Avian astrovirus caused mortality in pheasant (Phasianus colchicus, Linnaeus, 1758) farm in Hungary.

We present the clinical symptoms, pathologic lesions and diagnostic possibilities of the avian astrovirus-related mortality in a pheasant colony. In addition to enteritis in chicks, we also confirmed acute nephrosis. The genome section of the astrovirus was detected and verified by polymerase chain reaction (PCR) testing. After sequencing the isolated genome section based on BLAST driver analysis (601-base pair-long) avian astrovirus has the same ORF-1b gene as turkey astrovirus 1 (TAstV1).

Rainbow trout (Oncorhynchus mykiss, Walbaum 1792) adenocarcinoma investigation with various diagnostic imaging techniques.

Diagnostic imaging techniques provide a new aspect of the ante-mortem and post-mortem diagnostics in fish medicine. Ultrasonography, computed tomography (CT) and magnetic resonance imaging (MRI) can provide more information about the internal organs and pathognomic lesions. The authors used diagnostic imaging techniques to evaluate and describe the neoplastic malformation in a 3-year-old female rainbow trout (Oncorhynchus mykiss). The fish was examined with Siemens Somatom Definition AS + CT scanner and Siemens Biograph mMR scanner. The animal was lethargic and showed anorectic signs and muscular dystrophy. During the post-mortem investigation, histopathology and immunohistochemistry were also performed allowing us to identify the neoplasms. The results showed a large soft tissue mass in the first mid-intestine segment, which proved to be an adenocarcinoma. This subsequently led to digestion problems and absorption disorders. Immunohistochemically, neoplastic cells of carcinoma revealed E-cadherin and pancytokeratin positivity. This is the first study to report the use of MRI and CT for studying gastrointestinal adenocarcinoma in rainbow trout.

Structural similarity of human papillomavirus E4 and polyomaviral VP4 exhibited by genomic analysis of the common kestrel (Falco tinnunculus) polyomavirus.

Polyomaviruses are widely distributed viruses of birds that may induce developmental deformities and internal organ disorders primarily in nestlings. In this study, polyomavirus sequence was detected in kidney and liver samples of a common kestrel (Falco tinnunculus) that succumbed at a rescue station in Hungary. The amplified 5025 nucleotide (nt) long genome contained the early (large and small T antigen, LTA and STA) and late (viral proteins, VP1, VP2, VP3) open reading frames (ORFs) typical for polyomaviruses. One of the additional putative ORFs (named VP4) showed identical localization with the VP4 and ORF-X of gammapolyomaviruses, but putative splicing sites could not be found in its sequence. Interestingly, the predicted 123 amino acid (aa) long protein sequence showed the highest similarity with human papillomavirus E4 early proteins in respect of the aa distribution and motif arrangement implying similar functions. The LTA of the kestrel polyomavirus shared <59.2% nt and aa pairwise identity with the LTA sequence of other polyomaviruses and formed a separated branch in the phylogenetic tree among gammapolyomaviruses. Accordingly, the kestrel polyomavirus may be the first member of a novel species within the Gammapolyomavirus genus, tentatively named Gammapolyomavirus faltin.

Prevalence of red panda amdoparvovirus infection in European zoos.

Red panda amdoparvovirus (RPAV) was first described in captive red pandas (Ailurus fulgens) at a zoo in the United States in 2018. Subsequently, the prevalence of infection in zoos in the United States was reported to be 50%; however, RPAV prevalence outside the United States remains unstudied. This study was conducted to investigate the prevalence of RPAV in 134 red pandas from zoos in Europe. Overall, RPAV was detected with PCR in 21 of 62 zoos (33.9%), and the virus prevalence among individuals was estimated to be 24.2% (95% confidence interval, 17.4%-32.0%). Remarkably, adult females tested positive for RPAV more frequently than adult males. Zoos where RPAV was detected reported a significantly higher occurrence of alopecia (and clinical signs in general), whereas other commonly reported problems (fecal disorders and dental disease) showed no difference. A repeated pooled sampling of two positive individuals further showed that RPAV excretion in feces is intermittent, with the viral DNA being only detected on 8 out of 14 sampling days. The intermittent nature of excretion implies that RPAV prevalence may be higher than the estimated value.

