SARS-CoV-2 in Captive Nonhuman Primates, Spain, 2020-2023.

We conducted a serologic and molecular study to assess exposure of captive nonhuman primates (NHPs) to SARS-CoV-2 in Spain during the 2020-2023 COVID-19 pandemic. We found limited exposure of NHPs to SARS-CoV-2. Biosafety measures must be strictly maintained to avoid SARS-CoV-2 reverse-zoonotic transmission in the human-NHP interface.

Single or repeated immunization against GnRH fails to completely abolish spermatogenesis in dwarf bucks (Capra hircus).

In both captive wildlife and production animals is important to develop strategies for population control. Immunization against GnRH is an easy and inexpensive immunocastration method that reduces the concentration of testosterone and decreases sperm quality. However, its effectiveness depends on the species and repetition of the treatment. This study aimed to compare the effectiveness of a single treatment (initial immunization plus a booster with Improvac) vs repeated treatment (six doses of Improvac) to inhibit testicular function and maintain the contraceptive status during long periods in bucks. Three Dwarf bucks (Capra hircus) received two doses of Improvac, the first on Week 0, and the booster 4 weeks later (single immunization, group SI) while three Dwarf bucks received one dose of Improvac every 6 months during 3 consecutive years (repeated immunization, group RI). The other three Dwarf bucks remained untreated (control bucks, group CON). Bucks from RI had a greater decrease in scrotal circumference, testosterone concentration, male odor intensity, and sperm quality than SI bucks. However, there were no differences between SI and CON bucks in any of the variables studied. Overall, repeated treatment of Improvac decreased the testicular function of Dwarf bucks, although did not produce complete infertility. However, the repetition of the treatment produced more intensive negative effects, indicating that the strength of the effects of Improvac is rapidly lost in bucks.

Hepatitis E Virus Infections in Free-Ranging and Captive Cetaceans, Spain, 2011-2022.

Epidemiologic surveillance of hepatitis E virus in over 300 free-ranging and captive cetaceans in waters off Spain revealed extensive exposure to this pathogen. We suggest the persistent and widespread presence of hepatitis E in the marine environment off the coast of Spain may be driven by terrestrial sources of contamination.

Telomere shortening rate predicts species life span.

Telomere shortening to a critical length can trigger aging and shorter life spans in mice and humans by a mechanism that involves induction of a persistent DNA damage response at chromosome ends and loss of cellular viability. However, whether telomere length is a universal determinant of species longevity is not known. To determine whether telomere shortening can be a single parameter to predict species longevities, here we measured in parallel the telomere length of a wide variety of species (birds and mammals) with very different life spans and body sizes, including mouse (Mus musculus), goat (Capra hircus), Audouin's gull (Larus audouinii), reindeer (Rangifer tarandus), griffon vulture (Gyps fulvus), bottlenose dolphin (Tursiops truncatus), American flamingo (Phoenicopterus ruber), and Sumatran elephant (Elephas maximus sumatranus). We found that the telomere shortening rate, but not the initial telomere length alone, is a powerful predictor of species life span. These results support the notion that critical telomere shortening and the consequent onset of telomeric DNA damage and cellular senescence are a general determinant of species life span.

Immunocontraception of male and female giraffes using the GnRH vaccine Improvac®.

The aim of this study was to develop protocols for contraception in both sexes of giraffes (Giraffa camelopardalis) by using the GnRH vaccine Improvac®. We evaluated the success of immunization by analyzing fecal reproductive hormone metabolites in female (n = 20) and male (n = 9) giraffes. Endocrine analysis provided the basis for the successful immunization protocol, as well as for assessing long-term effects. Reliable reduction of fecal steroid metabolites to baseline levels in female giraffes was achieved with three, and in males with four or five injections at 4-week intervals. Effective booster injections were administered at 2-month intervals in the first year of treatment and at three to 4-month intervals in the following years. In addition to endocrine analysis, we determined vaccination efficacy in bulls by assessing testicular atrophy. Long-term (>2 years) use in females was often accompanied by prolonged periods of persistent corpus luteum activity, although normal cycles were not observed. Problems might occur with reversibility, because in a few males and females, even after more than 2 years since treatment had been stopped, fecal hormone metabolites have not returned to pretreatment levels. The results are somewhat ambiguous, as reproduction can be suppressed by use of Improvac®, but the question of reversibility remains unsolved.

Composition of marsupial zona pellucida: a molecular and phylogenetic approach.

