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.

Although much attention has been paid in the literature to the morphology of the tongue in various animal species, including some Felidae, it has not yet been described in detail in the vulnerable Neofelis nebulosa and Panthera leo bleyenberghi and the last concern Lynx lynx and Otocolobus manul. Therefore, the present study aimed to characterize the features of the tongue surface, lingual glands and lyssa in the above-mentioned four selected wild species of the Pantherinae and Felinae subfamilies. Macroscopic, histological, histochemical and ultrastructural analyzes were used in the present work. Comparative analyzes of the dorsal tongue surface showed the presence of mechanical lingual papillae on five subtypes of filiform papillae on the apex and body and conical papillae on the root of the tongue. Gustatory papillae in the four analyzed species were fungiform papillae and various numbers of vallate papillae. Foliate papillae were absent in P. leo bleyenberghi and L. lynx, while delicate smooth folds, which were separated by parallel grooves but without taste buds, were present in N. nebulosa. The vallate and foliate papillae were accompanied by lingual glands, which produced a serous secretion, whereas the mixed lingual glands of the lingual root were with a predominance of mucus secretions comparable to four captive Felidae species. In the median plane on the ventral surface of the apex under its epithelium and within the muscle fibers, the lyssa was also observed to a varying degree, with the least developed, and thought about the size of the entire tongue, was in P. leo bleyenberghi. The lyssa structure in the four species was dominated by adipose tissue. The obtained results contribute knowledge concerning the functional anatomy of the tongue in four selected Felidae species, especially in terms of comparative anatomy.

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.

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.

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.

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.