxigenic Escherichia coli strains isolated from diarrhoeic calves. Vet Microbiol, 66(4), 265-273.

10. Paton J.C. & Paton A.W. 1998. Pathogenesis and diagnosis of shiga toxin-producing Eschrichia coli infections. Clin Microbiol Rev, 11(3), 450-479.

11. Pepin M., Russo P. & Pardon P. 1997. Public health hazards from small ruminant meat products in Europe. Rev Sci Tech Off Int Epiz, 16(2), 415-425.

12. Rowe B., Gross R.J., Lindop R. & Baird R.B. 1974. A new E. coli O group O158 associated with an outbreak of infantile enteritis. J Clin Pathol, 27(10), 832.

13. Sackey B.A., Mensah P., Collison E. & Sakyi D.E. 2001. Compylobacter, Salmonella, Shigella and Escherichia coli in live and dressed poultry from metropolitan Accra. Int J Food Microbiol, 71(1), 1-28.

14. Savov D. 1963. Studies on colisepticaemia in chicks. IZV-Vet Inst. Zaraz Parazit Bolesti, Sofia, 9, 97-110.

15. Scallan E., Griffin P.M., Angulo F.J., Tauxe R.V., Hoekstra R.M. 2011. Foodborne illness acquired in the United States – unspecified agents. Emerg Infect Dis, 17(1), 16-22.

16. Stanescu V., Chirila F., Sahleanu C. & Vana V. 1992. Occurrence of E. coli serogroups in raw and pasteurized milk and in chicken meat pathogenic potential in man and animal. In Proc. 3rd World Congress on foodborne infections and intoxications, 16-19 June 1992, Robert von Ostertag-Institute, Berlin, 509-512 pp.

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SummaryThe objective of this study was the development of hatching protocols in controlled conditions to obtain juveniles, in order to restock and increase the resource of Sepia officinalis. The study was divided into the following phases: development and application of artificial surfaces at specific sites of the Molise coast in Italy; induction of eggs hatching and juveniles maintenance under controlled condition; juveniles introduction into specific sites and assessment their increment; experimental data elaboration. The obtained results concerned both the effectiveness of the artificial surfaces tasted during the study and the importance of the recovery of the eggs laid on artificial surfaces (artefacts and fishing gear) for preservation and the management of the Sepia officinalis resource. The induction tests conducted on eggs hatching under controlled conditions confirmed what described in the extant literature. Water salinity was detected as the only limiting factor, with values ≤ 20% related to the absence of hatching. The described practices for harvesting and induction of hatching for the production of juvenile cuttlefish may be endorsed by the operators at relatively low cost and throughout the year, with obvious economic benefits.

RiassuntoL’obiettivo del presente studio è stato la messa a punto di procedure di schiusa di uova di Sepia officinalis in condizioni controllate ai fini dell’ottenimento precoce di giovanili da utilizzare per l’incremento della risorsa di Sepia officinalis. Lo studio è stato articolato nelle seguenti fasi: messa a punto ed applicazione di manufatti artificiali in siti specifici della costa molisana; induzione della schiusa delle uova e mantenimento di una parte di giovanili in condizioni controllate; immissioni nei siti specifici di giovanili di seppia e valutazione dell’incremento degli stessi; elaborazione dei dati ottenuti nella fase sperimentale. I risultati hanno riguardato l’efficacia dei diversi substrati impiegati e l’importanza del recupero delle uova deposte sui substrati artificiali (manufatti ed attrezzi di pesca) ai fini della salvaguardia e della gestione della risorsa Sepia officinalis. Le prove di induzione della schiusa delle uova in condizioni controllate hanno confermato quanto descritto in letteratura: la salinità si è rilevata unico fattore limitante, infatti a valori ≤20% non si registra alcuna schiusa. Le pratiche di raccolta e di induzione della schiusa descritte potrebbero essere adottate dagli operatori a costi relativamente bassi per la produzione di giovanili di seppia, in periodi dell’anno diversi da quelli del ciclo naturale, con evidente vantaggio economico.

Nuove procedure per migliorarela deposizione e schiusa di uova di Sepia officinalis

Parole chiaveDeposizione uova,Gestione della risorsa,Induzione controllata,Manufatti artificiali,Protocolli di schiusa,Sepia officinalis.

KeywordsArtificial surfaces,Controlled induction,Hatching protocols,Management resource,Sepia officinalis,Spawning.

1 Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise 'G. Caporale',Centro Sperimentale Regionale per la Pesca ed Acquacoltura, Viale Marinai d’Italia 20, 86039 Termoli, Italy

* Corresponding author at: Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise 'G. Caporale',Centro Sperimentale Regionale per la Pesca ed Acquacoltura, Viale Marinai d’Italia 20, 86039 Termoli, Italy.


Nadia B. Barile*, Sabatino Cappabianca, Luigi Antonetti, Mariaspina Scopa, Eliana Nerone, Giuseppina Mascilongo, Sara Recchi & Antonio D’Aloise

New protocols to improve the deposition and hatching of Sepia officinalis’ eggs

368

Sepia officinalis decreased markedly. For example, in 2012 a decrease of 10% in terms of both quantity and price was registered [4,172 tonnes caught with a turnover of 35.94 million euro was detected with respect to 2011 (24)].

Several studies have been conducted (7, 10, 25, 27, 29) focusing on the distribution, biology, physiology and ethology of this species and on techniques for innovative fishing and protection (1, 6). Of particular interest are those studies related to the development of the resource such as the use of new artificial substrates for eggs collection (1). It has been previously reported (3) that the choice of deposition surfaces depends on female preferences: usually cuttlefishes lay eggs on long surfaces with a diameter lower than 10 mm. In this respect, the research analysing the conditions for embryos development and growth both in captivity and in nature is particularly worthwhile (12, 14, 15, 17, 18, 20, 22, 28).

