Bacteriological culture and direct PCR for detecting the Mycobacterium tuberculosis complex in the Italian eradication campaign: a decade of experience at the National Reference Laboratory.

AIMS: Our study evaluates the capacity of direct real-time PCR for detecting Mycobacterium tuberculosis complex (MTBC), with a focus on diagnostic performances and the feasibility of implementing this protocol in an eradication campaign. Specifically, we compare the effectiveness of the direct PCR method to various culture systems used by the Italian National Reference Laboratory over the last decade to detect MTBC. METHODS AND RESULTS: Bovine tissue samples were routinely tested and analyzed for bovine tuberculosis (bTB) confirmation using microbiological culture (solid and liquid media), histopathological analysis, and a direct PCR assay targeting IS6110, an insertion sequence specific to the MTBC that is widely used for tuberculosis diagnosis. The direct real-time PCR demonstrated a high concordance (K = 0.871) with microbiological culture, as well as good sensitivity (91.84%) and specificity (95.24%). In contrast, histopathology demonstrated lower concordance (K = 0.746) and performance levels (sensitivity 91.41%, specificity 82.88%). Liquid media promoted faster and more efficient growth of MTBC than solid media. M. bovis and M. caprae had the comparable ability to respond to the direct real-time PCR test and grow on the microbiological medium. CONCLUSIONS: This study confirms that direct real-time PCR can detect MTBC with high diagnostic accuracy within a few days. This study found no significant differences in performance between culture media and direct PCR for M. bovis and M. caprae.

Genomics insights into a Mycobacterium pinnipedii isolate causing tuberculosis in a captive South American sea lion (Otaria flavescens) from Italy.

Tuberculosis (TB) affects humans and other animals, and it is caused by bacteria within the Mycobacterium tuberculosis complex (MTBC). In this study, we report the characterisation of Mycobacterium pinnipedii that caused a TB case in a sea lion (Otaria flavescens) kept in an Italian zoo. The animal died due to severe, progressive disorders involving the respiratory and gastro-enteric systems and the skin. At necropsy, typical gross lesions referable to a TB generalised form were found. In particular, nodular granulomatous lesions were detected in the lungs and several lymph nodes, and colonies referable to Mycobacterium spp. were isolated from lung, mesenteric, and mediastinal lymph nodes. The isolate was identified by PCR as a MTBC, had a spoligotype SB 1480 ("seal lineage"), and was characterised and characterised by whole-genome sequencing analysis confirming that the MTBC involved was M. pinnipedii. The analysis of the resistome and virulome indicated the presence of macrolide and aminoglycoside resistance genes intrinsic in M. tuberculosis [erm-37 and aac(2')-Ic] and confirmed the presence of the region of difference 1 (RD1), harbouring the esxA and esxB virulence genes, differently from its closest taxon, M. microti. As for other MTCB members, M. pinnipedii infection can spill over into non-pinniped mammalian species; therefore, zoological gardens, veterinary practitioners, and public health officers should be aware of the hazard posed by tuberculosis from marine mammals. Since the isolate under study, as well as all available genomes of M. pinnipedii investigated in this study retains almost all the M. tuberculosis virulence genes, it could indeed cause infection, lesions, and disease in other animal species, including humans.

Influence of Anthropic Environmental-Related Factors on Erysipelas in Wild Boar.

Erysipelothrix rhusiopathiae (ER) is an old but still emerging zoonotic infection that is not yet completely understood. ER infects a wide range of species and wild boar is of significant interest because of their similarities to pigs, a known ER reservoir. Moreover, the increase of its densities and the limited data available about ER in this species should be considered. The need is to investigate whether wild boar could represent a risk of erysipelas at the wildlife-domestic-human interface. Here, 1067 sera and 149 tonsils of wild boar from five hunting districts in Northwest Italy were tested using ELISA and bacteriological culture, respectively. Using generalized linear models, we evaluated host and environmental factors influencing ER spread and dynamics. We found an ER seroprevalence of 69.4% among wild boar. Increased human density and pig farm density lead to an increase of ER seropositivity highlighting its association with anthropic environmental-related factors. The high ER percentage of isolation (34.2%) found in healthy wild boar suggests that this species can serve as a healthy carrier. This fact, together with the high seroprevalence, supports a role of wild boar as an ER reservoir. Potential zoonotic and economic risks should be considered in light of these data.

