The aim of this paper was to evaluate the degree of mycological air contamination and determine the taxonomic diversity of airborne fungi residing in the air of 20 different animal facilities in a zoological garden. The concentrations of fungi in the zoological garden were measured using a MAS-100 air sampler. The collected microorganisms were identified using the combination of molecular and morphological methods. The fungal concentration ranged from 50 to 3.65 × 104 CFU/m3 during the whole study. The quantitative analysis of the fungal aerosol showed that the obtained concentration values were lower than the recommended permissible limits (5 × 104 CFU/m3 for fungi). Environmental factors, including temperature and relative humidity, exerted a varying effect on the presence and concentration of isolated fungi. Relative humidity was shown to correlate positively with the concentration of fungal spores in the air of the facilities studied (rho = 0.57, p < 0.0021). In parallel, no significant correlation was established between temperature and total fungal concentration (rho =  - 0.1, p < 0.2263). A total of 112 fungal strains belonging to 50 species and 10 genera were isolated. Penicillium was the dominant genera, including 58.9% of total fungal strains, followed by Aspergillus 25.89%, Cladosporium 3.57%, Talaromyces 3.57%, Mucor 1.78%, Schizophyllum 1.78%, Syncephalastrum 0.89%, Alternaria 0.89%, Absidia 0.89%, and Cunninghamella 0.89%. Our preliminary studies provide basic information about the fungal concentrations, as well as their biodiversity in zoological garden. Further studies are needed to generate additional data from long-term sampling in order to increase our understanding of airborne fungal composition in the zoological garden.

Obligatory endoparasitic mites of the genera Halarachne Allman, 1847 and Orthohalarachne Newell, 1947 (Acari: Halarachnidae) parasitize different segments of the respiratory tract of marine mammals, including pinnipeds and sea otters, and infestations can cause asymptomatic to serious respiratory diseases. However, knowledge on biology, pathogenic potential and occurrence of halarachnid mites infesting pinnipeds, especially in captivity, is scarce. A two-year-old South American sea lion (Otaria flavescens Shaw, 1800) male, born and held at the Vienna Zoo, was anesthesized for routine pre-transport examinations, including computed tomography, bronchoalveolar lavage, and blood sampling. During the final phase of general anesthesia, the individual abruptly became apneic and died despite all attempts at resuscitation. At necropsy, 45 highly motile whitish millimeter-sized structures were macroscopically detected in the trachea, bifurcatio tracheae and main bronchi and were identified as adult stages of Orthohalarachne diminuata Doetschman, 1944 following morphological descriptions. After trepanation of the nasal cavity and sinus paranasalis, a total of 407 larval and 3 nymphal specimens distributed in clusters were detected. Macroscopically, sinus mucosa showed hyperemia and multiple petechial hemorrhages. Histopathological analyses of paranasal sinuses revealed mite cross-sections surrounded by sanioserous exudate and epithelial exfoliation. For the first time, O. diminuata was molecularly characterized and phylogenetically analyzed based on its 16S rDNA. Our study constitutes the first record of a severe O. diminuata infestation in captive O. flavescens and one of the few host-parasite records in general. We present clinical data and pathological results, the first scanning electron microscopic images of a O. diminuata larval stage and discuss the etiology of this autochthonous infestation, possible transmission pathways and detrimental effects. Further studies on biology and pathogenic effects of halarachnid mites, as well as on the development of non-invasive sampling techniques are essentially required for a better understanding of (ortho-)halarachnosis in pinnipeds held in zoological gardens.

UNASSIGNED: Entamoeba spp. are globally distributed zoonotic parasites that infect various hosts, among which non-human primates (NHPs) have been identified as one of the most common hosts of these parasites. Consequently, the infections of Entamoeba spp. in captive NHPs from Nanjing Hongshan Forest Zoo in China were investigated in order to assess their zoonotic potential.
UNASSIGNED: A total of 120 fresh fecal samples, including 19 species of NHPs, were collected from four breeding bases of the zoo from May to June 2019. The infections of six species of Entamoeba spp. were detected by PCR using the 16S or 18S rDNA-specific primers, and the positive samples were sequenced and analyzed.
UNASSIGNED: Entamoeba spp. were detected as positive in 59 NHPs fecal samples (49.17%), including five Entamoeba species: Entamoeba histolytica (7.50%), E. dispar (22.50%), E. coli (22.50%), E. chattoni (10.00%) and E. nuttalli (1.67%). Infection with one Entamoeba species was more common (35%) than co-infections (13.33%) or infections with three Entamoeba species (0.83%). There was a significantly higher prevalence rate of Entamoeba spp. in the species Pongo pygmaeus and Macaca mulatta than in Papio sp., Mandrillus sphinx, and Saimiri sciureus.
UNASSIGNED: Entamoeba spp. are highly prevalent in the NHPs raised in Nanjing Hongshan Forest Zoo. Therefore, attention should be paid to the development of containment strategies of Entamoeba spp. in this zoological garden.

