SARS-CoV-2 Surveillance between 2020 and 2021 of All Mammalian Species in Two Flemish Zoos (Antwerp Zoo and Planckendael Zoo).

The COVID-19 pandemic has led to millions of human infections and deaths worldwide. Several other mammal species are also susceptible to SARS-CoV-2, and multiple instances of transmission from humans to pets, farmed mink, wildlife and zoo animals have been recorded. We conducted a systematic surveillance of SARS-CoV-2 in all mammal species in two zoos in Belgium between September and December 2020 and July 2021, in four sessions, and a targeted surveillance of selected mammal enclosures following SARS-CoV-2 infection in hippopotamuses in December 2021. A total of 1523 faecal samples from 103 mammal species were tested for SARS-CoV-2 via real-time PCR. None of the samples tested positive for SARS-CoV-2. Additional surrogate virus neutralisation tests conducted on 50 routinely collected serum samples from 26 mammal species were all negative. This study is the first to our knowledge to conduct active SARS-CoV-2 surveillance for several months in all mammal species of a zoo. We conclude that at the time of our investigation, none of the screened animals were excreting SARS-CoV-2.

Pulmonary Aspergillosis in Humboldt Penguins-Susceptibility Patterns and Molecular Epidemiology of Clinical and Environmental Aspergillus fumigatus Isolates from a Belgian Zoo, 2017-2022.

Aspergillus fumigatus is the main causative agent of avian aspergillosis and results in significant health problems in birds, especially those living in captivity. The fungal contamination by A. fumigatus in the environment of Humboldt penguins (Spheniscus humboldti), located in a Belgian zoo, was assessed through the analysis of air, water, sand and nest samples during four non-consecutive days in 2021-2022. From these samples, potential azole-resistant A. fumigatus (ARAF) isolates were detected using a selective culture medium. A total of 28 veterinary isolates obtained after necropsy of Humboldt penguins and other avian species from the zoo were also included. All veterinary and suspected ARAF isolates from the environment were characterized for their azole-resistance profile by broth microdilution. Isolates displaying phenotypic resistance against at least one medical azole were systematically screened for mutations in the cyp51A gene. A total of 14 (13.6%) ARAF isolates were identified from the environment (n = 8) and from Humboldt penguins (n = 6). The TR34/L98H mutation was observed in all resistant environmental strains, and in two resistant veterinary strains. To the best of our knowledge, this is the first description of this mutation in A. fumigatus isolates from Humboldt penguins. During the period 2017-2022, pulmonary aspergillosis was confirmed in 51 necropsied penguins, which reflects a death rate due to aspergillosis of 68.0%, mostly affecting adults. Microsatellite polymorphism analysis revealed a high level of diversity among environmental and veterinary A. fumigatus isolates. However, a cluster was observed between one veterinary isolate and six environmental strains, all resistant to medical azoles. In conclusion, the environment of the Humboldt penguins is a potential contamination source of ARAF, making their management even more complex.

SARS-CoV-2 Infection in Captive Hippos (Hippopotamus amphibius), Belgium.

Two adult female hippos in Zoo Antwerp who were naturally infected with SARS-CoV-2 showed nasal discharge for a few days. Virus was detected by immunocytochemistry and PCR in nasal swab samples and by PCR in faeces and pool water. Serology was also positive. No treatment was necessary.

Semen collection, evaluation, and cryopreservation in the bonobo (Pan paniscus).

BACKGROUND: Captive breeding of bonobos (Pan paniscus) has proven to be successful, but maintaining genetic diversity remains a challenge. Cryopreservation of semen is an important potential tool to maintain genetic diversity by preserving current genetic material for future use, as well as facilitating the transport and exchange of genetic material. This study aimed to develop a protocol for semen collection and cryopreservation in the bonobo. Semen was collected from four healthy adult bonobos under general anesthesia during management translocation procedures. Semen collection utilizing urethral catheterization was not successful (n = 1), however, all males (n = 4) responded well to rectal probe electro-ejaculation. Immediately after collection, ejaculates were evaluated for color and admixtures, volume, motility, and concentration. Eosin-Nigrosin staining was prepared to evaluate morphology and viability. Ejaculates were split into two equal volumes and cryopreserved in two different extenders, using a one-step and a two-step approach. Ejaculates were gradually cooled to 4 °C in two hours, subsequently stored in liquid nitrogen vapor for twenty minutes (0.25 ml straws), and finally dropped into liquid nitrogen. RESULTS: Pre-freeze evaluation showed thick, white samples with an average ejaculate volume of 450 µl (100-1000 µl), total motility of 59% (40-80%), viability of 69% (38-85%) and 58% (46-72%) normal spermatozoa. Mainly head (22%) and tail (19%) defects were detected on the Eosin-Nigrosin stain. Ejaculates were highly concentrated, nevertheless, due to the coagulum that caused high viscosity and non-homogenous fractions, only estimations of concentration could be made (1000 million/ml). After 24 h of storage, the post-thaw evaluation showed a loss of quality with an average post-thaw total motility of 15% (5-25%) using the one-step freezing medium, and 19% (5-30%) using the two-step medium. Average post-thaw viability was 15% (4-24%) and 21% (15-29%), respectively. CONCLUSIONS: This report on ejaculates from bonobos obtained by rectal probe electro-ejaculation shows that semen parameters of this species are not completely similar to those of its sibling species, the chimpanzee. Further studies are necessary to develop an optimal protocol for the processing and cryopreservation of bonobo spermatozoa.