Evaluation of the Presence and Viability of Mycobacterium bovis in Wild Boar Meat and Meat-Based Preparations.

The aim of the present study is to provide information about the ability of Mycobacterium bovis to survive within wild boar (Sus scrofae) meat and meat-based preparations and the duration of this survival, and to consider the preservation of its infectious potential toward humans and animals. Meat samples were artificially contaminated with an M. bovis field strain and then stored at -20 °C, while two sausages batches were contaminated with the same field strain at two different concentrations, 105 CFU/g and 103 CFU/g, before storing them in proper conditions to allow for their ripening. A third sausage batch was contaminated by adding 2 g of wild boar lymph nodal tissue with active tuberculous lesions to the meat mixture. Bacteriological and biomolecular (PCR) methods were used to test the meat and sausage samples every 60 days and every 7-10 days, respectively. M. bovis was detected as still alive and viable on the frozen meat for the last test on the 342nd day, while from the sausage samples, M. bovis was isolated until 23 days after contamination. Our results indicate that M. bovis can stay alive and be viable for 23 days within sausages prepared with contaminated meat from infected wild boars. These products are usually eaten as fresh food after grilling, often cooking at a temperature that does not ensure complete inactivation of the pathogenic microorganisms present, which can pose a risk for humans to develop zoonotic tuberculosis.

Mycobacterium microti at the Environment and Wildlife Interface.

An unexpected high presence of Mycobacterium microti in wild boar in Northern Italy (Garda Lake) has been reported since 2003, but the factors contributing to the maintenance of this pathogen are still unclear. In this study, we investigated the presence of M. microti in wild rodents and in water and soil samples collected at wild boar aggregation areas, such as watering holes, with the aim of clarifying their role in M. microti transmission. In total, 8 out of 120 captured animals tested positive for the Mycobacterium tuberculosis complex (MTBC) as assessed by real-time PCR, and six samples were confirmed to be M. microti. A strain with a genetic profile similar to those previously isolated in wild boars in the same area was isolated from one sample. Of the 20 water and 19 mud samples, 3 and 1, respectively, tested positive for the presence of MTBC, and spacer oligotype SB0118 (vole type) was detected in one sample. Our study suggests that wild rodents, in particular Apodemus sylvaticus, Microtus sp. and Apodemus flavicollis, play roles in the maintenance of M. microti infections in wild boar through ingestion or by contact with either infected excreta or a contaminated environment, such as at animal aggregation sites.

Mycobacterium microti Infection in Wild Boar (Sus scrofa): Histopathology Analysis Suggests Containment of the Infection.

The European wild boar (WB) (Sus scrofa) population has rapidly expanded over the years, raising public health concerns over the species reservoir of several pathogens, including Mycobacterium microti (Mm), a Mycobacterium tuberculosis complex member. In this study, we aimed to investigate the Mm natural infection in WB in Lombardy and Emilia Romagna Italian regions by statistically evaluating the granulomatous lesions' histological features and Mm microbiological isolation. We analyzed 103 WB retropharyngeal and submandibular lymph nodes (LNs) for Mm identified by gyrB PCR-restriction fragment length polymorphism, and were retrospectively selected and histologically assessed. For each sample, Hematoxylin-eosin and Ziehl-Neelsen stained slides were evaluated. Considered histological variables were: the number of granulomas, size and maturational stage of granulomas, granulomas completeness within the section, number of multinucleated giant macrophages (MGMs), and acid-fast (AF) bacilli per granuloma. Furthermore, Mm microbiological results were also considered. Mm microbiological isolation was negatively influenced by granulomas maturation and positively affected by AF bacilli's presence within the section. Granuloma maturation was positively influenced by granuloma size and granuloma incompleteness and negatively affected by the number of granulomas in the section and the number of MGMs within the granuloma. The results indicate that granuloma maturation should ensures an efficient containment of Mm infection in the WB, suggesting that the intra-species transmission of the disease might be an unlikely event.