This study reports on a fatal case of a captive great grey owl infected with the West Nile virus (WNV) in the zoological garden Košice, eastern Slovakia (Central Europe). The tissue samples of the dead owl were used for virus isolation and genetic characterization. The novel isolate is genetically closer to Hungarian, Greek, and Bulgarian strains from the central/southern European clade of lineage 2 than to the strains previously isolated in Slovakia. Interestingly, it carries NS3-249P, a molecular virulence determinant associated with higher neurovirulence, which has not previously been observed in Slovakia. Subsequent serological investigation of the captive owls revealed additional seropositive animals, indicating local WNV transmission. Although no WNV-positive mosquitoes were found, the presence of the WNV principal vector Culex pipiens complex together with the described fatal case and further serological findings indicate an endemic focus of bird-neurovirulent WNV variant in the area.

Little information is currently available on the epidemiology of parasitic and commensal protist species in captive non-human primates (NHP) and their zoonotic potential. This study investigates the occurrence, molecular diversity, and potential transmission dynamics of parasitic and commensal protist species in a zoological garden in southern Spain. The prevalence and genotypes of the main enteric protist species were investigated in faecal samples from NHP (n = 51), zookeepers (n = 19) and free-living rats (n = 64) by molecular (PCR and sequencing) methods between 2018 and 2019. The presence of Leishmania spp. was also investigated in tissues from sympatric rats using PCR. Blastocystis sp. (45.1%), Entamoeba dispar (27.5%), Giardia duodenalis (21.6%), Balantioides coli (3.9%), and Enterocytozoon bieneusi (2.0%) (but not Troglodytella spp.) were detected in NHP. Giardia duodenalis (10.5%) and Blastocystis sp. (10.5%) were identified in zookeepers, while Cryptosporidium spp. (45.3%), G. duodenalis (14.1%), and Blastocystis sp. (6.25%) (but not Leishmania spp.) were detected in rats. Blastocystis ST1, ST3, and ST8 and G. duodenalis sub-assemblage AII were identified in NHP, and Blastocystis ST1 in zookeepers. Giardia duodenalis isolates failed to be genotyped in human samples. In rats, four Cryptosporidium (C. muris, C. ratti, and rat genotypes IV and V), one G. duodenalis (assemblage G), and three Blastocystis (ST4) genetic variants were detected. Our results indicate high exposure of NHP to zoonotic protist species. Zoonotic transmission of Blastocysts ST1 was highly suspected between captive NHP and zookeepers.

West Nile virus (WNV) and Usutu virus (USUV) are zoonotic arboviruses. These flaviviruses are mainly maintained in the environment through an enzootic cycle involving mosquitoes and birds. Horses and humans are incidental, dead-end hosts, but can develop severe neurological disorders. Nevertheless, there is little data regarding the involvement of other mammals in the epidemiology of these arboviruses. In this study, we performed a serosurvey to assess exposure to these viruses in captive birds and mammals in a zoo situated in the south of France, an area described for the circulation of these two viruses. A total of 411 samples comprising of 70 species were collected over 16 years from 2003 to 2019. The samples were first tested by a competitive enzyme-linked immunosorbent assay. The positive sera were then tested using virus-specific microneutralization tests against USUV and WNV. USUV seroprevalence in birds was 10 times higher than that of WNV (14.59% versus 1.46%, respectively). Among birds, greater rhea (Rhea Americana) and common peafowl (Pavo cristatus) exhibited the highest USUV seroprevalence. Infections occurred mainly between 2016-2018 corresponding to a period of high circulation of these viruses in Europe. In mammalian species, antibodies against WNV were detected in one dama gazelle (Nanger dama) whereas serological evidence of USUV infection was observed in several Canidae, especially in African wild dogs (Lycaon pictus). Our study helps to better understand the exposure of captive species to WNV and USUV and to identify potential host species to include in surveillance programs in zoos.

