Large Animal Review - Year 26, Number 3, June 2020 by E.V. Soc. Cons. a r.l.

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Poste Italiane spa - Spedizione in A.P. - D.L. 353/2003 (conv. in L. 27/02/2004 N. 46) art. 1, comma 1, DCB Piacenza - Concessionaria esclusiva per la pubblicità: E.V. Soc. Cons. a r.l. - Cremona

03/20

Bimonthly, Year 26, Number 3, June 2020

LAR

Large Animal Review

ISSN: 1124-4593

LARGE ANIMAL REVIEW is ranked in Citation Index (SciSearch®) Journal Citation Reports/Science Edition and CAB ABSTRACTS

ORIGINAL ARTICLES BOVINE • Aggiornamento sull’andamento del piano di controllo di Aflatossina M1 nel latte in Lombardia • Detection of digital and interdigital dermatitis in Holstein Friesian dairy cows by means of infrared thermography OVINE • The effects of non-genetic factors on the morphometric parameters of sheep placenta and the birth weight of lambs • Influences of maternal undernutrition on placental development and birth weight in sheep SWINE • On-farm risk factors associated with Salmonella in pig herds CASE REPORTS BOVINE • Ringworm by Trichophyton erinacei in calves: description of two Italian outbreaks • Palatoschisis, brachygnathia inferior and accessory mandibular mass in a Simmental breed cal A

SWINE • Ricerca del virus dell’epatite E (HEV) in cinghiali durante la stagione venatoria 2017/2018 e 2018/2019

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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INDEX

ORIGINAL ARTICLES

Anno 26, numero 3, Giugno 2020 Rivista indicizzata su: CAB ABSTRACTS e GLOBAL HEALTH IF (2017/2018): 0.26

BOVINE Aggiornamento sull’andamento del piano di controllo di Aflatossina M1 nel latte in Lombardia GIUSEPPE BOLZONI, SARA ARMORINI, CRISTINA BAIGUERA, GIORGIO ZANARDI

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Editor in chief: Massimo Morgante

Detection of digital and interdigital dermatitis in Holstein Friesian dairy cows by means of infrared thermography

Editorial Board 2019-2021: Anna Rita Attili - Roberto Bardini Francesca Bonelli - Marta Brscic Marco Colombo - Vincenzo Cuteri Antonella Dalle Zotte - Enrico Fiore Giovanni Franzo - Matteo Gianesella Elisabetta Giudice - Paolo Moroni Davide Ranucci - Antonia Ricci Giuseppe Stradaioli - Erminio Trevisi Managing Editor: Matteo Gianesella Technical Editor: Enrico Fiore

GIORGIA FABBRI, ENRICO FIORE, GIUSEPPE PICCIONE, ELISABETTA GIUDICE, MATTEO GIANESELLA, MASSIMO MORGANTE, LEONARDO ARMATO, ORTENSIO BONATO, SONIA GIAMBELLUCA, FRANCESCA ARFUSO

LARGE ANIMAL REVIEW è una rivista bimestrale pubblicata per favorire l’aggiornamento dei veterinari che si dedicano alla prevenzione e alla cura delle malattie degli animali da reddito e alla qualità e salubrità dei prodotti derivati.

Consiglio direttivo SIVAR 2020-2023 Mario Facchi (Presidente) Daniele Gallo (Presidente Senior) Alberto Ferrero (Vice-Presidente) Michela Conterbia (Segretario) Vito Loconte (Tesoriere) Alessandro Federici (Consigliere) Osvaldo Parolin (Consigliere) Chiara Musella (Consigliere) Mattia Bottacini (Consigliere) Giuseppe Argiolas (Consigliere) Edizioni SCIVAC Palazzo Trecchi - 26100 Cremona Tel. 0372/460440 Iscrizione registro stampa del Tribunale di Cremona n. 299 del 25/9/1995

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Influences of maternal undernutrition on placental development and birth weight in sheep AYSONDU MEHMET HANIFI, OZYUREK SELCUK

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SWINE

On-farm risk factors associated with Salmonella in pig herds ALESSIA DE LUCIA, FABIO OSTANELLO

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CASE REPORTS

BOVINE Ringworm by Trichophyton erinacei in calves: description of two Italian outbreaks FRANCESCO AGNETTI, ROSA CIAVARELLA, DEBORAH CRUCIANI, ERSILIA MARIA EPIFANIO, DANIELA GOLINELLI, PAOLA PAPA, ELISA SGARIGLIA, ANDREA VALENTINI, SILVIA CROTTI

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OVINE

The effects of non-genetic factors on the morphometric parameters of sheep placenta and the birth weight of lambs ANGELIKA BRZOZOWSKA, NATALIA WOJTASIAK, BARBARA BŁASZCZYK, TOMASZ STANKIEWICZ, MARTA WIECZOREK-DA˛BROWSKA, JAN UDAŁA

Direttore Responsabile Antonio Manfredi

Concessionaria esclusiva per la pubblicità E.V. Soc. Cons. a r.l. Palazzo Trecchi - 26100 Cremona Ufficio Pubblicità: Paola Orioli Tel. 0372/403539 - E-mail: info@sivarnet.it

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Palatoschisis, brachygnathia inferior and accessory mandibular mass in a Simmental breed calf UYGUR CANATAN, M. METİN ŞEN, ERENCAN ÖZFIRAT, MELİKE ÇETIN, HAKAN SALCI

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SWINE

Ricerca del virus dell’epatite E (HEV) in cinghiali durante la stagione venatoria 2017/2018 e 2018/2019 CHIARA MASOTTI, ROBERTA BATTISTINI, WALTER MIGNONE, ENRICA BERIO, MONICA DELLEPIANE, TIZIANA ANDREOLI, ELISABETTA RAZZUOLI, SIMONE PELETTO, PIERLUIGI ACUTIS, CHIARA BELTRAMO, PAOLA MODESTO, VALERIA LISTORTI, CARLO ERCOLINI, LAURA SERRACCA 149

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G. Bolzoni et al. Large Animal Review 2020; 26: 107-112

Aggiornamento sull’andamento del piano di controllo di aflatossina M1 nel latte in Lombardia

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GIUSEPPE BOLZONI1, SARA ARMORINI1, CRISTINA BAIGUERA1, GIORGIO ZANARDI1 1

Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna (IZSLER) - Via Bianchi, 9 25124 Brescia

RIASSUNTO La presenza di aflatossina M1 (AFM1) nel latte è dovuta all’ingestione da parte delle bovine di foraggi e mangimi contaminati da aflatossina B1 (AFB1), che viene metabolizzata e trasformata a livello epatico nel suo metabolita meno tossico. La produzione di aflatossine ad opera di funghi del genere Aspergillus è condizionata da vari fattori, quali specie fungina, substrato e ambiente. La regione Lombardia, con la sua elevata concentrazione di allevamenti di bovine da latte e una produzione pari al 43,3% di quella nazionale, è particolarmente sensibile e attenta al problema anche perché tra i foraggi che possono risultare contaminati il mais (insilato o in farine) rappresenta la componente maggiore della tipica razione delle bovine da latte. Considerata la disomogenea distribuzione dell’AFB1 negli alimenti zootecnici, il monitoraggio della presenza di AFM1 nel latte di massa aziendale risulta particolarmente importante per il controllo di questo tipo di contaminazione a livello di singolo allevamento. Dopo il periodo critico del biennio 2015-2016, appare interessante fornire un quadro sintetico della situazione osservata nel corso degli anni successivi, in particolare del periodo che va dal 2017 a metà del 2019. A tale scopo, sono stati valutati i dati ottenuti con tecnica ELISA (metodo di screening) su oltre 20.000 dei campioni di latte di massa aziendale, conferiti ai laboratori dell’Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, nel periodo di tempo tra il 2017 fino a tutto il primo semestre del 2019. Dall’osservazione dell’andamento dei dati ottenuti nel periodo in oggetto e dal confronto con quello delle annate precedenti (2012-2016), appare evidente il superamento della situazione di criticità e il mantenimento pressoché costante di bassi livelli medi di contaminazione. I dati ottenuti sono stati suddivisi in base alla tipologia dei campioni: non ufficiali (autocontrolli) ed ufficiali (realizzati dai Servizi Veterinari territoriali) ed anche in funzione dell’entità di contaminazione dei casi di non conformità per superamento del limite normativo (0,050 µg/kg). Dalla valutazione complessiva dei dati è possibile estrapolare informazioni utili alla realizzazione di programmi di monitoraggio continuo e diffuso e calibrare i programmi di controllo in risposta a situazioni, ad esempio, di contaminazioni generalizzate oppure localizzate sia per la prevenzione che, se necessario, per attuare interventi correttivi in caso di emergenze.

PAROLE CHIAVE Aflatossina M1, latte di massa, Lombardia, sicurezza alimentare.

INTRODUZIONE Le aflatossine sono metaboliti secondari prodotti da funghi del genere Aspergillus, soprattutto da A. flavus e A. parasiticus1. I cereali, in particolare il mais, rappresentano un ottimo substrato per la loro crescita e le condizioni climatiche di tipo caldo-umido, caratteristiche della zona della Pianura Padana, favoriscono contaminazione e produzione. La contaminazione del latte da aflatossina M1 (AFM1) deriva direttamente, per metabolizzazione epatica, da quella di aflatossina B1 (AFB1) in foraggi e mangimi. La temperatura, l’umidità e la disponibilità di acqua per l’irrigazione durante le fasi di crescita dei cereali, ma anche le condizioni ambientali durante le successive fasi di raccolta e stoccaggio dei prodotti (ad esempio: insilamento, macinazione, fioccatura), sono i principali punti critici per la produzione di tossine da parte dei funghi responsabili. A seconda delle condizioni climatiche, dei microrganismi coinvolti e del tipo di vegetale, la pro-

Corresponding Author: Giuseppe Bolzoni (giuseppe.bolzoni@izsler.it).

duzione delle tossine può avvenire già in campo durante la coltivazione oppure nelle fasi successive di stoccaggio; nei climi temperati, ad esempio, la contaminazione da A. flavus è molto spesso associata a danneggiamenti dei semi causati da insetti in condizioni di clima siccitoso, anche se la produzione di tossina avviene soprattutto nelle fasi post-raccolto2,3. Le alte temperature giocano un ruolo determinante nella riproduzione dei microrganismi nella fase pre-raccolto4,5, ma possono influenzarne l’evoluzione anche in quelle successive: la permanenza a 32-38 °C agevola l’infezione6 e, nell’arco di 16-24 giorni, la produzione di tossine7. È stato osservato anche che i funghi, che persistono nel suolo sotto forma di ascospore e macroconidi, sono favoriti nell’attacco al cereale quando, nel periodo immediatamente precedente la raccolta, si verificano condizioni di elevata umidità e piovosità8,9. Le condizioni di forte siccità e insufficiente irrigazione, inoltre, incrementano replicazione e contaminazione fungina dei cereali nelle fasi di crescita e possono essere ulteriormente peggiorate dalla compresenza della piralide del mais (Ostrinia nubilalis) che veicola le spore fungine e danneggia la pianta in condizioni di stress idrico10. Essendo evidentemente impossibile agire sulle condizioni climatiche, le strategie di prevenzione si possono indirizzare

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ad esempio sulla scelta degli ibridi di coltivazione, ma si concentrano soprattutto sul trattamento dei prodotti nella fase post-raccolta con la rapida essiccazione e la gestione delle condizioni microambientali di stoccaggio dei prodotti nell’industria mangimistica2,11. A livello di singolo allevamento particolare attenzione deve essere riservata alla gestione degli insilati ed al monitoraggio della contaminazione del latte nel corso dell’annata o in occasione dell’introduzione di nuove partite di prodotti commerciali. L’AFB1 è considerata il più potente cancerogeno epatico presente in natura12. Secondo la classificazione della International Agency for Research on Cancer (IARC) l’AFB1 appartiene al Gruppo 1 con effetti cancerogeni accertati, mentre AFM1 appartiene al gruppo 2B ed è definita come potenzialmente cancerogena per l’uomo, vale a dire che vi è una evidenza limitata di cancerogenicità nell’uomo e meno che sufficiente evidenza di cancerogenicità negli animali da esperimento, ovvero c’è inadeguata evidenza di cancerogenicità nell’uomo, ma c’è sufficiente evidenza di cancerogenicità negli animali da esperimento5,13. Per quanto riguarda la quantificazione del rischio nei prodotti lattiero-caseari è poi importante sottolineare la termoresistenza della AFM1 rispetto ai classici trattamenti di pastorizzazione del latte. In conseguenza di ciò, e in applicazione del principio di precauzione, il Reg (UE) n. 1881/200614 e successive modifiche, stabilisce il limite massimo tollerato di AFM1 negli alimenti, a 0,050 µg/kg per il latte. La Dir. 2002/32/CE15 fissa i limiti di aflatossine per l’alimentazione animale: 5 µg/kg per i mangimi composti per bovine da latte e 20 µg/kg per le materie prime. Inoltre vieta l’uso di agenti chimici per decontaminare le partite di prodotti contaminati e la possibilità di miscelare partite conformi con quelle non conformi. L’ingestione media di AFB1 dovrebbe essere inferiore a 40 µg/capo/die al fine di produrre latte con AFM1 < 0,050 µg/kg16. La regione Lombardia, con quasi il 30% delle bovine da latte del patrimonio nazionale (29,7% di 1.693.332, fonte Istat 2018) ed il 43,6% della produzione di latte (433.737 su 993.126 tonnellate, fonte Istat 2018) è da decenni particolarmente sensibile al problema, poiché il mais rappresenta il principale costituente della razione alimentare di quasi tutti i circa 4.000 allevamenti di pianura (in montagna prevale invece un’alimentazione tradizionale con periodo di pascolo estivo). Anche sulla base delle esperienze maturate negli ultimi anni, i sistemi di monitoraggio, verifica e intervento correttivo si sono notevolmente evoluti sia in termini di prevenzione (colture, stoccaggio delle materie prime, trattamenti di “ripulitura” in mangimificio) sia in termini di controllo degli alimenti zootecnici e del latte e derivati. La disomogenea distribuzione della aflatossina B1 nelle partite di alimenti zootecnici (materie prime, farine, insilati) determina però che spesso i campionamenti eseguiti sui prodotti foraggeri siano poco rappresentativi ed estremamente variabili nel tempo soprattutto nel caso di grandi partite di prodotto17,18. Proprio per questo l’importanza del controllo di AFM1 nel latte di massa aziendale (e/o di quello delle cisterne di raccolta in arrivo ai caseifici) è stato individuato come il più efficace indicatore sia per i monitoraggi preventivi che per i controlli puntuali e le verifiche successive ai casi di non conformità. Tempi relativamente brevi, generalmente circa 3-5 giorni, trascorrono tra la somministrazione dell’alimento, il rilievo della contaminazione del latte e l’eventuale intervento correttivo sulla razione. In particolare, AFB1 assun-

ta con l’alimento contaminato viene idrossilata a livello epatico e trasformata in AFM1 dopo circa 12 ore10. Il carry over varia dallo 0,3 al 6,2% a seconda della specie animale e anche dello stadio di lattazione, più intenso nella prima fase19. Dopo circa 72 ore di sospensione dell’assunzione il contenuto di aflatossina M1 nel latte diminuisce progressivamente ed in misura consistente18. Dopo un periodo difficile come il biennio 2015-2016, che ha visto, tra l’altro, la realizzazione del Piano Straordinario messo in atto dai Servizi Veterinari regionali20 e un significativo incremento delle attività di autocontrollo richieste ai produttori di latte ed all’industria di trasformazione, appare particolarmente interessante fornire un quadro sintetico della situazione osservata nel corso degli anni successivi, in particolare del periodo che va dal 2017 a metà del 2019.

MATERIALI E METODI I dati utilizzati per le seguenti valutazioni riguardano campioni di latte analizzati con metodica di screening (ELISA quantitativo) dai laboratori dell’IZSLER (Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna) nel periodo di tempo che va dal 2017 al primo semestre del 2019 (complessivamente oltre 20.000 campioni di latte di massa aziendale provenienti da oltre 3.000 allevamenti). Il Kit in uso presso i nostri Laboratori (I’screen AFLA M1, TECNA), presenta, secondo la validazione del Metodo di prova interno accreditato: campo di applicazione tra 0,005 e 0,100 µg/kg (gli esiti superiori al limite massimo, seppur quantificabili, vengono espressi come > 0,100), LOD di 0,0025 µg/L e LOQ di 0,005 µg/L, incertezza di misura estesa stimata al 22,6%. L’attività di campionamento comprende sia i campioni ufficiali prelevati dalle autorità sanitarie territoriali che quelli conferiti da singoli allevatori e da strutture casearie in applicazione dei piani di autocontrollo eseguiti nelle due regioni di competenza; quest’ultima componente afferisce in parte anche a vari altri laboratori accreditati nelle due regioni ed è pertanto soltanto parzialmente rappresentata dai dati che seguono. Sono esclusi anche i risultati ottenuti su un numero decisamente più limitato di campioni analizzati con metodiche di riferimento che sono riservate ai soli casi di campionamento ufficiale legale a seguito di positività o su sospetto. A scopo di confronto, nelle illustrazioni seguenti sono stati riportati anche i risultati ottenuti, con il medesimo tipo di attività, negli anni precedenti a partire dal 2012, per un totale di oltre 100.000 campioni analizzati.

RISULTATI E DISCUSSIONE Il quadro complessivo dell’attività e dei risultati ottenuti nel corso di questi ultimi anni, suddivisi per mesi, è riassunto in Grafico 1 che mostra l’andamento dei dati ottenuti nel periodo oggetto dello studio (2017-2019) e in quello ad esso antecedente (2012-2016); sono riportati il numero di campioni analizzati (102.314, linea rossa) e la contaminazione media in essi riscontrata (linea blu). La situazione attuale è direttamente confrontabile con quella osservata nei due recenti periodi di maggior criticità del 2012 e della fine 2015-inizio 2016 (con medie complessive rispettiva-

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G. Bolzoni et al. Large Animal Review 2020; 26: 107-112

mente di 30 e 25 ppt). L’andamento dell’ultimo biennio appare invece sostanzialmente costante con contaminazioni medie comprese tra 5 e 10 ppt che, anche considerando il limite minimo di sensibilità della metodica analitica (5 ppt), rappresentano un livello di tutta sicurezza. Va sottolineato che il periodo di maggior interesse di ogni anno è quello dell’ultimo quadrimestre, in quanto è in questo periodo che i foraggi provenienti dalle colture estive entrano

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progressivamente nel circuito commerciale sotto forma di farine, mangimi, o pastoni, oppure direttamente nella razione del singolo allevamento per l’apertura degli insilati di mais della nuova stagione. Nel Grafico 2 i campioni analizzati, con riferimento alla sola Regione Lombardia, sono divisi in base alla tipologia di appartenenza in non ufficiali (autocontrolli) ed ufficiali (realizzati dai Servizi Veterinari territoriali), e ne viene mostrata la

Grafico 1 - Andamento del valore medio di Aflatossina M1 in campioni di latte di massa (102.314 in totale) in regione Lombardia ed Emilia-Romagna.

Grafico 2 - Andamento trimestrale dei risultati delle analisi per ricerca di Aflatossina M1 nel latte di massa, suddivisi in campioni ufficiali e in autocontrollo.

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Grafico 3 - Andamento dei dati ottenuti da campioni di latte di massa non conformi per Aflatossina M1 in regione Lombardia analizzati fino a giugno del 2019 (totale campioni 69.776).

relativa quota di non conformità. Viene inoltre differenziata la categoria relativa ai campioni di “latte crudo destinato alla vendita diretta” che, seppur numericamente ridottissima, si contraddistingue per la definizione di un limite di conformità più restrittivo (0,03 µg/kg). Tale limite è stato definito in Regione Lombardia in relazione al fatto che questo prodotto giunge direttamente dal singolo allevamento al consumatore finale (senza la caratteristica miscelazione con quello di altri allevamenti tipica della normale filiera di trasformazione degli altri prodotti). Per quanto riguarda, in generale, le percentuali di non conformità è importante sottolineare che fanno riferimento ai campioni analizzati; il numero di allevamenti coinvolti è pertanto inferiore. Ciò deriva in particolare dal fatto che, a seguito di esiti non conformi o comunque elevati, negli stessi allevamenti si ripetono più volte i campionamenti nei giorni successivi come verifica delle azioni correttive applicate alla composizione della razione alimentare quotidiana. Dal medesimo grafico si può inoltre osservare come, tenuto conto della differente scansione temporale (anni/trimestri), il numero di campioni analizzati in regime di autocontrollo si sia mantenuto pressoché costante mentre ci sia stata una riduzione delle analisi relative ai campioni ufficiali, che deriva sostanzialmente dalla rimodulazione del Piano Straordinario conseguente al progressivo miglioramento della situazione generale nel corso del tempo. L’indicatore relativo alla percentuale di non conformità è quello previsto dal limite normativo di 0,050 µg/kg14 ovvero 50 ppt. Il Piano Regionale20, relativo sia ai controlli ufficiali che a quelli in regime di autocontrollo per mangimifici, industria casearia e allevamenti, ha però previsto alcuni specifici indicatori aggiuntivi che, per quanto riguarda la produzione primaria, possono essere così sintetizzati: – limite di 0,030 µg/kg (30 ppt) per il latte crudo destinato a vendita diretta in considerazione del rapporto diretto tra produttore e consumatore finale;

– limite di 40 ppt come livello di guardia (soglia di attenzione) che richiede interventi rapidi di controllo e correzione al fine di prevenire ulteriori incrementi della contaminazione3,4; – per il solo limite di non conformità è stata presa in considerazione l’incertezza di misura analitica, al fine di differenziare i casi di “contaminazione certa” (con blocco immediato della commercializzazione del latte e distruzione del prodotto come da Reg (CE) n. 1069/2009)21 da quelli di “contaminazione probabile” con necessità immediata di intervento correttivo della razione e verifica del rientro nei giorni a seguire (si veda in merito anche il parere n° 0019699 del 11/07/2016 emesso dal Laboratorio Nazionale di Riferimento dell’Istituto Superiore Sanità). Il Grafico 3 offre un ulteriore approfondimento in quanto rappresenta l’entità delle contaminazioni osservate nei campioni risultati non conformi nel corso del tempo. In base al contenuto di AFM1 osservato in tali campioni si sono voluti distinguere: – i casi border-line (valori compresi tra 51 e 65 ppt; linea azzurra) che, tenuto conto dell’incertezza di misura degli esiti, potrebbero ricadere probabilisticamente anche al di sotto del limite legale; – i casi di non conformità certa ma di entità modesta (valori compresi tra 65 e 99 ppt; linea rossa) che sono in genere rapidamente risolvibili con parziali modifiche della razione in azienda; – i casi di non conformità più consistente, che potrebbero rappresentare un problema reale anche per gli effetti sanitari per i consumatori (valori superiori a 100 ppt; linea verde). Ciò può offrire, in specifici momenti di attuazione del Piano, importanti indicazioni sull’entità del problema che, insieme alle valutazioni sulla diffusione geografica dei casi, permettono di modulare gli interventi di controllo e quelli di correzione nelle singole aziende (anche se ovviamente, dal punto di vista grafico, ciò è apprezzabile soltanto nei periodi di significativa presenza di campioni non conformi).