The zona pellucida (ZP) is an extracellular matrix that surrounds mammalian oocytes. In eutherians it is formed from three or four proteins (ZP1, ZP2, ZP3, ZP4). In the few marsupials that have been studied, however, only three of these have been characterised (ZP2, ZP3, ZP4). Nevertheless, the composition in marsupials may be more complex, since a duplication of the ZP3 gene was recently described in one species. The aim of this work was to elucidate the ZP composition in marsupials and relate it to the evolution of the ZP gene family. For that, an in silico and molecular analysis was undertaken, focusing on two South American species (gray short-tailed opossum and common opossum) and five Australian species (brushtail possum, koala, Bennett's wallaby, Tammar wallaby and Tasmanian devil). This analysis identified the presence of ZP1 mRNA and mRNA from two or three paralogues of ZP3 in marsupials. Furthermore, evidence for ZP1 and ZP4 pseudogenes in the South American subfamily Didelphinae and for ZP3 pseudogenes in two marsupials is provided. In conclusion, two different composition models are proposed for marsupials: a model with four proteins (ZP1, ZP2 and ZP3 (two copies)) for the South American species and a model with six proteins (ZP1, ZP2, ZP3 (three copies) and ZP4) for the Australasian species.

ATYPICAL PRESENTATION OF CRYPTOCOCCUS NEOFORMANS IN A KOALA (PHASCOLARCTOS CINEREUS): A MAGNETIC RESONANCE IMAGING AND COMPUTED TOMOGRAPHY STUDY.

Cryptococcosis is a worldwide and potentially fatal mycosis documented in wild and captive koalas ( Phascolarctos cinereus ) caused by Cryptococcus neoformans . Though mainly a subclinical disease, when the nasal cavity is affected, epistaxis, mucopurulent nasal discharge, dyspnea, and facial distortion may occur. This report describes a case of cryptococcosis in a koala where unilateral exophthalmos was the only evident clinical sign and magnetic resonance imaging and computed tomography findings are described. Both advanced imaging techniques should be considered as standard and complementary techniques for nasal cavity evaluation in koalas.

Antifungal Susceptibility Testing of Ascomycetous Yeasts Isolated from Animals.

Recent studies suggest that antifungal resistance in yeast isolates of veterinary origin may be an underdiagnosed threat. We tested a collection of 92 ascomycetous yeast isolates that were obtained in Spain from birds, mammals and insects for antifungal susceptibility. MICs to amphotericin B and azoles were low, and no resistant isolates were detected. Despite these results, and given the potential role of animals as reservoirs of resistant strains, continuous monitoring of antifungal susceptibility in the veterinary setting is recommended.

Water Sources in a Zoological Park Harbor Genetically Diverse Strains of Clostridium Perfringens Type A with Decreased Susceptibility to Metronidazole.

The presence of Clostridium perfringens in water is generally regarded as an indicator of fecal contamination, and exposure to waterborne spores is considered a possible source of infection for animals. We assessed the presence and genetic diversity of C. perfringens in water sources in a zoological park located in Madrid (Spain). A total of 48 water samples from 24 different sources were analyzed, and recovered isolates were toxinotyped, genotyped by fluorophore-enhanced repetitive polymerase chain reaction (rep-PCR) fingerprinting and tested for antimicrobial susceptibility. C. perfringens was recovered from 43.8 % of water samples and 50 % of water sources analyzed. All isolates (n = 70) were type A and 42.9 % were ß2-toxigenic (i.e., cpb2+), but none contained the enterotoxin-encoding gene (cpe). Isolates belonged to 15 rep-PCR genotypes and most genetic diversity (88 %) was distributed among isolates obtained from the same sample. Most isolates displayed intermediate susceptibility (57.1 %; MIC = 16 µg ml-1) or resistance (5.7 %; MIC ≥ 32 µg ml-1) to metronidazole. No resistance to other antimicrobials was detected, although some isolates showed elevated MICs to erythromycin and/or linezolid. Finally, a marginally significant association between absence of cpb2 and decreased susceptibility to metronidazole (MIC ≥ 16 µg ml-1) was detected. In conclusion, our results reveal a high prevalence of C. perfringens type A in the studied water reservoirs, which constitutes a health risk for zoo animals. The elevated MICs to metronidazole observed for genetically diverse isolates is a cause of additional concern, but more work is required to clarify the significance of reduced metronidazole susceptibility in environmental strains.

Aggressive behavior and hair cortisol levels in captive Dorcas gazelles (Gazella dorcas) as animal-based welfare indicators.