In the present study, we have developed and tested new artificial substrates for egg collection and hatching controlled induction in order to obtain organisms for resource restocking.


Identification of sitesThe seabed typology along the coast of Molise was evaluated through diving floor survey. The survey showed that the seabed is sandy, muddy or rocky-sandy. Cuttlefish most populated areas were chosen to place the artificial substrates: 2 areas were in the Northern part of Termoli city (‘Skyscraper’ and ‘Starfish’) and 2 in the Southern part (‘Rio vivo’ and ‘Campomarino Lido’).

Development and positioning of artificial substratesOn the basis of previous research (27), two easy handling and long lasting artificial substrates were produced to collect eggs and reduce fisheries losses (due to nets, ropes, etc.): modified cuttle-traps and modified grids, armed with ropes of various sizes (diameter of 8 mm, 10 mm and 12 mm) and different colours (red and white with black dots), with and without floatation devices (Figures 1, 2 and 3).

In May 2006, we placed the artificial substrates (modified cuttle-traps and modified grids) on the selected areas in the Northern part of Termoli, in order to evaluate their efficiency and, if necessary, endurance. Until November 2006, we weekly controlled the anchorage and the status of substrates and counted the laid eggs. In March 2007, we positioned 5 modified cuttle-traps at the ‘Starfish’ site for 6 months.

IntroductionThe progressive and increasingly worrying depletion of marine fishery resources occurred during the 20th century has resulted in a serious crisis for the entire fishing industry and the connected activities. The Molise small-scale fishery activity is mainly carried out within 3 miles from the coast. The maritime compartment of Termoli (Molise, Italy) involves 20 gross tonnage boats of less than 10 tonnes and is characterized by small, often family-run, businesses which employs about 130 people considering both those engaged in fishing and those participating in the induced activities (23). The small-scale fishery represents about a third of the entire commercial fleet of Molise.

Sepia officinalis (Linnaeus, 1758) is a common cuttlefish and is of particular importance among the species commercialized and caught within 3 miles from the coast. Using static gears (pots, fyke nets, trammel nets), the small-scale fishery is assiduously dedicated to the capture of this species, especially during the spawning period, which coincides with the warmer seasons (spring and autumn) with a peak in spring, in conjunction with the arrival in the coastal areas of the adult specimens from deeper water, and a peak in the autumn before the wintery migration of these molluscs to open sea (2, 16).

The reproductive period of Sepia officinalis lasts about 7 months (4, 21). The freshly laid eggs have a diameter between 5 and 9 mm and are fixed by the female to any type of natural (rocks, macroalgae, seagrass) and artificial (ropes, fishing gear, pieces of iron, etc.) substrate.

Development is direct, incubation lasts in average 1 month at 20 °C, however it can spam from 30 to 60 days, depending on the temperature. At the time of hatching the young cuttlefish, which measure about 10 mm (length of the mantle), are already able to hunt independently and assume quickly a benthic lifestyle. The cuttlefish present along the Atlantic coast reach a maximum size of 45 cm and weight of 4 kg, while in the Mediterranean basin they reach a maximum length of 35 cm (usually from 15 to 25 cm). The lifetime is generally between 18 and 24 months although some males can live longer (up to 36 months) (3).

For small fisheries, Sepia officinalis is of commercial importance and, consequently, it is necessary to put in place proper management procedures devoted to protect this resource and to increase presence of this species along the shores. Considering the 2004-2007 national catches, the common cuttlefish is 1 of the 9 species with the highest landed amount. During 2007, 7,500 tonnes of cuttlefish were landed in Italy, corresponding to 17% of small fishery catches (19). During the last 5 years, however the landed of


The deposition and hatching of Sepia officinalis’ eggs Barile et al.


Barile et al. The deposition and hatching of Sepia officinalis’ eggs

Induction of egg hatchingHatching test in aquaculture plantIn June 2006, we collected and transported about 2000 cuttlefish eggs to the local aquaculture plant named Ittica Molisana (Campomarino, Molise, Italy). The ropes with the egg clusters were placed vertically in special racks and were planted in a circular fiberglass pool of 20 m³ capacity in open circuit. During the test the water salinity, due to problems related to plant water intake, decreased significantly (<18‰). Therefore, it was deemed useful to transfer the experiments in laboratory to follow the hatching in small volumes: a batch of about 100 eggs was collected, transferred to the laboratory and planted in 2 racks placed in 2 tanks of 60 l.

Hatching test in hatcheryIn the period January-March 2007, the hatcheries for incubation of large volumes of eggs were set up and prepared, under controlled conditions of temperature and salinity at Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Centro Sperimentale Regionale per la Pesca ed Acquacoltura of Termoli. The hatchery (horizontal trough) is composed of 2 rectangular tanks with bottom flat and 220 l of capacity, each containing 7 racks with a maximum capacity of 1,500/2,000 eggs corresponding to about 1.5 kg of weight.

The eggs retrieval concerned 5 artefacts placed on the site ‘Starfish’ and was conducted with the cooperation of Termoli small fishery operators. Eggs placed in the tanks were taken between March the 27th and 9th April the 19th 2007, at a temperature of 12-15°C and transported to the laboratory.

In May 2008, 5 modified cuttle-traps were placed in the ‘Campomarino Lido’ site. The substrates were kept there until July 2008 when, due to scanty deposition, they were moved to the ‘Starfish’ site and left there until November 2008. In October 2008, 8 modified cuttle-traps were placed in the ‘Skyscraper’ and ‘Rio Vivo’ sites. The activities of these traps lasted until November the 10th 2008, when all substrates were removed and taken to laboratory for storage and winter maintenance.

In February 2009, 5 modified cuttle-traps were placed in the ‘Starfish’ site until May 2009.

Surveys of Sepia officinalis catchesThe assessment of the amount of cuttlefish along Termoli coastline was determined by daily catches operated by small commercial fishing. The amount of cuttlefish was assessed according to the area and distribution. Despite the fact that the resource is available throughout the year, investigations concerned only spawning periods (March-October 2007 and February-May 2008).