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Grafico 4 - Andamento dei valori medi di concentrazione di aflatossina M1 in campioni di latte bovino e di specie diversa dal bovino.

Un’ultima rappresentazione che vogliamo fornire, quasi soltanto come curiosità considerato il numero limitato di campioni (1.847 in totale), riguarda gli esiti ottenuti su campioni di latte di altre specie animali nel medesimo intervallo temporale (2012-2019) (Grafico 4). Se in passato questo tipo di controlli era effettuato raramente, negli ultimi anni, anche in relazione alle situazioni di crisi del settore bovino, le richieste al laboratorio sono cresciute. Va ovviamente ricordato che, seppure il latte di capra, bufala e pecora sia normalmente destinato all’alimentazione umana (in stragrande maggioranza dopo trasformazione in formaggi), in Regione Lombardia la produzione risulta decisamente limitata rispetto ad altre aree geografiche. Tenuto conto del numero decisamente limitato di campioni (che può fortemente condizionare il valore medio osservato in un singolo periodo) ed estendendo il limite di conformità di 50 ppt anche alle altre specie, potremmo concludere che, anche nei periodi di emergenza, il coinvolgimento di questi settori produttivi sembra essere decisamente episodico: – 1 campione di latte di bufala non conforme su 441 controllati (0,23%); – 17 campioni di latte di capra non conformi su 976 controllati (1,71%); – 2 campioni di latte non conformi tra pecora, asino e cavallo su 430 controllati (0,46%). Oltre alle differenze di razione alimentare rispetto ai bovini (meno significative nel caso del bufalo rispetto alle altre specie), anche le differenze metaboliche possono infatti avere un ruolo determinante nella trasformazione in AFM1 delle eventuali AFB1 dei foraggi (in particolare nel caso degli equidi monogastrici).

CONCLUSIONI L’insieme dei risultati ottenuti conferma l’importanza di realizzare sistemi di monitoraggio e controllo, estesi e ripetuti

nel corso dell’anno, al fine di valutare questo tipo di contaminazione del latte. Sia la dinamica nel tempo che le possibili differenze in diverse aree territoriali geografiche possono infatti presentare fortissime variazioni: nel primo caso in relazione alle condizioni climatiche e nel secondo anche per l’immissione in bacini commerciali di singoli impianti di produzione di foraggi o mangimi contaminati. Soltanto con programmi di monitoraggio continuo e diffuso è quindi possibile percepire situazioni di incremento generalizzato o, in alternativa, focalizzato in alcune aree del valore medio di contaminazione. Da queste informazioni è poi possibile calibrare i programmi di controllo ufficiale sia in termini preventivi che di intervento in emergenza, in funzione del rischio reale che si profila. È proprio da questo tipo di informazioni che è stato deciso di incrementare i controlli del 2016 e di ridurli nel primo semestre del 2017. L’esperienza maturata in quasi un ventennio ci ha del resto insegnato che l’ottimizzazione dei costi e dell’efficienza di un sistema di controllo dipendono molto più dall’organizzazione del sistema durante le situazioni di “normalità” piuttosto che dall’intensità degli interventi nelle fasi di emergenza. Anche per questo possiamo definire gli anni presi in considerazione (2017-2019) come “non problematici” senza ovviamente illuderci per questo di poter prevedere l’andamento dei prossimi anni (a cominciare ovviamente dal secondo semestre dell’anno in corso), ma anzi confermando l’assoluta necessità di proseguire con il sistema di monitoraggio attuale.

❚ Update on the aflatoxin M1 contamination trend in Lombardy Region

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Aggiornamento sull’andamento del piano di controllo di Aflatossina M1 nel latte in Lombardia

SUMMARY The contamination of milk by aflatoxin M1 (AFM1) depends on the presence of aflatoxin B1 (AFB1) in animal feed, especially corn. The surveillance of AFM1 levels in bulk milk is an indirect but rapid and efficient indicator of the possible contamination of feed. The 2016 was a critical year for that concern the contamination of cereals by AFB1 leading to the implementation of a special control plan and an increase of own checks plans. In this contest, a summary overview of the situation of the years following 2016, in particular the period from 2017 to first six months of 2019, seems to be required. Approximately 20,000 milk samples (official samples taken in the scope of the extraordinary regional control plan and samples conferred to our laboratories by farmers and milk enterprises in self-control plans applications) were analyzed by an ELISA quantitative method. Comparing the data obtained in different years, the trend obtained shows that, after the critical years 2012 and 2015, during the evaluated period (2017-2019) the levels of AFM1 in milk remained almost unchanged and well below the legal limit of 0.050 µg/kg. Furthermore, the samples analyzed were split into two categories according to type (official samples and samples collected from the own checks plans) and for each group the corresponding non-compliance was assessed. The results obtained confirmed the importance to carry out monitoring and control systems, continuous and repeated during the time, with the aim to assess the contamination of milk by aflatoxins. Only in this way is it possible to detect a general or limited to some areas increase of the cereal contamination. Working on this base, it is possible to coordinate official control programs, as preventive as emergency action plans, in relation to the risk assessed.

KEY WORDS Aflatoxin M1, bulk tank milk, Lombardy Region, food safety.

Bibliografia 1. Nidhina N., Bhavya M.L., Bhaskar N., Muthukumar S.P., Murthy S. (2017). Aflatoxin production by Aspergillus flavus in rumen liquor and its implications. Food Control 71: 26-31. 2. Miller D. J. (1995). Fungi and mycotoxins in grain: Implications for stored product research. Journal of Stored Products Research 31(1): 1-16. 3. Lee U.S., Jang H.S, Tanaka T., Oh Y.J., Cho C.M., Ueno Y. (1987). Effect of milling on decontamination of Fusarium mycotoxins nivalenol, deoxynivalenol, and zearalenone in Korean wheat. Journal of Agriculture and Food Chemistry 35: 126-129. 4. Marsh P.B., Simpson M.E., Craig G.O., Donoso J., Ramey H., Jr. (1973). Occurrence of aflatoxin in cotton seeds at harvest in relation to location of growth and field temperatures. Journal of Environmental Quality 2: 276-281.

5. Simpson M.E., Batra L.R. (1984). Ecological relations in respect to a boll rot of cotton caused by Aspergillus flavus. In Toxigenic Fungi: Their Toxins and Health Hazard, Eds. Kurceta H. and Uneo Y., 24-32, Elsiever, Amsterdam. 6. Jones R.K., Duncan H.E., Payne G.A., Leonard K.J. (1980). Factors influencing infection by Aspergillus flavus in silk inoculated corn. Plant Disease 64: 859-863. 7. Payne G.A. (1983). Nature of field infection of corn by Aspergillus flavus. In: Aflatoxin and Aspergillus flavus in corn. Ed. Diener U.L., Asquith R.L., Dickens J.W., 16-19, Department of Research Information, Alabama Agricultural Experiment Station, Auburn University, Auburn. 8. Haouet M.N., Altissimi M.S. (2003). Micotossine negli alimenti e micotossicosi animale e umana. Webzine Sanità Pubblica Veterinaria n. 18. 9. Jard G., Liboz T., Mathieu F., Guyonvarc’h A., Lebrihi A. (2011). Review of mycotoxin reduction in food and feed: from prevention in the field to detoxification by adsorption or transformation. Food Additives & Contaminants: Part A 28(11): 1590-1609. 10. Brera C. Cinque domande sulle aflatossine. http://old.iss.it/binary/efsa/ cont/Aflatossine_Brera.pdf 11. Chatterjee D., Chattopadhyay B.K., Mukherje S.K. (2008). Storage deterioration of maize having pre harvest infection with Aspergillus flavus. Letters in Applied Microbiology. 11(1): 11-14, June 2008. 12. Mc Kean C., Tang L., Billam M., Tang M., Theodorakis C.W., Kendall R.J., Wang J. (2005). Comparative acute and combinative toxicity of aflatoxin B1 and T-2 toxin in animals and immortalized human cell lines. Journal of Applied Toxicology 26(2): 139-47. 13. International Agency of Research on Cancer (IARC) (2012). Monographs on the Evaluation of Carcinogenic Risks to Humans. In: IARC Monographs 100: 225-248. 14. Regolamento (CE) n. 1881/2006 della Commissione, del 19 dicembre 2006, che definisce i tenori massimi di alcuni contaminanti nei prodotti alimentari (Testo rilevante ai fini del SEE). 15. Direttiva (CE) n. 32/2002 del Parlamento europeo e del Consiglio, del 7 maggio 2002, relativa alle sostanze indesiderabili nell’alimentazione degli animali. 16. Xiong J.L., Wang Y.M., Nennich T.D., Li Y., Liu J.X. (2015). Transfer of dietary aflatoxin B1 to milk aflatoxin M1 and effect of inclusion of adsorbent in the diet of dairy cows (2015). J. Dairy Sci. 98: 2545-2554. 17. Bertocchi L., Scalvenzi A., Santini S., Fusi F. (2012). Aflatossine dal mais al latte? La siccità alimenta i timori. Informatore zootecnico 15: 6-18. 18. Fusi F., Scalvenzi A., Angelucci A., Bolzoni G., Bertocchi L. (2013). Aflatossine: La fase di allerta non può dirsi conclusa. Informatore Zootecnico 9: 54-60. 19. Veldman, A., Meijs, J., Borggreve, G., Heeres-van der Tol, J. Carry-over of aflatoxin from cows’ food to milk. Anim. Sci. 1992, 55, 163-168. 20. D.g.r. 30 marzo 2016 - n. X/4984. Approvazione del «Piano regionale straordinario di sorveglianza del rischio aflatossine nella catena alimentare di produzione del latte e dei prodotti a base di latte. BURL Serie Ordinaria n. 14 - Lunedì 04 aprile 2016. 21. Regolamento (CE) n. 1069/2009 del Parlamento europeo e del Consiglio, del 21 ottobre 2009, recante norme sanitarie relative ai sottoprodotti di origine animale e ai prodotti derivati non destinati al consumo umano e che abroga il regolamento (CE) n. 1774/2002 (regolamento sui sottoprodotti di origine animale). 22. EFSA (European Food Safety Authority) (2018). Effect on public health of a possible increase of the maximum level for ‘aflatoxin total’ from 4 to 10 µg/kg in peanuts and processed products thereof, intended for direct human consumption or use as an ingredient in foodstuffs. EFSA Journal 16(2):51-75 doi: 10.2903/j.efsa.2018.5175

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GIORGIA FABBRI1, ENRICO FIORE1, GIUSEPPE PICCIONE2, ELISABETTA GIUDICE2, MATTEO GIANESELLA1, MASSIMO MORGANTE1, LEONARDO ARMATO3, ORTENSIO BONATO3, SONIA GIAMBELLUCA1, FRANCESCA ARFUSO2 1

Department of Animal Medicine, Productions and Health (MAPS), University of Padua, Viale dell’Università, 35020, Padua, Italy 2 Department of Veterinary Sciences, University of Messina, Polo Universitario dell’Annunziata, 98168, Messina, Italy 3 Veterinary Freelance

SUMMARY Diseases of the bovine foot are a serious threat to dairy cows welfare and productivity. Commonly divided in infectious and noninfectious depending on their aetiology, infectious lesions include digital dermatitis, interdigital dermatitis, heel horn erosion, and foot rot, whereas the most common non-infectious lesions are sole ulcer, toe ulcer, sole haemorrhage, and white line disease. Infrared thermography (IRT) has been adopted in livestock studies for different analyses such as metabolic responses to thermal stress and the diagnosis of inflammatory processes. The aim of this study was to evaluate the potential usefulness of infrared thermography as a non-invasive tool to rapidly screen digital and interdigital dermatitis (DD and ID) in dairy cows. Fortyeight healthy cows and forty-eight cows affected by DD and ID on central and interdigital regions of the hind feet were enrolled. Feet were cleansed to reduce biases and artifacts and left to dry for five minutes to restore normal blood flow. Thermography images of the hind feet were then collected using a digital infrared camera. Foot temperature was measured in four regions: central area of the hind foot (R1), interdigital area of the hind foot (R2), lateral (R3) and medial (R4) claw in the hind foot. Higher temperature values were found in the central (R1) and interdigital area (R2) compared to lateral (R3) and medial (R4) areas in both healthy and diseased cows (P<0.001). Moreover, cows affected by DD and ID showed higher foot temperature values compared to healthy cows in the R1 and R2 regions (P<0.001). Results from the present study show that IRT could be a useful diagnostic tool for the detection of DD and ID in dairy cows. The obtained results suggest that IRT could contribute in defining the localization of areas of increased inflammation and could be useful for veterinary podologists, permitting to act directly on the lesion detected by thermography rather than on the whole foot.

KEY WORDS Dairy cows; digital dermatitis; foot temperature; infrared thermography; interdigital dermatitis.

INTRODUCTION Lameness represents one of the main causes of decreased productive performance and impaired animal welfare in the dairy industry. Foot lesions are commonly categorized according to their aetiology into infectious and non-infectious lesions1,2. Infectious lesions include digital dermatitis, interdigital dermatitis, heel horn erosion, and foot rot, whereas the most common non-infectious lesions are sole ulcer, toe ulcer, sole haemorrhage, and white line disease. Digital and interdigital dermatitis are a dynamic and multifactorial infectious foot disease with increasing prevalence in many countries3,4. The precise aetiology of digital and interdigital dermatitis is unknown. The marked susceptibility of lesions to topical antibiotics5, as well as the isolation of spirochetes and proteobacteria from the lesions caused by digital and interdigital

Corresponding Author: Giuseppe Piccione (giuseppe.piccione@unime.it).

dermatitis, respectively suggest that bacteria have an important role in the development of these diseases6. Early detection of digital and interdigital dermatitis is the first step towards therapeutic resolution and reduction of reservoirs of infection within the herd7,8. Therefore, the need to make a rapid and simple diagnosis remains a key feature in treating and controlling lameness in dairy cows. Traditionally, locomotion scoring is used to identify developing diseases of limb and foot or for clinical follow-up. This approach, besides being timeconsuming to be used on the whole herd, may not always be suitably sensitive to identify foot lesions9. Moreover, dermatitis is often present without any sign of locomotion impairment, and lameness does not appear until the lesion becomes severe10. Infrared thermography (IRT) has been adopted in animal production studies for different analyses such as metabolic responses to thermal stress11 and the diagnosis of inflammatory processes12,13. The use of IRT to recognize lameness in cattle has increased largely in the last years since it represents a non-invasive method, ease of automation and low cost14. The newest generation of IRT cameras is capable of measuring temperature in real-time.

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The equipment is light, portable and temperature detection shows great sensitivity, which is significant in recording the heat generated by animals’ skin15. Additionally, the camera software permits analysis of temperature data in every surface or area of the thermogram. Therefore, IRT can be useful to detect thermal abnormalities in animals by characterizing changes in their skin temperature16. The skin surface is a highly efficient radiator, a fact that permits to detect infrared emissions of the skin and to map temperature distributions in a non-invasive manner17. IRT permits to detect even small changes in temperature with precision and without the need for physical contact with the animals18, and has therefore become important as a safe assessment method in experiments. Aim of this study was to evaluate the potential usefulness of infrared thermography as a non-invasive tool to rapidly screen digital and interdigital dermatitis in dairy cows.

MATERIALS AND METHODS Farm and animal selection The study was conducted in an intensive Italian dairy herd located in North Italy (45° 38’ N; 10° 87’ E) at an altitude of 68 m. The selected farm had 200 Holstein Friesian dairy cows in lactation. The herd production was in an average of 8412±1426 kg of milk per cow in 310±11 days. Farms made a dry period of 55 days and a period of steaming-up of 15 days before calving. Cows were on a maintenance claw-trimming scheme before the study, thus receiving routine hoof trimming twice a year in the farm (-70±15 days before and +50±15 days after calving). Scheduled hoof trimming sessions were performed by a veterinarian professional claw trimmer. Forty-eight healthy cows (35±10 months; mean body weight 630±74 kg) and forty-eight cows affected by foot diseases (39±11 months; mean body weight 626±95 kg) were enrolled in the study. All cows were placed in an up-right chute equipped with a headlock gate and a manually operated rope foot lift at 55±10 days in milk. Each limb was raised with a rope fastened on the middiaphysis of the third metatarsal bone. All feet were cleansed and trimmed to remove stained, overgrown hoof horn tissue following guidelines of the Dutch Technique19. During trimming sessions, one leg at a time was raised to preserve as much as possible animals’ welfare and reduce their stress. A complete clinical examination was performed based on the cow’s reaction to palpation. The disease status of the foot was confirmed using a thorough clinical examination. In the cases where both hind feet were compromised, the one with higher clinical relevance was selected based on the reaction to palpation and pain. Moreover, all lame cows were also checked and recorded. The same procedure was performed also for healthy cows. Hind feet of enrolled animals were cleaned with cold water to remove dirt and left to dry. Sick cows were affected by digital dermatitis (DD) and interdigital dermatitis (ID), with DD and ID lesions localized in central and interdigital regions of the hind foot. Selected cows were separated from the herd and introduced in a crush where the affected foot was nursed. Protocols of animal husbandry and experimentation were reviewed and approved in accordance with the standards recommended by the Guide for the Care and Use of Laboratory Animals and Directive 2010/63/EU for animal experiments.

Infrared thermography and thermogram analysis Thermography images of hind feet were collected from each animal using a digital infrared camera (ThermaCam P25 Model, Flir Systems, Boston, MA, USA). All feet were cleaned and trimmed to remove dirt before the acquisition of thermal images to reduce biases and artifacts. After washing and drying of the feet, a five-minute timeout period was set to allow blood flow to return to its pre-wash state. All thermographic imaging was performed in a closed, indoor environment, while the animals were restrained in the stall in a standing position. Mean ambient temperature was recorded at the IRT time points and it showed a mean value of 15.7±3.61 °C. All images were scanned at the same distance (0.7 m) from the subject to reduce the effects of environmental factors on thermographic readings. The settings of the camera were as follows: temperature range: 10-40 °C; emissivity of skin: 0.98; reflected air temperature (Trifl): 20 °C; distance between the camera and skin surface (Dist): 0.7 m; and view field (FOV): 23°. The detector consisted of a focal plane array (FPA) uncooled microbolometer with the following specifications: 320 × 240 pixels resolution, thermal sensitivity of 0.08 °C (at 30 °C), spatial resolution (IFOV) of 1.3 mrad, spectral range between 7.5 and 13 m accuracy ±2 °C. Automatic corrections based on user input were conducted for reflected ambient temperature, distance, relative humidity, and atmospheric transmission. Foot temperature was measured in four specific regions of interest: central area of the hind foot (R1), interdigital area of the hind foot (R2), lateral (R3) and medial (R4) claw in the hind foot. The absolute mean temperature of each region of interest was calculated using thermography software (Thermacam Researcher Basic 2.8 software, FLIR, Wilsonville, Oregon, USA).

Statistical analysis All data were tested for normality of distribution using Kolmogorov-Smirnov test. All data were normally distributed (P>0.05) and statistical analysis was performed. Two-way analysis of variance (ANOVA) was applied to evaluate the significant change of temperature among the selected foot areas and whether significant differences in foot temperatures occur between healthy cows and cows affected by foot diseases. When significant differences were found Bonferroni’s post hoc comparison was applied. The statistical analysis was performed using the STATISTICA software package (STATISTICA 7 Stat Software Inc., Tulsa, Oklahoma).