Infection by Mycobacterium caprae in three cattle herds in Emilia-Romagna Region, Northern Italy.

Bovine tuberculosis (bTB) is a contagious chronic disease associated with progressive emaciation (starvation) and tubercles (granuloma) formation commonly caused by Mycobacterium bovis. In cattle, M. caprae may also be responsible for bTB. In EU, human tuberculosis due to M. bovis had a notification rate of 0.04 cases per 100,000 inhabitants in 2017, but data did not include M. caprae infections. From September 2018 to April 2019, bTB outbreaks were investigated in three neighbouring dairy cattle herds in Parma province, Northern Italy. Parma municipality belongs to an officially free of bovine tuberculosis (OTF) Italian region. Official testing on cattle herds, performed every three years as legally required, revealed no positive animals. Tubercular lesions were found during the post mortem (PM) examination of slaughtered cattle and M. caprae genotype SB0418/VNTR 4,3,5,3,4,5,2,2,4, 3,15,5 was isolated. This report confirms the crucial importance of PM veterinary inspection at slaughterhouse, despite the OTF status of cattle herds.

Acute lameness in a cat with disseminated mycobacteriosis.

Mycobacterium avium infection was diagnosed in an adult cat showing acute lameness of the right hind limb, enlargement of the right popliteal lymph node and two cutaneous nodular lesions of the right chest wall. Conventional radiography of the proximal tibia showed a proliferative osteolytic lesion. Cytological examination of the right popliteal lymph node and the nodular skin lesions fine needle aspiration smears, demonstrated granulomatous inflammation with many negative staining bacilli within macrophages or in smears background. The diagnosis was confirmed by Ziehl­Neelsen staining of the smears and the identification of mycobacteria was performed by microbiological and molecular methods. Histopathology performed after the necropsy revealed disseminated mycobacteriosis with granulomatous mesenteric lymphadenitis, granulomatous pneumonia, hepatitis and tibial osteomyelitis. M. avium is a well­known agent of gastro­enteric, respiratory or disseminated disease in immunocompromised cats but there are few cases reported in literature of bone involvement in systemic mycobacteriosis.

Types of pesticides involved in domestic and wild animal poisoning in Italy.

Data obtained from samples of poisoned domestic and wild animals sent for toxicological evaluation during the period between 2005 and 2014 have been analyzed. Among the 4606 tested samples, the majority of which were collected in the northern regions of Italy, 2006 (43.55%) were found positive for pesticides. Analytical detections were performed via solvent extraction followed by separation and chromatographic characterization and all the methods applied for the toxicological investigations were developed by IZSLER. Insecticides, mainly represented by acetylcholinesterase inhibitors (carbamates 17.55%, n = 352; organophosphates 15.15%, n = 304) and organochlorines (29.21%, n = 586), were found to be the first category of pesticides involved in intoxications, in both domestic and in wild animals, followed by rodenticides (anticoagulant rodenticides 21.09%, n = 423; zinc phosphide 2.59%, n = 52; chloralose 0.95%, n = 19 and thallium 0.15%, n = 3) and molluscicides (metaldehyde 6.63%, n = 133). Second and third generation anticoagulants (bromadiolone and brodifacoum) were the most represented (10.52%, n = 211) but also first generation compounds (i.e. coumatetralyl and warfarin) were still found responsible of intoxications. Even if some pesticides are frequently involved in domestic animal poisoning (i.e. metaldehyde and strychnine), they did not show the same diffusion in wild animals. In particular, unlike domestic species, cyanide and pyrethroids have not been found responsible of intoxications in wild animals. Interestingly, a great number of positive samples involved banned pesticides like α- (14.41%, n = 289) and ß- (14.16%, n = 284) endosulfan, carbofuran (5.73%, n = 115), methamidophos (9.47%, n = 190), strychnine (6.23%, n = 125) but, on the other hand, many positives were due to the exposure to commercially available products (i.e. metaldehyde and anticoagulant rodenticides). Thus, together with measures aimed to reduce illegal uses, educational campaigns and a wider range of compounds to detect would be beneficial in order to address the issue of animal poisoning, which besides has also repercussions on environmental and public health.