Presence of Broad-Spectrum Beta-Lactamase-Producing Enterobacteriaceae in Zoo Mammals.

Broad-spectrum beta-lactamase (BSBL)-producing Enterobacteriaceae impose public health threats. With increased popularity of zoos, exotic animals are brought in close proximity of humans, making them important BSBL reservoirs. However, not much is known on the presence of BSBLs in zoos in Western Europe. Fecal carriage of BSBL-producing Enterobacteriaceae was investigated in 38 zoo mammals from two Belgian zoos. Presence of bla-genes was investigated using PCR, followed by whole-genome sequencing and Fourier-transform infrared spectroscopy to cluster acquired resistance encoding genes and clonality of BSBL-producing isolates. Thirty-five putatively ceftiofur-resistant isolates were obtained from 52.6% of the zoo mammals. Most isolates were identified as E. coli (25/35), of which 64.0% showed multidrug resistance (MDR). Most frequently detected bla-genes were CTX-M-1 (17/25) and TEM-1 (4/25). Phylogenetic trees confirmed clustering of almost all E. coli isolates obtained from the same animal species. Clustering of five isolates from an Amur tiger, an Amur leopard, and a spectacled bear was observed in Zoo 1, as well as for five isolates from a spotted hyena and an African lion in Zoo 2. This might indicate clonal expansion of an E. coli strain in both zoos. In conclusion, MDR BSBL-producing bacteria were shown to be present in the fecal microbiota of zoo mammals in two zoos in Belgium. Further research is necessary to investigate if these bacteria pose zoonotic and health risks.

Elephant Endotheliotropic Herpesvirus Is Omnipresent in Elephants in European Zoos and an Asian Elephant Range Country.

Elephant endotheliotropic herpesviruses (EEHVs) may cause acute, often lethal, hemorrhagic disease (EEHV-HD) in young elephants. Prevalence of EEHV in different elephant populations is still largely unknown. In order to improve diagnostic tools for the detection of EEHV infections and to obtain insight into its spread among elephants, we developed novel ELISAs based on EEHV1A gB and gH/gL. Performance of the ELISAs was assessed using sera from 41 European zoo elephants and 69 semi-captive elephants from Laos, one of the Asian elephant range countries. Sera from all (sub)adult animals tested (≥5 years of age) showed high reactivity with both gB and gH/gL, indicating that EEHV prevalence has been highly underestimated so far. Reactivity towards the antigens was generally lower for sera of juvenile animals (1 > 5 years). Only one (juvenile) animal, which was sampled directly after succumbing to EEHV-HD, was found to be seronegative for EEHV. The two other EEHV-HD cases tested showed low antibody levels, suggesting that all three cases died upon a primary EEHV infection. In conclusion, our study suggests that essentially all (semi-)captive (sub)adult elephants in European zoos and in Laos carry EEHV, and that young elephants with low antibody levels are at risk of dying from EEHV-HD.

Identification of fungal isolates by MALDI-TOF mass spectrometry in veterinary practice: validation of a web application.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable method to identify fungal isolates. The success of this approach relies on the availability of exhaustive databases, but the latter were built with a focus on human pathogens. We assessed a large in-house database of reference spectra and a dedicated web application for their suitability for use in veterinary laboratories. A panel of 290 mold and yeast isolates representing 69 different fungal species was isolated from various animals (including pets, cattle, and zoo animals) and identified using both MALDI-TOF MS and conventional techniques. The performance of the 2 methods was compared, and identifications were confirmed by DNA sequencing. MALDI-TOF MS allowed distinction between some closely related species and achieved 89% correct identification at the species level. In comparison, only 60% of the isolates were correctly identified with conventional approaches. Using this online application, MALDI-TOF MS thus appears to be a relevant alternative for the identification of fungal isolates encountered by animal health professionals.