RESULTS All the obtained results are expressed as means ± standard deviation (M±SD). Figure 1 shows a representative IRT of a foot affected by DD and ID, with the selected regions of interest. As reported in Figure 2, statistical analysis showed higher temperature values (P<0.001) in the central (R1) and interdigital area (R2) compared to lateral (R3) and medial (R4) areas of the hind feet of both healthy and diseased cows. Differences were also found in the regions R1 and R2, with cows affected by DD and ID showing higher foot temperature values compared to healthy cows (P<0.001). In particular, the temperature of central areas of diseased cows hind feet showed a difference (∅) of 2.80 °C and an increase of 10.15% respect to healthy cows; whereas the temperature values recorded from

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Figure 1 - Representative infrared thermal image of hind foot, with the regions of interest (R1, central area; R2, interdigital area; R3, lateral claw; R4, medial claw), of healthy cow (a), cow affected by digital (b) and interdigital (c) dermatitis - Thermal scale is included on the right side of the image.

the interdigital areas of diseased cows showed a difference (Δ) of 2.70 °C and an increase of 9.82% respect to healthy cows.

DISCUSSION

ed that IRT was reliable in detecting elevated temperatures associated with foot lesions10, external factors such as the environmental or facilities temperature and animal dirt may influence the foot temperature using IRT. Thoroughly cleaning cows’ feet with water had a cooling effect10. In the present study, the IRT detections were done within the neutral zone for dairy cattle31 and in closed barns at a controlled temperature without exposure to any direct sunlight or detectable airflow. In addition, all thermographic images were obtained after foot cleaning and drying and a five-minute timeout period was set to allow blood flow to return to its pre-wash state. The results obtained in the present study indicated that the temperature measured in the four selected regions of foot showed different values both in cows affected by foot diseases than in

Efforts to reduce lameness in dairy cattle remain a priority as the industry strives to enhance animal welfare and productivity. Stress, especially during the transition period, is an undesirable aspect of livestock production as it often results in immune dysfunction and increased likelihood of infection20. Early lactation can be a seriously challenging event for the severe negative energy balance that high-yielding dairy cows can develop21, this combination of stress, immune dysfunction and metabolic demand induced by parturition and beginning of milk production may help explain the increase in infectious foot lesions. This stress and immunosuppression interaction likely contributed to the high proportion of events diagnosed as foot rot22. In recent years, application of new diagnostic technologies in the dairy field such as non-reproduction related ultrasound and IRT has improved greatly23, and the experiments on animals performed with the use of IRT have become very popular in livestock14,24-27. In some researches of skin temperature detection, it has been proved that the intensity of infrared radiation emitted is directly proportional to the metabolic processes occurring in some related surfaces and is associated with a simultaneous increase in blood supply to a given area28. The blood flow of the body surface depends on the relationship between the autonomic nervous system, the local vessel constriction and vessel-dilation mediators29. In literature, it is been reported that cow hooves infected with the foot-and-mouth disease virus IRT was apFigure 2 - Mean values ± standard deviation (±SD) of hind foot temperatures measured plied, and show a marked increase in the tem- at the different regions of interest (R1, central area; R2, interdigital area; R3, lateral claw; perature of sick animals before clinical symp- R4, medial claw) in healthy cows and in cows with foot lesions together with the statistitoms appeared30. Although it is demonstrat- cally significant differences found.

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healthy cows. In particular, higher amount of infrared radiation, indicative of temperature, was emitted from the central and the interdigital areas of hind foot than from the lateral and medial claw in the hind foot. This could be due not only to the different vascularization and tissue metabolic activity of the foot regions, but also, and probably more importantly to the amount of keratinization, which may be associated to the lower temperatures found in the sole regions32. Cows affected by DD and ID showed higher temperatures in the central and interdigital regions of the foot compared to the foot of cows without diseases. Our findings agree with previous studies carried out on cows affected by hoof diseases reporting a rise in the values of temperature in cows with hoof diseases respect to healthy animals10,26. Inflammation or infectious conditions usually lead to a rise in the underlying circulation and tissue metabolic rate25 resulting in a localized increase in the surface temperature of the affected areas, detected by IRT17,33.

CONCLUSION In conclusion, the results obtained in this study show that IRT could be a useful diagnostic tool in screening for DD and ID presence in dairy cows. In particular, DD and ID cause an increase in foot temperature measured by IRT in both the central and interdigital regions of affected feet, compared to feet without lesions. The study emphasizes the potential usefulness of IRT as a reliable, practical and non-invasive tool for the detection of hoof lesions in dairy cows, and suggests that IRT could contribute in defining the localization of increased inflammation and/or injury area. The application of IRT might be useful to veterinary podologists in detecting foot lesions. The early detection of foot diseases is likely to be valuable in the prevention of further progression and in early effective treatment. In this regard, the thermography recording could allow coordinating lameness monitoring plans to control and manage foot lesions to enhance dairy performance and animal welfare within each farm.

Disclosure statement No conflict of interest was reported by the authors.

Acknowledgments The research was funded by the University of Padua, CPDA134009/13.

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8. Leach K.A., Tisdall D.A., Bell N.J., Main D.C., Green L.E. (2012). The effects of early treatment for hindlimb lameness in dairy cows on four commercial uk farms. Vet J, 193: 626-632. 9. Flower F.C., Weary D.M. (2006). Effect of hoof pathologies on subjective assessments of dairy cow gait. J Dairy Sci, 89: 139-146. 10. Stokes J.E., Leach K.A., Main D.C., Whay H.R. (2012). An investigation into the use of infrared thermography (IRT) as a rapid diagnostic tool for foot lesions in dairy cattle. Vet J, 193: 674-678. 11. Paim T.P., Borges B.O., Lima P.M.T. (2013). Thermographic evaluation of climatic conditions on lambs from different genetic groups. Int J Biometeorol, 57: 59-66. 12. Schrank M., Bonsembiante F., Fiore E., Bellini L., Zamboni C., Zappulli V., Stelletta C., Mollo A. (2017). Diagnostic approach to fibrocystic mastopathy in a goat: termographic, ultrasonographic, and histological findings. Large Anim Rev, 23: 33-37. 13. Hovinen M., Siivonen J., Taponen S., Hänninen L., Pastell M., Aisla A.M., Pyörälä S. (2008). Detection of clinical mastitis with the help of a thermal camera. J Dairy Sci, 91: 4592-4598. 14. Alsaaod M., Schaefer A.L., Büscher W., Steiner A. (2015). The role of infrared thermography as a non-invasive tool for the detection of lameness in cattle. Sensors, 15: 14513-14525. 15. Perazzi A., Iacopetti I., Stelletta C., Fiore E. (2016). Bilateral glaucoma in a Tibetan goat: clinical and thermographic findings. Large Anim Rev, 22: 281-283. 16. McManus C., Tanure C.B., Peripolli V., Seixas L., Fischer V., Gabbi A.M., Menegassi S.R.O., Stumpf M.T., Kolling G.J., Dias E., Costa J.B.G. Jr. (2016). Infrared thermography in animal production: an overview. Comput Electron Agric, 123: 10-16. 17. Purohit R.C., Hudson R.S., Riddell M.G., Carson R.L., Wolfe D.F., Walker D.F. (1985). Thermography of the bovine scrotum. Am J Vet Res, 46: 23882392. 18. Bowers S., Gandy S., Anderson B., Ryan P., Willard S. (2009). Assessment of pregnancy in the late-gestation mare using digital infrared thermography, Theriogenology, 72: 372–377. 19. Toussaint-Raven E. (1989). Cattle foot care and claw trimming. Ipswich: Farming Press. 20. Overton T.R., Waldron M.R. (2004). Nutritional management of transition dairy cows: Strategies to optimize metabolic health. J Dairy Sci, 87: 105119. 21. Fiore E., Perillo L., Piccione G., Gianesella M., Bedin S., Armato L., Giudice E., Morgante M. (2016). Effect of combined acetylmethionine, cyanocobalamin and α-lipoic acid on hepatic metabolism in high-yielding dairy cow. J Dairy Res, 83: 438-441. 22. DeFrain J.M., Socha M.T., Tomlinson D.J. (2013). Analysis of foot health records from 17 confinement dairies. J Dairy Sci, 96: 7329-7339. 23. Banzato, T., Fiore, E., Morgante, M., Manuali, E., Zotti, A. (2016). Texture analysis of B-mode ultrasound images to stage hepatic lipidosis in the dairy cow: A methodological study. Res Vet Sci, 108: 71-75. 24. Bortolami A., Fiore E., Gianesella M., Corrò M., Catania M., Morgante M. (2015). Evaluation of the udder health status in subclinical mastitis affected dairy cows through bacteriological culture, somatic cell count and thermographic imaging. Polish J Vet Sci, 18: 799-805. 25. Alsaaod M., Syring C., Dietrich J., Doherr M.G., Gujan T., Steiner A. (2014). A field trial of infrared thermography as a noninvasive diagnostic tool for early detection of digital dermatitis in dairy cows. Vet J, 199: 281-285. 26. Alsaaod M., Büscher W. (2012). Detection of hoof lesions using digital infrared thermography in dairy cows. J Dairy Sci, 95: 735-742. 27. Stelletta C., Gianesella M., Vencato J., Fiore E., Morgante M. (2012). Thermographic applications in veterinary medicine. In: Prakash, R.V. (ed) Infrared Thermography. 117-140, In Tech, China. 28. Fita K., Dobrzy ski M., Całkosi ski I., Dudek K., Bader-Orłowska D. (2007). The usefulness of the thermography in medical-dental diagnostic - the author’s experiences. Ann Acad Med Stetin, 53: 34-38. 29. Całkosi ski I., Dobrzy ski M., Halo A., Fita K., Całkosi ska M., Majda J. (2007). Humoral-circulatory response in the somato-vegetative reflex caused by pain factors. Postepy Hig Med Dosw, 61: 331-337. 30. Rainwater-Lovett K., Pacheco J.M., Packer C., Rodriguez L.L. (2009). Detection of foot-and-mouth disease virus infected cattle using infrared thermography. Vet J, 180: 317-324. 31. Roenfeldt R. (1998). You can’t afford to ignore heat stress. Dairy Herd Management 35: 6-12. 32. Rodríguez A.R., Olivares F.J., Descouvieres P.T., Werner M.P., Tadich N.A., Bustamante H.A. (2016). Thermographic assessment of hoof temperature in dairy cows with different mobility scores. Livest Sci, 184: 92-96. 33. Gianesella M., Arfuso F., Fiore E., Giambelluca S., Giudice E., Armato L., Piccione G. (2018). Infrared thermography as a rapid and non-invasive diagnostic tool to detect inflammatory foot diseases in dairy cows. Pol J Vet Sci, 21: 299-305.

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The effects of non-genetic factors on the morphometric parameters of sheep placenta and the birth weight of lambs

119

ANGELIKA BRZOZOWSKA1*, NATALIA WOJTASIAK1, BARBARA BŁASZCZYK1, TOMASZ STANKIEWICZ1, MARTA WIECZOREK-DĄBROWSKA2, JAN UDAŁA1 1

Department of Animal Reproduction Biotechnology and Environmental Hygiene, West Pomeranian University of Technology, Szczecin; 29 Klemensa Janickiego Street, 71-270 Szczecin 2 National Research Institute of Animal Production, Kraków, Experimental Department, Kołbacz, 1 Warcisława Street, 74-106 Stare Czarnowo

SUMMARY The objective of this study was to determine morphometric parameters of sheep placenta and the birth weight of lambs and their relationship with the type of pregnancy, litter sex and age of ewes. Placenta was obtained directly after delivery from Pomeranian sheep (n=128), including single (n=99) and multiple (n=29) pregnancies. For twin pregnancies, monochorionic and dichorionic placentas were taken into account. The following were determined: lamb birth weight (BWL), placental weight (PW), placental length (PL), placental width (WP), cotyledons number (CN), cotyledons weight (CW), mean diameter of the cotyledon (MDC), umbilical cord diameter (DUC). One-way ANOVA variance analysis was used for statistical comparisons and the Pearson and/or Spearman’s correlation coefficient for correlation analysis. The primiparous were shown to have significantly lower PW and MDC (p<0.05) compared to older sheep. On the other hand, in the case of PL (p<0.05) and CN (p<0.01), which were significantly higher in the primiparous compared to multiparous. A significantly higher BWL was found in the male lambs than in the female lambs (p<0.05). Significantly higher PW, MDC (p<0.01) and CW (p<0.05) were found in the placenta from which male lambs were born. There was also a significantly higher BWL in male lambs (p<0.05) born from dichorial twin pregnancy. CW was significantly higher in the dichorial placenta from which the male lambs were born (p<0.05). Comparing the placenta from single and twin dichorial pregnancies, it was shown that type of pregnancy had a significant impact on the development of some placental indices and the birth weight of lambs. Significantly heavier lambs were born from single pregnancy (p<0.01). Also PW and PL (p<0.01) and CN (p<0.01) were significantly higher in single pregnancy placentas. In turn, MDC (p<0.05) was significantly higher in twin placental dichorial pregnancies. Monochorial placenta were characterized by significantly larger PW and CW (p<0.05) from which the male lambs were born. The obtained results showed that morphometric parameters of sheep placenta and birth weight of lambs depend on the type of pregnancy, litter sex and age of ewes. These results may be helpful in assessing postpartum placenta in this animal species. In addition, recorded differences in placenta parameters and birth weight of lambs may be useful in ultrasound assessment of placental and fetal development during pregnancy.

KEY WORDS Cotyledon, litter size, placenta, lamb, sheep.

INTRODUCTION The results regarding reproductive parameters are of key importance in sheep breeding. They particularly concern the assessment of the course of pregnancy, whose proper development may affect the health of newborn lambs. Among many factors, placenta plays the key role1. It starts when an embryo is implanted and from that moment, it play a significant role in the development of pregnancy2. This is where the morphological and functional analysis of the placenta originated. It has been shown that some morphometric parameters of the placenta can directly affect fetal growth and development, and thus the success of pregnancy3. On the example of sheep pla-

Corresponding Author: Angelika Brzozowska (angelika_brzozowska@zut.edu.pl).

centa, it was confirmed that its certain indicators can directly contribute to placental insufficiency, and thus constitute one of the factors that determine the mortality of newborn livestock4. Therefore, this type of work is important in the diagnosis and monitoring of pregnancy, especially in vivo5. Currently, ultrasound is increasingly used for this purpose, the use of which is associated with the ability to correctly interpret it 5. Therefore, you should first learn about morphology of the placenta, including actual dimensions of its structures, which is best done by direct testing6,7, e.g. non-invasive, using the placenta expelled immediately after delivery. This type of work can have practical significance, especially in controlling pregnancy and increasing productivity in large livestock farms5. They can also be helpful for breeders in preparing for deliveries from multiple pregnancies or the occurrence of heavy births and other complications during this period, which results in minimizing infant mortality5. However, previous studies have not tak-

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Table 1 - The number of male lambs and female lambs born and died up to 2 weeks of age from single and multiple pregnancies. Lambs alive1

Type of pregnancy (n)

male lambs

female lambs

male lambs

female lambs

16 8

16 12

2 -

Single (n= 99) dichorial (n=18) monochorial (n=10)

Twin (n=28)

dead2

Triplet (n=1) 1

live born lambs dead lambs born and those that did not survive up to 2 weeks of age

Table 2 - Mean (±SD) birth weight of lambs and morphometric parameters of sheep placenta from single pregnancies in ewes of different ages. Parameters

Age of ewes I (1 year; n=20) 1

II (2-5 years; n=41) 2

III (5> years; n=33) 3

Statistically significant differences

BWL (g)

Mean ± SD Range

5375.00±767.69 3600.00-6500.00

5226.83±712.05 3800.00-6900.00

5163.64±648.47 3800.00-6500.00

PW (g)

Mean ± SD Range

305.45±68.71 200.00-403.00

307.24±66.10 196.00-494.00

318.85±78.06 187.00-482.00

1<3; p<0.05

PL (cm)

Mean ± SD Range

153.06±17.57 126.70-185.00

141.12±17.39 116.00-181.40

143.23±21.39 106.60-214.00

1>2; p<0.05

WP (cm)

Mean ± SD Range

54.03±7.77 37.50-69.00

55.88±7.90 40.00-76.70

57.07±9.85 40.70-74.90

Mean ± SD Range

86.20±14.64 55.00-113.00

69.24±17.91 37.00-98.00

70.27±18.06 40.00-107.00

1>2,3; p<0.01

CW (g)

Mean ± SD Range

92.82±39.75 43.00-166.00

102.39±23.53 53.00-135.00

96.83±36.54 33.50-170.00

MDC (cm)

Mean ± SD Range

2.09±0.36 1.45-2.68

2.33±0.46 1.65-3.95

2.24±0.24 1.73-2.89

1<2; p<0.05

DUC (cm)

Mean ± SD Range

0.64±0.11 0.50-0.80

0.64±0.11 0.50-0.90

0.62±0.13 0.40-0.90

Statistical significance of p < 0.01; p < 0.05; SD - standard deviation; NS - no significant; BWL - lamb birth weight; PW - placental weight; PL - placental length; WP - placental width; CN - cotyledons number; CW - cotyledons weight; MDC - mean diameter of cotyledons; DUC - umbilical cord diameter.

en into account all the morphometric parameters of the placenta that can be tested, as well as the relationships between them, taking into account fetal and maternal factors. One of the aspects not yet discussed in the available literature is the analysis of sheep’s placenta in multiple pregnancy, especially twin occurring frequently in sheep8, 9, taking into account the classification into a monochorial placenta, having one chorion and a common placenta for developing fetuses and dichorial, where they are two chorions and completely separate placenta for each fetus in twin pregnancy10. It is also worth examining the effect of fetal sex on the placenta11 and mother’s age, with which reproduction parameters are known to change12,13. Therefore, it is so important to include the foregoing aspects in the analysis of placenta, which are not fully explained in the available literature. The purpose of this study was to examine morphometric parameters of the sheep placenta de-

pending on the type of pregnancy, litter sex and age of ewes, and to analyze the birth weight of lambs.

MATERIAL AND METHODS The study was carried out on Pomeranian sheep kept on an organic sheep farm in the Experimental Plant of the National Research Institute of Animal Production in Kołbacz (Kołbacz, Poland: latitude 53°30’ N). The Pomeranian sheep are the main breed in the population of meat and wool sheep raised in the north-western region of Poland. This breed is included in the livestock genetic resources conservation program. In the study, sheep were kept in a pasture-alcove system, under conditions of uniform feeding. Nutrition was carried out according to standards adapted for this species, based on fodder, and oth-

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Table 3 - Mean (± SD) birth weight of lambs and morphometric parameters of sheep placenta from single female and male pregnancies. Parameters

Sex of lambs female (n=51)

male (n=43)

Statistically significant differences

BWL (g)

Mean ± SD Range

5082.35±613.42 3800.00-6500.00

5418.60±756.64 3600.00-6900.00

1<2; p<0.05

PW (g)

Mean ± SD Range

294.24±68.53 187.00-494.00

330.74±68.45 196.00-482.00

1<2; p<0.01

PL (cm)

Mean ± SD Range

146.23±19.99 117.00-214.00

142.22±18.33 106.60-185.00

WP (cm)

Mean ± SD Range

54.49±7.87 37.50-68.00

57.58±9.17 41.00-76.70

Mean ± SD Range

73.10±18.04 37.00-107.00

73.35±19.13 37.00-113.00

CW (g)

Mean ± SD Range

89.61±33.07 33.50-166.00

111.59±26.33 67.00-170.00

1<2; p<0.05

MDC (cm)

Mean ± SD Range

2.18±0.43 1.45-3.95

2.33±0.30 1.74-3.15

1<2; p<0.01

DUC (cm)

Mean ± SD Range

0.61±0.12 0.40-0.90

0.66±0.11 0.50-0.90

Table 4 - Mean (± SD) birth weight of lambs and morphometric parameters of sheep placenta from dichorial twin pregnancies including lambs sex. Parameters

Sex of lambs female (n=16)

male (n=16)

Statistically significant differences

BWL (g)

Mean ± SD Range

3800.00±307.06 3400.00-4300.00

4141.67±716.63 2400.00-4900.00

1<2; p<0.05

PW (g)

Mean ± SD Range

242.25±32.13 213.00-292.00

274.00±74.18 197.00-436.00

PL (cm)

Mean ± SD Range

110.90±21.48 75.80-141.40

123.46±27.62 91.00-187.40

WP (cm)

Mean ± SD Range

54.73±6.72 44.20-64.90

52.67±8.69 39.00-68.80

Mean ± SD Range

50.00±20.23 35.00-98.00

58.67±21.29 36.00-102.00

CW (g)

Mean ± SD Range

64.50±2.12 63.00-66.00

87.86±9.96 69.00-96.00

1<2; p<0.05

MDC (cm)

Mean ± SD Range

2.50±0.39 2.00-2.96

2.42±0.29 2.01-2.89

DUC (cm)

Mean ± SD Range

0.60±0.14 0.40-0.80

0.67±0.10 0.60-0.90

er forages and concentrate feeds, depending on the time of year (summer and winter nutrition). In the grazing season from May to October, sheep grazed in the meadow, and in the fold they

received oats, hay and straw. In the winter, from November to April, the sheep stayed in the fold, where they were fed with oats, straw and hay. The animals had constant access to water and

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salt licks. Sheep were mated during their natural breeding season (September) in a natural way, no oestrus synchronization methods were used. Duration of pregnancy was determined based on the day of mating and the success of mating was confirmed by transrectal ultrasound (USG scanner 480, Pie Medical, linear probe with frequency of 7.5 MHz) and vaginal mucus resistance measurements (ohmmeter DraminĚ ski, Poland)14. To distinguish single and multiple pregnancy, abdominal ultrasound was performed (USG scanner EDAN U50, sectoral probe with frequency of 5 MHz). A total of 128 sheep were included in the study.