Figure 1. Metallic grid with different ropes.

Figure 2. Cuttletraps.

Figure 3. Cuttletraps with 8 mm ropes.



> 8; breeding density: 8000 nauplii/l, concentration dissolved oxygen: > 7 mg/l). In the growth phase, Artemia salina was fed on phyitoplankton coltures (Chaetoceros calcitrans, Phaeodactylum tricornutum and Tetraselmis suecica, Isochrysis galbana, Dunaliella tertiolecta) and yeast (Saccharomyces cerevisiae), after 16 days adults of Artemia salina were obtained to be used as food. This feeding mode has been selected on the basis of previous studies (5, 8, 9, 11, 13).

Releasing of post-hatching juvanilesThe cattlefish juveniles obtained in the laboratory hatching trial performed in 2007 were released in the ‘Starfish’ site near the bottom sea at 4 different sowing times 72-96 hrs after the hatching event. The site was selected as a poorly productive site for Sepia officinalis according to local the fishery operators.

In July and September 2007, 4 sea samplings were performed in the restocking area to evaluate the efficacy of the releasing. The fishing was carried out with gillnet fine mesh of 22 mm and 400 m in length sailed after 48 hrs. For each sampling, all captured Sepia officinalis juveniles were counted and measured.

StatisticsIn order to assess the effect of the different types of artificial substrate on the amount of laid eggs, analysis of variance (ANOVA one-way, type of artificial substrate) was performed on the data concerning both the eggs deposited on the racks and on the modified pots at the ‘Starfish’ site in 2006. Furthermore, in order to evaluate the effect of the different size of the ropes on the amount of laid eggs, an analysis with a single source of variability (diameter of the ropes) was performed on dataset relating to depositions number on modified cuttle-traps.

Data of hatching tests with a salinity of 20‰, 25‰, 30‰ and 35‰ conducted in the aquaria during 2008 and 2009 were analysed (ANOVA one-way, salinity) in order to evaluate the effect of this parameter on the number of hatched eggs. When differences were significant, a post hoc Tukey's test was conducted.

The analysis was conducted on transformed data (square root) using the SPSS 22.0 software.

Results and Discussion

Selection of the substratesThe endurance and efficiency of ‘grids’ and ‘modified cuttle-traps’ were compared; grids had the disadvantage of poor handling, high production costs and tendency of silting also with low surges,

After a few hours of conditioning, eggs were transferred into tanks with water at a temperature of 20 ± 1°C, salinity of 38 ± 1‰ and dissolved oxygen concentration above 8 mg/l. The eggs were counted, measured (length and width) and positioned in the racks (Table I).

Hatching test in acquariumFour hundred eggs (divided in 8 lots consisting of 50 eggs each) were taken from the traps placed in the ‘Starfish’ site on September the 12th and on October the 10th 2008. The collected eggs were transported to the laboratory to carry out 2 hatching tests under controlled conditions. In both tests, eggs were placed in 4 small racks suspended in 4 aquaria with a capacity of 80 l. In the first test of hatching, the following parameters were set: temperature of 20 ± 1°C and salinity of 20‰, 25‰, 30‰ and 35‰. The second test was performed at 2 values of salinity (25‰ and 30‰) and at 2 temperatures (20 ± 1°C and 25 ± 1°C). In May 2009, a test was repeated under the same experimental conditions of the first hatching test conducted in 2008.

Maintaining of juveniles under controlled conditions In the maintenance phase, juveniles were daily fed ad libitum with live food consisting of adult Artemia salina. They were produced by incubation of cysts under controlled conditions (salinity: 26.5‰, temperature: 27 ± 1°C, pH: > 8, surface brightness: 1,800/2,000 lux; density of incubation: 1.5 g of cysts/litre) with a final hatching percentage close to 70% after 36 hrs. The nauplii were then transferred into cylinder-conical tanks of 250 l capacity equipped with aeration (salinity: 37‰; temperature: 25 ± 1°C, pH:

Table I. Eggs positioning for hatching tests in hatchery during 2007.

N° rack Eggs number Introduction date

1 1,762 27/03/2007

2 790 28/03/2007

3 1,001 29/03/2007

4 2,839 30/03/2007

5 1,309 02/04/2007

6 2,532 05/04/2007

7 1,032 18/04/2007

8 2,381 19/04/2007

9 2,026 19/04/2007

10 899 19/04/2007

11 1,502 19/04/2007



In addition, statistical analysis showed that egg deposition was significantly greater on 8 mm diameter ropes than on those with a diameter of 10 cm and 12 cm (Tukey test, p <0.001). Whereas, the number of eggs laid on ropes with a 10 cm and 12 cm diameter was not significantly different (Tukey test, p <0.001). Finally, the egg amount on different artificial substrates varied according to the monitoring period (Figure 4).

In 2007, the deposition found on 8 mm diameter rope traps placed on the ‘Starfish’ site was greater than that reported in the 2006 monitoring (1,871 vs 571 eggs on average).

In 2008, the trials included ‘Starfish’, ‘Campomarino Lido’, ‘Rio Vivo’ and ‘Skyscraper’ sites (Figures 5, 6, 7, 8). The highest number of eggs was laid on the ‘Starfish’ site (1,878, 218, 63 and 450 eggs on average, for ‘Starfish’, ‘Campomarino Lido’, ‘Rio Vivo’ and ‘Skyscraper, respectively’).

Furthermore, the amount of eggs laid on ‘Starfish’ site during late winter-early spring of 2009 was much lower than the one collected in same period of 2007

whereas modified cuttle-traps, showed a reduced tendency of silting and reduced resistance to oxidative processes.