Mycobacterium caprae in a dairy farm in Northeast Italy.

Veneto region, Northeast Italy, has been declared officially free from bovine tuberculosis since 2008, although the disease is sporadically detected in association with cattle trade. In September 2015, bovine tuberculosis was detected in a dairy cattle farm of the region, in a holding with 69 animals. The herd underwent single intradermal tuberculin testing as part of the regional surveillance plan, and 24 animals resulted positive. Mycobacterium caprae was evidenced in 22 samples, further genotyped by PCR-based assays, as Allgäu type. Epidemiological investigation reported that sixteen animals were introduced from an officially tuberculosis free Member State in previous years. Nevertheless, spoligotyping and multilocus variable tandem repeat analysis (MLVA) indicated that M. caprae was strictly related to the strain circulating in 2007-2009 in Trento province, although no at-risk contacts were described. M. caprae is a zoonotic pathogen and further analyses are warranted in order to control its spread and impact on public health and animal trade.

Mycobacterium genavense Infection in a Domestic Ferret (Mustela putorius furo).

Mycobacterium genavense infection was diagnosed in an adult ferret with ptosis of the left eye, a proliferative lesion of the conjunctiva of the nictitating membrane, conjunctival swelling, and tumefaction of the periorbital tissues with a watery ocular discharge and the presence of a retrobulbar mass. The diagnosis was based on characteristic cytology of the retrobulbar mass and left mandibular lymph node that revealed granulomatous inflammation. Ziehl-Neelsen staining showed the presence of positive acid-fast bacilli in the cytoplasm of the macrophages. The diagnosis was confirmed by sequence analysis of the 16S rRNA gene amplified by using a multiplex polymerase chain reaction from a fresh lymph node biopsy. Therapy with marbofloxacin, rifampicin, and clarithromycin was recommended for 6 months and after this period, the veterinarian who was treating the ferret reported the disappearance of clinical signs. Six months after the end of the antibiotic treatment, the symptoms described previously reoccurred. Confirmatory laboratory tests were not performed but a recurrence of M genavense infection was suspected and the veterinarian, in agreement with the owner, euthanized the ferret.

Pesticide incidence in poisoned baits: A 10-year report.

The incidence of pesticides in poisoned baits recovered from 2005 to 2014 in Italian northern regions of Emilia Romagna and Lombardy was analysed. A total of 956 baits tested positive for pesticides during the study period. In 9.3% (n=89) of the baits analysed more than one toxic compound was present. Insecticides (53.2%) proved to be the pesticides most commonly involved followed by rodenticides (34.3%), molluscicides (12.3%) and herbicides (0.2%). Among insecticides, the anticholinesterases (organophosphates and carbamates) were the most frequently detected (59.1%, n=412) followed by organochlorines (39.8%; n=277). Anticoagulants proved to be the rodenticides most commonly involved (78.5%; n=353) with many baits containing brodifacoum or bromadiolone, while metaldehyde was the most used molluscicide (88.2%; n=142). A high number of baits contained banned pesticides such as endosulfan, methamidophos, carbofuran and strychnine indicating that the restrictions and the bans on the use of toxic and highly toxic pesticides are not accompanied by a lower use in poisoned baits. The high incidence of banned pesticides detected in the present survey is extremely worrying and poses the question of where and how these compounds were obtained. Efforts have to be made to reduce this illegal practice and its environmental impact. Moreover, education and awareness campaigns should be instituted.

Infectious agents identified in aborted swine fetuses in a high-density breeding area: a three-year study.