Data collection Placentas were obtained immediately after deliveries. The following types of placenta were obtained from single pregnancies and twin pregnancies. In twin pregnancies there were: monochorial and dichorial (Fig. 1). After delivery, the sex and birth weight of lambs (BWL) were determined (veterinary weight; MENSOR WE15P2-A, Poland). In the first stage, placentas obtained from single (n=94) and twin (n=16) pregnancies, from which live lambs were born, were used for comparative analyses. On the other hand, the parameters of placentas obtained from monochorial twin pregnancies (n=10) were subjected to a separate analysis due to the type of placenta, which prevents comparative analysis with other types of placenta. Due to the presence of one large placenta, the total birth weight of lambs (TBWL) was determined for a given monochorial placenta. According to the classification given by Steven15, monochorial placentas include chorionic sacs: joined by avascular tips and completely fused with placental vascular asymmetry, and without asymmetry of the placental vessels. The study also took into account the survival of lambs, distinguishing a group of live-born lambs and a group of dead lambs up to 2 weeks of age. Placenta characteristics were determined based on the following parameters: placenta weight (PW), placenta length (PL), pla-

centa width (WP), number of cotyledons (CN), the weight of cotyledons (CW), mean cotyledon diameter (MDC), umbilical cord diameter (DUC). The length of the placenta was measured using a non-stretchy cord according to the method described by Vernunft et al.16. Each placenta was placed on the countertop with the fetal surface outside and the umbilical cord arranged centrally. Estimation of organ length was done along the main curvature that was opposite the umbilical cord base (Fig. 2). Necrotic placenta elements were not included in the measurements. In turn, determination of the placenta width was based on the method given by Winder et al.17. The measurement was taken at the widest point of the placenta after it was cut along the curvature opposite the umbilical cord (Fig. 2). Then the number of cotyledons was estimated and isolated from the placenta, then the diameters of randomly selected 10 cotyledons from the placenta were measured8. Cotyledon diameters were estimated by averaging the length and width (cm) measurements18 using a caliper (Fig. 3). Then the obtained values were averaged and the mean diameter of the cotyledons per test placenta was obtained. All isolated cotyledons from the placenta were weighed using the same scale as placental and lamb weight measurements. The umbilical cord diameter was measured using a caliper.

Statistical analysis Statistical analysis of the study results was performed using the Statistica program version 13.3 (StatSoft, Poland). Mean values and standard deviations were calculated. One-way analysis of variance (ANOVA) was used to determine the significance of differences between the normal distribution means. The Duncan test was used as the post-hoc test. For variables that did not meet the parameters of parametric tests, the non-parametric KruskalWallis test was used to analyze the variance. A multiple comparison test was used as the post-hoc test. Correlation analysis was done by calculating Pearson and/or Spearmanâ&#x20AC;&#x2122;s correlation coefficients. The level of statistical significance was p <0.05.

Figure 1 Type of placentas in twin pregnancies: A - monochorial placenta; B - dichorial placenta.

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Figure 2 Representative picture of sized ovine placentas of one litter; the line demonstrates the measurement of placental length (A) and width (B); C-cotyledon.

RESULTS Table 1 presents numbers of pregnant sheep, pregnancy types, live and dead lambs up to two weeks of age. The sex of lambs was also taken into account. The number of sheep with single pregnancy was more than three times higher than the number of sheep with multiple pregnancies. In the examined sheep were born more male lambs than female lambs. In twin pregnancies, there were more dichorial than monochorial ones. The mortality rate of lambs was similar in both sexes (Table 1). Table 2 presents the average values of morphometric parameters of placenta and birth weight of lambs from single pregnancies depending on the age of the ewes. There were significant differences in placental weight, with the lowest weight for the youngest ewes, and the highest for sheep over 5 years of age. The length of nulliparous placenta was greater than that of older sheep, but significant differences were noted between nulliparous placenta and ewe placenta at the age of 2-5 years. Nulliparous placenta also had more cotyledons than the older sheep placenta. On the other hand, the mean diameter of the cotyledon was smaller than in older sheepâ&#x20AC;&#x2122;s placentas, with significant differences between the cotyledons of nulliparous placenta and the cotyledons of sheep aged 2-5 years. In the remaining parameters, no significant differences were found between the examined groups of ewes (Table 2). The average birth weight of lambs and morphometric parameters of the placenta from single pregnancies, taking into account the sex of lambs, is given in Table 3. Male lambs had a significantly higher birth weight than female lambs. Also, the placental weight from male pregnancies was significantly higher than from female pregnancies. Similarly, the differences in cotyledon weight and mean diameter of cotyledon were shaped. However, no statistical differences were found in other parameters (Table 3). Table 4 shows the average values of analyzed parameters of the

placenta and birth weight for female and male lambs born from dichorial twin pregnancies. The birth weight of male lambs was significantly higher than that of female lambs. The differences in cotyledon weight were similar. However, no significant differences were found in other parameters examined (Table 4). Table 5 presents the results regarding the comparison of the birth weights of lambs and placental morphometric parameters between single and twin dichorial pregnancies. Lambs from single pregnancies had a significantly higher birth weight than lambs with twin dichorial pregnancy. The placenta weight was also significantly higher in single pregnancies than in lambs from dichorial twin pregnancies. The differences in placental length and the number of cotyledons were similar. In turn, the mean diameter of cotyledon was significantly larger in twin dichorial placentas than in single pregnancies. No significant dif-

Figure 3 - Measurement of cotyledon using a caliper.

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Table 5 - Mean (± SD) birth weight of lambs and morphometric parameters of sheep placenta from single and twin dichorial pregnancies. Parameters

Type of pregnancy single (n=94)

dichorial twin (n=32)

Statistically significant differences

BWL (g)

Mean ± SD Range

5236.17±699.44 3600.00-6900.00

4005.00± 601.29 2400.00-4900.00

1>2; p<0.01

PW (g)

Mean ± SD Range

310.94±70.53 187.00-494.00

261.30±61.81 197.00-436.00

1>2; p<0.01

PL (cm)

Mean ± SD Range

144.42±19.26 106.60-214.00

118.17±25.38 75.80-187.40

1>2; p<0.01

WP (cm)

Mean ± SD Range

55.85±8.61 37.50-76.70

53.54±7.79 39.00-68.80

Mean ± SD Range

73.21±18.45 37.00-113.00

55.20±20.79 35.00-102.00

1>2; p<0.01

CW (g)

Mean ± SD Range

98.10±32.19 33.50-170.00

82.67±13.45 63.00-96.00

MDC (cm)

Mean ± SD Range

2.25±0.38 1.45-3.95

2.45±0.33 2.00-2.96

1<2; p<0.05

DUC (cm)

Mean ± SD Range

0.63±0.12 0.40-0.90

0.64±0.12 0.40-0.90

ferences were found in other parameters (Table 5). Table 6 presents the average total birth weight of twin lambs and placental parameters from monochorial twin pregnancies. The average placental weight from female twin pregnancies was significantly lower than from male twin pregnancies. In contrast, the weight of cotyledons was significantly higher in placenta from male twin pregnancies than from female ones. No statistical differences were found in other parameters (Table 6). Table 7 shows correlation coefficients between the tested morphometric parameters of the placenta, and the age of ewes and the birth weight of lambs. It was shown that the birth weight of lambs was positively correlated with placental weight, placental length, cotyledon number and weight. There were no significant correlations between the age of ewes and placental parameters (Table 7).

DISCUSSION Morphometric parameters of the sheep placenta were examined depending on the type of pregnancy, litter sex and age of ewes, as well as the analysis of lambs birth weight. The obtained results confirmed that the foregoing factors had an impact on some morphometric parameters of the placenta and the birth weight of newborn lambs. Some results of research on given parameters were similar to those observed in the works of other authors. Additional results were also obtained that could expand knowledge in this area of sheep research. In this study, no impact of sheep age on the weight of newborn offspring was found. Similar observations were noted in studies of Karakus and Atmaca19. On the other hand, other authors showed that age of ewes no affects the birth weight of lambs20.

This study showed that the number of placental cotyledons decreased with increasing age, and the mean diameter of cotyledon increased. Results regarding the number of cotyledons were similar to those obtained in another study21, however they differed from the results obtained by Kolosov et al.3 and Pettigrew22. It can be presumed that in older sheep, the decrease in the number of cotyledons and the increase in the cotyledon diameter with age can lead to minimizing losses in the exchange of nutrients between the mother and the fetus. Parraguez et al.4 showed similar conclusions regarding the number and diameter of cotyledons. It is known that cotyledons as fetal placenta (placentomes) are responsible for nutrition of the fetus, due to the penetration of metabolites from these structures from the mother23. It is possible that a smaller amount of cotyledons may affect the weight of lambs born to older sheep than by nulliparous. Confirmation of this hypothesis can be the results of this work. As the pregnancy progresses, the size of cotyledons may change due to nutritional requirements of the fetus23. It can be assumed that the larger diameter of cotyledon is intended to increase the penetration of nutrients into the fetus. It can be assumed that this is not enough to offset the smaller number of cotyledons per placenta. Thus, it can affect the birth weight of lambs. This study also noted significant differences in placental length between primiparous and older sheep. Placental length had highest values in youngest sheep. However, there is no analysis of this indicator in sheep available in the literature, which is why it is so important to continue research in this area. Significant differences in some placenta parameters and birth weight were noted between female and male lambs in single pregnancies. It was shown that placental weight, cotyledon

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Table 6 - Mean (± SD) total birth weight of lambs from monochorial twin pregnancies and morphometric parameters of placenta. Parameters

Twin female (n=6)

male (n=4)

Statistically significant differences

TBWL (g)

Mean ± SD Range

7966.67±801.66 6500.00-8600.00

7875.00±309.57 7600.00-8300.00

PW (g)

Mean ± SD

435.33±77.75

523.75±37.62

1<2 ; p<0.05

Range

332.00-540.00

485.00-573.00

PL (cm)

Mean ± SD Range

203.82±35.85 167.50-271.50

203.90±15.47 182.00-215.40

WP (cm)

Mean ± SD Range

57.82±3.37 53.00-61.00

56.10±10.01 44.50-64.90

Mean ± SD Range

95.33±11.00 80.00-108.00

105.25±14.50 88.00-120.00

CW (g)

Mean ± SD Range

153.75±61.45 112.00-244.00

223.00±31.11 201.00-245.00

1<2 ; p<0.05

MDC (cm)

Mean ± SD Range

2.34±0.34 1.96-2.94

2.34±0.24 2.04-2.61

ADUC (cm)

Mean ± SD Range

0.63±0.08 0.50-0.70

0.68±0.10 0.60-0.80

Statistical significance of p < 0.01; p < 0.05; SD - standard deviation; NO - no significant; TBWL - total lamb birth weight; PW - placental weight; PL - placental length; WP - placental width; CN - cotyledons number; CW - cotyledons weight; MDC - mean diameter of cotyledons; ADUC - average umbilical cord diameter.

weight and their diameters were affected by the sex of the lambs. Similar results were obtained in the studies of other authors8,24. It is possible that this is due to the different requirements of the male and female fetus25,26. The male lambs, due to their higher birth weight, compared to female lambs, need more nutrients delivered through the placenta. Therefore, it may increase placental weight, cotyledon weight, and mean cotyledon diameter in the placenta where the fetus was male. The study also analyzed twin dichorial placentas, where cotyledons had a larger weight in the placenta from which the male lambs were born. As in the case of single pregnancy, the weight of cotyledons was greater in placenta from dichorial twin pregnancies from which male lambs were born. Similarly, the birth weight of lambs was higher for male twins. Other authors’ conclusions indicated that the size and weight of the litter affect the weight of the placenta and the size of cotyledons27. The smaller number and weight of cotyledons in twin pregnancy dichorial placentas compared to single pregnancy placentas confirm these assumptions. In contrast, the increase in cotyledon diameter in twin pregnancies compared to those with single pregnancies may be the result of the placenta trying to minimize losses in nutrient transport to twin fetuses, caused by a reduced amount of cotyledons in each part of the dichorial placenta per fetus. Previous studies showed that twin pregnancies caused changes in maternal and fetal physiology differently from single pregnancies28. In this study, differences were noted in the influence of litter size on individual morphometric parameters of the placenta. Some authors suggested that in cases where the placenta was burdened with multiple pregnancy, specific interactions occurred between developing offspring11. There may be uneven competition for already limited resources in the twin placen-

ta, which may subsequently lead to differences in the birth weight of twin lambs. This study also considers the second type of monochorial twin placenta in pregnancy. Monochorial placenta due to their structure carry a greater risk of anastomosis of placental vessels 29. It is possible that the occurrence of anastomoses of these vessels limits the access to nutrients to one of the twin fetuses, and thus affects its development and birth weight30. The available literature has not yet included the classification of twin placenta into dichorial and monochorial in sheep, and thus the morphometric parameters of the placenta in this aspect have not been analyzed. That is why it is so important to continue re-

Table 7 - Correlation coefficients between placental morphometric parameters and the age of ewes and lamb weight. Age of ewes Age of ewes

Lamb birth weight

0.04

Placental weight

0.13

0.75**

Placental length

0.03

0.72**

Placental width

0.07

0.15

Cotyledons number

-0.10

0.54**

Cotyledons weight

0.23

0.69**

Mean diameter of cotyledons

0.17

0.004

Umbilical cord diameter

-0.14

0.14 (*) p<0.05; (**) p<0.01

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The effects of non-genetic factors on the morphometric parameters of sheep placenta and the birth weight of lambs

search and extend the analysis to include placental types and their possible impact on the morphometric parameters of the placenta and on fetal development.

CONCLUSIONS The obtained results indicate that processes in the body of a ewe during pregnancy occur on many levels. These processes are directed in particular at creating favorable conditions for fetal development, which in turn affects the survival of newborns. It can be assumed that the proper course of pregnancy, including the proper development of the placenta and obtaining its best parameters can affect success in the delivery period. It is similar with birth weight in farm animals, which has a great impact on the survival and further development of the offspring30. The obtained results showed that the morphometric parameters of sheep placenta and the birth weight of lambs depend on the type of pregnancy, litter sex and age of ewes. These results should be helpful in assessing postpartum placenta in this animal species. In addition, recorded differences in placenta parameters and birth weight of lambs may be useful in ultrasound assessment of placental and fetal development during pregnancy.

References 1.

Ocak S., Ogun S., Gunduz Z., Onder H. (2015). Goat placental efficiency determination by comparing litter weight to the surface area of the cotyledons. Anim Reprod, 12: 920-926. 2. Degrelle S.A., Murthi P., Evain-Brion D., Fournier T., Hue I. (2011). Expression and localization of DLX3, PPARG and SP1 in bovine trophoblast during binucleated cell differentation. Placenta, 32: 917-920. 3. Kolosov Y.A., Klimenko A.I., Vasilenko V.N., Shirokova N.V., Getmantseva L.V., Kolosov A.Y., Aboneev V.V., Chizhova L.N., Marchenko V.V., Mikhailenko A.K., Aboneev D.V. (2017). Some Biological Characteristics and Prediction of Sheep Productivity at Different Variants of Breed Selection. Online J Biol Sci, 17: 343-347. 4. Mellor D.J., Stafford K.J. (2004). Animal welfare implications of neonatal mortality and morbidity in farm animals. Vet J, 168: 118-133. 5. Medan M.S., Abd El-Aty A.M. (2010). Advances in ultrasonography and its application in domestic ruminants and other farm animals reproduction. J Adv Res, 1: 123-128. 6. Kaulfuss K.H., May J., Süss R., Moog U. (1997). In vivo diagnosis of embryo mortality in sheep by real-time ultrasound. Small Ruminan Res, 24: 141-145. 7. Carr D.J., Aitken R.P., Milne J.S., David A.L., Wallace J.M. (2011). Ultrasonographic assessment of growth and estimation of birthweight in late gestation fetal sheep. Ultrasound Med Biol, 37: 1588-1595. 8. Fernandes C.E., Cigerza C.F., dos Santos Pinto G., Miazi C., Barbosa-Ferreira M., Ferreira-Martins C. (2013). Parturition characteristics and uterine involution in native sheep from brazilian pantanal. Ci Anim Bras, 14: 245-252. 9. El-Tarabany A.A. (2012). Physiological changes in ewes conceived single or twins fetuses related with survivability of lambs. Arab J Nucl Sci Appl, 45: 224-235. 10. Casser E., Israel S., Boiani M. (2019). Multiplying embryos: experimental monozygotic polyembryony in mammals and its uses. Int J Dev Biol, 63: 143-155.

11. Korsten P., Clutton-Brock T., Pilkington J.G., Pemberton J.M., Kruuk L.E.B. (2009). Sexual conflict in twins male co-twins reduce fitness of female Soay sheep. Biol Lett, 5: 663-666. 12. Jitendra P., Ravi D.P., Ritesh K.S. (2013). Morphological study of placenta in normal uncomplicated pregnancy in Gujarat region. Int J Biol Medical Res, 4: 3155-3158. 13. Udainia A., Jain M.L. (2001). Morphological study of placenta in pregnancy induced hypertension with its clinical relevance. J Anat Soc India, 50: 24-27. 14. Błaszczyk B., Stankiewicz T., Udała J. (2013). The intravaginal electrical impedance measurements in monitoring estrous cyclicity, the length of the breeding season and ovarian cysts in goats. Small Ruminan Res, 113: 109-114. 15. Steven D.H.: Placental vessels of the foetal lamb. J Anat 1968, 103, 539552. 16. Vernunft A., Maass M., Brüssow K.P. (2018). Placental characteristics of german landrace sows and their relationships to different fertility parameters. Czech J Anim Sci, 63: 339-346. 17. Winder R.N., Krishnaveni V.G., Veena R.S., Hill C.J., Karat S.L.C., Thorn-Burg L.K., Fall D.H.C., Barker P.J.D. (2011). Mother’s lifetime nutrition and the size, shape and efficiency of the placenta. Placenta, 32: 806810. 18. Paraguez V.H., Atlagich M., Diaz R., Cepeda R., González C., De los Reyes M., Bruzzone M.E., Behn C., Raggi L.A. (2006). Ovine placenta at high altitudes: Comparison of animals with different times of adaptation to hypoxic environment. Anim Reprod Sci, 95: 151-157. 19. Karakus F., Atmaca M. (2016). The effect of ewe body condition at lambing on growth of lambs and colostral specific gravity. Arch Anim Breed, 59: 107-112. 20. Aktaş A.H., Doğan Ş. (2014). Effect of live weight and age of Akkaraman ewes at mating on multiple birth rate, growth traits, and survival rate of lambs. J Vet Anim Sci, 38: 176-182. 21. Dwyer C.M., Calvert S.K., Farish M., Donbavand J., Pickup H.E. (2005). Breed, litter and parity effects on placental weight and placentome number, and consequences for the neonatal behaviour of the lamb. Theriogenology, 63: 1092-1110. 22. Pettigrew E.J., Hickson R.E., Morris S.T., Lopez-Villalobos N., Pain S.J., Kenyon P.R., Blair H.T. (2019). The effects of birth rank (single or twin) and dam age on the lifetime productive performance of female dual purpose sheep (Ovis aries) offspring in New Zealand. PLoS ONE, 14: 1-14. 23. Liu Y., Li H., Sha Q., Hai R., Wang Y., Song Y., Gao F. (2018). Effects of maternal undernutrition on the growth, development and antioxidant status of ovine placentome subtypes during late pregnancy. Theriogenology, 110: 96-102. 24. Ocak S., Emsen E., Köycegiz F., Kutluca M., Önder H. (2009). Comparison of placental traits and their relations to litter size and parit weight in sheep. J Anim Sci, 87: 3196-3201. 25. Lampl M., Gotsch F., Kusanovic J.P., Gomez R., Nien J.K., Frongillo E.A., Romero R. (2010). Sex differences in fetal growth responses to maternal height and weight. Am J Hum Biol, 22: 431-443. 26. Broere-Brown Z.A., Baan E., Schalekamp-Timmermans S., Verburg B.O., Jaddoe V.W.V., Steegers E.A.P. (2016). Sex-specific differences in fetal and infant growth patterns: a prospective population-based cohort study. Biol Sex Differ, 65: 1-9. 27. Kaulfuss K.H., Schramm D., Berttram M. (2000). Effect of genotype, age of dams, litter size, birth weight and rams on morphological parameters of the placenta in sheep. Dtsch Tieraerztl Wochenschr, 107: 269-275. 28. Vonnahme K.A., Arndt W.J., Johnson M.L., Borowicz P.P., Reynolds L.P. 2008. Effect of morphology on placentome size, vascularity and vasoreactivity in late pregnant sheep. Biol Reprod, 79: 976-982. 29. Mellor D.J. (1983). Nutritional and placenal determinants of foetal growth rate in sheep and consequences for the newborn lamb. Br Vet J, 139: 307324. 30. Longo L.D., Reynolds L.P. (2010). Some historical aspects of understanding placental development, structure and function. Int J Dev Biol, 54: 237255.