3D Reconstruction of the Blood Supply in an Elephant's Forefoot Using Fused CT and MRI Sequences.

Being the largest still-living terrestrial mammal on earth, an elephant's feet play an important role in its health status. The musculoskeletal structures in the forefoot are well described in the literature, but information about vascularization is limited. The novel aim of this work is to provide anatomical guidance to structures found in the forefoot, focusing on the arterial system. Initially, native CT and MRI sequences were taken of the left forefoot of a deceased 6-year-old female Asian elephant; the foot was then filled with an iodine-containing contrast medium through the a. mediana and the CT scans were repeated in the same position. The images obtained were processed with 3D Slicer software for the 3D reconstruction of the bones and arteries. The results clearly showed the palmar blood supply of the forefoot. A so far undescribed vessel was revealed, stemming from the a. metacarpea, supplying the first digit and the digital cushion. The course of the deep palmar arch's terminal section was also established. This paper provides the first description of the exact disposition of the arteries in the palmar aspect of an elephant's forefoot and may be used in planning surgeries in clinically affected animals.

Dermatologic Aspects of Zoo Mammal Medicine.

The article deals with the primary aspects of how to maintain healthy integument in zoo mammals and in particular why husbandry-related health problems can occur in general in a zoologic setting. While working with these species we are often faced with diagnostic challenges, which may include a general approach (often requiring anesthesia or medical training), lack of "normal" references, and difficulties, especially if the cutaneous signs are not a primary ailment, but a manifestation of a generalized disease (eg, immune-suppression). The different etiologies of skin problems are discussed with clinical examples.

Home Range of the Caspian Whipsnake Dolichophis caspius (Gmelin, 1789) in a Threatened Peri-Urban Population.

Semi-natural environments within cities can provide habitats for vulnerable reptile species. Better understanding of their habitat use and home range sizes is important for their conservation. We investigated the spatial ecology of Caspian whipsnakes (Dolicophis caspius) in a peri-urban habitat in Budapest, Hungary. We used radiotelemetry to track five adult snakes and analyzed their microhabitat preferences, home range sizes and daily movements. The Caspian whipsnakes intensively utilized areas covered with woody vegetation, with a high density of hibernacula. The tracked snakes used an area of 40.15 ha during the activity period from spring to autumn, but for the winter, they withdrew to a central area of 1.75 ha, abundant in hibernacula. During the activity period the individual home range sizes varied between 6.1 and 15.5 ha, estimated using the minimum convex polygon (MCP); however, for the entire datasets of the individuals, the adaptive kernel method gave the highest mean (13.8 ha), while the LoCoH-R yielded the smallest home ranges (5.19 ha). We found that the average daily displacement for the different individuals ranged between 12.6 and 36.6 m during their main activity season. In the study area, the whipsnakes currently have enough space for foraging, but the restricted spatial distribution of hibernacula, which is mainly available in the central dry rocky forest and partly in the shrubby areas, can limit the extent of the suitable habitat. Human activities and anthropogenic disturbances, especially around hibernacula, may exert pressure on this peri-urban snake population.

MANDIBULAR FRACTURES IN GIRAFFES (GIRAFFA CAMELOPARDALIS) IN EUROPEAN ZOOS.

Following a case of mandibular fracture in a giraffe (Giraffa camelopardalis) at the Budapest Zoo, a survey was conducted across European zoological institutions with the support of the European Association of Zoos and Aquariums Giraffe Ex situ Programme (EAZA Giraffe EEP) to help identify the incidence, potential causes, and management of such injuries. Two hundred sixteen zoos keeping giraffes were invited to participate in the survey. Eighty-six responses were received, 14 of which (16.3%) reported jaw fractures with various causes in their giraffes. Of these responses, hay racks, where the muzzle of the giraffe could fit between the grid of feeding contraptions, were associated with mandibular fractures in seven cases (50.0%). Most giraffes were managed surgically (10/14; 71.4%), two were managed medically (14.3%), one was euthanized because of the severity of the injury (7.1%), and one was only diagnosed with a mandibular fracture postmortem. One giraffe died owing to postanesthetic complications; all other individuals had a full recovery. Most mandible fractures occurred in relatively young giraffes (between 5 wk and 8 yr with an average of 3.4 yr). Based on these findings, facilities with giraffe should evaluate their hay feeders for mandible entrapment risk, especially if they house young individuals.