Data of 2006 deposition events on grids and modified cuttle-traps have been compared in order to assess the effect of the different types of substrates on the amount of laid eggs. Considering that a unique deposition event was recorded in the ‘Skyscraper’ site (150 eggs on July the 25th 2006), statistical analyses were performed only with respect to the deposition data of the ‘Starfish’ site. Significant differences were recorded in number of eggs laid on different substrates, with significantly higher values obtained on traps compared to grids (one-way ANOVA, p <0.001). Consequently, the ‘modified cuttle-trap’ a parallelepiped of 30x45x120 cm dimension, iron built with a diameter of 10 mm was selected for the following trials.

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uniform distribution in the inshore areas (Table II). In contrast, in 2008, a more uniform spatial distribution was observed (Table III). Considering sample average value for each survey, the amount of cuttlefish caught in the Northern Termoli areas was higher than the one recorded in the previous year and almost similar to that captured in the Southern areas.

Induction of egg hatchingTest of hatching induction conducted in 2006 at the Ittica Molisana aquaculture farm highlighted that, in order to have an optimal hatching rate, the chemical-physical parameters such as water sea temperature and salinity should be within specific ranges (18-25°C for temperature and 30-35‰ of salinity). In fact, when water salinity decreased significantly (< 18‰) due to water intake problems, eggs regressed in size and eggs’ maturation stopped. Consequently, the test was stopped and 100 eggs were transferred to laboratory, where complete hatching occurred in 10 days. Juveniles were fed on adult Artemia salina after 24 hrs for about 3 days and were released in the ‘Starfish’ site 4 days after hatching.

With reference to the induction test conducted in the hatchery during March 2007, the first hatching events occurred after about 30 days from incubation and continued for 19 days. Hatching rate was 35% for a total of 6,351 live organisms (Figure 9).

This study also demonstrated that salinity affects the hatching rate. In fact, the amount of hatched eggs significantly changed according to different salinity levels (Tukey test, p <0.05). Minimum values of hatching (0%) was recorded at a salinity of 20‰, while maximum (> 90%) at 30‰ (Figures 10 and 11).

In tests conducted in aquarium at 2 salinities levels (25‰ and 30‰) and 2 temperatures (21°C and 25°C), hatching percentages ranged from 84% (for the batch incubated at 21°C and 25‰ of salinity) and 98% (for the batch incubated at 21°C and 30‰ of salinity) (Figure 12).

Furthermore, comparing tests at 25‰ of salinity, egg hatching started after 17 and 11 days, respectively at temperatures of 21 and 25°C, while in tests at salinity of 30‰, it started after 11 and 7 days respectively at 21 and 25°C. These data suggest that temperature affects the length of incubation period, as reported in previous studies (15).

Finally, evaluating different percentages of hatching obtained in tests conducted in 2007, 2008 and 2009 at similar conditions (20°C, 35-38‰), it is noteworthy that hatching values obtained in hatchery (35%) were notably lower than those in aquarium (80% in 2008 and 84% in 2009).

(946 vs 1,878 eggs on average). This was probably due to adverse weather and sea conditions.

Surveys of Sepia officinalis catchesConsidering the surveys conducted in 2007, the Northern part of Termoli fishing areas were less profitable than those located in the Southern part of the city. Sepia officinalis showed a not

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Figure 8. Eggs laid number on cuttletraps in 'Skyscraper' site during 2008.

Table II. Catched specimens during 2007 samplings.

Catch area N° catched specimens

Average of specimens in each sampling

Saccione(Southern Coast) 1,900 95

Vallone 2 miles(Southern Coast) 4,000 200

Rio vivo(Southern Coast) 260 13

Cala Saraceni Sud(Northern Coast) 1,120 56

Cala Saraceni Nord (Northern Coast) 1,640 82

Modenese(Northern Coast) 1,800 90

Table III. Catched specimens during 2008 samplings.

Catch area N° catched specimens

Average of specimens in each sampling

Vallone 2 miles(Southern Coast) 3,624 151

Porto di Campomarino(Southern Coast ) 4,056 169

Cala Saraceni Nord(Northern Coast) 3,360 140

Torre diroccata(Northern Coast) 3,216 134

Modenese(Northern Coast) 4,224 176



in this study under controlled conditions, confirmed what has already been reported in the extant literature: the water salinity was the unique limiting factor, in fact hatching events were not recorded at salinity lower than 20‰.

The techniques of collecting and hatching induction described in this study could be adopted by operators in order to lower the costs for the production of juveniles throughout the year independently from the natural cycle, with obvious economic benefits.

Finally, it would be useful to develop protocols for the growth and marking of specimens, in order to provide more accurate quantitative estimates of the increasing of resource following restocking.

Introduction of post-hatching juvenilesDuring samplings carried out 2 and 4 months after introduction, about 600 specimens of Sepia officinalis with size compatible to the 6,321 individuals previously introduced were captured. Considering that the restocking site had been previously reported by fishing operators as a non-productive area, specimens could be presumably attributed to the juveniles entered.

ConclusionsThe results of this study provided information on effectiveness of different substrates and importance of recovery of eggs laid on artificial substrates for preservation and management of Sepia officinalis resource. The tests of hatching induction, carried out

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Figure 10. Hatching trends during 2008 experimental tests at 20‰, 25‰, 30‰ and 35‰ of salinity.

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Figure 12. Hatching trends during 2008 experimental tests at temperature of 21°C, 25°C and salinity of 25‰, 30‰.



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SummaryThis short communication reports the results of a bluetongue sero-surveillance conducted in the Pithoragarh hills of Uttarakhand in India during the autumn of 2011. Unclotted blood and serum samples were collected from 51 goats for detection of bluetongue virus (BTV) antigen and antibodies. Of the 51 collected samples, 18 (35%) were positive to an indirect ELISA and 33 (64%) resulted positive to a BTV ELISA antigen. From a strong antigen-positive blood sample, a BTV was isolated (named as PTG-13) on cell culture and was subsequently confirmed as BTV-1 by RT-PCR and partial sequencing of genome segment-2. The goat serum samples were found to contain high titer of neutralising antibodies against BTV-23, nonetheless the virus could not be isolated. Interestingly, no neutralizing antibodies were detected against PTG-13 or other BTV-1 isolate, which suggests that sampling was probably done before the development of neutralizing antibodies against PTG-13 virus in the host. Isolation of BTV-1 (PTG-13) and presence of BTV-23 neutralizing antibodies in serum samples indicate that goats were probably infected with BTV-1 and 23 in different periods.