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Influences of Maternal Undernutrition on Placental Development and Birth Weight in Sheep

127

AYSONDU MEHMET HANIFI1, OZYUREK SELCUK2 1

Malatya Turgut Ozal Universty, Akcadag Vocational School, Veterinary Department, Malatya, Turkey, +090 422 417 14 10 mhaysondu@ozal.edu.tr 2 Erzincan Binali Yildirim Universty, Cayirli Vocational School, Veterinary Department, Erzincan, Turkey, +090 446 311 35 15 sozyurek@erzincan.edu.tr

SUMMARY The aim of this study was to determine the effects of maternal feeding status on placental characteristics and lamb birth weight in mid-gestational period in sheep. The study was carried out in a private sheep farm in Erzincan during the lambing period of 2018 (39°80’ N, 40°03’ E and 1617 m above sea level). In our study, Morkaraman ewes with 50 heads in 3rd lactation was used. Sheep were divided into two groups. The first group was fed only on pasture until the 80th day of pregnancy; the second group is feed 100% of the daily energy needs of ration between 30-80. days. Although live weight differences between groups were statistically insignificant in mating and 80th days. On the 30th day and at birth there was a statistically significant difference. In our study, significant differences were found between the groups in terms of BW (p <0.01), PE and ACSA (p <0.05). In our study, lamb birth weight was 11.3% higher in the treatment group. The highest BW, PE and ACSA were observed in the treatment group. The results obtained from this study showed that maternal nutrition level between 30th and 80th day was effective on birth weight (P <0.01), placental weight, placental activity and average cotyledon surface area (P <0.01). While there was no significant correlation between birth weight and placental and cotyledon characteristics in the control group, a positive correlation (r =, 829, P <0.01) was found between birth weight and placental weight (r =, 465, P <0.05) and cotyledon number in the treatment group. These data show that in the livestock industry, the manipulation of the mother in terms of nutrition in the prenatal period or the prevention of restricted feeding based only on pasture will affect birth weight, newborn losses, average daily live weight gain, market weight, healthy meat production economy and profitability.

KEY WORDS Placenta, cotyledon, sheep, nutrition, ACSA.

INTRODUCTION Feeding in sheep during pregnancy has a significant effect on fetal and placental development. According to fetal programming theory, the fetus gains protective adaptation for the postnatal period in the uterus during pregnancy. Therefore, the failure of nutrition in the prenatal period may harm the developmental adaptation of the lamb. This is called intrauterine growth restriction (IUGR). IUGR is the deterioration in the growth and development of the fetus and hence organs of the animal during pregnancy. In addition, IUGR may cause negative consequences that permanently alter the offspring’s structure, physiology, metabolism and postnatal growth1. Placenta growth in sheep starts approximately on the 30th day of pregnancy (Symonds et al., 2007) and it is completed by 100 days2. Therefore, it has been reported that 30-80 days are sensitive for placental development in the feeding of pregnant sheep3. In general, restricted nutrition during pregnancy leads to significant reductions in placental growth4. It has also been reported that poor nutrition from 28 days to 78-80 days, when maximum placental growth occurs, reduces placental mass5 and

Corresponding Author: Ozyurek Selcuk (sozyurek@erzincan.edu.tr).

placenta size6. Changes in placental growth may cause low birth weight at the end of pregnancy due to the high correlation between placental weight and fetal weight7. When feeding is considered to have an important effect on placental development and to have considerable effect placental characteristics on lamb birth weight, it is concluded that feeding during the period of placental development is important. In many parts of the world, the normal mating period in sheep coincides with autumn and early winter, when pasture quality declines and nutrient intake is poor. Therefore, it is thought that sheep in Turkey are faced with malnutrition during pregnancy and this situation results in poor placental and fetal development. Recently, interest in “fetal programming” has increased, and study of effect of prenatal maternal nutrition on postpartum have become widespread. The aim of this study was to determine the effects of maternal nutrition during mid-gestation on placental characteristics and lamb birth weight in sheep.

MATERIALS AND METHODS The study was conducted in a private sheep farm in Erzincan during the lambing period of 2018 (39°80’ N, 40°03’ E and 1617 m above sea level). In this study, 50 Morkaraman ewes in 3rd lactation was used. Starting 15 days before the mating until the

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and gender of the lambs were recorded. Although the Morkaraman breed was usually single birthing, two sheep with twin births were not included in the study. The distribution of lambs by sex was 13 males and 10 females in the control group and 11 males and 12 females in the treatment group. Width, length and depth measurements were taken on 30 samples representing small and large cotyledons taking into account the vascularity in each placenta. In the observations of placentas, cotyledon number (CN), cotyledon density (CD), placental weight (PW), placental efficiency (PE) and total cotyledon surface area (TCSA) were determined. PE refers to the ratio of birth weight to placental weight (PE = BW / PW). CD is the ratio of cotyledon number to placental weight (CD = CN / PW). TCSA was calculated according to the following formula: [((CWi + CL) / 4) ×2] × 3.14 (π) × TCN. Average cotyledon surface area (ACSA) was obtained by dividing TCSA by cotyledon number8. The effect of the groups on placenta and cotyledon characteristics was analyzed using a general linear model (GLM) procedure in SPSS using a completely randomized design. In addition, gender was added as cofactor in the model to adjust placental and cotyledon characteristics between groups. Pearson correlation was used in 95% confidence interval to determine the relationships between placenta and cotyledon characteristics.

30th day of pregnancy, all sheep were given 650 gr/day of barley addition to the pasture. The hand mating method was applied in the mating. At 08:00 am and 16:00 pm, 5 rams that have mating ram mark participated in the herd and the sheep have ovulating were hand-mated and recorded. Then, starting from the 30th day of pregnancy, sheep was divided into two groups with 25 sheep in each group considering sheep live weight. The first group (control group; C) was grazed on pasture for 10 hours a day and no additional feeding was done (extensively). The second group was housed in a closed barn (treatment group; T) and ration that corresponding to 100% of daily energy requirement according to live weight was given (89% DM, 23.4% crude protein and 10.7 MJ ME / kg DM). In addition, ad libitum good quality alfalfa (86.1% DM, 15.4% crude protein and 8.1 MJ ME / kg DM) were given. Live weights were determined at the beginning of the mating, 30th, 80th day and before birthing. The ration amount to be given in each weighing period was recalculated according to live weight. From the 80th day onwards, both groups were combined and fed with a ration that corresponded to 100% of the daily energy requirement until birth in a closed barn. After birthing, the placentas were collected and they were weighed after removal of the liquid. In addition, birth weight

Table 1 - Effect of groups on placental traits (X ± SD)

Mean

BW (kg)

PW (g)

ACSA (cm2)

4,67±0,85

437,1±6,3

56,20±2,8

11,44±0,4

0,13±0,0

10,93±0,4

7,76±0,1

Group

Control

4,42±0,35

425,0±27,6

55,4±4,3

10,40±0,5

0,13±0,0

11,06±1,3

7,25±0,2

Treatment

4,92±0,24

449,2±16,4

57,0±12

12,48±1,8

0,14±0,0

10,80±1,4

8,27±0,8

Means with different superscript in each column (a, b) differ significantly; ns=not significant, *: P<0.05, **:P<0.01

Table 2 - Effect of groups on cotyledon traits (X ± SD) CNs

CNm

CNl

CL (cm)

CWi (cm)

CDe (cm)

Mean

3,18±0,5

45,4±3,9

7,20±1,5

2,71±0,0

2,22±0,0

0,99±0,0

Group

Control

3,80±2,0

46,20±6,1

5,40±3,0

2,53±0,1

2,08±0,0

0,99±0,1

Treatment

2,57±1,9

44,71±8,5

9,00±6,2

2,89±0,3

2,37±0,2

0,98±0,1

Means with different superscript in each column (a, b) differ significantly; ns=not significant, *: P<0.05

Table 3 - Pearson correlation coefficient of placental and cotyledon traits in control group BW

CWi

,299

,189

-,259

,106

,771**

-,291

-,396

,869**

-,325

,802**

CWi

-,163

,761

CDe

-,075

,661* **

,228

-,849

,408

-,688

-,831**

-,695*

-,011

-,648*

,888**

-,536

-,624

-,533

,704*

,055

-,049

-,826

-,181

-,105

-,147

,026

-,624

ACSA

,002

,791**

-,345

,980**

,951**

,862**

-,768**

-,591

*: P<0.05, **:P<0.01

,883**

CDe

-,153

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Table 4 - Pearson correlation coefficient of placental and cotyledon traits in treatment group BW

CWi

,465*

,829**

,021

-,458*

-,084

-,755*

CWi

-,202

,022

-,440

CDe

-,412

-,134

-,548

,702

-,195

-,049

,060

-,397 *

CDe

,692 ,073 ,074

-,619

,371

-,165

,750

-,825

-,381

-,706

,672

-,801*

,048

-,923**

,534

,071

,565

-,108

-,690

,482

-,221

-,698

ACSA

-,383

-,041

-,679*

,947

,887

,374

*: P<0.05, **:P<0.01

RESULTS The live weight change graphic of sheep during pregnancy is given in Figure 1. The live weights of both groups (control and treatment) were found to be similar during the the mating and 30th day. However, there was a difference in favor of treatment group at 80th day (p<0,05). The control and treatment groups had a live weight of 59.14 ± 1.61 and 57.88 ± 1.77 kg respectively in mating. Until 30 days of pregnancy from mating, control group and treatment group provided 5.8% (3,42 kg) and 7.2% (4,18 kg) live weight gain, respectively. In the following period, the treatment group gained 6.3% live weight and reached 65.96 ± 1.85 kg on the 80th day, whereas the control group lost 6.2% live weight and decreased to 58.92 ± 1.94 kg. From day 80, both groups were taken into the same feed and the control group and the treatment group showed a live weight gain to 63.41 ± 2.23 kg and 71,06 ± 2.41 kg, respectively. The effect of the groups on birth weight and placental characteristics is given in Table 1. As can be seen in Table 2, significant differences were found between the groups in terms of BW (p <0.01), PE and ACSA (p <0.05) (Table 2.). In our study, lamb

Figure 1 - Live weight change during pregnancy (kg)

birth weight was 11.3% higher in the treatment group. The highest BW, PE and ACSA were observed in the treatment group. PW and CN were not different between the groups. In terms of CNl, CL and CWi in favor of the treatment group, the differences were determined (p <0.05) (Table 2). The CL and CWi were higher 14.2% and 13.9%, respectively, in treatment groups. There was no difference between the groups in terms of CNs, CNm and CN1. Although CNs and CNm were higher in the control group, CN1 was 66.7% higher in the treatment group. The results of Pearson correlation analysis showing the relationship between birth weight, placenta and cotyledon characteristics for both control and treatment groups were given in Tables 3 and 4, respectively. While there was no significant correlation between birth weight and placental and cotyledon characteristics in the control group, a positive correlation was found between birth weight and placental weight (r =,465, P<0.05) and number of cotyledons (r =,829, P<0.01) in the treatment group. There was no relationship between BW and ACSA in both groups. Positive correlation was determined between PW and CL (r=,771, p<0.01), CWi (r=,761, p<0.05), CDe

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(r=,661, p<0.05) and ACSA (r=,791, p<0.01) and negative correlation was determined between PE (r=-,849, p<0.01) and CD (r=,th 648, p<0.05). In the treatment group, there was no correlation between PW and other factors (Table 4).

nipulation of the mother in terms of nutrition in the prenatal period or the prevention of restricted feeding based only on pasture will affect birth weight, newborn losses, average daily live weight gain, market weight, healthy meat production economy and profitability.

DISCUSSION References In both groups, it is thought that the increase in live weight from the mating period until the 30th day of pregnancy is based on the flushing. For control group the loss of 6,2% live weight in the pasture was result of that the pasture was insufficient to meet the daily energy needs of the sheep. Thomas et al.9 reported that in West America, ewes grazing on pasture without additional feeding, the pasture supplies 50% less nutrient requirements than the National Research Council’s (NRC) recommended consumption. In both groups, the increase in live weight from the 80th day of pregnancy to birthing is due to the growth of the fetus. The results are consistent with a research study on Angora goats10. The results of this study show that maternal nutrition level between 30-80 days is effective on birth weight (P <0.01), PW, PE and ACSA (P <0.01). These observations are consistent with the view that maternal dietary restriction during pregnancy may affect placental development and thereby reduce placental weight and size2;6;11;12. Also, Muñoz et al.13 reported that sheep that received 200% of their daily energy requirement during mid-pregnancy had heavier lambs than received 100% and 60%. In addition, McGregor10 reported that the birth weight increased by 0.3 kg for each 10 kg increase in doe live weight on the 137th day of pregnancy. But contrary to the results found, Fahey et al.14, Daniel et al.15 and Sen and Onder16 found that the mother’s nutritional level during mid-pregnancy did not have an effect on the lamb’s birth weight. Ocak et al.17 on sheep, Konyali et al.8 on goats reported a positive correlation between PW with CN and CD in their studies. However, in this study, no correlation was found between PW with CN and CD in both groups. When the correlations between placental characteristics in the treatment group were analyzed, it supports the opinion that nutrition in middle pregnancy bring on an increase in birth weight. As a result of the nutritional amount, the number of cotyledons increased in the treatment group and the increase in placental weight lead to an increase in birth weight. Also, the negative correlation between PW and PE observed in this study supports the findings of Ocak and Onder18 for goats. In the control group, there was no correlation between birth weight and placental weight similar to Ocak et al.19, whereas in the treatment group, positive correlation was determined similar to Sen and Onder16. Also, relationship between birth weight and placental weight is similar to the findings of Sen et al.20. In treatment group, the positive correlation between BW and CN obtained in this study are in agreement with past studies in beef cattle and sheep19;21;22.

CONCLUSION This study showed that only pasture-based feeding between 3080 days of pregnancy reduced the mother’s live weight during mid-gestation and resulted in lower lamb birth weights (11% lighter). These data show that in the livestock industry, the ma-

1. Gootwine E., Spencer T.E., Bazer, F.W. (2007). Litter-size-dependent intrauterine growth restriction in sheep. Anim, 1(4), 547-564. 2. Redmer D.A., Wallace J.M., Reynolds L.P. (2004). Effect of nutrient intake during pregnancy on fetal and placental growth and vascular development. Domestic Anim Endocrinology, 27, 199-217. 3. McCrabb G.J., Hosking, B.J., Egan, A.R. (1992). Changes in the maternal body and fetoplacental growth following various lengths of feed restriction during mid- pregnancy in sheep. Aust. J. Agric. Res, 43(6), 14291440. 4. Wu G.Y., Bazer F.W., Cudd T.A., Meininger C.J., Spencer T.E. (2004). Maternal nutrition and fetal development. J Nutr, 134(9), 2169-2172. 5. Symonds M.E., Stephenson T., Gardner D.S., Budge H., 2007. Long-term effects of nutritional programming of the embryo and fetus: mechanisms and critical windows. Reprod. Fertil. Dev, 19, 53-63. 6. Clarke L., Heasman L., Juniper D. T., Symonds M. E. (1998). Maternal nutrition in early-mid gestation and placental size in sheep. Br J Nutr, 79(4), 359-364. 7. Mellor D. J., Murray L. (1982). Effects of long-term undernutrition of the ewe on the growth-rates of individual fetuses during late pregnancy. Res Vet Sci, 32(2), 177-180. 8. Konyali A., Tolu C., Das G., Savas T. (2007). Factors affecting placental traits and relationships of placental traits with neonatal behaviour in goat. Anim Reproduc Sci, 97, 394-401. 9. Thomas D.M., Clapp J.F., Shernce S.A. (2008). Fetal energy balance equation based on maternal exercise and diet. J R Soc Interface, 5:449-55. 10. McGregor B.A. (2016). The effects of nutrition and parity on the development and productivity of Angora goats: 1. Manipulation of mid pregnancy nutrition on energy intake and maintenance requirement, kid birth weight, kid survival, doe live weight and mohair production. Small Rumin Res, 145: 65-75. 11. Osgerby J.C., Wathes D.C., Howard D., Gadd T.S. (2002). The effect of maternal undernutrition on ovine fetal growth. J Endocr, 73: 131-142. 12. Wu G., Bazer F.W, Wallace J.M., Spencer T.E. (2006). Board-invited review: intrauterine growth retardation: implications for the animal sciences. J Anim Sci, 84:2316-37 13. Muñoz C., Carson A.F., McCoy M.A., Dawson L.E., O’Connell N.E., Gordon A.W. (2009). Effect of plane of nutrition of 1- and 2-year-old ewes in early and mid-pregnancy on ewe reproduction and offspring performance up to weaning. Anim, 3, 657-669. 14. Fahey A.J., Bramel J.M., Parr T., Buttery P.J. (2005). The effect of maternal undernutrition before muscle differentiation on the muscle fiber development of the newborn lamb. Sci. J. Anim. Sci, 83, 2564-2571. 15. Daniel Z.C.T.R., Brameld J.M., Craigon J., Scollan N.D., Buttery P.J. (2007). Effect of maternal dietary restriction during pregnancy on lamb carcass characteristics and muscle fiber composition. Sci. J. Anim, 85, 1565-1576. 16. Sen U., Onder H. (2016). Poor placental traits reduce kid birth weight in young Saanen dams in the first parity. Turkish J Vet Anim Sci, 40(5), 554561. 17. Ocak S., Emsen E., Koycegiz F., Kutluca M., Onder H. (2009). Comparison of placental traits and their relation to litter size and parity weight in sheep. J Anim Sci, 87(10), 3196-3201. 18. Ocak S., Onder H. (2011). Placental traits and maternal intrinsic factors affected by parity and breed in goats. Anim Reproduct Sci, 128, 45-51. 19. Ocak S., Ogun S., Onder H. (2013). Relationship between placental traits and maternal intrinsic factors in sheep. Anim. Reprod, 139(1-4), 31-37. 20. Sen U., Sirin E., Kuran M. (2013). The effect of maternal nutritional status during mid-gestation on placental characteristics in ewes. Anim. Reprod, 137.1-2: 31-36. 21. Echternkamp S.E. (1993). Relationship between placental development and calf birth weight in beef cattle. Anim Reprod Sci 32: 1-13. 22. Dwyer C.M., Calvert S.K., Farish M., Donbav J., Pickup H.E. (2005). Breed, litter and parity effects on placental weight and placentome number, and consequences for the neonatal behavior of the lamb. Theriogenology, 63, 1092-1110.

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On-farm risk factors associated with Salmonella in pig herds

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ALESSIA DE LUCIA, FABIO OSTANELLO* Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum - UniversitĂ di Bologna

ABSTRACT In the pig production Salmonella infections are cause of concern for two major reasons. The first is the clinical disease in pigs and the second is that pigs can be infected with a broad range of Salmonella serotypes which can potentially contaminate pork products and pose a threat to human health. In Europe, salmonellosis is the second most frequent zoonoses for number of confirmed human cases and number of hospitalizations. After eggs, the consumption of contaminated pork meat and meat-product is the major cause of human outbreaks. According to the most recent survey conducted on pig farms in the EU in 2008, Italy was among the top five countries with the highest prevalence around 51.2% in breeding farms and 43.9% in production farms. These finding highlighting the need to investigate the risk factors in pig farms that should be managed to maintain a low prevalence. Pigs are susceptible to most Salmonella serotypes and although S. Typhimurium and its monophasic variants are the most common, a large variety of other serotypes are also reported in surveillance studies at farm level. Low Salmonella prevalence in pig herd is associated with a lower contamination pressure at the slaughterhouse reducing the occurrence of cross-contamination of carcasses during the slaughter process and, subsequently, the likelihood that human cases of salmonellosis will occur. This review focuses on risk factors in farms and biosecurity measures that can help to control pig important pathogens at the same time as reducing the within-farm prevalence of Salmonella. The main factors influencing Salmonella infection in pigs can be grouped into four different categories: farm hygiene, feeding practices, herd and health management. However, there is no universal protocol that all pig herds can put into place to minimize the risk of disease introduction or spread. Each farm is unique for host susceptibility, management, facilities, and other influential factors. Biosecurity measures, cleaning and disinfection, feed practices, as well as vaccination are often mentioned as the intervention categories with the greatest potential to reduce Salmonella prevalence in pig farms. The information included in this review may persuade the farmers that improving good hygiene practices and animal management would result in economic rewards. The efforts to control Salmonella in farms could also help to reduce infection by other porcine pathogens.

KEY WORDS Salmonella; pig farms; risk factors; biosecurity measures, food safety.

INTRODUCTION In the pig production Salmonella infections are cause of concern for two major reasons. The first is clinical disease in pigs (septicaemic salmonellosis associated with Salmonella Choleraesuis and enterocolitis associated with the broad host range serotype such as S. Typhimurium and its variant S. (1,4,[5],12:i:-), and the second is that pigs can be infected with several Salmonella serotypes that can potentially contaminate pork products and pose a threat to human health. Salmonella is the second most common zoonotic gastrointestinal bacteria in Europe with 91,857 confirmed human cases in 2018 1. However, this data represents just the tip of the iceberg as only the most serious cases are reported to the health department. Many other cases are not diagnosed because not all ill persons seek medical care and at healthcarelevel not all the cases are reported to public health officials. Therefore, a more collaborative approach between human and veterinary medicine, in the context of the â&#x20AC;&#x153;one healthâ&#x20AC;?

