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.

Comparison of Cortisol Concentrations in Different Matrices in Alpine Ibex (Capra ibex) at the Zoo.

The usefulness of blood collection using venipuncture versus kissing bugs or medicinal leeches and the collection of saliva, faeces, hair, urine, and tears for measuring "immunoreactive" C (iC) concentration in Alpine ibexes was verified using commercial enzyme immunoassays. The mean value of serum C was highest in serum collected using venipuncture and lowest in serums collected using kissing bugs. Statistically significant differences were observed between venipuncture and kissing bugs and between leeches and kissing bugs. However, no statistically significant difference was found in C concentrations between samples collected with venipuncture and those collected with leeches. The highest mean value of C concentration was measured in serum (all three methods), followed by that in hair and faeces, and the lowest mean value was found in saliva. Statistically significant differences were found between saliva and faeces samples and between saliva and hair samples. The difference between the concentrations for faeces and hair was not statistically significant. A significant difference in C concentration between males and females was found in saliva. A significant difference in C concentration among different ages was measured in serum obtained using venipuncture in all three groups and in faeces between the groups older than ten years and younger than 10 months. Highly significant differences in C concentrations were also found between hair sampled in summer and hair sampled in autumn. Collecting tear and urine samples is a laborious procedure and is therefore less acceptable for C determination. Due to the small number of samples, statistical values are not given for these two matrices.

Validation of Medicinal Leeches (Hirudo medicinalis) as a Non-invasive Blood Sampling Tool for Hematology and Biochemistry Profiling in Mammals.

Blood sampling is a challenging procedure in many captive animals. Although manual restraint or anesthesia are usually possible, they entail intense stress and a high risk of injuries or organ failure. Blood sampling using medicinal leeches (Hirudo medicinalis) represents a promising non-invasive alternative to venipuncture; however, leech blood meal was to date used only for qualitative analyses such as genetic or serological screenings. Hence, the aim of this study was to evaluate the suitability of the leech blood sampling method for quantification of hematological and biochemical parameters. Medicinal leeches were manually applied on 67 zoo animals of eleven species, and control blood samples were obtained by venipuncture of the jugular vein. The leeches drew up to 20 ml of blood in 20 to 55 min. Although most hematological and biochemical parameters were significantly altered in leech-derived samples, their values showed strong (r = 0.62-0.79; 10/24 parameters) to very strong (r > 0.8; 13/24 parameters) correlations with venipuncture in all blood parameters, except for sodium (r = 0.39). As the parameter alterations and correlations were similar among species, simple cross-species regression formulas were sufficient to correct the alterations, thereby ensuring good repeatability between leeches and venipuncture in most parameters. Our data thus suggest that medicinal leeches can be used as a reliable non-invasive and stress-reducing alternative to standard venipuncture, even for quantitative assays. This opens new opportunities for a significant improvement to animal welfare in zoological gardens, conservation programmes, and ecophysiological research, where quantification of blood parameters is often needed.

Health Status and Stress in Different Categories of Racing Pigeons.

The influence of different stress parameters in racing pigeon flocks, such as the presence of diseases and environmental conditions at the time of the races, were described. A total of 96 racing pigeons from 4 pigeon flocks were examined, and health monitoring was carried out. No helminth eggs and coccidia were found. Trichomonas sp. was confirmed in subclinical form. Paramyxoviruses and avian influenza viruses were not confirmed, but circovirus infections were confirmed in all flocks. Chlamydia psittaci was confirmed in one flock. Blood samples were collected, and HI antibody titers against paramyxoviruses before and 25 days after vaccination were determined. To improve the conditions during racing and the welfare of the pigeons, critical points were studied with regard to stress factors during the active training season. Serum corticosterone levels were measured in the blood serum of four different categories of pigeons from each flock. Corticosterone levels were almost twice as high in pigeons from the category that were active throughout the racing season, including medium- and long-distance racing, compared to the other three categories that were not racing actively. Within five hours of the finish of a race, the average serum corticosterone level was 59.4 nmol/L in the most physically active category. The average serum corticosterone level in this category remained at 37.5 nmol/L one month after the last race.