RiassuntoNella presente comunicazione sono riportati i risultati sulla presenza del virus della bluetongue (BTV) nell’area Pithoragarh, Stato di Uttarakhand, India, nell’autunno del 2011. Lo studio ha riguardato 51 campioni di sangue intero e siero di capra analizzati per rilevare la presenza di BTV e relativi anticorpi. Quando testati in laboratorio, 18 (35%) dei 51 campioni di siero sono risultati positivi all'ELISA indiretta mentre 33 (64%) campioni di sangue sono risultati positivi all'ELISA antigene per BTV. Dai campioni fortemente positivi è stato inoltre possibile far crescere il virus su tessuto colture (PTG-13). L’appartenenza del ceppo isolato al sierotipo 1 del BTV è stata confermata attraverso RT-PCR e sequenziamento parziale del genoma del segmento-2. Non è stato possibile invece isolare il sierotipo 23 nonostante i campioni di siero mostrassero titoli anticorpali elevati nel confronti di questo sierotipo. È possibile che il mancato rilevamento di anticorpi per PTG-13 o per altri ceppi di BTV-1 sia dovuto al fatto che il prelievo negli animali coinvolti nell'indagine sia stato effettuato prima che gli anticorpi per PTC-13 potessero svilupparsi. La presenza contemporanea di BTV-1 e di anticorpi per BTV-23 indica che gli animali testati sono stati infettati da BTV-1 e BTV-23 in momenti diversi.

Virus della bluetongue sierotipo 1 in caprenell’area di Pithoragarh, Stato di Uttarakahand, India

Parole chiaveBluetongue virus sierotipo-1,BTV-23,Capra,India.

KeywordsBluetongue virus serotype-1,BTV-23, Goat,India.

Division of Virology, Indian Veterinary Research Institute, Mukteswar Campus,Dist. Nainital 263138, Uttarakhand, India

* Corresponding author at: Division of Virology, Indian Veterinary Research Institute,Mukteswar Campus, Dist. Nainital 263138, Uttarakhand, India.


Bimalendu Mondal*, Bhoopendra Bisht, Sanchay K. Biswas & Karam Chand

Bluetongue virus serotype-1 in goatsin the Pithoragarh area of Uttarakahand, India

SHORT COMMUNICATION


specific sandwich ELISA (s-ELISA) as described elsewhere (5). Out of 51 samples, 33 (64%) were positive, 11 of which showed high OD values (s-ELISA OD 1.00-1.25, negative control OD 0.22). The highest rate of the BTV antigen prevalence (87.5%) was noticed in Swakot Village, where a large number of goats died due to PPR a month before sampling. Lower prevalences were found in Kalsin (53.5%) and Sintoli Village (42.8). All the samples with the highest OD values originated from Swakot Village. Out of 51 serum samples, 18 (35%) were found positive for BTV antibody as tested by a group-specific indirect ELISA (9). In 12 animals both BTV antigen and antibodies were found.

Five samples were randomly chosen among the 11 blood samples with the highest OD values and were tested for virus isolation. The virus could successfully be isolated on BHK-21 cells from 1 sample (No. PTG-13) only, the isolate was named as PTG-13. At third passage, cytopathic effects (CPE) was clearly evident 24 hrs post infection (hpi). It was characterised by rounding and ballooning of cells (Figure 1). By 48 hpi, a degeneration was observed, aggregation and death of cells resulting into detachment from the surface of culture vessel. Aliquots of infected cell lysate of each passage were tested for BTV antigen by s-ELISA (5). Antigen was barely detectable at first passage. However, in subsequent steps more cells showed CPE and antigen quantity was also found to increase (Figure 1). The bluetongue antigen detection assay (s-ELISA) provides serological evidence that the virus isolate PTG-13 was a BTV. For further investigation, double stranded RNA (dsRNA) was extracted and purified from virus-infected cell culture (at third passage of virus)

Bluetongue is endemic in India. Outbreaks are frequent in sheep although domestic ruminants have high level of seropositivity throughout the country (6, 9, 23). Several serotypes of BTV have been isolated from different states of India, especially from the regions where sheep population is more abundant. Bluetongue virus serotype-1 was isolated from sheep and Culicoides vectors (11, 12, 20), and serotype-specific antibodies were detected against several serotypes including BTV-1 and 23 (7) in the North-Western part of the country (i.e. the states of Rajasthan and Gujarat). Seroprevalence and virus isolation results suggest that BTV-1 is the predominant serotype in the Northern and North-Western parts of India as recently reported (4, 8). In 1995, BTV-23 was isolated from sheep at foot-hills of Dehradun District of Uttarakhand State (18). The virus circulated in the region and 2 years later it caused an outbreak in sheep in a nearby place (Rishikesh). No other bluetongue outbreak, due to serotype-23 or any other serotype, has been subsequently reported in this region.

A nation-wide programme - All India Network Programme on Bluetongue - was then established in order to monitor and control the presence of bluetongue in domestic ruminants in the sub-Himalayan Indian plains and in the Deccan. Due to peculiarities of the terrain, very little BTV sero-surveillance studies have been conducted in the past in the hilly states of Northern India, especially in the state of Uttarakhand. The aim of this study was to assess the BTV situation in this region.