Full-Genome Characterization of Simian T-Cell Leukemia Virus Type 1 Subtype b from a Wild-Born Captive Gorilla gorilla gorilla with T-Cell Lymphoma.

There are four lineages of primate T-cell lymphocytic viruses (human T-cell lymphocytic virus [HTLV]/simian T-cell lymphocytic virus [STLV]), which are further divided into subtypes. To date, there is only one full-length HTLV-1 subtype b genome available. Here, we report the genome of a new STLV-1 subtype b from a 43-year-old male gorilla with T-cell lymphoma.

Encephalomyocarditis virus in a captive Malayan tapir (Tapirus indicus).

A 5-month-old female captive Malayan tapir (Tapirus indicus) died suddenly without preceding symptoms. Gross necropsy revealed numerous white circular and linear foci in the myocard. Differential diagnosis all turned out negative, except for encephalomyocarditis virus. Histopathology revealed mineralisation of myocardial cells and interstitial infiltration of lymphocytes, plasma cells and less neutrophils. Encephalomyocarditis virus was detected by PCR. Although encephalomyocarditis virus occurs in many mammals, this is the first published description of this virus in a Malayan tapir.

Long-term assessment of glucosuria in captive okapi (Okapia johnstoni) after a dietary change.

Glucosuria in okapis (Okapia johnstoni) was first documented in 1980, yet the etiology remains unclear. In August 2006, an attempt to lower glucosuria in captive okapi by diet modification (omitting all fruit and adding unmolassed beet pulp) was started at the Antwerp Zoo. To study the possible relationship between glucosuria and diet, stress, and/or pregnancy, four okapis were monitored over a period of 4.5 yr. One animal, born in 2006, became glucosuric near the age of three. Three okapis were adults at the start of the study and had been glucosuric for more than 5 yr. The glucose/creatinine urinary ratio values of these four glucosuric animals did not change considerably over time despite dietary changes. Stress did not appear to influence glucosuria in these okapi. Urinary ratio decreased during the second half of pregnancy in two females. In conclusion, the diet change did not reduce glucosuria, but pregnancy appeared to lower urinary glucose in okapis.

Combining ethidium monoazide treatment with real-time PCR selectively quantifies viable Batrachochytrium dendrobatidis cells.

Detection of the lethal amphibian fungus Batrachochytrium dendrobatidis relies on PCR-based techniques. Although highly accurate and sensitive, these methods fail to distinguish between viable and dead cells. In this study a novel approach combining the DNA intercalating dye ethidium monoazide (EMA) and real-time PCR is presented that allows quantification of viable B. dendrobatidis cells without the need for culturing. The developed method is able to suppress real-time PCR signals of heat-killed B. dendrobatidis zoospores by 99.9 % and is able to discriminate viable from heat-killed B. dendrobatidis zoospores in mixed samples. Furthermore, the novel approach was applied to assess the antifungal activity of the veterinary antiseptic F10(®) Antiseptic Solution. This disinfectant killed B. dendrobatidis zoospores effectively within 1 min at concentrations as low as 1:6400.

Antibacterial therapeutics for the treatment of chytrid infection in amphibians: Columbus's egg?

BACKGROUND: The establishment of safe and effective protocols to treat chytridiomycosis in amphibians is urgently required. In this study, the usefulness of antibacterial agents to clear chytridiomycosis from infected amphibians was evaluated. RESULTS: Florfenicol, sulfamethoxazole, sulfadiazine and the combination of trimethoprim and sulfonamides were active in vitro against cultures of five Batrachochytrium dendrobatidis strains containing sporangia and zoospores, with minimum inhibitory concentrations (MIC) of 0.5-1.0 µg/ml for florfenicol and 8.0 µg/ml for the sulfonamides. Trimethoprim was not capable of inhibiting growth but, combined with sulfonamides, reduced the time to visible growth inhibition by the sulfonamides. Growth inhibition of B. dendrobatidis was not observed after exposure to clindamycin, doxycycline, enrofloxacin, paromomycin, polymyxin E and tylosin. Cultures of sporangia and zoospores of B. dendrobatidis strains JEL423 and IA042 were killed completely after 14 days of exposure to 100 µg/ml florfenicol or 16 µg/ml trimethoprim combined with 80 µg/ml sulfadiazine. These concentrations were, however, not capable of efficiently killing zoospores within 4 days after exposure as assessed using flow cytometry. Florfenicol concentrations remained stable in a bathing solution during a ten day period. Exposure of Discoglossus scovazzi tadpoles for ten days to 100 µg/ml but not to 10 µg florfenicol /ml water resulted in toxicity. In an in vivo trial, post metamorphic Alytes muletensis, experimentally inoculated with B. dendrobatidis, were treated topically with a solution containing 10 µg/ml of florfenicol during 14 days. Although a significant reduction of the B. dendrobatidis load was obtained, none of the treated animals cleared the infection. CONCLUSIONS: We thus conclude that, despite marked anti B. dendrobatidis activity in vitro, the florfenicol treatment used is not capable of eliminating B. dendrobatidis infections from amphibians.