Corresponding Author: Fabio Ostanello (fabio.ostanello@unibo.it).

perspective is necessary. The consumption of pig meat constitutes a source of human Salmonella infections 1. Consequently, the main purpose of controlling Salmonella is to reduce prevalence within herds, prevent dissemination of Salmonella during later stages of production (i.e. to rearing and fattening pigs) and improve transport and slaughter hygiene. This dissemination may lead to Salmonella contamination of pig meat and consequently to human disease (Figure 1). In Europe, after egg and egg products, bakery products and mixed food, consumption of pork and products thereof has been identified as 4rd most important source of Salmonella in human food-borne outbreaks 1. For the pig chain, monitoring of Salmonella laid down by the meat hygiene criteria regulation (Commission Regulation EC - No 2073/2005). This monitoring is based on the bacteriological analysis of the carcasses samples. Although, in case of a positive sample, it is difficult to establish whether tissue contamination originated from an infected farm or from crosscontamination at the slaughterhouse 2. The subclinical and largely widespread Salmonella infection across the different production stages in pig farms makes that control at slaughterhouse level difficult and costly. However, some European

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Figure 1 - Progress of infected or contaminated pigs (red) and Salmonella-free pigs (white) from farm to the slaughterhouse. Salmonellafree pigs can become infected or contaminated (red/white) during transport, in the holding pens, and by cross-contamination in the slaughter line (adapted from Arguello et al.82).

countries such as Danmark and German set up national control programmes based on serological testing of blood or meat juice samples collected from pigs at the abattoir 3, 4. According to the serological results, pig farms are classified in one of three herd-levels 3, 5. Highly infected herds, assigned to level 2 or 3, are supported by the national governments to reduce the infection load of their herd. Additionally, these farms are subjected to penalty fees to cover the expenses of the special hygienic precautions that have to be taken at the slaughterhouse when pigs from herd level 3 are slaughtered. Farmers are therefore motivated to apply better control measures to reduce Salmonella prevalence and avoid the financial consequences 3. Pigs are susceptible to most Salmonella serotypes and although S. Typhimurium and its monophasic variants are the most common serotypes reported, a large variety of other serotypes can be isolated from pig farms 6. It is widely acknowledged that infection in pigs is typically not associated with clinical disease, and pigs acting as â&#x20AC;&#x153;asymptomatic carriersâ&#x20AC;? 7, 8. However, some serovars such as S. Choleraesuis or S. Typhimurium and its monophasic variant along with other invasive serotypes (e.g. S. Enteritidis. S. Dublin, S. Newport) may result in clinical disease and septicemic episodes 9. The impact of Salmonella on-farm performance is still not clear 10. An American study reported that finisher pigs with high Salmonella prevalence had feed conversion rates above the median when compared with herds with lower prevalence 11 A more recent study from UK showed that pig farm with a low Salmonella seroprevalence (<10%) had a higher slaughter live weight (mean 103.7 kg), compared with conventionally control farm (mean 93.8 kg) 12. Despite these findings, to date, there is no enough evidence to said that a

lower Salmonella prevalence could result in higher productivity 10. Although, Salmonella infection is often associated with other diseases such as postweaning multisystemic wasting syndrome (PMWS), resulting in decreased productivity 13. Due to the lack of clinical infectious disease and economic losses, farmers and pig owners, do not see the need to intervene in order to reduce its prevalence at farm level as a priority. Likewise, the lack of any financial incentives or penalties in the majority of the EU member states may have led to the perception that Salmonella infection in pigs is of lesser importance compared to the other pig diseases or Salmonella in poultry 10. According to the most recent survey conducted on pig farms in the EU in 2008, Italy was among the top five countries with the highest prevalence around 51.2% in nucleus and multiplier farms and 43.9% in production farms 14. Several studies have shown that there is a strong association between Salmonella intestinal carriage of pigs at the herd level and the contamination pressure at the abattoir 15, 16. Therefore, on-farm interventions and good control measures applied at farm aimed to reduce the prevalence of Salmonella positive pigs should prevent further spread within the production chain. This review focuses on risk factors in pig farms and biosecurity measures that can help to control important pig pathogens at the same time as reducing the within-farm prevalence of Salmonella. Several risk factors are associated with Salmonella prevalence in the herds. Following the classification used by some authors 17, 18 the main risk factors are grouped into 4 different categories (Figure 2): 1. Farm hygiene: cleaning and disinfection (C&D), boots,

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rodents, animal vector, humans and vehicles, and manure management. 2. Feeding practices: type of feeding (pellets or meal; particle size, dry or wet) and acidification of feed. 3. Herd management: herd size, type of farm, all in/all out, quarantine and housing systems (type of pen and wall separation). 4. Health management: herd health status, vaccination and antibiotic treatments.

FARM HYGIENE Cleaning and disinfection practices It appears to be common sense that cleaning and disinfection (C&D) practices of pig pens are an essential part of any effective on-farm disease control regimen 10. Salmonella-free pigs housed in a contaminated environment are likely to become infected 19. However, the elimination of Salmonella from farm buildings is complex due to the robustness of the organism which is able to persist in the environment from months to years, especially when protected by residual organic matter (e.g dust and faeces) 20. There are different types of disinfectant commercially available and effective against Salmonella, such as quaternary ammonium compounds (QAC), products containing glutaraldehyde or formaldehyde, iodine based compounds or chlorocresols and peroxygen or peracetic acidbased compounds 21. However, their effectiveness may be severely compromised if not properly applied. Disinfectants can be easily inactivated by the presence of organic matter or overdiluted if used before the surfaces are completely dried 10. In addition, most farm buildings have crevices and cracks in floors, ceilings and walls which are particularly difficult to clean properly 22. It is important to mention that some types of pig house material

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can be more challenging to clean. Concrete is a common material used in pig accommodation and its rough surface is more likely to have a high level of residual contamination compared with smooth surfaces 23. For careful and systematic C&D procedures also the tool and farm equipment (feeders and drinkers), are of paramount importance. Moreover, the presence of rodent vectors on a farm can hamper the effectiveness of any C&D procedures, as Salmonella-carrying rats and mice can re-contaminate the cleaned surfaces and recycle the infection from one batch to the next 21. The general idea is that only a frequent cleaning regimen after every movement of pig group and linked with all in/all out (AIAO) management could lead to sustainable success 10. A recent study showed that an increased frequency and efficiency of cleaning practices on-farm reduced the prevalence of Salmonella Typhimurium shedding at the time of slaughter. However, the authors found that these efforts alone were not sufficient to eliminate the infection from the pig population 24. To control the infection in pigs and reduce the level of Salmonella shedding under the infection dose (103 CFU/g), it is necessary to combine the cleaning procedures to other appropriate measures such as vaccination and acid treatment of feed 24.

Boot dips Similarly, to the C&D, the use of boot dips containing disinfectant is a procedure commonly seen as good biosecurity measures. The correct use of boot dips is associated with a lower prevalence of Salmonella infection 10. Ideally, the footwear should be wash first before dipping to remove organic matter that severely depletes the disinfect activity. On the other hand, the incorrect use of boots and boot dips could potentially become a risk factor for Salmonella and a source of spreading the infection through the farm 25, 26. Commonly in farms, the boot dips are not efficacious and became inactive due to contamination with fecal matter, mistakes with the dis-

Figure 2 - Main risk factors associated with Salmonella prevalence within herds.

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infectant concentration used, or not respecting biocidal expiry dates 27. Environmental factors such as hot weather and rain may also influence the efficacy of the dips by increased evaporation or dilution by rainwater. The use of a cover for the dips may help to prevent environmental factors from compromising the dips. It is important to frequently change and replenish the dips at least twice a week 27. If not used correctly the efforts are vain and result in wasting resources, time and money.

Animal vectors

and slaughterhouses and can transfer pathogens from one farm to another, acting as mechanical vectors 39. Due to the considerable risk that they represent if possible should be farm dedicated. Otherways, a rigorous biosecurity regimen of vehicle disinfection is essential. This required an adequate wheel wash and spray disinfection or a dedicated disinfection station, but these procedures are expensive and not commonly applied even in large pig farms. Alternatively, any external vehicles (lorries and visitor’s cars as well) should not be allowed to enter into the clean areas of the farm and parked at least 300 meters away from animal buildings 10.

Since members of the Salmonella are notorious for their ability to infect a wide range of hosts, contact with other animal species (domestic, wild or synanthropic) can increase the pig’s risk of infection 28, 29. The role of carrier vectors that can contribute to the horizontal transmission of Salmonella and other pathogens (e.g PCV2, PRV, Toxoplasma gondii, Lawsonia intracellularis) has been well documented. Rodents, wild birds, insects, and pets (dogs, and cats) are common inhabitants living on pig farms that can all potentially introduce and transmit Salmonella by direct contact with pigs or via faecal contamination of feed, drinkers or farm equipment 30. Among them, wild birds and synanthropic rodents (mice and rats) are of particular importance 30, 31. A recent study showed that wild birds can be infected with the same serotype of pigs, suggesting that wild birds are capable of recycling infection and contributing to the persistence of Salmonella between batches of pigs 32. Rodents can acquire and persistently carry Salmonella infection for several months 33, 34. On farms, rodents are considerate very efficient vectors and amplifiers of Salmonella infection, due to their high Salmonella-carriage and their good reproductive capacity. Mouse droppings can have up to 104 CFU/g of Salmonella and a single mouse could shed 100 fecal pellets per day 33, 35. Pest control on-farm is of paramount importance due to their ability in transmitting several pig pathogens but also because of the economic damage that they can cause to the farm infrastructure and the amount of feed that they can consume 36. Poison and traps are the most common methods of rodent control. An efficient pest control rodent should have an integrated approach which includes the use of rodenticides and strict biosecurity procedures focussed on repairing facility structure (holes, door seals, etc.), disposal of dead animals and waste and removal of pig’s leftover feed 35. Moreover, training the farm staff to respond more quickly and thoroughly to signs of rodents can be very helpful 10.

Manure can be a source of many important pig’s infections such as swine dysentery (infection with Brachyspira spp.), classical swine fever, foot and mouth disease, porcine reproductive and respiratory syndrome (PRRS), Streptococcus suis, Escherichia coli and Salmonella. The risk of introduction and spreading infections increases considerably if manure from other farm facilities is spread within 3.2 km of a pig farm 10. Pig slurry should be stored as long as possible and under controlled conditions (10.5°C for 84-112 days) to reduce or kill Salmonella and other organisms irrespective of the initial load39, 40. Several technologies are designed in order to reduce microbial concentrations contained in the pig manure by 90 to 99% 41. Manure treatment systems are generally physical, biological and chemical or a combination of all three treatments and rely on anaerobic digestion, composting, and separation technologies 35, 41. Well-designed farm facilities should avoid the accumulation of feces and contact between animals and their waste. A French study showed that a frequent removal of the sow’s manure during the lactation period decreases the risk of Salmonella infection in piglets. The same authors reported that emptying the pit below slatted floors after that a batch of sows was removed is a protective factor against Salmonella 18. Types of floor which decrease pig contact with fecal material and decrease fecal-oral transmission among pigs should be preferred. A lower Salmonella prevalence was found in pigs housed on fully slatted floors, were feces immediately flow away in the manure pit, than in pigs raised on the flush gutter floor 42. While pen with earth floors have been associated with a higher Salmonella prevalence 43. Today it is generally accepted that solid or partially slatted cause a higher risk for Salmonella compared with fully slatted floors 30, 31, 44.

Manure management

Humans and vehicles as vectors Biosecurity-related practices regarding visitors and vehicles have been reported to be useful in lowering the risk of Salmonella infection in pigs farm 29. Moreover, other important pig pathogens can be carried by humans, for example, Mycoplasma hyopneumoniae and foot and mouth disease virus have been found to survive in humans for 30 and 11 hours respectively 37. Therefore, often pig farms demand that visitors 24 to 48-hours pig free and to shower before access into the farm. Good practice as the presence of areas where visitors can change clothes and footwear before entering help to reduce the risk of spread Salmonella into pig areas. Similarly, because humans can act as mechanical and biological vectors, access to toilets and handwashing have a protective effect against Salmonella for pigs and people as well 11, 38. Transport vehicles are constantly in contact with other farms

FEEDING PRACTICES Type of feeding Feed-practices are considered to be of maximum importance to control and reduce Salmonella prevalence within herds. The feed nature (meal wet or dry), proportions of ingredients and particle size have been reported to influence the risk of Salmonella infection in pigs 12, 29, 38. There is a general consensus among authors that dry meal is preferred to pelleted feed and wet feed is preferred over dry feed. The principle behind this is that both meal and liquid feed reduce the intestine pH creating an inhospitable environment for Salmonella 45. Many studies available have proved the protective effect of wet feed compared to dry feed 12, 18, 38, 46, 47. However, it is important to highlight that liquid feed alone with no acidic condiction

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achieved by fermentation, is not sufficient to provide protection 25. On the contrary, the use of trough feeding water without the fermentation step was reported to be a risk factor for Salmonella infection 46. Additionally, a ration with 25% of barley and wheat (such as maize, beet, manioc), have been associated with a reduction of Salmonella infection in pigs 48, 49. The feed particle size may be responsible for higher Salmonella prevalence in pigs. Small particle fractions used to make pellets are considered a risk factor due to the quicker transit passage through the pig digestive tract which does not result in a low intestinal pH. While the slower gastric passage and the lower gastric pH when pigs are fed a coarsely versus with finely ground feed are reported to be protective 50. It is important to mention that the animal feed can be a source of introducing Salmonella serovars and resistant bacteria into pig farms 29, 51, 52. Feed ingredients may be accidentally contaminated by Salmonella during growth, harvesting, transport, or storage. Additionally, on-farm feedstuff contamination, in particular of cereals, can result from the through contact with wildlife or synanthropic animals that defecate in crop or storage facilities or by the usage of contaminated equipment 53, 54. A multi-country survey carried out in five EU countries detected Salmonella from feedstuff in 17.6% of pig farms and 6.9% of all feed samples, highlighting the role of feed as one of the major sources of persisting infection in pigs 29.

Acidification of feed Several studies have investigated the action of substances as organic acids, probiotics, prebiotics, formaldehyde, essential oils and bacteriophages, regarding their ability to modify the gut environment to prevent Salmonella colonization. Among those interventions, the acidification with organic acids added to the feed or drinking water seems to be the most popular. Formic and propionic are the main acids used as inhibitors against Salmonella however their efficacy is reported to be inconsistent 55. The beneficial effect is highly variable and depends on the type of organic acid administered, the inclusion rate and the physical and chemical form of the product (acid or acid salt) 51, 52. For example, the administration of lactic acid to weaner 56 and grower 57 pigs and formic acid to finisher pigs 58 were found to have a beneficial effect against Salmonella. Contrary, in another study, the acid-supplemented feed to weaners did not decrease the prevalence of Salmonella in their feces 59. It has been hypothesized that also the high level of bacteria contamination, the presence of other concurrent diseases in herd, the wrong compound administration (not for a sufficiently long period) and the â&#x20AC;&#x153;acid tolerance responseâ&#x20AC;? acquired from the organism may also contribute to the failure of the activity of the organic acids 10, 51 . In conclusion, the problems associated with the use of acid substances such as clogging of drinkers, fungal growth, corrosion of the equipment, along with the uncertainty regarding their effect, make the advice difficult in terms of costbenefit 10.

HERD MANAGEMENT Herd size, types of farm and all in/all out (AIAO) management The herd size is considered a controversial risk factor for Sal-

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monella. Large companies are associated with a higher risk of infection due to practices of mixing pigs, which may happen most frequently in large herds 60. On the contrary, there are observations that suggest that Salmonella can be more prevalent in small or medium-size herds 47. Large farms can be very well managed with good C&D procedures or other practices successful in controlling Salmonella prevalence such as batch farrowing and AIAO management 61. The herd size is also associated with livestock management aspects that can potentially increase the risk. Large companies may need to replace or purchase animals but they can also operate as a closed herd breeding their own replacements. As widely accepted that operate a closed herd system is desirable in order to prevent the introduction of infected animals into the farm. In this type of farm, pis are generally exposed to farm-resident Salmonella strains and can acquire a herd immunity able to control the infection reducing the carrying and shedding of specific resident strains 10. However many pig producers consider the closed system impractical and they regularly introduce pigs into the main farm. In Denmark, a strategy to minimize the risk associated with this animal flow is to import pigs from farms having an equal or superior health status 62. Some authors also report that having more than two suppliers was a risk factor 38, 63. However, pig flow practices if associated with strict age group segregation and AIAO policies can result in an efficient strategy to decrease Salmonella exposure and infection 29. The AIAO is generally thought one of the most important interventions to reduce disease in pig farms. Although there are not many studies that have specifically proved the association of this practice with the reduction of Salmonella prevalence. Canadian and French studies showed that strict application of AIAO procedures have lent to a decreasing of Salmonella infection 18, 64, 65. In contrast, American and Danish studies did not found in the AIAO system a protective effect against Salmonella. Despite all these contradictory findings, minimizing the animal movement around the farm, strict management of group of pigs as well as the presence of quarantine facilities are considered of paramount importance for the successful control of Salmonella and other diseases.

Quarantine, isolation and pen partition The isolation of the new pigs on arrival is often ignored. Quarantine facilities allow animals to recover from the stress of transport, adapt to a new environment and showing symptoms of any incubating infections in a contained environment 39. This isolation period is also an opportunity to run laboratory tests, challenge with resident pathogens and perform vaccination if appropriate. Regarding Salmonella the majority of the authors recommend keeping the replacement animals in isolation for six weeks with no cross-contact with the main unit 39. Where possible, quarantine building should be located at 100 to 150 meters far away from the main farm and it should be run as a completely independent unit with its own farm equipment, and waste management system. It is preferable to have their own farm staff in charge of managing the quarantined animals, if not possible the animals should be visited at the end of the working day 10. Isolation premises should adopt AIAO systems and operated with strict biosecurity measures with particular attention on C&D procedures between batches of pigs and the use of different protective

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clothing before entering and after leaving the isolation building 38. In fattening farms where the pigs are introduced to multiple age sites, the use of quarantine arrangements is not always practicable. There, it is important to have a good separation between buildings housing animals from different ages and minimize the use of any common equipment between different risk categories 10. Authors agree that the snout contact between pigs in neighboring pens is considered a significant risk factor. Pens allowing for nose-to-nose contact between pigs had 2.2 times higher odds to be Salmonella-positive than pens without such contact 30, 66. Closed partitions between pens are feature inherent to farm design and not easy to change. However, pen partition is an important barrier to prevent the seepage of contaminated material between adjacent pens and the spread of pig important pathogens, therefore it should be taken into account during the design of new barns 30, 67.

HEALTH MANAGEMENT Herd health status It has been said that the high health status of herd (e.g membership in specific free) is associated with a lower Salmonella risk of infection, suggesting the presence of synergy between control pig pathogens and control of Salmonella 10, 29. Diarrhea problems caused by pathogens that damage or disrupt the gut and its flora may predispose or affect Salmonella infection 68. Likewise, co-infection with the other pig pathogens itself could play a role in Salmonella epidemiology, probably due to interference with the host’s immune response 12. Lawsonia intracellularis infection was associated with an increased prevalence of pigs shedding Salmonella at the end of the fattening period 18. A recent study found that the vaccination against L. intracellularis was able to reduce the shedding of S. Typhimurium in co-infected pigs 69. PRRSV infection was also identified as a risk factor for Salmonella shedding due to the ability of the virus to induce immunodepression leading pigs more susceptible to Salmonella contamination and multiplication 18. A couple of researches investigated the interactions between parasites and bacteria, suggesting that nematodes in particular Oesophagostomum spp. 70 and Ascaris suum 71 might play an important role in the dynamics of Salmonella infections. The associations between the herd health status and Salmonella prevalence may reflect the overall expertise and management practices of the pig farmers 29. In many ways, Yersinia and Salmonella have similar behavior and some practices like limiting the mixing of pig batches during the fattening period appear to be effective in controlling both infections 17. As previously stated, Salmonella infection is often subclinical and thus pig owners do not usually see the need to intervene, as they consider Salmonella problem a consumer’s responsibility by “proper cooking”. Nonetheless, if actions against Salmonella would result in better control of other serious pig pathogens, that might motivate the farmers to improve Salmonella controls 10.

