Herpesvirus and Subsequent Usutu Virus Infection in a Great Grey Owl (Strix nebulosa) at the Ljubljana Zoo, Slovenia.

Herpesvirus (HV) has been known to cause disease in owls, with various clinical signs and outcomes for the last several decades. The HV DNA polymerase gene was detected in oropharyngeal and cloacal swabs of a male great grey owl (Strix nebulosa) in a zoological collection in Ljubljana, Slovenia. In the following 4 months, despite continuous HV detection in swabs, no clinical signs with a clear link to HV disease were observed. Hepatoprotective and immunostimulant therapies applied during this period did not prevent HV shedding. Therefore, peroral antiviral therapy with acyclovir (150 mg/kg q24 h for seven days) was performed, and the owl tested negative at the next sampling and remained negative for the next 8 months. After that, the owl again tested positive for HV presence, and the same protocol with antiviral therapy was performed. After 3 weeks with a negative test for HV presence, without any clinical signs of illness, the owl suddenly died because of Usutu virus (USUV) infection. Among all the owls at the zoo, interestingly, only the HV-positive great grey owl died because of USUV infection. The USUV sequence detected and obtained in this study clusters together with other Europe 2 sequences detected in neighboring countries. Our study shows the potential of acyclovir therapy in the prevention of herpesvirus shedding and, moreover, lowering the possibility for spreading HV to other owls and birds. To the best of our knowledge, this is the first report of HV presence and USUV infection in a great grey owl in Slovenia.

Encephalitozoon cuniculi Infection of Domestic Rabbits (Oryctolagus cuniculus) in Slovenia between 2017 and 2021.

Encephalitozoon cuniculi is a microsporidial parasite that primarily infects domestic rabbits (Oryctolagus cuniculus). It is the causative agent of encephalitozoonosis, a disease with an internationally recognized seroprevalence among rabbits. This study determines the presence, clinical manifestation, and serological status of encephalitozoonosis in pet rabbits in Slovenia using various diagnostic procedures. From 2017 to 2021, 224 pet rabbit sera were collected and tested for encephalitozoonosis with the indirect immunofluorescence assay. Immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against E. cuniculi were confirmed in 160 (65.6%) cases. Most seropositive rabbits suffered from neurological clinical signs or signs of gastrointestinal disorders such as recurrent hypomotilities, chronic weight loss, cachexia, or anorexia, and fewer showed clinical signs related to the urinary system or phacoclastic uveitis. A quarter of the positively tested rabbits presented without clinical signs. Hematological and biochemical blood analysis confirmed that seropositive animals had elevated globulin and deviated albumin levels in comparison to the normal reference values of non-infected animals. Furthermore, rabbits with neurological clinical signs showed statistically significant higher levels of globulins and total protein. Sixty-eight whole-body radiographs and thirty-two abdominal ultrasound reports were analyzed, looking for changes in the shape or size of the urinary bladder, presence of urinary sludge or uroliths, and any abnormalities related to the kidneys (shape, size, or nephrolites). The results suggest that neurological defects in the urinary bladder caused by E. cuniculi lead to a distended urinary bladder and consequently dysuria, incontinence, urine scalding, and sludgy urine.

Detection of Herpesviruses in Wild Bird Casualties in Slovenia.

The complete host range of avian herpesviruses in wild birds is unknown, and information about nucleotide sequences is available only in limited cases. The aim of this study was to detect the presence of herpesviruses in wild birds and to gain more information about their phylogenetic relationship. Oropharyngeal and cloacal swabs from 447 wild birds from 15 different orders presented as wildlife casualties were examined for herpesvirus presence with PCR targeting a fragment of the DNA polymerase gene. Herpesviruses were detected in oropharyngeal and/or cloacal swabs in 34 (7.5%) birds belonging to 11 species from six different avian orders: Accipitriformes, Charadriiformes, Columbiformes, Falconiformes, Passeriformes, and Strigiformes. The results of phylogenetic analysis showed that various herpesviruses sequences are present in the wild bird population. Some herpesviruses are host species-specific, whereas in some cases very similar sequences were detected through different avian orders, which confirms findings that herpesviruses are not always restricted to bird species. It seems that herpesvirus transmission could occur by predation from avian prey, and even by superpredation-for example, large owls, such as the Eurasian eagle owl (Bubo bubo) or Ural owl (Strix uralensis), preying on smaller raptors. This can lead to greater infection exposure and is in line with the fact that raptors were the most infected species group. Nevertheless, the individual or simultaneous detection of herpesviruses in oropharyngeal and cloacal swabs shows that both swab samples should be used for herpesvirus detection in wild birds.