Reproductive failure in sows is one of the most important factors affecting pig breeding. Many reproductive disorders are linked to both environmental factors and infectious agents. The goal of our study was to determine the presence of pathogens that are known to cause abortion, considering a set of conditioning factors, such as seasonality and pregnancy period. A large number of aborted fetuses (1,625 fetuses from 140 farms) from a high-density breeding area in northern Italy was analyzed for a period of 3 years. The pigs were diagnosed based on direct (culture, PCR) or indirect (enzyme-linked immunosorbent assay) evidence. An infectious etiologic agent was found in 323 of 549 cases of abortion (58.8%). These included viral agents (Porcine circovirus-2, 138/323; Porcine reproductive and respiratory syndrome virus, 108/323; porcine parvovirus, 20/323; pseudorabies virus, 6/323; and Encephalomyocarditis virus, 3/323) and bacteria (Escherichia coli, 64/323; Streptococcus sp., 63/323; Staphylococcus sp., 5/323; Pasteurella sp., 3/323; Shigella sp., 1/323; and Yersinia sp., 1/323). This study describes the prevalence of infectious agents involved in reproductive failure in a high-density swine population. The data can be useful to swine breeders, practitioners, and medical specialists in monitoring animal health and in supervising the breeding process.

Surveillance of Mosquitoes and Selected Arthropod-Borne Viruses in the Context of Milan EXPO 2015.

From 1 May 2015 to 31 October 2015 over 20 million visitors from all over the world visited the Universal Exhibition (EXPO) hosted by Milan (Lombardy region, Italy), raising concerns about the possible introduction of mosquito-borne diseases from endemic countries. The entomological surveillance protocol performed in Lombardy over the last three years was implemented in the EXPO area and in the two major regional airports using both Center for Disease Control CO2 and Biogents Sentinel traps. This surveillance aimed to estimate the presence and densities of putative vectors, and also to support investigations, including the vector species involved and area of diffusion, on the local spread of Chikungunya, Dengue and West Nile viruses (WNV) by competent vectors. From 3544 mosquitoes belonging to five different species, 28 pools of Culex spp. and 45 pools of Aedes spp. were screened for the presence of WNV, and for both Chikungunya and flaviviruses, respectively. The entomological surveillance highlighted a low density of potential vectors in the surveyed areas and did not reveal the presence of Chikungunya or Dengue viruses in the local competent vectors inside the EXPO area or in the two airports. In addition, the surveillance reported a low density of Culex spp. mosquitoes, which all tested negative for WNV.

Identification and Characterization of Fringilla coelebs Papillomavirus 1 (FcPV1) in Free-living and Captive Birds in Italy.

A papillomavirus (PV) was identified by negative-staining electron microscopy in skin lesions of two bird species (Fringillidae) in Italy. Genetic analyses revealed an FcPV1 with a low genetic variability in the E6, E7, E1, E2, and L1 genes and the long control region when compared to the FcPV1 reference strain.

West Nile Virus Surveillance in 2013 via Mosquito Screening in Northern Italy and the Influence of Weather on Virus Circulation.

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle.

Long-Term Surveillance of Aujeszky's Disease in the Alpine Wild Boar (Sus scrofa).

Although wild boar can act as a persistent Aujeszky's disease (AD) reservoir, limited data are available on long-term epidemiology in free-ranging wild boar living in areas where industrial swine herds are limited. Hence, this study provides crucial information, which fills this knowledge gap, on the natural dynamics of AD infection. From 3260 sera sampled during eight hunting seasons, 162 (4.97%) were tested positive. Factors, including the animal's age class, and the sampling year, had significant effects on the probability of the wild boar being seropositive, while wild boar mean abundance per area, yearly abundance and the total number of pig farms, as well as interactions among age, year and sex, were not significant. In particular, a positive trend of seroprevalence was observed over the years, with values ranging from 2.1 to 10.8%. This long-term surveillance showed an increase in seroprevalence with a higher probability of being seropositive in older individuals and the independence of wild boar seropositivity from the likelihood of contact with pigs in the area.

Lead, cadmium and organochlorine pesticide residues in hunted red deer and wild boar from northern Italy.