In the beginning of 2009, subsequently to an outbreak of peste des petits ruminants (PPR) in the Pithoragarh area, some goat blood and serum samples were sent to our laboratory for detection of peste des petits ruminants virus (PPRV) antibodies. The sera resulted positive for PPRV antibodies. Serum and blood samples were also examined for BTV antibody/antigen and a number of samples were found positive. This prompted a further investigation on BTV seroprevalence on a fairly large number of samples from the area. In November 2011 serum and EDTA (Ethylenediaminetetraacetic acid) blood samples were collected from 51 goats of 3 villages around the town of Pithoragarh (altitude 1,635 m) in Uttarakhand State (16 from Swakot Village, 28 from Kalsin Village and 7 from Sintoli Village). They were brought to laboratory in ice and stored properly until further investigations were conducted. Incidentally, 1 month prior to sampling, an outbreak of PPR in goats was reported in these villages causing the death of 34 goats in the Swakot Village alone according to data received from the local veterinarians.

Presence of BTV antigen was tested in EDTA blood samples by a polyclonal antibody-based group-

Bluetongue virus serotype-1 in goats in India Mondal et al.

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Figure 1. Detection of BTV antigen by sandwich enzyme-linked immunosorbent assay in the infected cell lysates. The cells were infected with PTG-13 virus and supernatants were collected after each passage for the assay. P0 indicates goat blood sample and P1-P5 indicates passage number of the virus isolate. Inset: Cytopathic effects (rounding and ballooning) produced by PTG-13 virus on BHK-21 cells at 24 hpi.

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Serum neutralisation test (SNT) was done by microneutralisation assay as described in (15) to detect neutralising antibodies against BTV-1 and 23 in goat sera. Ten serum samples from those goats which showed high OD values in the BTV ELISA antigen test were used against 200 TCID50 of each of the following viruses: BTV-1 [isolates PTG-13 and MKD-18 (4)] and BTV-23 Dehradun isolate (17). Out of 10, 6 samples were from Swakot village (PTG-1, 5, 10, 11, 12 and 14) including the sample number PTG-13, which yielded the isolate BTV-1 PTG-13, and 4 samples were from Kalsin Village (PTG-17, 18, 24 and 31). It was interesting to note that no neutralising antibodies were detected in any of the above goat sera (including the sample No. PTG-13) against PTG-13 and MKD-18. However, high level of neutralising antibodies against BTV-23 Dehradun isolate was detected in all serum samples, including the sample No. PTG-13, at 1:128 dilution and in 6 samples at 1:256 dilution.

In this study, a BTV-1 (PTG-13) has been isolated on cell culture from an asymptomatic goat. No neutralising antibodies against the PTG-13 virus or MKD-18 isolate of BTV-1 were detected in goat sera. Furthermore, high level of neutralising antibodies was detected against BTV-23. Based on the previous reports of virus isolation (3, 4, 8, 12, 13, 17, 20, 22), it could be considered that prevalence of BTV-1 and BTV-23 is higher in the North-Western states and foot hills of Uttarakhand (Figure 3) and so, we have attempted to search neutralizing antibodies in the goat sera against these 2 serotypes. However, antibodies against other BTV serotypes could have been present in the goats involved in this study; similarly viraemia caused by other serotypes could not be excluded. The presence of BTV-1 viraemic animal suggests that BTV-1 was circulating at the time of sampling. The infection in goats with 2 serotypes, in fact, could have occurred in distinct periods. Failure to isolate BTV-23 may be due to absence of the virus in the animals at the time of sampling. The appearance of BTV-23 neutralising antibodies in the blood circulation coincided with the decline in virus titre. In general, the peak of bluetongue viraemia occurs in the first 2 weeks of infection, before the appearance of neutralising antibodies, and then the virus titre drops rapidly to a low level when infection persists for a month or more (14, 26).

It was possible to isolate the PTG-13 virus from goat blood because sampling was done probably within the first 2 weeks of infection (when the viraemia was at the peak) before appearance of neutralising antibody against PTG-13 virus. This also explains the absence of neutralising antibody in the goat serum samples against the PTG-13 virus. Isolation of BTV from blood is more successful when sample is collected at peak viraemia. However, in the later phase of infection virus isolation is possible

using Tri reagent (Sigma, St. Louis, MO, USA) according to the protocol described by Attoui et al. (1). The dsRNA was resolved by polyacrylamide gel electrophoresis (PAGE) and then stained with silver nitrate (10, 25). Ten bands, typical of BTV, or any Orbivirus (18) genome, were observed to have migrated according to their size (Figure 2a).

Polyacrylamide gel electrophoresis, silver staining and s-ELISA all confirmed PTG-13 as a BTV. The serotype of the virus was determined by RT-PCR coupled with sequencing of a fragment of genome segment-2 (seg-2). Considering the endemicity of BTV-1 in the Northern part of India, PCR was performed on PTG-13 virus with primers specific to seg-2 of the same serotype as per the procedure described in (4). A fragment of 828bp size (nucleotide position 50-878 bp) of seg-2 was amplified from PTG-13 virus and was visualised on agarose gel electrophoresis (Figure 2b). The PCR product was cloned and sequenced. BLAST (Basic Local Alignment Search Tool http://www.ncbi.nlm.nih.gov/) analysis of the sequence revealed 99% nucleotide identity (data not shown) with the seg-2 of recent BTV-1 isolates MKD-18 (GenBank accession No. HM014236), SKN-7 (JN558348) and SKN-8 (JN558349). PCR and partial sequencing of seg-2 confirmed PTG-13 as BTV-1.


Mondal et al. Bluetongue virus serotype-1 in goats in India

Figure 2. Polyacrylamide gel electrophoresis and polymerase chain reaction.(A) BHK cells (in 75 cm2 flask) were infected with the PTG-13 virus, and viral genomic dsRNA was extracted and resolved in 10% polyacrylamide gel (5h, 120 V) and silver-stained.Ten dsRNA segments, typical of BTV (or any Orbivirus) genome, were observed (segments are numbered).(B) Agarose gel electrophoresis of PCR product.Amplification of segment-2 of PTG-13 virus was done by RT-PCR using BTV-1 VP2 (segment-2) gene-specific primers. An 886-bp (nucleotide position 7-893 of segment-2) product was amplified (lane-2) using primers described by Biswas et al. 2010 (4). Lane-1 contains 1Kb ladder DNA molecular weight marker.