Ensuring welfare in captive wild animal populations is important not only for ethical and legal reasons, but also to maintain healthy individuals and populations. An increased level of social behaviors such as aggression can reduce welfare by causing physical damage and chronic stress to animals. Recently, cortisol in hair has been advanced as a non-invasive indicator to quantify long-lasting stress in many species. The sensitivity of social behavior and hair cortisol concentration was evaluated in several groups of dorcas gazelles (Gazella dorcas). Four different groups of gazelles from three different zoos were observed and the expression of intra-specific affiliative and negative social behaviors was assessed across the different groups. Hair samples were taken from sub-groups of animals and analyzed for cortisol concentrations. Significant differences between groups of dorcas gazelles were found in frequency of negative social behavior and hair cortisol concentration. Despite the low sample size, these two parameters had a positive Spearman correlation coefficient (rs = +0.80, P = 0.20). These results suggest that hair cortisol levels are sensitive to differences in the social structure of dorcas gazelles. Zoo Biol. 35:467-473, 2016. © 2016 Wiley Periodicals, Inc.

DETECTION OF COXIELLA BURNETII INFECTION IN A SAHARAWI DORCAS GAZELLE (GAZELLA DORCAS NEGLECTA).

Coxiella burnetii, the causative agent of Q fever, can infect a wide range of host species, but limited information exists on the occurrence and implications of infection in wild species. This study describes a natural infection in a population of dorcas gazelles ( Gazella dorcas ) from a zoo. A 9-yr-old male Saharawi dorcas gazelle ( Gazella dorcas neglecta) tested positive on enzyme-linked immunosorbent assay (ELISA) and fecal polymerase chain reaction (PCR). Despite treatment with oxytetracycline, the animal did not clear the infection after 6 mo, as confirmed by a PCR test on a semen sample. This is the first report of a Saharawi dorcas gazelle infection with C. burnetii and the first time that C. burnetii was detected in semen from a zoo animal, suggesting the possibility of venereal transmission in captive wild species. This may have major implications for management of zoo populations, particularly in endangered species.

Central corneal thickness and intraocular pressure in captive black-footed penguins (Spheniscus dermersus).

OBJECTIVE: To determine the central corneal thickness (CCT) by ultrasonic pachymetry and the effect of these values on the measurements of intraocular pressures (IOP) with rebound tonometry (TonoVet(®) ) in a captive flock of black-footed penguins (Spheniscus dermersus). Variations in CCT by age and weight, and variations in IOP by age were compared. ANIMAL STUDIED: Both eyes of 18 clinically normal black-footed penguins (Spheniscus dermersus) were used. PROCEDURE: The IOP was measured by the TonoVet(®) in both eyes of all the penguins. CTT measurements were performed 5 min later in all eyes using an ultrasound pachymeter. RESULTS: The mean IOP values ± SD were 31.77 ± 3.3 mm Hg (range of mean value: 24-38). The mean CCT values were 384.08 ± 30.9 µm (range of mean value: 319-454). There was no correlation between IOP and CCT values (P = 0.125). There was no difference in CCT measurements by age (P = 0.122) or weight (P = 0.779). A correlation was observed (P = 0.032) between IOP values and age. The coefficient of correlation was negative (ρ = -0.207). CONCLUSIONS: Ultrasound pachymetry has shown to be a reliable and easy technique to measure CCT in penguins. No correlation was observed between IOP and CCT values in this study. IOP showed a significant but weak decrease as age increased in the black-footed penguin.

Characterisation of cataracts and other ophthalmic findings in various species of captive penguins.

BACKGROUND: The identification of ophthalmic diseases that affect vision and/or cause discomfort should be a priority in captive penguins to improve their overall health and quality of life. METHODS: A routine ophthalmological examination was performed on 80 clinically normal penguins (160 eyes), and any lesions observed were recorded. RESULTS: Ocular lesions were frequent (56% of penguins), with cataracts being the most common (48.8% of penguins). Cortical cataracts (63% of eyes) and posterior subcapsular cataracts (27.4%) were the most commonly occurring. All maturation stages were found; incipient cataracts (52.1% of eyes with cataracts) were predominant, while Morgagnian cataracts (8.2%) were the least frequent. A correlation existed between lenticular changes and increasing age. Uveitis was present in 43.8% of eyes with cataracts, and ectropion uveae was the predominant clinical sign. Other ocular findings included blepharitis (3.8% of all eyes), corneal leukoma (5.6%) and posterior lens subluxation (7.5%). LIMITATIONS: The small number of birds of some species prevented the comparison of ophthalmic findings between species. CONCLUSION: This study corroborates the high prevalence of ocular lesions in captive penguins. Cataracts were frequent and age related. Most cataracts were cortical, and the predominant maturation stage was incipient. Lens-induced uveitis was a common finding. Lowered intraocular pressure was related to cataract formation.