Molecular epidemiological study on ticks and tick-borne protozoan parasites (Apicomplexa: Cytauxzoon and Hepatozoon spp.) from wild cats (Felis silvestris), Mustelidae and red squirrels (Sciurus vulgaris) in central Europe, Hungary.

BACKGROUND: Among live wild mammals adapted to urban and peri-urban habitats in Europe, members of the families Felidae, Mustelidae and Sciuridae deserve special attention as pathogen reservoirs because all of these families include members that are kept as pets. We report here the results of our study on two important groups of tick-borne protozoan parasites in ticks and tissues of wild cats, mustelids and red squirrels. METHODS: DNA was extracted from the tissues of carnivores (wild cats, mustelids; n = 16) and red squirrels (n = 4), as well as from ixodid ticks (n = 89) collected from these hosts. These DNA extracts were screened for piroplasms and Hepatozoon spp. using conventional PCR analysis and sequencing. In addition, 53 pooled samples of 259 questing Haemaphysalis concinna ticks were evaluated for the presence of Hepatozoon DNA, followed by phylogenetic analyses. RESULTS: One wild cat was found to be coinfected with Cytauxzoon europaeus and a new genotype of Hepatozoon felis, and two additional wild cats were infected with H. felis from a different phylogenetic group. In mustelids, Hepatozoon martis and two further Hepatozoon genotypes were detected. The latter clustered separately, close to others reported from eastern Asia. In addition, Hepatozoon sciuri was detected in red squirrels. Morphologic and molecular analyses verified eight tick species. One wild cat was infected with a H. felis genotype that was significantly different from that in Ixodes ricinus females infesting this cat. Only three pools of questing H. concinna nymphs tested positive for Hepatozoon, one of which contained H. martis. CONCLUSIONS: This study provides the first evidence of the occurrence of any Cytauxzoon species and of three Hepatozoon species in Hungary. In addition to H. martis, two further mustelid-associated Hepatozoon genotypes were detected, one of which was new in terms of phylogenetic and broader geographical contexts. This may be the first indication that H. felis genotypes from both of its phylogenetic groups occur in Europe. This also appears to be the first evidence of H. felis and C. europaeus coinfection in felids in Europe, and of autochthonous H. felis infection in wild cats north of the Mediterranean Basin. New tick-host associations were also observed in this study. Based on the results, H. felis and H. martis might survive transstadially in I. ricinus and H. concinna, respectively.

A novel gammapolyomavirus in a great cormorant (Phalacrocorax carbo).

In this study, the complete genome of a novel polyomavirus detected in a great cormorant (Phalacrocorax carbo) was characterized. The 5133-bp-long genome of the cormorant polyomavirus has a genomic structure typical of members of the genus Gammapolyomavirus, family Polyomaviridae, containing open reading frames encoding the large and small tumor antigens, viral proteins 1, 2, and 3, and the X protein. The large tumor antigen of the cormorant polyomavirus shares 45.6-50.4% amino acid sequence identity with the homologous sequences of other gammapolyomaviruses. These data, together with results of phylogenetic analysis, suggest that this cormorant polyomavirus should be considered the first member of a new species within the genus Gammapolyomavirus, for which we propose the name "Phalacrocorax carbo polyomavirus 1".

Novel Circoviruses from Birds Share Common Evolutionary Roots with Fish Origin Circoviruses.