BACKGROUND: Culicoides biting midges are biological vectors of arboviruses including bluetongue virus (BTV), Schmallenberg virus (SBV) and African horse sickness virus (AHSV). Zoos are home to a wide range of \'at risk\' exotic and native species of animals. These animals have a high value both in monetary terms, conservation significance and breeding potential. To understand the risk these viruses pose to zoo animals, it is necessary to characterise the Culicoides fauna at zoos and determine which potential vector species are feeding on which hosts.
METHODS: Light-suction traps were used at two UK zoos: the Zoological Society of London (ZSL) London Zoo (LZ) and ZSL Whipsnade Zoo (WZ). Traps were run one night each week from June 2014 to June 2015. Culicoides were morphologically identified to the species level and any blood-fed Culicoides were processed for blood-meal analysis. DNA from blood meals was extracted and amplified using previously published primers. Sequencing was then carried out to determine the host species.
RESULTS: A total of 11,648 Culicoides were trapped and identified (n = 5880 from ZSL WZ; n = 5768 from ZSL LZ), constituting 25 different species. The six putative vectors of BTV, SBV and AHSV in northern Europe were found at both zoos and made up the majority of the total catch (n = 10,701). A total of 31 host sequences were obtained from blood-fed Culicoides. Culicoides obsoletus/C. scoticus, Culicoides dewulfi, Culicoides parroti and Culicoides punctatus were found to be biting a wide range of mammals including Bactrian camels, Indian rhinoceros, Asian elephants and humans, with Culicoides obsoletus/C. scoticus also biting Darwin\'s rhea. The bird-biting species, Culicoides achrayi, was found to be feeding on blackbirds, blue tits, magpies and carrion crows.
CONCLUSIONS: To our knowledge, this is the first study to directly confirm blood-feeding of Culicoides on exotic zoo animals in the UK and shows that they are able to utilise a wide range of exotic as well as native host species. Due to the susceptibility of some zoo animals to Culicoides-borne arboviruses, this study demonstrates that in the event of an outbreak of one of these viruses in the UK, preventative and mitigating measures would need to be taken.

One captive musophagid bird at a zoological garden in Japan showed clinical symptoms and was found to be infected with avian haemosporidia. We subsequently collected blood from all musophagid birds kept in the garden and examined for avian haemosporidia using both microscopic and molecular examination. Only Haemoproteus gametocytes were observed in the blood of two Guinea turaco (Tauraco persa). Three genetic lineages of Haemoproteus were identified from three Guinea turacos and one genetic lineage of Leucocytozoon was identified from a grey plantain-eater (Crinifer piscator). Detected Haemoproteus lineages were all identical and completely different from those previously reported in Japan, suggesting that these birds were infected in their original habitat. This is the first record of Haemoproteus infection in Guinea turacos.

The detection and documentation of pathogenic Leptospira serovars in wild captive and zoological garden animals are scarce in literature from Nigeria. The knowledge of the prevalence of prevalence of pathogenic Leptospira serovars in these animals as a zoonotic risk to workers, zoo visitors and the general public is essential. This investigation was carried out on archival kidney and liver samples of captive and Zoological Garden animals (66) of an institutional facility, submitted for necropsy to the Department of Veterinary Pathology between the periods of 2010-2015. The gross diagnosis reports were obtained from the necropsy records, detection of pathogenic Leptospira serovars was by Warthin Starry silver staining and immunohistochemistry techniques using standard methods. Six samples out of the sixty-six samples were positive for leptospira four samples were positive by silver stain method, while two samples were positive by immunohistochemistry. In this study, serovar Pomona and grippotyphosa were detected in the foxes while serovar Pomona was detected in the horse. This study has revealed the presence of pathogenic leptospires in some captive wild and zoological garden animals.

The epidemiological cycle of zoonotic phlebotomine-borne Leishmania infantum is a complex system in which domestic animals and wildlife interact and participate in its maintenance and transmission. In this study, we combined entomological surveillance, xenomonitoring of L. infantum and identification of host feeding sources of engorged females to investigate the potential contribution of a periurban wildlife park to leishmaniosis in neighbouring residential areas. Overall, 7,309 sand flies were collected in 111 trap-days during the summers of 2016-2018 in an endemic area in south-east Spain. Five different sand fly species were captured, with Phlebotomus perniciosus, the main L. infantum vector in this region, representing the most common species. Sand fly distribution was spatially heterogeneous in terms of species, sexes and female physiological stage (unfed, gravid and engorged females) and related to host distribution and management, and environmental features. None of the 602 sand flies analysed for L. infantum infection by kinetoplast real-time PCR were positive. We used molecular tools to identify the vertebrate hosts of sand flies and identified 17 host species, mainly mammals. Human DNA was not identified in engorged sand flies. This study provides evidence that wildlife parks in south-east Spain are ideal grounds for sand fly vectors but do not necessarily increase L. infantum infection risk to humans and dogs living in surrounding residential areas. This is probably because vectors feed mostly on non-L. infantum competent hosts and this should be investigated for a better understanding of the contribution of wildlife parks to the local epidemiology of L. infantum.