Prevalence of methicillin-resistant Staphylococcus aureus in mammals of the Royal Zoological Society of Antwerp, Belgium.

Contrary to the numerous reports on methicillin-resistant Staphylococcus aureus (MRSA) in domestic animals, only three articles concerning zoo animals are documented in the literature. A skin infection of an African elephant (Loxodonta africana) calf was most likely acquired from an infected caretaker. Another zoo detected MRSA in the rumen content of a mouflon (Ovis aries), and, in a third facility, it was reported in a fistulous wound at the coronary band of a digit of an Indian rhinoceros (Rhinoceros unicornis). In the present study, which lasted 13 months and involved 93 different individual mammals that belonged to 40 species and 19 families housed in the Royal Zoological Society of Antwerp, Belgium, this study reports the absence of MRSA in swabs of nostrils, skins, conjunctiva, vulva, abscess, and arm rests in public spaces. Samples were enriched overnight and inoculated on a selective chromogenic medium.

Microsatellite typing of avian clinical and environmental isolates of Aspergillus fumigatus.

Aspergillosis is one of the most common causes of death in captive birds. Aspergillosis in birds is mainly caused by Aspergillus fumigatus, a ubiquitous and opportunistic saprophyte. Currently it is not known whether there is a link between the environmental isolates and/or human isolates of A. fumigatus and those responsible for aspergillosis in birds. Microsatellite typing was used to analyse 65 clinical avian isolates and 23 environmental isolates of A. fumigatus. The 78 genotypes that were obtained were compared with a database containing genotypes of 2514 isolates from human clinical samples and from the environment. There appeared to be no specific association between the observed genotypes and the origin of the isolates (environment, human or bird). Eight genotypes obtained from isolates of diseased birds were also found in human clinical samples. These results indicate that avian isolates of A. fumigatus may cause infection in humans.

Molecular identification of Entamoeba spp. in captive nonhuman primates.

This study describes the molecular identification of 520 Entamoeba-positive fecal samples from a large and diverse population of captive nonhuman primates (NHP). The results revealed the presence of Entamoeba histolytica (NHP variant only), E. dispar, E. moshkovskii, E. hartmanni, E. coli, and E. polecki-like organisms.

Molecular characterisation of Giardiaduodenalis in captive non-human primates reveals mixed assemblage A and B infections and novel polymorphisms.

Giardia is frequently detected in stools of non-human primates (NHP). However, a molecular identification has been rarely applied to Giardia isolates from NHP, and the distribution of the zoonotic assemblages A and B remains unclear. Moreover, little is known about the genetic variability among the isolates, although this may contribute to the elucidation of the different transmission pathways, including the role of NHP as a reservoir for human giardiasis. Therefore, 258 Giardia samples from 31 NHP species housed in nine zoological gardens and one sanctuary in Belgium and The Netherlands were characterised based on an assemblage-specific PCR targeting the triose phosphate isomerase (tpi) gene to identify both assemblage A and B infections. In addition, a multi-locus sequencing approach based on the glutamate dehydrogenase, the tpi and the beta-giardin genes was used to examine both the genetic variability and the ability to allocate these isolates to different NHP groups. Overall, assemblage B was the most prevalent (78.6%), but mixed assemblage A and B infections occurred in 32.7% of the samples. Sequencing of the isolates revealed the presence of new polymorphisms for both assemblages and at the three loci examined. The majority of the assemblage B isolates could not be grouped into recently described sub-assemblages, particularly at the tpi gene. Isolates could only be allocated to a specific group when polymorphisms of the three loci were combined. The results confirm that NHP are a potential reservoir for zoonotic transmission and advocate the use of assemblage-specific primers in molecular epidemiological surveys, as mixed infections are likely to be underestimated. The high level of heterogeneity within assemblages indicates that a revised nomenclature of these sub-assemblages is needed, but points out the potency of a multi-locus sequencing approach to unravel the complex epidemiology of Giardia duodenalis.

