Circoviridae Survey in Captive Non-Human Primates, Italy.

Circoviruses (CVs) and cycloviruses (CyVs), members of the family Circoviridae, have been identified only occasionally in non-human primates (NHPs). In this study, we investigated the presence and genetic features of these viruses in 48 NHPs housed in the Bioparco-Rome Zoological Garden (Italy) and in the Anima Natura Wild Sanctuary Semproniano (Grosseto, Italy), testing fecal, saliva, and serum samples with a broadly reactive consensus nested PCR able of amplifying a partial region of the replicase (Rep) gene of members of the family Circoviridae. Viral DNA was detected in a total of 10 samples, including a saliva swab and 9 fecal samples collected, respectively from five Japanese macaques (Macaca fuscata) and four mandrills (Mandrillus sphinx), with an overall prevalence of 18.7% (9/48). On genome sequencing, five strains revealed the highest nucleotide identity (98.3-98.6%) to a CyV strain (RI196/ITA) detected in the intestinal content of a Maltese wall lizard (Podarcis filfolensis) in Italy. Although the origin of the Italian NHP strains, genetically distant from previously detected NHP CyVs, is uncertain, our results also highlight that the virome of captive animals is modulated by the different dietary and environmental sources of exposure.

Plasmodium matutinum Transmitted by Culex pipiens as a Cause of Avian Malaria in Captive African Penguins (Spheniscus demersus) in Italy.

Avian malaria is a parasitic disease of birds caused by protozoa belonging to the genus Plasmodium, within the order Haemosporida. Penguins are considered particularly susceptible, and outbreaks in captive populations can lead to high mortality. We used a multidisciplinary approach to investigate the death due to avian malaria, occurred between 2015 and 2019, in eight African penguins (Spheniscus demersus) kept in two Italian zoos located in central Italy, and situated about 30 km apart. We also provided information about the presence and circulation of Plasmodium spp. in mosquitoes in central Italy by sampling mosquitoes in both zoos where penguin mortalities occurred. In the eight dead penguins, gross and histopathological lesions were consistent with those previously observed by other authors in avian malaria outbreaks. Organs from dead penguins and mosquitoes collected in both zoos were tested for avian malaria parasites by using a PCR assay targeting the partial mitochondrial conserved region of the cytochrome b gene. Identification at species level was performed by sequencing analysis. Plasmodium matutinum was detected in both dead penguins and in mosquitoes (Culex pipiens), while Plasmodium vaughani in Culex pipiens only. Parasites were not found in any of the PCR tested Aedes albopictus samples. Based on our phylogenetic analysis, we detected three previously characterized lineages: Plasmodium matutinum LINN1 and AFTRU5, P. vaughani SYAT05. In Culex pipiens we also identified two novel lineages, CXPIP32 (inferred morphospecies Plasmodium matutinum) and CXPIP33 (inferred morphospecies P. vaughani). Significantly, LINN1 and AFTRU5 were found to be associated to penguin deaths, although only LINN1 was detected both in penguins (along the years of the study) and in Culex pipiens, while AFTRU5 was detected in a single penguin dead in 2017. In conclusion, in our study Plasmodium matutinum was found to cause avian malaria in captive penguins kept in Europe, with Culex pipiens being its most probable vector. Our results are in agreement with previous studies suggesting that Culex pipiens is one of the main vectors of Plasmodium spp. in Europe and the Northern Hemisphere. Zoos maintaining captive penguins in temperate areas where Culex pipiens is abundant should be well aware of the risks of avian malaria, and should put every effort to prevent outbreaks, in particular during the periods when the number of vectors is higher.

Death of captive-bred vultures caused by flunixin poisoning in Italy.

Among non steroidal anti-inflammatory drugs (NSAIDs) diclofenac is considered the main cause for the decline of vulture populations in the Indian subcontinent since the '90 s. Chemical analysis showed high levels of flunixin (31,350 µg/kg) in beef which three captive Gyps vultures fed on, later dying with severe visceral gout. Levels in dead vultures' organs and tissues ranged from 4 to 38.5 µg/kg. The typical lesions and the concentrations found in beef indicate flunixin as the cause of death. This is the first observational study which correlates the concentration of flunixin in the meat ingested with that found in tissues of vultures.