Antibiotic treatments and vaccination The usage of antibiotics is another controversial risk factor. Prophylactic antibiotic treatment or the use of antibiotics

growth promoter (e.g tylosin) during the fattening period was observed to enhance the risk of Salmonella shedding 12, 47, 72 . A German epidemiological survey found that pigs treated with antibiotics had a five times higher chance to be seropositive for Salmonella compared with the untreated animals 44. All the authors agree on the plausible explanation for this intriguing finding, suggesting that the alteration effect of antibiotics on the normal protective gut may have favored the colonization of endogenous pathogens. In contrast, two American studies found a higher prevalence of Salmonella in antimicrobial-free production systems compared with conventional ones 73, 74. Despite these studies, there is still no compelling evidence to show that antibiotics can help to lower Salmonella prevalence. Conversely, probiotics and prebiotics may help to reduce the infection by promoting protective gut endogenous flora 75, 76. The use of vaccination might be a suitable alternative to the use of antimicrobials for controlling the Salmonella infection cycle in pig farms. Currently, many Salmonella vaccines induce an antibody response against the lipopolysaccharide layer of the bacterial wall, and thus the DIVA status (differentiation of infected from vaccinated animals) is not possible 77, 78. This can potentially interfere with herd-level serological monitoring programmes for Salmonella. To overcome these limitations, a strategy could be to vaccine only to the sows rather than finisher pigs, aimed at providing passive immune protection to whose piglets without interfering with any monitoring serological programs in place 77. In general serovar-specific vaccines have been reported to be the more effective, this raised the issue of the low cross-protection against more than one serogroup. In farms where multiple serovars are present, the efficacy of the vaccination may be compromised and can cause the emergence and spread of the other serovars 79. Ideally, should be prioritized the control of serovars that pose the highest risk to humans. In fact, it has been speculated that vaccination of pigs with S. Typhimurium together with good biosecurity measure help to reduce Salmonella prevalence in pigs and the proportion of “carrier” animals at the slaughterhouse 80. It is important to note that vaccination is not a cheap strategy and alone is not sufficient to eliminate the infection 81. There is a general consensus that an holistic approach covering different interventions such as vaccination, biosecurity measures, C&D procedures and feeding practices is required in order to reduce the prevalence of Salmonella in pigs.

CONCLUSION Control of Salmonella in pig meat as a public health problem should be based on individual State epidemiological situations covering the whole production chain from farm to fork. Owing to the characteristics of the infections and the ability to survive in the environment, the Salmonella control onfarm should be considered as part of a holistic plan. Effective control strategies should combine different key interventions of biosecurity, C&D, feed practices and vaccination. Hygiene is extremely important in intensive pig production. Despite careful and systematic C&D procedures, good hygiene practice also involves physical segregation of animal groups and the prevention of re-contaminate by vectors, personnel and equipment. The use of acidified feed and water

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may further potential benefits but highly expensive for the farmer. Moreover, there is a need for a more complete evaluation of their efficacy not always consistent. In the era of antimicrobial resistance, any control strategy should also include the reduction of antimicrobial treatments. The use of the current vaccines against Salmonella for pigs might be a suitable alternative to antibiotic use. However, further investigations are needed to avoid interference between vaccination and serological monitoring schemes. Even when productivity benefits can be demonstrated, it can still be difficult to motivate farmers to apply improved standards. There is therefore a need for a greater understanding of the drivers and barriers involved in promotion of voluntary improvement of farm standards.

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Ringworm by Trichophyton erinacei in calves: description of two Italian outbreaks

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FRANCESCO AGNETTI1, ROSA CIAVARELLA1, DEBORAH CRUCIANI1, ERSILIA MARIA EPIFANIO1, DANIELA GOLINELLI2, PAOLA PAPA1, ELISA SGARIGLIA1, ANDREA VALENTINI1, SILVIA CROTTI1 1 2

Istituto Zooprofilattico Sperimentale Umbria e Marche “Togo Rosati”, Via G. Salvemini 1, 06126 Perugia, Italy Bio98 SRL, Via L. Frapolli 21, 20133 Milano, Italy

SUMMARY Trichophyton erinacei is a zoonotic dermatophyte mainly isolated from hedgehogs. In other hosts (human or animals) it can cause skin infections, with typical clinical features of dermatophytosis. It has never been reported in cattle in which T. verrucosum is frequently isolated, particularly in calves. The present case report describes two outbreaks (case A and case B) of cattle ringworm caused by T. erinacei in Northern Italy; the first case involved a group of 5 month old Bruna Alpina calves, while the second one concerned a group of 24 months (approximate) Italian Frisona cows. Herd A was subjected to a regular vaccination protocol against T. verrucosum, but the calves involved in the outbreak had not yet been vaccinated. Crusts, alopecic and pruritic skin lesions, appeared about 4 months before the clinical visit, were mainly distributed on the head, trunk and flanks in all the animals involved. The veterinarian of farm B detected abdomen skin lesions compatible with dermatophytosis about 2 weeks after the appearance of the disease in the animals. Animal and human hairs/crusts samples were collected for mycological investigations by cultural and molecular methods (PCR and sequencing). The colonies observed showed macroand microscopical features attributable to T. mentagrophytes, however the strain has been confirmed as T. erinacei by molecular analysis. The described cases showed that T. erinacei can cause bovine ringworm, with clinical features completely comparable to those expressed by T. verrucosum. The use of vaccination in some animals of herd A appears to have contributed to their protection against the mycosis, as demonstrated by the fact that only non-vaccinated animals showed T. erinacei skin lesions. The use of different laboratory diagnostic methods was essential to reach a correct fungal identification. Furthermore, it was possible to confirm that this is the first Italian isolation of T. erinacei from calves.

KEY WORDS Trichophyton erinacei; ringworm; calves; outbreak.

INTRODUCTION T. erinacei is a zoonotic dermatophyte mainly isolated from the hedgehogs, which are often in a role of asymptomatic carriers1; in other hosts (human or animals) it can cause skin infections, with typical clinical features of dermatophytosis2. Although it has commonly been considered belonging to the T. mentagrophytes complex, recent studies allow classification as a different species3. This report describes two outbreaks of dermatophytosis due to T. erinacei occurred in two Italian cattle farms.

MATERIALS AND METHODS The outbreaks involved a group of 14 Bruna Alpina calves (farm A) and a group of 40 Italian Frisona cows (farm B). Animals of farm A were represented by 5-month old calves, bred in a mountain farm in North-Western Italy and composed of about 60 animals. These calves were housed in outdoor cages. Animals of farm B were about 24 months old and

Corresponding Author: Francesco Agnetti (f.agnetti@izsum.it).

were bred in a farm composed of 200 specimens, located in North-Eastern Italy. Cows of this farm were housed in typical external fences with sheds for night shelters. In both two cases, multifocal circular, crusted and mild thickened areas of alopecia, highly pruritic, 1-4 cm in diameter were noticed which appeared about 4 months before the clinical visit. The lesions were widely distributed to the head, trunk, and flanks (Figure 1, 2). Herd A, according to the breeder, was normally subjected to a regular vaccination protocol against T. verrucosum, but the calves involved in the outbreak had not yet been vaccinated. On the contrary, herd B was never vaccinated for T. verrucosum. The latter case also involved a veterinarian, who developed two inflamed and ellipsoidal skin lesions on the abdomen, compatible with dermatophytosis, after about two weeks the onset of the disease in animals (Figure 3). Hair and crusts samples from animals of farm A and B and from the vet involved were collected for mycological investigations, through cultural and molecular methods. The sampling areas were previously decontaminated with alcohol 70% and sterile pliers were used for the collection. Samples were inoculated into Dermasel Agar and into Sabouraud with thiamin and inositol plates (specific for the growth of T. verrucosum), then incubated at 25°±1°C for 3-4 weeks and daily observed. At the same time, samples were investigated

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Figure 1 - Skin lesions in a calf of the farm A: multifocal circular, crusting and mild thickened areas of alopecia, distributed to the head, trunk, and flanks. Figure 3 - Abdomen skin lesions of the veterinarian involved in the farm B outbreak.

Figure 2 - Skin lesions in a cow of the farm B, similar in appearance to those of the animal in Figure 1, but more distributed to the trunk and flanks.

through molecular techniques (PCR and sequence analysis), following the protocol of Cafarchia et al.4. To this purpose, after an initial step of lysis with lysozyme, DNA was extracted using the QIAamp DNA mini kit® (Qiagen), according to manufacturer’s instructions. DNA was then subjected to heminested-PCR. The primers used were DMTF18SF1 (5’CCAGGGAGGTTGGAAACGACCG-3’) and DMTF28SR1 (5’-CTACAAATTACAACTCGGACCC-3’) for the one-step PCR and DMTF18SF1 and DMTFITS1R (5’-CCGGAACCAAGAGATCCGTTGTTG-3’) for the second step. They we-

re designed to the region ITS+ of nuclear ribosomal DNA. PCRs and the following DNA amplifications (first and second step) were performed according to Cafarchia et al.4. PCR products were analysed by 2% agarose gel electrophoresis with Midori Green Advance DNA stain® (NIPPON Genetics) and purified using the QIAquick® PCR Purification Kit (Qiagen), following the manufacturer’s instructions. The identity of dermatophites were revealed by sequence analysis with primers DMTF18SF1 and DMTFITS1R, using BigDye® Teminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Consensus sequences were created by BioEdit Sequence Alignment Editor software v 7.0.9.0 and then aligned against Genbank database. From all the samples examined, growth of white, flat, powdery, with lemon-yellow reverse colonies has been observed on both culture media, within 7-10 days (Figure 4A). At microscopy, smooth-walled, two to six-celled, clavate, variable in size macroconidia and hyaline, smooth-walled, and predominantly spherical in shape microconidia were observed. (Figure 4B). Sequence analysis showed a degree of similarity of 99% with T. erinacei, with an e-value: 0.0 (accession number MF153407.1). Therefore, the final fungal identification was found to be T. erinacei. A spontaneous regression of the disease in both herds started approximately one month after the clinical visit. The human lesions began to regress after about two weeks of treatment with azole compounds.

DISCUSSION Dermatophytosis by T. erinacei is mainly related to contact with quills and underbelly of hedgehogs1. Although isolation of T. erinacei has been reported in other animals - hunting dogs, wild rodents and brown hares5,6,7, there is no prior evidence, as far as the authors are aware, of this fungal infection being detected in calves. Cattle are subject to skin fungal in-

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would be interesting to carry out further mycological investigations on hair samples from these species, in order to isolate any strain of T. erinacei. Other important factors which could be considered in the transmission of T. erinacei into the investigated farms could be breeding management errors such as the lack of sanitation in routine procedures or factors related to the immunity status of the animals. Generally, the use of preventive measures i.e. vaccination, plays an important role in the prevalence of the T. verrucosum infection in bovine. On the contrary, no specific immunity derived from natural exposure to T. erinacei was demonstrated, like in other animal species12. From the laboratory perspective, the use of different diagnostic methods was essential: molecular investigations allowed the correct fungal identification surpassing the culture method, proving to be a useful adjunct tool in the mycological diagnostics, thus enhancing the epidemiological knowledge of the organism.

CONCLUSIONS A

Figure 4 - Macroscopical (A) and microscopical (B) features of T. erinacei (light microscope, 40X).

fections from T. verrucosum, the main ringworm agent in ruminants, also described in other species8,9. Therefore, it is possible to say that the outbreaks described here represent the first T. erinacei infection in calves in Italy. In keeping with other animal fungal diseases, the potential for transmission to humans should not be underestimated. Breeders, veterinarians and/or livestock workers are the occupational categories most at risk of contracting the infection and of disseminating it to other people not directly in contact with animals. This potential was recognized when the veterinarian demonstrated abdominal skin lesions compatible with dermatophytosis approximately 2 weeks after the appearance of the disease in the animals. Laboratory examinations confirmed the role of T. erinacei as a cause of the human disease, in accordance with similar studies conducted in other countries10,11. In the outbreaks described, it was not possible to establish for certain a direct transmission of the fungus from hedgehogs to cattle; although the farms are located near to wooded areas, according to the breeders, there should be no hedgehogs or, at least, not close to the fences. However, once can hypothesize the presence of other animals that play the role of intermediate hosts between the hedgehogs and the calves, developing the mycotic disease and then remaining potential spore carriers. These alternative hosts to the hedgehog could play the simple role of asymptomatic spore carriers with no disease effect. Wild ungulates, red foxes, wild boars or rodents could be animals acting as a suitable reservoir of the fungus in the areas where the farms are located. Therefore, it

In conclusion, the described outbreaks have demonstrated the disease potential of the dermatophyte T. erinacei in animal species other than hedgehog and allow the authors to state that they are the first cases of cattle dermatophytosis by T. erinacei in Italy.

References 1. Heatley J.J. (2009). Hedgehogs. In: Manual of Exotic Pet Practice, St. Louis: Saunders Elseviers, 433-455. 2. Schauder S., Kirsch-Nietzki M., Wegener S. et al. (2007). From hedgehogs to men. Zoophilic dermatophytosis caused by Trichophyton erinacei in eight patients. Hautarzt, 58:62-67. 3. Monod M., Fratti M., Mignon B. et al. (2014). Dermathophytes transumi par les animaux domestiques. Rev Med Swiss, 10:749-753. 4. Cafarchia C., Gasser R.B., Figueredo L.A., Weigl S., Danesi P., Capelli G., Otranto D. (2013). An improved molecular diagnostic assay for canine and feline dermatophytosis. Med Mycol, 51(2):136-143. 5. Bond R. (2010). Superficial veterinary mycoses. Clin Dermatol, 28:226236. 6. Otčenášek M., Hubálek Z., Sixl W. (1980). Survey of dermatophytes in the hair of small mammals from Austria. Folia Parasit, 27:83-87. 7. Nardoni S., Papini R., Gallo M.G. et al. (2010). Survey on the role of brown hares (Lepus europaeus, Pallas 1778) as carriers of zoonotic dermatophytes. Ital J Anim Sci, 9:126-128. 8. Agnetti F., Righi C., Scoccia E. et al. (2014). Trichophyton verrucosum infection in cattle farms of Umbria (Central Italy) and transmission to humans. Mycoses, 57:400-405. 9. Mitra S.K., Sikdar A., Das P. (1998). Dermatophytes isolated from selected ruminants in India. Mycopathologia, 142:13-16. 10. Perrier P., Monod M. (2015). Tinea manuum caused by Trichophyton erinacei: first report in Switzerland. International Journal of Dermatology, 54:959-960. 11. Drira I., Neji S., Hadrich I. et al. (2015). Tinea manuum due to Trichophyton erinacei from Tunisia. Journal de Mycologie Médicale, 25:200203. 12. Chermette R., Ferreiro L., Guillot J. (2008). Dermatophytosis in animals. Mycopathol, 166:385-405.

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Palatoschisis, brachygnathia inferior and accessory mandibular mass in a Simmental breed calf

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UYGUR CANATAN, M. METİN ŞEN, ERENCAN ÖZFIRAT, MELİKE ÇETIN, HAKAN SALCI Bursa Uludag University, Faculty of Veterinary Medicine, Department of Surgery, 16059 Bursa - Turkey

SUMMARY Congenital anomalies in calves are encountered in different regions of the body. Generally, anomalies are seen in fronto-nasal, maxillar or mandibular regions. In addition several pathologies such as meningocele, cleft palate, nasal dermoid cyst, gingival vascular hamartoma are reported on the head. The aim of the present case report was to describe clinical and surgical result of palathoschisis, brachygnathia inferior and accessory mandibular mass in a Simmental calf. A 3-day-old male calf was referred with the complaint of milk discharge from the nose after suckling and an atypical structure in the mouth. At clinical examination, submandibular lymph nodes were swollen and physical examination results were normal. Oral examination revealed a cleft palate (palatoschisis), short lower jaw (brachygnathia inferior) and a pedunculated accessory mandibular mass midway between palatum durum and molle. However, the calf presented a good suction reflex. The accessory mandible was connected to the cleft palate and contained tooth-like structures. At palpation, the end portion of this mass was hard while its peduncle was soft in consistency. Radiographic exams on latero-lateral and ventro-dorsal projections of the cranium were carried out. The patient was subjected to surgical treatment; it was sedated with xylazine chloridrate (0.1 mg/kg im) and general anaesthesia was induced with ketamine chloridrate (4 mg/kg im). After tracheal intubation, general anesthesia was maintained with 2% isoflurane plus oxygen. The accessory mandible was removed by dissecting the surrounding tissues. Congenital cleft was closed through application of n° 0 suture silk with simple interrupted stitches. On the following day, the calf was discharged from the clinic with a postoperative recommended therapy including antibiotics (penicillin-streptomycin, 1×1.5 cc, im, for 5 days), analgesic (flunixin meglumin, 1.1 mg/kg, iv, for 3 days) and oral antiseptic (10% glycerin iode) drugs. According to the information obtained from the owner on the 15th postoperative day, calf ’s appetite was normal and chewing movements were reported to be relatively good. As a result, multiple anomaly including palatoschisis, brachygnathia inferior and accessory mandible in a calf may occured in clinical practice and as in this case, the observed pathology was corrected by surgical intervention, and postoperative results were favorable.

KEY WORDS Accessory mandible, brachygnathia inferior, calf, palatoschisis, surgical repair.

CASE HISTORY Different types of congenital anomalies are observed in farm animals. Congenital anomalies in calves are encountered between 0.2-3.6%1,2. In general, the causes of the anomalies are reported as genetic and environmental factors3,4. Particularly, at the beginning of organogenesis, maternal protein and vitamin deficiencies, infectious diseases, radiation, folic acid deficiency, teratogens such as lupine and endocrine disorders lead to anomaly formation1,3,5. Among congenital anomalies, fronto-nasal, maxillar and mandibular defects are observed in the facial region. The cleft palate (palatoschisis) may be of different size in the palatum durum and palatum molle of calves. Depending on the size and location of the cleft, nasal turbinates may be seen from the congenital cleft and nasal regurgitation is the most typical finding in these cases. Aspiration pneumonia is en-

Corresponding Author: Hakan Salci (hsalci@uludag.edu.tr).

countered due to milk aspiration6. Diagnosis is made on the basis of anamnesis and oral examination4. Brachygnathia inferior is defined as the mandible being shorter than the maxilla. This anomaly is a cranio-facial defect caused by homozygous recessive genes with incomplete penetration and various degrees of expression. It is mostly observed in horses, sheep, goats and cows 7. Congenital masses are rarely observed in cattle that these masses may have different appearance and shape1. Although many pathologies are seen on the cranial region8,9, meningocele is the most common congenital cranial anomaly in calves10. In this case report, it was aimed to report palatoschisis, brachygnathia inferior and accessory mandibular mass observed in a Simmental calf. A 3-day-old, 52 kg body weight, male Simmental breed calf was referred to Bursa Uludag University Faculty of Veterinary Medicine, Department of Surgery Clinics with the complaint of a swelling in the month. In the anamnesis, the owner informed that the calf was born normally and he had an abnormal mass in the mouth of calf. In addition, while the calf was feeding, there was difficulty swallowing and milk was nasally discharging

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Palatoschisis, brachygnathia inferior and accessory mandibular mass in a Simmental breed calf Table 1 - Hematological results of the case. Parameters

Values

Reference

WBC (109/L)

9.19

4-12

LYM (109/L)

6.42

2.5-7.5

MON (109/L)

0.22

0-0.84

NEU (109/L)

2.25

0.6-6.7

EOS (109/L)

BAS (109/L)

RBC (10 /L)

7.56

5-10

HGB (g/dl)

10.9

8-15

HCT (%)

33.20

24-46

MCV (fl)

40-60

MCH (pg)

11.5

11-17

MCHC (g/dl)

32.8

30-36

PLT (10 /L)

278

100-800

PCT (%)

0.13

WBC: White blood cell LYM: Lymphocytes, MON: Monocytes, NEU: Neutrophil, EOS: Eosinophil, BAS: Basophil, RBC: Red blood cell, HGB: Hemoglobin, HCT: Hematocrit, MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration, PLT: Platelet, PCT: Plateletcrit.

Figure 1 - Clinical appearance of the case, star: accessory mandibular mass, arrow: palatoschisis (cleft palate).

after sucking. The referred veterinarian had administered some drugs to support the calf healthy status, and then the calf was brought to our clinics. At physical examination, vital parameters (pulsation: 108/min, respiration: 24/min, body temperature: 37.6, capillary refilling time: 1 sec, mucous membrane color: normal) were normal but submandibular lymph nodes were swollen. Inspection revealed a cleft that begins at the middle of the hard palate and separated the soft palate longitudinally, and a formation covered with tooth-like structures on the left side of the cleft, with a mandibular appearance associated with soft tissue (Figure 1). During occlusion, the mandible was shorter than the maxilla (brachygnathia inferior). At palpation, it was determined that the end part of this tissue was hard and the bottom part

had soft consistency. In hematological examination, the results were in the normal reference values (Table 1). Radiologically, lateral and ventrodorsal radiographs of the cranium showed multiple radiopaque tooth-like structure on the mass and anomalous portion of the mandible relative to the maxilla (Figure 2). Apart from oral pathologies, no other pathology was found clinically and radiologically in the calf. After informing the owner about the pathology, a surgical intervention was decided to be performed. Calf was sedated with xylazine chlorhydrate (0.1 mg/kg, im) (AlfazyneÂŽ, Egevet, TURKEY) and ketamine chlorhydrate (4 mg/kg, im) (AlfamineÂŽ, Egevet, TURKEY) was administered for induction. After tracheal intubation, general anesthesia was maintained with 2% isoflurane plus oxygen. Oral examination was repeated under general anesthesia and the size of the cleft and the position of the mandibular mass were determined. It was observed that the mass was attached to the left side of the soft palate and the cleft on the hard palate was also extending

Figure 2 - In the lateral (A) and ventrodorsal (B) radiographs of cranium, brachygnathia inferior (arrow) and accessory mandibular mass (dashed line).