Occurrence and molecular characterization of Cryptosporidium and Giardia in captive wild ruminants in Belgium.

Both Cryptosporidium and Giardia are frequently found in the stool of domestic ruminants, especially young animals. Wild ruminants are also host to these protozoa, but the prevalence of these parasites in both free-ranging and captive nondomesticated ruminants needs to be further investigated. Moreover, the role of wild ruminants serving as reservoirs for these zoonotic parasites remains unclear. Therefore, a cross-sectional survey was conducted to estimate the occurrence of Cryptosporidium and Giardia in captive wild ruminants younger than 6 mo and to determine the potential of these animals to serve as reservoirs for these zoonotic parasites. A total of 67 captive wild ruminants belonging to 21 different animal species at the Antwerp Zoo (Belgium), along with 82 American bison (Bison bison) on a commercial breeding farm, were sampled for the detection of Cryptosporidium and Giardia, using a commercial immunofluoresence assay (Merifluor Cryptosporidium/Giardia IFA). The Cryptosporidium prevalence was 7.5% in the Antwerp Zoo animals and 3.7% in the bison from the breeding farm. All but two of the Cryptosporidium-positive animals were younger than 1 mo of age. Molecular characterization by amplification of the 70-kDa heat-shock protein and the 18S ribosomal DNA gene identified Cryptosporidium parvum in four animals of the Antwerp Zoo. The prevalence of Giardia was 8.9% in the Antwerp Zoo animals and 23.2% in the bison calves. Most Giardia-positive animals were older than 1 mo of age. Molecular characterization on the beta-giardin gene and the triose phosphate isomerase gene identified Giardia duodenalis assemblage A in the Antwerp Zoo and both G. duodenalis assemblage A and assemblage E in the bison calves. These findings indicate that both protozoan parasites are prevalent in captive wild ruminants and that these animals can serve as a potential reservoir for zoonotic transmission.

Gastrointestinal protozoa in non-human primates of four zoological gardens in Belgium.

Gastrointestinal parasites are important infectious causes of diarrhoea in captive non-human primates (NHP). However, prevalence data of gastrointestinal parasites in zoological gardens are scarce. Therefore, a cross-sectional survey was conducted to estimate the occurrence of gastrointestinal parasites in NHP of four zoological gardens in Belgium. Between August 2004 and April 2006, 910 faecal samples were collected from 222 animals housed in 39 groups. The 31 species involved were representatives of prosimians, New World (NW) monkeys, Old World (OW) monkeys and apes. Because individual sampling was impossible, a statistical simulation was performed to estimate a sufficient sample size. All samples were microscopically examined after an acetic acid-ether concentration. Differences in host species susceptibility were examined by non-parametric tests. Entamoeba spp. (44%) and Giardia spp. (41%) were the most prevalent species. Other parasites detected were Endolimax nana (36%), Chilomastix mesnili (21%), Balantidium coli (13%), Trichuris spp. (10%), Iodamoeba bütschlii (5%) and Strongyloides spp. (5%). Parasites for which a significant difference in susceptibility at the level of host taxonomy was noted were Entamoeba spp. (p<0.001) and C. mesnili (p<0.05). Samples containing Entamoeba spp. were the most prevalent in OW monkeys (p<0.0083). Samples collected from OW monkeys contained the highest number of parasite species (p<0.0083).

A DNA sequence capture extraction method for detection of Mycobacterium avium subspecies paratuberculosis in feces and tissue samples.

Culturing of Mycobacterium avium subspecies paratuberculosis (Map) remains difficult and is time consuming. An alternative for the rapid detection of Map in samples is PCR. We have developed a sensitive DNA-extraction method based on sequence capture for the rapid detection of M. avium subspecies paratuberculosis by PCR in fecal and tissue samples. The method detected 10(2)Map/g feces using spiked samples, and reached a diagnostic sensitivity of 33,7% compared to 22% for culture. Analysis of tissue samples gave 65 polymerase chain reaction (PCR)-positive (42.2%) and 49 culture-positive samples (31.8%). Therefore, the detection limit of the DNA-extraction is the same as previously reported for culture, the PCR assay could detect more positive samples than the culture method.