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even in the presence of neutralising antibodies; because bluetongue virus is strongly cell-associated during viraemia, and there is strong evidence that this cellular association protects circulating virus from elimination by neutralising antibodies during prolonged infection (2). Due to this fact, we have selected viraemic animals for SNT (Serum neutralization test) with the expectation that both virus (BTV) and its neutralizing antibody would be present together in blood. In this case, the detection of both virus and its neutralising antibodies in the same animal would have been a strong evidence of infection. However, we could not detect BTV-1 antibodies in the viraemic animals but the chance to find BTV-1 antibodies would have been higher in non-viraemic animals.

It is worth mentioning that highest rate of BTV antigen prevalence (87.5%) and the highest OD values in the BTV ELISA antigen were observed in the goats of Swakot Village where a heavy mortality was reported due to PPR. It seems that there is a correlation between PPR infection and surge of BTV antigen (or virus) in small ruminants if the latter virus is already present in the host. In several occasions, BTV has been isolated from sheep or goats naturally infected with the PPRV (4, 18) and in such cases of mixed infection, the PPR symptoms become prominent (19). The surge of BTV antigen or virus may be due to flare up of virus that was already present inthe goats (in this study) because of stress or immunosuppressive conditions. The role of PPRV for suppressing the immune system of the goats should not be ignored in this case. A few cases of mixed viral infections in sheep and goats involving PPRV and orf virus, goat pox virus or BTV have been reported in India and other countries (16, 17, 24). Morbilliviruses, in general, have been shown to cause suppression of the host immune system (27, 28). Recent studies show that experimental PPRV infection in goats can lead to marked suppression of host immune response accompanied by severe leucopaenia (21).

BTV-23 was isolated more than 15 year ago from Dehradun and Rishikesh, and at present, seems to have established its presence at Pithoragarh (a place at hills about 220 Km North-East to Dehradun) as evidenced in this study. Bluetongue virus serotype-1, which normally circulates in the North Indian valleys, has now invaded the hills. Finally, the isolation of BTV-1 (PTG-13) from goat blood, the presence of high titre neutralising antibodies in goat sera against BTV-23 and the absence of neutralising antibody against PTG-13 virus suggest that goats were naturally infected with BTV-1 and 23 in 2 moments in time.

AcknowledgmentThis work has been supported by ICAR All India Network Project on Bluetongue (AINP-BT). Authors would like to extend their gratitude tothe Head of the Division of Virology, IVRI Mukteswar for providing facilities in which this study was conducted.



Figure 3. Map showing the North-Western states of India where strains of BTV-1 and 23 have been isolated. Bluetongue virus serotype-1 was isolated from Hisar (12), Avikanagar (20), Mathura (isolate MKD-18, 4), Jasdan (isolate SKN-7, 8), SK Nagar (Sardarkrushinagar, isolate SKN-8, 8) and Pithoragarh (isolate PTG-13, this study). Bluetongue virus serotype-23 was isolated from Dehradun (17) and Rishikesh (22).JK: Jammu & Kashmir; HP: Himachal Pradesh; PB: Punjab; UK: Uttarakhand; HR: Haryana; RJ: Rajasthan; UP: Uttar Pradesh; GJ: Gujarat.


Mondal et al. Bluetongue virus serotype-1 in goats in India

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16. Martrenchar A., Zoyem N. & Diallo A. 1997. Experimental study of mixed vaccine against peste des petits ruminants and capripox infection in northern Cameroon. Small Rumin Res, 26, 39-44.

17. Mehrotra M.L., Shukla D.C., Singh K.P., Khanna P.N. & Saikumar G. 1995. Isolation of bluetongue virus from an outbreak of Morbillivirus infection in sheep andgoat. Ind J Comp Microbiol Immunol Infect Dis, 16, 135-136.

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21. Rajak K.K., Sreenivasa B.P., Hosamani M., Singh R.P., Singh S.K., Singh R.K. & Bandyopadhyay S.K. 2005. Experimental studies on immunosuppressive effects of peste des petits ruminants (PPR) virus in goats. Comp Immunol Microbiol Infect Dis, 28, 287-296.

22. Ramakrishnan M.A., Singh K.P., Pandey A.B., Singh R., Nandi S. & Mehrotra M.L. 2005. Genomic diversity of Indian isolates of bluetongue virus. Acta Virologica, 49, 285-286.

23. Ravishankar C., Krishnan Nair G., Mini M. & Javaprakasan V. 2005. Seroprevalence of bluetongue virus antibodies in sheep and goats in Kerala State, India. Rev Sci Tech, 24, 953-958.

24. Saravanan P., Balamurugan V., Sen A., Sarkar J., Sahay B., Rajak K.K., Hosamani M., Yadav M.P. & Singh R.K. 2007. Mixed infection of peste des petits ruminants and orf on a goat farm in Shahjahanpur, India. Vet Rec, 160, 410-412.

25. Squire K.R.E., Chuang R.Y., Osburn B.I., Knudson D.L. & Doi R.H. 1983. Rapid methods for comparing the double-stranded RNA genome profiles of bluetongue virus. Vet Microbiol, 8, 543-553.

26. Tessaro S.V. & Clavijo A. 2001. Duration of bluetongue viremia in experimentally infected American bison. J Wild Dis, 37, 722-729.

27. Wohlsein P., Wamwayi H.M., Troutwein G., Pohlelz J., Liess B. & Barrett T. 1995. Pathomorphological



and immunohistological findings in cattle experimentally infected with rinderpest virus isolates of different pathogenicity. Vet Microbiol, 44, 144-147.