Circoviruses occur in a variety of animal species and are common pathogens of mammalian and avian hosts. In our study internal organ samples of wild birds were processed for screening of circoviral sequences. Two novel viruses were identified and characterized in specimens of a little bittern and a European bee-eater that suffered from wing injuries, were weakened, had liver or kidney failures, and finally succumbed at a rescue station. The 1935 nt and 1960 nt long viral DNA genomes exhibited a genomic structure typical for circoviruses and were predicted to encode replication-associated protein in the viral strand, and a capsid protein in the complementary strand of the replicative intermediate DNA form. The genome of the newly described viruses showed 37.6% pairwise identity with each other and ≤41.5% identity with circovirus sequences, and shared a common branch with fish, human and Weddel seal circoviruses in the phylogenetic tree, implying evolutionary relationship among the ancestors of these viruses. Based on the results the little bittern and European bee-eater circoviruses represent two distinct species of the Circovirus genus, Circoviridae family.

Gastrointestinal Parasitism in Przewalski Horses (Equus ferus przewalskii).

PURPOSE: Przewalski's horse (Equus ferus przewalskii) is a subspecies of Equus ferus classified as endangered by the International Union for Conservation of Nature (IUCN), whose gastrointestinal (GI) parasitism has been studied in different populations, in situ and ex situ, during the last decades. We aimed to summarize the information available on the subject. METHODS: In this review, we collected information from distinct published literature, interpreted it, compared it and related the Przewalski's horse biology and ecology with different findings. RESULTS: Many similarities can be found between the Przewalski's horse populations and the domestic horses regarding their gastrointestinal parasite community. However, not all the GI parasites described in the domestic horse have been reported in Przewalski's horse's surveys. The most prevalent parasites found in Przewalski's horses belong to the Strongylidae family, namely the Cyathostominae. CONCLUSIONS: More continuous monitoring studies should be carried on in different in situ and ex situ locations to consistently support the one-off surveys. It might be necessary to better correlate the parasitological findings of this subspecies with other animals that share the same habitat and ecosystem, for instance, intermediate and reservoir hosts, from different animal taxonomic groups.

PREVALENCE OF SALMONELLA SPECIES, CLOSTRIDIUM PERFRINGENS, AND CLOSTRIDIUM DIFFICILE IN THE FECES OF HEALTHY ELEPHANTS (LOXODONTA SPECIES AND ELEPHAS MAXIMUS) IN EUROPE.

Pathogenic Salmonella spp., Clostridium perfringens, and Clostridium difficile have been reported to infect and cause severe enteritis and enterotoxemia in African (Loxodonta spp.) and Asian elephants (Elephas maximus). However, little information exists on whether healthy elephants carry and possibly shed these gastrointestinal organisms. This study was conducted to investigate the prevalence of all three bacteria in feces from healthy elephants in European zoos. Bacterial identification was performed by selective culture on fecal samples and a polymerase chain reaction (PCR) amplification protocol, on the basis of primers targeting the hilA gene (Salmonella spp.), the cpa gene (C. perfringens), and the tpi gene (C. difficile) from deoxyribonucleic acid extracted from elephant feces. The PCR protocol was validated prior to initiation of the investigation. Fecal samples collected from 50 African and 86 Asian elephants originating from 30 European zoologic institutions were investigated. The PCR validation revealed detection limits ranging from 104 to 106 colony-forming units per gram of feces of each gene. Only C. perfringens (one type A and two type E) was detected in the initial sampling (2.2%, three Asian elephants), whereas no Salmonella spp. or C. difficile was detected. At a follow-up sampling from C. perfringens-positive animals and relatives, 2 mo after the initial sampling, three animals were culture positive for Salmonella enterica spp. enterica. All positive samples were obtained with bacterial culture, whereas no PCR reactions were positive. Despite carrying these pathogens, all culture-positive animals were clinically healthy and did not develop signs of gastrointestinal disease during the study period. The findings indicate that prevalence of Salmonella spp., C. perfringens, and C. difficile in feces from healthy Asian and African elephants in Europe is very low.

Influencing factors on the foot health of captive Asian elephants (Elephas maximus) in European zoos.