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U. Canatan et al. Large Animal Review 2020; 26: 145-148

Figure 3 - Postoperative image of calf, PD: palatum durum.

to the soft palate. After the calf was placed ventrodorsally on the operation table, antisepsis was provided inside the mouth and the area was limited by means of sterile covers. The accessory mass was excised from the region where it was attached to the cleft on the palate, and was dissected from the surrounding tissues. During the removal of the mass from the area where it was attached to the soft palate, some mucosal tissue on the mass was separated and this tissue was used as a flap to close the cleft in the palate with nÂ° 0 absorbable suture material (Figure 3). At day 1 postoperative, 5% dextrose and 0.9% isotonic NaCl solutions were given 2 times a day intravenously, and the calf was fed 2 times a day with 2 liters of milk with a stomach catheter. Postoperative penicillin-streptomycin (1.5 ml, im, one a day) (Vetimisin, Vetas, TURKEY), flunixin meglumin (1.1 mg/kg, iv) (Fulimed, AlkeÂŽ, TURKEY) and oral antiseptic (10% glycerin iode, Mervet, Turkey) treatments were administered. These medications and oral feeding with stomach catheter for 5 days were recommended to the calf owner, and then the calf was discharged one day later. According to the information obtained from the owner on the 15th postoperative day, the calf was able to feed and drink water normally and chewing movements were reported to be relatively good.

DISCUSSION Congenital anomalies arise from developmental problems in the embryonal period5. In recent years, up to 300 of congenital anomalies have been reported in worldwide literatures11. Herein, a case with different anomalies such as palatoschisis,

147

brachygnathia inferior and mandibular anomaly was detected and reported in a Simmental calf. The specific cause of most of the anomalies observed in animals is not clear. Anomalies include many pathological conditions from single organ disorders to multiple organ failure1. The facial clefts are due to lack of fusion3,5,12 and are usually seen in the lip, chin and palate12. The palate and cleft lip are usually associated with other disorders such as arthrogryposis, congenital joint contracture, muscular dystrophy3. Palatoschisis occurred in Sharole and Hereford cows with arthrogryposis4. Palatoschisis and brachygnathia inferior are two different developmental diseases observed in calves13. As a result of clinical and radiological examinations, no pathology other than the head region was observed. In our case, the reason of the pathologies we encountered in the head region is not known exactly, but when the health status of the region where the animals live is evaluated, it is thought to be genetically related to the lack of fusion in the maxilla. Palatum molle is an anatomical structure which plays a vital role in oral and pharyngeal phase of swallowing, preventing leakage with tongue and preventing aspiration of liquid and solid foods. It provides a mechanical and physical barrier between oral and nasal cavity4. In palatoschisis, the clinical findings are observed immediately after birth and during the suckling. Bilateral nasal discharge, cough and dysphagia are characteristic findings. In addition, passive immune transfer failure, aspiration pneumonia, growth retardation, chronic bilateral nasal discharge and food regurgitation are seen in patient with palatoschisis. Sometimes nasal turbinates are also observed in the oral cavity during inspection4,6. Palatoschisis is diagnosed by oral examination and it is possible to monitor the patency by means of endoscopy4. Our case was presented to our clinic with good general condition with bilateral nasal discharge and dysphagia. Surgical repair of the defect is performed4. There are laryngotomy, mandibular symphysiotomy, pharyngotomy and intraoral surgical approach techniques to close the cleft palate. The use of techniques varies according to the size of the cleft and mandibular symphysiotomy is said to be the best approach method in large clefts14. In addition, treatment should be directed considering the secondary pathologies of the patient such as malnutrition, aspiration pneumonia and passive immune transfer failure. There are few cases with positive results from treatment, but postoperative complications are possible4. Postoperative complication rate due to saliva is high in cases with cleft palate. These complications include dehiscence of the surgical wound, aspiration pneumonia, osteomyelitis and soft tissue infection14. Depending on the size of the cleft in the palate, the opening in the hard and soft palate can be surgically corrected3. In a case with multiple anomalies undergoing surgical intervention, the majority of the opening was closed and no complications were encountered, but the animal died 2 days later due to complications related to other anomalies11. Uncomplicated recovery was achieved by surgical intervention in another calf shaped Cheiloschisis15. Patients can survive for a long time after successful operative intervention2. Accessory mandible and palate cleft cases observed in humans are corrected by surgical repair16. In this case, the mandibular mass was removed with the operative intervention and the opening of the palate was repaired. Due to the small size of the cleft palate and the mass associated with the soft palate, it reduced the likelihood of complications in the postoperative period. It was reported by the owner that mouth movements, feed and water

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Palatoschisis, brachygnathia inferior and accessory mandibular mass in a Simmental breed calf

intake improved in 15 days postsurgery. Brachygnathia inferior may be associated with different anomalies such as polidactylism17 and hydrocephalus18 in calves. Dental problems, malnutrition, growth retardation and even death may occur due to the fact that chewing is abnormal. Generally, genetic selection should be done very carefully in flocks with such animals17. In our case, mouth movements were found to be better than preoperative, and calf growth was reported to be normal.

10.

11.

12.

References 1. Hobbenaghi R., Dalir-Naghadeh B., Nazarizadeh A. (2015). Congenital multi-organ malformations in a Holstein calf. Vet Res Forum, 6(3): 257-260. 2. Usta Z., Distl O. (2017). Congenital cleft lip-jaw-palate and cleft palate in German Holstein Calves with Common Ancestry. Erciyes Üniv Vet Fak Derg, 14(1): 73-80. 3. Oryan A., Shirian S., Samadian M.R. (2011). Congenital craniofacial and skeletal defects with arthrogryposis in two newborn male Holstein Friesian calves. Comp Clin Pathol, 20: 43-46 4. Smolec O., Vnuk D., Kos J., Brkljaca Bottegaro N., Pırkıc B. (2010). Repair of cleft palate in a calf using polypropylene mesh and palatal mucosal flap: a case report. Vet Med (Praha), 55, 2010 (11): 566-570 5. Herath U.T.K., Horadagoda N.U., De Silva L.N.A., De Silva D.D.N. (2002). Palatoschisis (cleft palate) and associated facial anomalies in a fresian calf. S.L. Vet J, 49(1A): 9-14. 6. Blowey R., Weaver A.D. (2011). Color Atlas of Diseases and Disorders of Cattle, 3rd ed., 1-11, Mosby Elsevier. 7. Shatab M.S., Kumar N., Singh M., Kumar S., Sekhon M.S., Dhindsa S.S. (2018). Brachygnathia (parrot mouth) inferior calf in a murrah buffalo. Buffalo Bull, 37(1): 101-103. 8. Hobbenaghi R., Dalir-Naghadeh B., Nazarizadeh A. (2015). Coinci-

13.

14.

15.

16.

17.

18.

dence of congenital infiltrative facial lipoma and lingual myxoma in a newborn Holstein calf. Iran J Vet Res, 16(3): 306-309. Yayla S., Beytut E., Kılıç E., Cihan M., Aydın U., Karakurt E. (2016). Congenital gingival vascular hamartoma in a calf. Harran Üniv Vet Fak Derg, 5(1): 66-69. Soundararajan C., Chockalingam P., Nagarajan K., Ananda Raja R., Arulprakash M. (2018). Cranial meningocoele in a crossbred calf. Dairy and Vet Sci J, 6(3): 1-2. Abdelhakiem M.A.H., Elrashidy M.H. (2017). A retrospectıve study of the congenital anomalies of the axial and appendicular skeleton in cow calves. Assiut Vet Med J, 63(153): 88-99. Moritomo Y., Tsuda T., Miyamoto H. (1999). Craniofacial Skeletal Abnormalities in Anomalous Calves with Clefts of the Face. J Vet Med Sci, 61(10): 1147-1152. Griffith J.W., Hobbs B.A., Manders E.K. (1987). Cleft palate, brachygnathia inferior and mandibular oligodontia in a holstein calf. J Comp Path, 97: 95-99. Minter L.J., Karlin W.M., Hickey M.J., Byron C.R. (2010) Surgical repair of a cleft palate in an American Bison (Bison Bison). Journal of Zoo and Wildlife Medicine, 41(3): 562-566. Gangwar A.K., Devi K.S., Singh A.K., Katiyar N., Patel G., Srivastava S. (2014) Congenital anomalies and their surgical correction in ruminants. Advances in Animal and Veterinary Science, 2(7): 369-376. Borzabadi-Farahani A, Gross J, Sanchez-Lara PA, Yen S.L.K. (2013). An unusual accessory mandible and a submucosal cleft palate-A case report and review of the literature. The Cleft Palate Craniofac J, 50(3): 369-375. Manokaran S., Palanisamy M., Prakash S., Selvaraju M., Ezakial Napolean R. (2018). Unilateral polydactylism with brachygnathism in a calf. Indian Vet J, 95(05): 69-70. Sunil T.P., Vikram R., Chaudhary G.R., Nayanakumara S.R., Kumar A., Balamurugan B., Kharayat N.S., Suthar A., Sanjukumar B.S., Sagar M.A. (2016). Dystocia in cattle due to hydrocephalıc fetus concurrent with brachygnathism and ankylosed limbs: a case report. Int J Sci Environ Technol, 5(3): 1275-1278.

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C. Masotti et al. Large Animal Review 2020; 26: 149-151

Ricerca del virus dell’epatite E (HEV) in cinghiali durante la stagione venatoria 2017/2018 e 2018/2019

149

CHIARA MASOTTI1, ROBERTA BATTISTINI1, WALTER MIGNONE2, ENRICA BERIO2, MONICA DELLEPIANE3, TIZIANA ANDREOLI3, ELISABETTA RAZZUOLI4, SIMONE PELETTO5, PIERLUIGI ACUTIS5, CHIARA BELTRAMO5, PAOLA MODESTO4, VALERIA LISTORTI1, CARLO ERCOLINI1, LAURA SERRACCA1 1

Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Sezione La Spezia - La Spezia - Italy, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Sezione Imperia - Imperia - Italy, 3 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Sezione Savona - Savona - Italy, 4 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Sezione Genova - Genova - Italy, 5 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta - Torino - Italy 2

SUMMARY An increasing number of Hepatitis E virus (HEV) cases are currently recognized in many European countries. HEV is an emerging disease in industrialized countries. HEV is an important cause of human acute viral hepatitis worldwide, where the infection is acquired probably through ingestion of contaminated food, in addition to travel-related cases. In Europe, outbreaks have been reported linked to the consumption of pork liver sausages and wild boar meat. It has been shown that wildlife can serve as natural reservoirs of HEV. In Europe, especially in region where game meat is widely consumed, wild boars (Sus scrofa) are considered as the main wildlife reservoir of zoonotic Hepatitis E virus genotypes. HEV is classified into eight genotypes. Genotypes 3 and 4 are considered zoonotic and are responsible for sporadic cases of food-borne disease due to the consumption of contaminated raw or undercooked meat. Genotypes 3 mainly circulates in domestic pigs and wild boars, which are the main sources of infection for humans. To assess the potential risk of zoonotic transmission of HEV from wild boars, an epidemiological survey has been conducted during the 2017-2018 e 2018-2019 hunting seasons in the Liguria region. Liver samples of 560 wild boars were analyzed for HEV RNA by realtime RT-PCR; positive samples were then sequenced and submitted to phylogenetic analysis. The results of the survey revealed the presence of HEV in 4.4% (25/560) of the examined wild boar’s livers. Phylogenetic analysis classified the isolates as HEV genotype 3, highlighting the risk related to the consumption of wild boar-derived products for humans. HEV sequences belonged to HEV subtypes 3a, 3chi, 3c, and 3f, and 3* (unclassified). Our results indicate that HEV is circulating in wild boars among the considered game species in north-western Italy and suggest a potential zoonotic risk related to handling and/or consumption of raw or undercooked meat and products made of the liver from this species.

KEY WORDS Hepatitis E Virus, wild boar, foodborne transmission, prevalence.

INTRODUZIONE Il virus dell’epatite E (HEV) è un Orthoherpevirus A appartenente alla famiglia Hepeviridae, di piccole dimensioni (27-34 nm) a simmetria icosaedrica, privo di envelope. Ha un genoma a singolo filamento di RNA di circa 7,2 Kb. Il genoma è costituito da una regione non tradotta 5’ (UTR) seguita da tre regioni codificanti (ORF 1, 2, 3) e una seconda regione non tradotta poliadenilata all’estremità 3’1. I ceppi che infettano l’uomo appartengono alla specie Orthoherpevirus A e sono stati identificati otto genotipi, di cui HEV-1, HEV-2, HEV-3 e HEV-4 sono principalmente causa di patologia nell’uomo2. HEV è, infatti, responsabile di una forma autolimitante di epatite acuta infettiva che di solito si risolve in 2-6 settimane. I genotipi 1 e 2 hanno come ospite unicamente l’uomo e sono associati ad infezioni ed epidemie in Asia, Africa e Messico. I genotipi 3 e 4 sono zoonotici, in quanto colpiscono sia gli animali (suini, cinghiali, cervi) che l’uomo. Il genotipo 3 circola principalmente in Europa, Stati Uniti e Giappone, mentre il genotipo 4 è stato identificato in Asia (Cina, GiapCorresponding Author: Chiara Masotti (chiara.masotti@izsto.it).

pone e India) e più raramente in Europa3. Il genoma di HEV è stato, inoltre, riscontrato in diversi tessuti e organi di suini, cervi e cinghiali. Nell’ultimo decennio, l’epatite E è diventata una delle principali priorità nel campo della ricerca dei virus di origine alimentare, per il fatto che le sono stati attribuiti più di 3 milioni di casi clinici rilevati ogni anno e l’infezione di circa 20 milioni di persone4. La malattia è considerata endemica nei Paesi in via di sviluppo, dove si manifesta con episodi epidemici generalmente associati al consumo di acqua contaminata, mentre nei paesi industrializzati i casi di epatite E nell’uomo erano considerati sporadici e legati a viaggi in Paesi dove la malattia è endemica1. HEV rappresenta una minaccia emergente nei Paesi industrializzati, poiché indagini epidemiologiche hanno dimostrato un incremento della prevalenza e dell’incidenza di HEV sia nell’uomo che negli animali e hanno identificato suini e cinghiali come possibile fonte di infezione umana attraverso il consumo di carne cruda o poco cotta, specialmente nelle regioni in cui la carne di selvaggina è ampiamente consumata. L’esclusione della carne e degli organi degli animali infetti per la produzione di cibo durante l’esame ispettivo delle carni è ostacolata dal fatto che suini domestici e cinghiali, nonostante siano altamente suscettibili all’infezione da HEV, non

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sviluppano segni di epatite infettiva5,6. Tra le specie selvatiche, il cinghiale è considerato il principale reservoir dell’HEV e svolge un importante ruolo nella trasmissione del virus in Europa. È stato dimostrato sperimentalmente, infatti, che i cinghiali infetti da HEV possono trasmettere l’infezione ad altri animali, come ai suini7,8. Questo gioca un ruolo importante, specialmente nei Paesi in cui l’allevamento estensivo di suini è diffuso, perché è facilitato il contatto tra suini domestici e specie simpatriche, aumentando così il rischio di trasmissioni inter-specie9. La conoscenza dello stato sanitario della fauna selvatica è quindi indispensabile, oltre che per minimizzare l’effetto diretto sulla salute umana derivante dal consumo di selvaggina cacciata, anche per studiare la diffusione sul territorio di patologie a carattere zoonosico. Nel presente lavoro sono stati riportati i dati relativi alla presenza e alla caratterizzazione di HEV nelle popolazioni di cinghiali presenti sul territorio ligure durante la stagione venatoria 2017/2018 e 2018/2019, grazie ai piani regionali presenti per il controllo sanitario della fauna selvatica.

MATERIALI E METODI Durante l’attività venatoria 2017/2018 e 2018/2019 sono stati raccolti 280 campioni di fegato da cinghiali provenienti in pari numero dalle quattro province della Regione Liguria (Imperia, Savona, Genova e La Spezia) per stagione venatoria, per un totale di 560 campioni complessivi durante le due stagioni di caccia citate. I fegati sono stati analizzati individualmente: 25 mg di tessuto sono stati posti in 300 microlitri di tampone fosfato salino e successivamente omogenati mediante tissuelyser10 ed analizzati con tecniche di biologia molecolare. L’estrazione dell’RNA è stata effettuata mediante il kit QIAamp Cador Pathogen Mini Kit (QIAGEN) seguendo il protocollo della ditta di produzione. L’RNA ottenuto è stato analizzato con una metodica di one-step real-time RT-PCR11. I campioni risultati positivi alla real-time RT-PCR sono stati poi sottoposti a sequenziamento ed analisi filogenetica dopo riamplificazione con una RT-nestedPCR, utilizzando due set di primer degenerati per la regione 5’ della ORF212. L’albero filogenetico è stato costruito da un dataset di sequenze parziali (135 bp) della regione 5’ ORF2 dei campioni risultati positivi all’HEV e di sequenze disponibili in GenBank, utilizzando il software MEGA7, mediante il metodo Neighbor-Joining (NJ) basato sul modello Kimura-2 parametri. La robustezza dell’inferenza filogenetica è stata testata utilizzando l’analisi di bootstrap con 1.000 pseudorepliche2.

RISULTATI L’RNA di HEV è stato rilevato nel 4,4% (25/560) dei fegati di cinghiale testati e rispettivamente nel 4,6% (13/280) durante il 20172018 e 4,2% (12/280) nel 2018-2019. I dati da noi ottenuti dall’analisi effettuata sulle due stagioni di caccia evidenziano positività per HEV in tutte le province liguri con le seguenti prevalenze: 7,8% (11/140) Imperia; 2,9% (4/140) Savona; 2,1% (3/140) Genova; 5% (7/140) La Spezia. Le tabelle 1 e 2 riportano questi dati suddivisi anno per anno. Il sequenziamento genico e l’analisi filogenetica (Fig. I) hanno permesso di identificare i campioni positivi come appartenenti al genotipo 3, sottotipi 3a (n=1), 3c (n=2), 3f (n=2), 3* (non classificati; n=8) durante la stagione venatoria 2017-2018, e sottotipi 3a (n=6), 3f (n=3), 3* (non classificati; n=3) durante la stagione del 2018-2019.

Figure 1 - Albero filogenetico della sequenza parziale della regione 5’ ORF2 di HEV. I campioni dello studio sono indicati (♦ stagione venatoria 2017/2018; ● stagione venatoria 2018/2019). Sequenze di altri isolati italiani da uomo, suino e cinghiale sono indicati dal simbolo ▲.

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Ricerca del virus dell’epatite E (HEV) in cinghiali durante la stagione venatoria 2017/2018 e 2018/2019

Tabella 1 - Prevalenza dell’epatite E suddivisa per provincie della regione liguria e stagione venatoria 2017-2018. Provincia

N° animali testati

N° animali positivi

Prevalenza (%)

Savona (SV)

2,9

Imperia (IM)

10,0

La Spezia (SP)

4,3

Genova (GE)

1,4

Totale

280

4,6

Tabella 2 - Prevalenza dell’epatite E suddivisa per provincie della regione liguria e stagione venatoria 2018-2019. Provincia

N° animali testati

N° animali positivi

Prevalenza (%)

Savona (SV)

2,9

Imperia (IM)

5,7

La Spezia (SP)

5,7

Genova (GE)

2,9

Totale

280

4,3

DISCUSSIONE E CONCLUSIONI I risultati ottenuti mostrano dati di prevalenza analoghi a quelli di uno studio effettuato su cinghiali in Piemonte durante la stagione venatoria 2012-2013 dove la prevalenza si attesta intorno al 4,9%13; in altre regioni italiane studi condotti durante la stagione venatoria 2014-2015 hanno mostrato una prevalenza del 33,5% nel Lazio14, 10,1% in Abruzzo e 12,4% in Campania, mentre in Calabria nessuno degli animali testati è risultato positivo per la presenza di RNA di HEV9. Le prevalenze riportate in altri paesi europei variano dal 10,1% in Spagna15 al 18% in Romania16. Tali differenze sono probabilmente da imputare alle diverse condizioni epidemiologiche nelle diverse zone (densità, dimensioni e composizione delle popolazioni considerate e contatti con suini domestici)12. Durante le stagioni venatorie precedenti sono stati riportati diversi studi sulla prevalenza di HEV effettuati nella popolazione di cinghiali nel territorio ligure. Nel periodo 2012-2014 la prevalenza dell’RNA di HEV trovata nella provincia di Imperia è del 2,3%17. Nella stagione 2013-2015 l’RNA di HEV è stato rilevato nel 3,9% (17/430) dei pool di fegati di cinghiali testati18. Il nostro studio, in linea con i dati riscontrati in precedenti analisi condotte, nel territorio ligure, evidenzia un’attiva circolazione di HEV nelle popolazioni di cinghiali liguri esaminate. L’analisi filogenetica ha dimostrato che i ceppi virali rilevati sono classificabili come HEV genotipo 3, evidenziando inoltre che le sequenze liguri appartenenti ai sottotipi HEV 3c e 3f formano dei cluster con sequenze identificate da cinghiali in Lazio19. È importante infine sottolineare che il genotipo 3, riscontrato nei campioni, è considerato zoonotico e quindi può rappresentare un rischio di infezione per le categorie professionali esposte e per il consumo di carni crude (insaccati) o poco cotte e di prodotti a base di fegato, dal momento che tale genotipo è quello maggiorente identificato nei paesi industrializzati e comprende ceppi virali che colpiscono sia l’uomo che gli animali.

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