Detection of Antibodies against Tick-Borne Encephalitis Virus in Zoo Animals Using Non-Invasive Blood Sampling with Medicinal Leeches (Hirudo medicinalis).

Reports on non-invasive blood sampling are limited, and there are only a few studies on using kissing bugs (Reduviidae) and medicinal leeches (Hirudo medicinalis) for hematology and biochemistry testing in various zoo animal species. The aim of this study was to evaluate the usefulness of non-invasive blood sampling with medicinal leeches for arbovirus epidemiological investigations in various animal species from one zoo collection. Medicinal leeches were manually applied on 35 animals of 11 species. Control blood samples were obtained by venipuncture of the jugular vein. Antibodies to tick-borne encephalitic virus (TBEV) were detected by using the immunoenzymatic method or an immunofluorescent assay (IFAT), depending on the animal species. One of the 35 animals (2.9%) was seropositive (Ovis aries), whereas the rest of the samples were seronegative in both methods of sampling (non-invasive by leeches vs. invasive by venipuncture). Blood sampling using medicinal leeches showed promising results. It is likely a good alternative to other more complex and invasive methods, and it can provide significant advancement in blood sampling for preventive medicine and epidemiological studies in zoo animals.

Detection of Antibodies Against Tick-Borne Encephalitis Virus and Other Flaviviruses in a Zoological Collection in Slovenia.

Monitoring infectious diseases is one of the most important pillars of preventative veterinary medicine in zoological collections. The zoo environment offers a great variety of different animal species living in proximity and in contact with small wild animals and vectors (e.g., ticks and mosquitos). In this context, tick-borne encephalitis virus (TBEV), Usutu virus (USUV), and West Nile virus (WNV) causing vector-borne diseases are emerging pathogens that raise concern. The aim of the study was to detect antibodies to selected flaviviruses in various animal species in the Ljubljana Zoo, Slovenia. In total, 874 sera from 96 animal species were tested for antibodies to TBEV by enzyme-linked immunosorbent assays (ELISA); positive samples were confirmed by a virus neutralization test (VNT) using TBEV, WNV, and USUV antigens. Antibodies to TBEV were detected by ELISA in 3.9% (34/874) of zoo animals, with 4% (30/753) in mammals and 5% (4/86) in birds; the sera of reptiles (n = 34) and amphibians (n = 1) were negative. Antibodies to TBEV were confirmed by VNT in 11 mammals; one bird was positive for both WNV and USUV. The mixture of exotic animal species and their contact with wild animals and vectors such as ticks and mosquitos suggest that screening of infectious diseases in zoo animals might provide good insight into the epizootological situation of the area. This is the first survey of TBEV, WNV, and USUV in a zoological collection in Slovenia.

Biosurveillance of Selected Pathogens with Zoonotic Potential in a Zoo.

Monitoring of infectious diseases is one of the most important pillars of preventive medicine in zoos. Screening for parasitic and bacterial infections is important to keep animals and equipment safe from pathogens that may pose a risk to animal and human health. Zoos usually contain many different animal species living in proximity with people and wild animals. As an epidemiological probe, 188 animals (122 mammals, 65 birds, and one reptile) from a zoo in Slovenia were examined for selected pathogens. Antibodies to Toxoplasma gondii and Neospora caninum were detected by ELISA in 38% (46/122) and 3% (4/122) of mammals, and in 0% (0/64) and 2% (1/57) of birds, respectively; the reptile (0/1) was negative. A statistically significant difference in T. gondii prevalence was found in Carnivora compared to Cetartiodactyla and primate antibodies to Encephalitozoon cuniculi were detected by IFAT in 44% (52/118) of mammals and 20% (11/56) of birds, respectively; the reptile (0/1) was negative. Herbivores had a higher chance of being infected with E. cuniculi compared to omnivores. Antibodies to Chlamydia abortus and Coxiella burnetii were not detected in any of the 74 tested zoo animals. The sera of 39 wild rodents found in the zoo were also examined; they were negative for all three parasites. The parasite T. gondii was detected by PCR in the tissue of two mute swans (Cygnus olor), three eastern house mice (Mus musculus), one yellow-necked field mouse (Apodemus flavicollis), and one striped field mouse (A. agrarius). Positive samples were genotyped by a single multiplex PCR assay using 15 microsatellite markers; one sample from a mute swan was characterized as type II. This micro-epidemiological study offers a better understanding of pathogens in zoo animals and an understanding of the role of zoos in biosurveillance.