Seroprevalence for norovirus genogroups GII and GIV in captive non-human primates.

Noroviruses (NoVs) are a major cause of epidemic gastroenteritis in children and adults. Several pieces of evidence suggest that viruses genetically and antigenically closely related to human NoVs might infect animals, raising public health concerns about potential cross-species transmission. The natural susceptibility of non-human primates (NPHs) to human NoV infections has already been reported, but a limited amount of data is currently available. In order to start filling this gap, we screened a total of 86 serum samples of seven different species of NPHs housed at the Zoological Garden (Bioparco) of Rome (Italy), collected between 2001 and 2017, using an enzyme-linked immunosorbent assay (ELISA) based on virus-like particles (VLPs) of human GII.4 and GIV.1 NoVs. Antibodies specific for both genotypes were detected with an overall prevalence of 32.6%. In detail, IgG antibodies against GII.4 NoVs were found in 18 Japanese macaques (29.0%, 18/62), a mandrill (10.0%, 1/10), a white-crowned mangabey (16.6%, 1/6) and in an orangutan (33.3%, 1/3). Twelve macaques (19.3%, 12/62), five mandrills (50.0%, 5/10), two chimpanzees (100%, 2/2) and a white-crowned mangabey (16.6%, 1/6) showed antibodies for GIV.1 NoVs. The findings of this study confirm the natural susceptibility of captive NHPs to GII NoV infections. In addition, IgG antibodies against GIV.1 were detected, suggesting that NHPs are exposed to GIV NoVs or to antigenically related NoV strains.

Serological and molecular investigation for hepatitis E virus (HEV) in captive non-human primates, Italy.

Hepatitis E virus (HEV) is the leading cause of human enterically-transmitted viral hepatitis occurring around the world both as outbreaks and as sporadic cases. Non-human primates (NHPs) have been experimentally infected with HEV, but few studies have been reported about natural infection in wild-living and zoo monkeys. In order to provide a more complete picture on the epidemiology of HEV in NHPs living in controlled environment, we investigated the presence of HEV by screening serologically and molecularly a historical collection of 86 sera from seven different species of primates housed at the Zoological Garden (Bioparco) of Rome, Italy. By using an enzyme-linked immunosorbent assay based on the recombinant capsid protein of a Gt3 HEV strain, IgG antibodies were detected in three macaques (4.8%; 3/62) and in a white-crowned mangabey (16.6%; 1/6), with an overall prevalence of 4.6% (4/86). This positivity was confirmed when assessed the sera by western blotting. Rescreening the sera for IgM and viral RNA, all the samples resulted negative. Also, HEV RNA was not found when 17 stool samples were analyzed by RT-PCR. Although these results suggest that none of the monkeys housed at the Bioparco of Rome in the 17-year time frame spanning 2001 to 2017 developed acute or at least sub-acute HEV disease, the detection of IgG antibodies demonstrated that animals living in this setting were exposed to HEV or to antigenically related viruses.

Tongue Squamous Cell Carcinoma in a European Lynx (Lynx Lynx): Papillomavirus Infection and Histologic Analysis.

Oral squamous cell carcinoma (SCC) is a common finding in domestic and wild felids. Only two cases of oral SCC have been reported in Lynx species (Lynx rufus and Lynx canadensis), at mandibular and gingival sites. In this study, we describe the first report of tongue SCC in a 15 years old female European lynx (Lynx lynx), along with viral investigations. Necropsy and histological analysis were performed and the presence of papillomavirus (PV) infection was investigated by ultrastructural and molecular methods. The lardaceous mass at tongue level was histologically diagnosed as moderately differentiated SCC. Typical microscopical features of SCC were also found in the retropharyngeal lymph node and at the pulmonary level. Neither viral DNA by PCR, nor viral particles by transmission electron microscopy were found. Despite that PV infection is associated with Felidae, this work reports the first description of tongue SCC in Lynx species, but no evidence of PV infection, suggesting that PV may not be involved in development of SCC in bobcat species.