Wild Animals in Captivity: An Analysis of Parasite Biodiversity and Transmission among Animals at Two Zoological Institutions with Different Typologies.

We have conducted a 10-year-long coprological study of the animals housed in two zoological institutions (ZooAquarium and Faunia, Madrid, Spain) to assess the parasite biodiversity, prevalence, and their relation with host class, diet, and enclosure type (soil type and level of isolation from wild fauna). A total of 4476 faecal samples from 132 mammal species and 951 samples from 86 avian species were examined. The results indicated that only 12.8% of avian species had parasites at least once during the study period, whereas 62.1% of mammal species tested positive. Predominantly, protists (Entamoeba, flagellates, and ciliates) and nematodes (mainly Trichuris) were identified in the findings. Carnivorous species were primarily infected by nematodes, while herbivorous and omnivorous species were mainly infected by protists. The number of infected herbivorous and omnivorous species was significantly greater than carnivorous species. Differences were observed based on soil type (artificial, natural, mixed) and isolation level (isolated/accessible), but these differences were not statistically significant. Several parasites (Entamoeba spp., Giardia spp., Balantidoides coli, Trichuris spp.) could potentially be transmitted between humans and some mammals and birds. Regular animal analyses and a personnel health program in the institutions would minimise transmission risks between zoo animals, wildlife, and humans.

Conjunctival squamous cell carcinoma in a reindeer (Rangifer tarandus tarandus).

An 8-year-old female adult reindeer (Rangifer tarandus tarandus) was referred to the Veterinary Hospital of Madrid for evaluation of a conjunctival mass on the left eye which had been present for about 2 months. A surgical excision was performed and biopsy material submitted for light microscopic evaluation which confirmed the diagnosis of conjuctival squamous cell carcinoma. Nuclear p53 immunolabeling was found in 52% of the neoplastic cells. Follow-up examination at 12 months postsurgery did not reveal recurrence of this neoplasm. Conjunctival squamous cell carcinoma has not been reported previously in reindeer and seems to have similar characteristics to the one existing in bovine species.

Metabolic bone disease in juvenile koalas (Phascolartcos cinereus).

Due to climate restrictions in parts of North America and Europe, koalas (Phascolarctos cinereus) are housed indoors. Koala young (joeys) raised indoors are susceptible to the development of metabolic bone disease (MBD) due to a lack of exposure to natural ultraviolet light to themselves and their female parents (dams). In this retrospective study, radiographs from 27 koala joeys born at four zoos in North America and two zoos in Europe were evaluated for signs of MBD. Eight of the joeys were radiographically diagnosed with MBD and four additional joeys were considered suspect MBD cases; in two joeys absence or presence of MBD could not be determined. All joeys had mild to severe hip and shoulder dysplasia. There were significant associations between a lack of exposure to UV light and MBD development and between MBD and the degree of severity of hip and shoulder dysplasia. It is recommended to house breeding female koalas and their joeys outdoors whenever possible.

Presence of Aspergillus fumigatus with the TR34/L98H Cyp51A mutation and other azole-resistant aspergilli in the air of a zoological park.

Antifungal-resistant fungi, including Aspergillus fumigatus and other Aspergillus species, pose an urgent threat to human and animal health. Furthermore, the environmental route of azole resistance selection due to the widespread use of azole fungicides in crop protection and other applications is a major public health issue. Although environmental surveillance of fungi is frequently performed in many zoological parks and wildlife rehabilitation centers, the antifungal susceptibility of recovered isolates is only rarely analyzed, which precludes a clear assessment of the threat posed by these fungi to captive animals. In this study, we assessed the presence of airborne azole-resistant Aspergillus spp., including the so-called 'cryptic species' (i.e., species which are phenotypically similar to more well-known aspergilli but clearly constitute different phylogenetic lineages) in a zoological park located in the city of Madrid, Spain. In general, our results revealed a low prevalence A. fumigatus and cryptic aspergilli with decreased susceptibility to azoles. However, we detected an A. fumigatus isolate with the TR34/L98H mutation in the gene encoding the lanosterol 14α-demethylase (Cyp51A), consisting of a tandem repeat of 34 base pairs in the promoter region and a lysine to histidine substitution at codon 98. Notably, this TR34/L98H mutation has been linked to the environmental route of azole resistance selection, thus highlighting the 'One Health' dimension of the emerging problem of antifungal resistance. In this context, continuous environmental surveillance of azole-resistant aspergilli in zoological parks and other similar animal facilities is recommended.