A Sentinel Serological Study in Selected Zoo Animals to Assess Early Detection of West Nile and Usutu Virus Circulation in Slovenia.

Monitoring infectious diseases is a crucial part of preventive veterinary medicine in zoological collections. This zoo environment contains a great variety of animal species that are in contact with wildlife species as a potential source of infectious diseases. Wild birds may be a source of West Nile virus (WNV) and Usutu (USUV) virus, which are both emerging pathogens of rising concern. The aim of this study was to use zoo animals as sentinels for the early detection of WNV and USUV in Slovenia. In total, 501 sera from 261 animals of 84 animal species (including birds, rodents, lagomorphs, carnivores, ungulates, reptiles, equids, and primates) collected for 17 years (2002-2018) were tested for antibodies to WNV and USUV. Antibodies to WNV were detected by indirect immunofluorescence tests in 16 (6.1%) of 261 animals representing 10 species, which were sampled prior to the first active cases of WNV described in 2018 in Slovenia in humans, a horse, and a hooded crow (Corvus cornix). Antibodies to USUV were detected in 14 out of 261 animals tested (5.4%) that were positive prior to the first positive cases of USUV infection in common blackbirds (Turdus merula) in Slovenia. The study illustrates the value of zoological collections as a predictor of future emerging diseases.

Outbreak of Human Metapneumovirus Infection in Zoo, Slovenia.

We report a case of human metapneumovirus infection that spread from humans to chimpanzees and back to humans. Bronchopneumonia developed in 4 of 6 members of a chimpanzee family, and 2 subsequently died. The chimpanzees' keeper also became ill. Sequencing showed 100% identity between virus sequences from chimpanzees and the keeper.

RETROSPECTIVE STUDY OF CAUSES OF ANIMAL MORTALITY IN LJUBLJANA ZOO 2005-2015.

Zoos harbor large collections of diverse species, aiding in both conservation and education, as well as research in multiple scientific fields. However, the most common causes of death in zoo animals around the world remain unclear because few extensive reports or reviews are published on this topic. This information could greatly improve preventive veterinary medicine in zoologic gardens. This study provides a retrospective overview of the causes of death of animals from the Ljubljana Zoo in the years 2005-2015. During this period, a total of 353 animals were submitted for necropsy, of which 244 were mammals, 85 were birds, and 25 were reptiles. The causes of deaths were divided into infectious diseases (38%), dysfunctions of individual organs (20%), traumas (13%), parasitosis (7%), reproductive disorders (6%), metabolic disorders (3%), neoplastic disease (4%), and intoxications (4%). In some cases, the cause of death was unable to be determined (7%), most commonly because of autolysis of the body. The results of this retrospective study bring a general overview of the epizootiologic situation in the Ljubljana Zoo over an 11-yr period and valuable information to other zoos to optimize preventative plans and diagnostics.

Tear production, intraocular pressure and conjunctival bacterial flora in selected captive wild ruminants.