The objectives of the present study were to assess heavy metal cadmium (Cd), lead (Pb) and organochlorine pesticide concentrations in tissues of red deer (Cervus elaphus) and wild boar (Sus scrofa) from nine hunting areas and to evaluate related risk factors for the host animal. Over a period of 2 years, a total of 1055 and 210 masseters, 424 and 201 livers, 642 and 152 kidneys were collected from wild boar and red deer, respectively, and concentrations of Cd, Pb and organochlorine pesticides were determined. Comparing the two species, Cd concentration in the kidney (3.72 mg/kg), liver (0.67 mg/kg) and muscle (0.02 mg/kg) of wild boar was found to be significantly higher than in the organs of red deer (1.02 mg/kg in the kidneys, 0.07 mg/kg in the liver and 0.006 mg/kg in muscle). Mean Pb concentrations were found to be similar in both animals, with 0.39, 0.52 and 2.60 mg/kg detected in the wild boar kidney, liver and muscle, respectively, and 0.24, 0.21 and 2.04 mg/kg in the respective organs of the red deer. No difference in concentrations were found based on age class, location of tissue sample or contaminant in the case of wild boar. By contrast, a significantly lower Cd concentration was found in the kidney of the young red deer. The search for organochlorine pesticides in both red deer and wild boar produced negative results with values below the limits of detection. Due to the high levels of renal Cd and muscle Pb detected in wild boar and red deer, further research needs to be carried out in an effort to identify the source of contamination and preserve the health of animals and humans.

Suitability of a Salmonella control programme based on serology in slaughter heavy pigs.

The key component of most European pig Salmonella control programmes is the classification of herds according to seroprevalence at slaughter. The objectives of this study were to estimate the true Salmonella seroprevalence, and investigate the association between the true status of infection and serology in slaughter heavy pigs. Blood of 3340 pigs was collected and tested with ELISA. From 385 pigs, also lymph nodes and cecal content were collected for bacteriology. Analysis was performed in a Bayesian framework. Results showed that a large proportion of pigs was serologically positive (herd seroprevalence 93% and within-herd seroprevalence higher than 81% in half of herds at cut-off 10 OD%). The association between the true status of infection and serology was not significant, and therefore the classification of heavy pig herds according to seroprevalence at slaughter would not be suitable to reduce the risk of introducing Salmonella into the food chain.

Field and experimental data indicate that the eastern cottontail (Sylvilagus floridanus) is susceptible to infection with European brown hare syndrome (EBHS) virus and not with rabbit haemorrhagic disease (RHD) virus.

The eastern cottontail (Sylvilagus floridanus) is an American lagomorph. In 1966, it was introduced to Italy, where it is currently widespread. Its ecological niche is similar to those of native rabbits and hares and increasing overlap in distribution brings these species into ever closer contact. Therefore, cottontails are at risk of infection with the two lagoviruses endemically present in Italy: Rabbit Haemorrhagic Disease virus (RHDV) and European Brown Hare Syndrome Virus (EBHSV). To verify the susceptibility of Sylvilagus to these viruses, we analyzed 471 sera and 108 individuals from cottontail populations in 9 provinces of north-central Italy from 1999 to 2012. In total, 15-20% of the cottontails tested seropositive for EBHSV; most titres were low, but some were as high as 1/1280. All the cottontails virologically tested for RHDV and EBHSV were negative with the exception of one individual found dead with hares during a natural EBHS outbreak in December 2009. The cottontail and the hares showed typical EBHS lesions, and the EBHSV strain identified was the same in both species (99.9% identity). To experimentally confirm the diagnosis, we performed two trials in which we infected cottontails with both EBHSV and RHDV. One out of four cottontails infected with EBHSV died of an EBHS-like disease, and the three surviving animals developed high EBHSV antibody titres. In contrast, neither mortality nor seroconversion was detected after infection with RHDV. Taken together, these results suggest that Sylvilagus is susceptible to EBHSV infection, which occasionally evolves to EBHS-like disease; the eastern cottontail could therefore be considered a "spill over" or "dead end" host for EBHSV unless further evidence is found to confirm that it plays an active role in the epidemiology of EBHSV.