First isolation of the anamorph of Kazachstania heterogenica from a fatal infection in a primate host.

We describe the isolation of the anamorph of the ascomycetous yeast Kazachstania heterogenica from a fatal infection in a 2 year, 9-month-old female white-handed gibbon (Hylobates lar). The yeast was observed in histological sections (lung and intestine) and co-isolated with the bacterium Escherichia coli from different internal organs. This is the first report of the recovery of this yeast from a fatal infection in a primate host.

DNA integrity and viability of testicular cells from diverse wild species after slow freezing or vitrification.

Introduction and objective: Cryopreservation of testicular tissues offers new possibilities to protect endangered species, genetically valuable individuals or even the fertility potential of prepubertal individuals who have died unexpectedly. However, the use of this technique still remains a challenge. In this study, slow freezing and vitrification of testicular tissue was investigated to find out which cryopreservation method could better preserve the viability and DNA integrity of testicular germ cells in diverse wild species. Methods: Testes were obtained post-mortem from 18 artiodactyls (wild boar, roe deer, dwarf goat, mhor gazelle, European mouflon, African forest buffalo, Malayan tapir, dorcas gazelle, Iberian ibex, gnu, red river hog), 5 primates (colobus monkey, capuchin monkey, mandrill), 8 carnivores (gray wolf, Persian leopard, binturong, European mink, American black bear, suricata), and 2 rodents (Patagonian mara). The testicles belonged to adult individuals and were cut into small pieces and cryopreserved by needle immersed vitrification or uncontrolled slow freezing using a passive cooling device. After warming or thawing, testicular tissues were enzymatically digested and two germ cell types were differentiated based on their morphology: rounded cells (spermatogonia, spermatocytes, and early spermatids) and elongated cells (elongated spermatids and spermatozoa). Cell viability was assessed by SYBR-14/propidium iodide while DNA fragmentation by TUNEL assay with fluorescence microscope. Results and discussion: Our preliminary results revealed that our uncontrolled slow freezing method better preserved the viability and DNA integrity of elongated cells than vitrification. Such trend was observed in all species, being significant in artiodactyls, carnivores, and primates. Similarly, the viability and DNA integrity of rounded cells was also better maintained in primates by uncontrolled slow freezing, while in carnivores, vitrification by needle immersion showed better results in this type of cells. In artiodactyls and rodents both techniques preserved the viability of rounded cells in a similar manner, although the DNA integrity of these cells was greater after needle immersed vitrification in artiodactyls. Conclusions: In conclusion, the effectiveness of each cryopreservation method is affected by the phylogenetic diversity between species and cell type.

Monitoring of bluetongue virus in zoo animals in Spain, 2007-2019.

Bluetongue (BT) is an emerging and re-emerging communicable vector-borne disease of animal health concern. A serosurvey was performed to assess exposure to BT virus (BTV) in zoo animals in Spain and to determine the dynamics of seropositivity in longitudinally sampled individuals during the study period. Serum samples were collected from 241 zoo animals belonging to 71 different species in five urban zoos (A-E) in Spain between 2007 and 2019. Twenty-four of these animals were longitudinally surveyed at three of the sampled zoos (zoos B, C and E) during the study period. Anti-BTV antibodies were found in 46 (19.1%; 95% CI: 14.1-24.1) of the 241 captive animals analysed by commercial ELISA. A virus neutralization test confirmed specific antibodies against BTV-1 and BTV-4 in 25 (10.7%; 95% CI: 6.7-14.6) and five (3.0%; 95% CI: 0.3-4.0) animals, respectively. Two of the 24 longitudinally sampled individuals (one African elephant (Loxodanta africana) and one aoudad (Ammotragus lervia)) showed anti-BTV antibodies at all samplings, whereas seroconversions were detected in one mouflon (Ovis aries musimon) in 2016, and one Asian elephant (Elephas maximus) in 2019. To the best of the authors' knowledge, this is the first large-scale survey on BTV conducted in both artiodactyl and non-artiodactyl zoo species worldwide. The results confirm BTV exposure in urban zoo parks in Spain, which could be of animal health and conservation concern. Circulation of BTV was detected in yearling animals in years when there were no reports of BTV outbreaks in livestock. Surveillance in artiodactyl and non-artiodactyl zoo species could be a valuable tool for epidemiological monitoring of BTV.