Pathological lesions of feet occur frequently in captive elephant populations. To improve foot health, it is important to identify risk factors associated with such pathologies. Several previous studies have analyzed potentially influencing factors but were limited, for example, by small sample sizes. This study analyzed the relationship between 87 independent variables and the foot health score of 204 Asian elephants (Elephas maximus) in European zoos using bivariate correlation, multivariable regression models, and principal component analysis (PCA). Correlation and regression tests revealed significant results for 30 different variables, mainly with small effect sizes. Only three variables were significant in more than one test: sex, time spent indoors, and time spent on hard ground, with lower scores (i.e. less or less severe pathological lesions) in females, and when less time is spent indoors or on hard ground. Due to small effect sizes and differing results of the statistical tests, it is difficult to determine which risk factors are most important. Instead, a holistic consideration appears more appropriate. A biplot of the PCA shows that factors representing more advanced husbandry conditions (e.g. large areas, high proportions of sand flooring) were associated with each other and with decreased foot scores, whereas indicators of more limited conditions (e.g. high proportions of hard ground, much time spent indoors) were also associated with each other but increased the foot score. In conclusion, instead of resulting from just one or two factors, reduced foot health might be an indicator of a generally poorer husbandry system.

Full genome sequence analysis of a novel adenovirus from a captive polar bear (Ursus maritimus).

The presence of a novel adenovirus (AdV) was detected by PCR and sequencing, in the internal organs of a captive polar bear that had died in the Budapest zoo. The virus content of the samples proved to be high enough to allow for conventional Sanger sequencing on PCR-amplified genomic fragments. With this approach, the sequence of the entire genome of the putative polar bear adenovirus 1 (PBAdV-1) was obtained. Although the genome was found to be short, consisting of 27,952 base pairs merely, with a relatively balanced G + C content of 46.3 %, its organisation corresponded largely to that of a typical mastadenovirus. Every genus-common gene could be identified except that of protein IX. The short E3 region of the PBAdV-1 consisted of two novel, supposedly type-specific ORFs only, whereas no homologue of any of the E3 genes, usually conserved in mastadenoviruses, such as for example that of the 12.5 K protein, were present. In the E4 region, only the highly conserved gene of the 34 K protein was found besides two novel ORFs showing no homology to any known E4 ORFs. In silico sequence analysis revealed putative splicing donor and acceptor sites in the genes of the E1A, IVa2, DNA-dependent DNA polymerase, pTP, 33 K proteins, and also of U exon protein, all being characteristic for mastadenoviruses. Phylogenetic calculations, based on various proteins, further supported that the newly-detected PBAdV is the representative of a new species within the genus Mastadenovirus, and may represent the evolutionary lineage of adenoviruses that coevolved with carnivorans.

Sex determination using circulating cell-free fetal DNA in small volume of maternal plasma in elephants.

The genetic sexing of animals having long gestation periods offers significant benefits in regard to breeding management among their populations living in captivity. In our study, a new increased-sensitivity PCR method for fetal sexing was developed and tested successfully on elephants, from only a small volume of maternal plasma. Suitable sensitivity was obtained by using short, reduced amplicon lengths with fluorescent labelling for capillary electrophoresis detection. The fundamental principle for this technique was based on the detection of two Y-specific markers (AmelY and SRY), the presence of which indicates the mother is carrying a male fetus and the absence of these markers designates a female fetus. As a reaction control, the X-chromosomal marker (PlpX) was used. To the best of our knowledge, this is the first report on this topic, confirming the presence of fetal cell-free DNA from the plasma of a pregnant captive elephant, and demonstrating a new opportunity for non-invasive assessment in fetal sex determination.

FOOT HEALTH OF ASIAN ELEPHANTS (ELEPHAS MAXIMUS) IN EUROPEAN ZOOS.