OBJECTIVE: Evaluation of tear production (Schirmer's tear test, STT) and measurement of intraocular pressure (IOP) were performed in a population of captive wild ungulates in a Slovenian ZOO during routine annual health check. ANIMALS STUDIED: In total, 10 fallow deer (Dama dama), 25 mouflons (Ovis aries musimon), 20 alpine ibexes (Capra ibex), and three alpine chamois (Rupicapra rupicapra) were included in the study. METHODS: Tear production was performed by Schirmer's tear test, IOP was measured with an applanation tonometer, and ophthalmological examination was conducted with slit-lamp biomicroscopy and indirect ophthalmoscopy. Conjunctival swabs were taken and submitted for aerobic bacteriology and for detection of Chlamydia spp. and Mycoplasma spp. tested by PCR. RESULTS: Average tear production (in mm/min) was 17.8 ± 3.16 for fallow deer, 17.9 ± 3.87 for mouflons, and 11.7 ± 3.87 for ibexes. Mean intraocular pressure (IOP, in mm Hg) was 14.1 ± 2.48 for fallow deer, 14.9 ± 2.20 for mouflons, and 13.1 ± 2.43 for ibexes. For chamois, average tear production and IOP were 14.5 ± 3.0 and 10.2 ± 2.5, respectively; this is the first record of STT I and IOP in chamois. Bacteriological swabs were positive for bacteria in 100% of the fallow deer, 56% of mouflons, 35% of ibexes, and 100% of chamois. Gram-positive bacteria were predominant. Moraxella spp., Chlamydia spp., and Mycoplasma spp. were not detected. CONCLUSION: The reported values were obtained in animals under manual restraint only to be applicative in similar conditions.

An eight-year survey of the intestinal parasites of carnivores, hoofed mammals, primates, ratites and reptiles in the Ljubljana zoo in Slovenia.

Problems with parasitic infections and their interspecies transmissions are common in zoological gardens and could pose serious health damage to captive animals. This study presents results of eight-year monitoring of intestinal parasites in animals from Zoo Ljubljana, Slovenia. A total of 741 faecal samples from 40 animal species were collected two to four times per year and examined microscopically. Intestinal parasites were detected in 45% of samples, with detection of helminths (Cestoda, Nematoda - Ascaridida, Enoplida, Strongylida, Oxyurida, Rhabditida and Trichurida) and protists (Apicomplexa and Ciliophora) in 25% and 13% of samples, respectively; mixed infection was found in 7% of samples. The mostly infected were ungulates (61%), followed by reptiles (44%), ratites (29%), primates (22%) and carnivores (7%). During the observation period, the number of infected animal species increased from 8 to 25. This is the first long-term monitoring study of intestinal parasites in zoo animals from Slovenia. Routine monitoring of parasitic infection and regular deworming and hygienic measures are necessary to prevent gastrointestinal infections in captive animals.

Occurrence of Bacterial and Viral Pathogens in Common and Noninvasive Diagnostic Sampling from Parrots and Racing Pigeons in Slovenia.

Airborne pathogens can cause infections within parrot (Psittaciformes) and pigeon (Columbiformes) holdings and, in the case of zoonoses, can even spread to humans. Air sampling is a useful, noninvasive method which can enhance the common sampling methods for detection of microorganisms in bird flocks. In this study, fecal and air samples were taken from four parrot holdings. Additionally, cloacal and oropharyngeal swabs as well as air samples were taken from 15 racing pigeon holdings. Parrots were examined for psittacine beak and feather disease virus (PBFDV), proventricular dilatation disease virus (PDDV), adenoviruses (AdVs), avian paramyxovirus type-1 (APMV-1), avian influenza virus (AIV), Chlamydia psittaci (CP), and Mycobacterium avium complex (MAC). MAC and AdVs were detected in three parrot holdings, CP was detected in two parrot holdings, and PBFDV and PDDV were each detected in one parrot holding. Pigeons were examined for the pigeon circovirus (PiCV), AdVs, and CP; PiCV and AdVs were detected in all investigated pigeon holdings and CP was detected in five pigeon holdings.