28. Yanagi Y., Cubitt B.A. & Oldstone M.B.A. 1992. Measles virus inhibits mitogen induced T-cell proliferation but does not directly perturb the T-cell activation process inside the cell. Virology, 187, 280-289.

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LIBRI/Book reviewsa cura di Manuel Graziani

Da sempre i Servizi Veterinari Pubblici sono una componente poco conosciuta del Servizio Sanita-rio Nazionale, le loro prestazioni vengono spesso fruite in modo inconsapevole sia dalla popolazio-ne che dall’amministrazione pubblica. È pleonastico ricordare che si tratta di attività sanitarie il cui scopo prevalente è quello di tutelare la salute animale e umana e di promuovere la produttività e il benessere delle popolazioni animali. Ma vale la pena ribadire che tali attività hanno un ruolo assai rilevante anche sotto il profilo economico e sociale. Questo corposo volume fa il punto sulle com-petenze che le diverse componenti professionali devono possedere. Un volume importante per comprendere la realtà veterinaria italiana, pubblica e privata, nato dal lavoro congiunto di figure di primo livello della medicina veterinaria del Paese: i curatori sono tutti docenti del Dipartimento di Scienze Mediche Veterinarie dell’Università di Bologna, convinti assertori del ruolo sociale della Medicina veterinaria, del concetto di Medicina/Salute unica e della necessità di migliorare l’aspetto formativo. I professori Battelli, Baldelli, Ostanello e Prosperi fanno parte di quella che viene defini-ta la “Scuola bolognese di Sanità Pubblica Veterinaria” il cui fondatore è stato Adriano Mantovani (scomparso a marzo dello scorso anno), unanimemente considerato il Padre della SPV italiana e di cui vengono qui pubblicati 3 scritti e una breve scheda biografica che lo ricorda.

Il volume è suddiviso in tre parti allo scopo di fornire al lettore un percorso interpretativo logico che si sviluppa verso la comprensione delle competenze e dei campi di attività dei Servizi Veterinari nel nostro Paese, fino a mettere in luce le sfide attuali e future che la SPV deve affrontare. Particolare attenzione viene posta nei confronti dell’evoluzione che tali attività sanitarie potranno avere nel prossimo futuro, in riferimento ai cambiamenti socio-economici, alle attività di produzione e com-mercializzazione di alimenti di origine animale, alle emergenze di tipo sanitario, alle nuove istanze dei consumatori e degli amministratori pubblici. La prima parte “La storia e i concetti” si sofferma in particolare sull’evoluzione della SPV in Italia, sulle zoonosi, sul rapporto uomo-animale anche in ambito urbano con l’ultimo contributo relativo all’igiene urbana veterinaria. La seconda parte “Le attività” passa in rassegna i Servizi Veterinari pubblici del Paese, i NAS, gli Enti scientifici (tra cui IIZZSS e ISS) fino a trattare attività quali la sicurezza alimentare, le emergenze non epidemiche, il benessere animale e la pet therapy. Nella terza e ultima parte “Le sfide” ci si interroga sul ruolo sociale della medicina veterinaria, sul concetto di medicina unica, sull’etica nella sperimentazione animale, sulla formazione, l’educazione e la comunicazione.

(Bononia University Press, pp. 422, € 30,00)www.buponline.com

(a cura di) Giorgio Battelli, Raffaella Baldelli, Fabio Ostanello, Santino Prosperi

Gli animali, l'uomo e l'ambiente

383

LIBRI/Book reviewsa cura di Manuel Graziani

Una raccolta di racconti, o meglio di “storie” ma anche di “geografie” riprendendo l’azzeccato titolo scelto dal dott. Raffaele Mastrocola per la sua prima opera di narrativa autobiografica.

Racconti di vita professionale e squarci di vita rurale nel solco del veterinario-scrittore britannico James Herriot. Qui, però, non siamo nell’elegante campagna inglese bensì in quella abruzzese, forte, gentile e certamente più genuina. Tra aneddoti comici, un’ironia delicata e a tratti debor-dante, l’autore ci racconta la sua vita professionale circondata da clienti pittoreschi, per lo più an-ziani, di un’umanità rurale oramai quasi dimenticata, con un’attenzione nostalgica per il periodo dell’infanzia e la formazione universitaria a Bologna (compresi i “rapporti” telefonici con Vasco Rossi e le cene “Da Vito”).

Una lettura gradevole e, perché no, utile ai giovani veterinari che troveranno tra le pagine consigli su diagnosi e terapie convalidate attraverso una lunga esperienza condotta sul campo, anche a beneficio umano, come nel caso del sedano selvatico che l’autore definisce un “antibiotico natu-rale, un toccasana per le vie urinarie”. Un altro racconto di particolare interesse è quello intitolato “Quel benedetto femore” con Mastrocola che narra con trasporto come è riuscito a trovare la so-luzione ad un caso complesso non attingendo ad un manuale di scienza veterinaria, bensì ad un libro di narrativa di James Herriot, suo vero e proprio faro personale: “Ho sempre avuto simpatie per i racconti di vita professionale. Il mio grande ispiratore è stato James Herriott! Oggi a distanza di oltre trenta anni dall’inizio della mia attività, sento la necessità di condividere le mie esperienze, le mie emozioni ed i miei ricordi con altri. La miscellanea dei miei racconti è una raccolta di ‘ricordi giovanili’, vita professionale ed il diario di un mio viaggio in moto d’epoca fatto in Australia.”

Sì perché l’autore, tra i suoi mille interessi, ha anche quello della meccanica e una passione smo-data per le moto d’epoca.

(Booksprint Edizioni, pp. 242, € 15,00)www.booksprintedizioni.it

Raffaele Mastrocola

Storie e geografie di un veterinario di campagna

Veterinaria Italiana, Volume 49 (4), October-December 2013

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