Foot problems are a common concern in elephant husbandry. Studies on this topic with sample sizes greater than 100 animals have only been carried out in North America. We investigated foot health of 243 Asian elephants (Elephas maximus) in 69 European institutions. During on-site visits between August 2016 and July 2017, standardized pictures were taken of each elephant's nails and pads. The pictures were analyzed with respect to pathological lesions (i.e. nail cracks, abscesses), care issues (i.e. minor abnormalities, which are easily resolvable with routine foot work), and pad structure. Of all analyzed nails and pads, 35.6% revealed varying degrees of pathological lesions, with minor nail cracks and overgrown cuticles with attachment to the nails being most frequently observed. The most lateral nail (N5) on both front feet demonstrated the highest percentage of pathological lesions, providing support to a separate study showing that the mean peak pressure of an elephant's foot occurs along the most lateral digits; however, this was not observed along the most lateral nail (N5) of the rear feet. Three (of 243) elephants did not show any pathological lesions in their feet. The most common issues requiring foot care were fissures in the nail sole. The structure of the pads was categorized in four grades reflecting the percentage of surface marked by sulci. These four grades occurred at nearly equal frequency. Pearson product moment correlations revealed no significant association between the frequency of care issues and pathological lesions per nail. Despite this finding, it may be prudent to implement husbandry protocols that could alleviate commonly observed pathological and care foot issues in captive Asian elephants. A standardized approach to evaluate elephant foot health will provide a more objective way to monitor responses to management and medical decisions and ultimately contribute to the overall wellbeing of elephants in human care.

Adenoviruses of the most ancient primate lineages support the theory on virus-host co-evolution.

The scarcity or complete lack of information on the adenoviruses (AdVs) occurring in the most ancient non-human primates resulted in the initiation of a study for exploring their abundance and diversity in prosimians and New World monkeys (NWMs). In order to assess the variability of these AdVs and the possible signs of the hypothesised virus-host co-evolution, samples from almost every family of NWMs and prosimians were screened for the presence of AdVs. A PCRscreening of 171 faecal or organ samples from live or dead, captive or wild-living prosimians and NWMs was performed. The PCR products from the gene of the IVa2 protein were sequenced and used in phylogeny calculations. The presence of 10 and 15 new AdVs in seven and ten different species of prosimians and NWMs was revealed, respectively. Phylogenetic analysis indicated that the tentative novel AdVs cluster into two separate groups, which form the most basal branches among the primate AdVs, and therefore support the theory on the co-evolution of primate AdVs with their hosts. This is the first report that provides a comprehensive overview of the AdVs occurring in prosimians and NWMs, and the first insight into the evolutionary relationships among AdVs from all major primate groups.

Micrococcoides hystricis gen. nov., sp. nov., a novel member of the family Micrococcaceae, phylum Actinobacteria.

A Gram-stain-positive bacterium, designated TSL3T, was isolated from faeces of a porcupine, Hystrix indica, from the Budapest Zoo and Botanical Garden, Hungary. On the basis of 16S rRNA gene sequence analysis, the strain is phylogenetically related to the family Micrococcaceae. The highest 16S rRNA gene sequence similarity was found with Micrococcus terreus V3M1T (96.50 %) followed by Arthrobacter humicola KV-653T (96.43 %). Cells of strain TSL3T were aerobic, non-motile and coccoid-shaped. The main fatty acids were anteiso-C15 : 0 (54.4 %), iso-C16 : 0 (18.2 %) and iso C15 : 0 (9.7 %). The major menaquinone was MK-7, and the polar lipid profile included phosphatidylglycerol, diphosphatidylglycerol, dimannosylglyceride, trimannosyldiacylglycerol, phosphatidylinositol, three unknown phospholipids and two unknown glycolipids. Strain TSL3T showed the peptidoglycan structure A4alpha l-Lys - Gly - l-Glu. The DNA G+C content of strain TSL3T was 58.4 mol%. Phenotypic and genotypic characterisation clearly showed that strain TSL3T could be differerentiated from the members of other genera in the family Micrococcaceae. According to these results, strain TSL3T represents a novel genus and species, for which the name Micrococcoides hystricis gen. nov., sp. nov. is proposed. The type strain is TSL3T (=DSM 29785T=NCAIM B. 02604T).

Novel adenovirus detected in kowari (Dasyuroides byrnei) with pneumonia.

A male kowari (Dasyuroides byrnei) originating from a zoo facility was delivered for post mortem evaluation in Hungary. Acute lobar pneumonia with histopathologic changes resembling an adenovirus (AdV) infection was detected by light microscopic examination. The presence of an AdV was confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Although the exact taxonomic position of this novel marsupial origin virus could not be determined, pairwise identity analyses and phylogenetic calculations revealed that it is distantly related to other members in the family Adenoviridae.