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.

Evaluation of Welfare in Commercial Turkey Flocks of Both Sexes Using the Transect Walk Method.

The study was conducted between March and September 2019 in six meat-type turkey flocks with similar management standard procedures using the transect walk method. The concept of the method is based on visual observation of the birds while slowly walking across the entire farm in predetermined transects. Each flock was evaluated at three different times during the fattening cycle: at 3 to 4, 12 to 13, and 19 to 20 weeks of age, and total number of males and females that were immobile or lame, had visible head, vent, or back wounds, were small, featherless, dirty, or sick, had pendulous crop, or showed aggression toward birds or humans were recorded. At each visit, NH3 and CO2 were measured within the facilities. In the first assessment, the most frequently observed welfare indicators were small size (0.87%) and immobility (0.08%). Males showed a significantly higher prevalence of small size (p < 0.01), sickness (p < 0.05), and dirtiness (p < 0.1) compared to females. In the second assessment, the most common findings in both sexes were dirtiness (1.65%) and poor feather condition (1.06%), followed by immobility (0.28%). Males were significantly dirtier (p < 0.001), had more immobile birds (p < 0.01) and birds with vent wounds (p < 0.1), but had fewer sick birds (p < 0.05). In the last assessment, an increase in immobile, lame, sick, and dead birds was recorded, indicating an increase in health problems. Higher CO2 (3000 and 4433 ppm) and NH3 (40 and 27.6 ppm) values were noted only at the first assessment in two facilities. Further analyses showed that slightly elevated NH3 and CO2 levels did not influence the occurrence of welfare indicators. This study is the first description of the welfare of commercial turkey flocks in Slovenia.

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.

Transmission of SARS-CoV-2 from Human to Domestic Ferret.

We report a case of natural infection with severe acute respiratory syndrome coronavirus 2 transmitted from an owner to a pet ferret in the same household in Slovenia. The ferret had onset of gastroenteritis with severe dehydration. Whole-genome sequencing of the viruses isolated from the owner and ferret revealed a 2-nt difference.

DETECTION OF HERPESVIRUSES IN PASSERINE BIRDS CAPTURED DURING AUTUMN MIGRATION IN SLOVENIA.

Herpesviruses (HVs) were detected by PCR in the cloacal swabs of 0.76% (4/525) clinically healthy free-living passerine birds from 32 different species captured in mist nets in Slovenia during the 2014 and 2017 autumn migrations. Herpesviruses were detected in the Eurasian Blackcap (Sylvia atricapilla), the Common Blackbird (Turdus merula), and the Eurasian Blue Tit (Cyanistes caeruleus). Phylogenetic analysis of partial DNA polymerase gene nucleotide sequences of the HV strains showed a distant relationship with other alphaherpesviruses of birds. In the phylogenetic tree, the HVs detected were clustered together with HV detected in Sulphur-crested Cockatoo and Neotropic Cormorants, as well as with known HVs such as gallid HV1, psittacid HV1 and HV2, and passerine HV1. Different sequences of HVs with relatively low identity were detected in our study, suggesting that different HVs were circulating in passerines sampled during the autumn migration in Slovenia.

Spontaneous Tumors and Non-Neoplastic Proliferative Lesions in Pet Degus (Octodon degus).

In recent years, degus (Octodon degus), rodents native to South America, have been becoming increasingly popular as pet animals. Data about neoplastic diseases in this species are still sparse and mainly limited to single-case reports. The aim of this study was to present neoplastic and non-neoplastic proliferative changes in 16/100 pet degus examined at the Veterinary Faculty University of Ljubljana from 2010 to 2015 and to describe the clinic-pathological features of these lesions. Twenty different lesions of the integumentary, musculoskeletal, genitourinary and gastrointestinal systems were diagnosed: amongst these were 13 malignant tumors, six benign tumors, and one non-neoplastic lesion. Cutaneous fibrosarcoma was the most common tumor (7/16 degus). It was detected more often in females (6/7 degus) and lesions were located mainly in hind limbs. The gastrointestinal tract was frequently affected, namely with two malignant neoplasms - an intestinal lymphoma and a mesenteric mesothelioma, four benign tumors - two biliary cystadenomas, an oral squamous papilloma and a hepatocellular adenoma, and a single non-neoplastic proliferative lesion. In one animal, two organic systems were involved in neoplastic lesions.

Electrochemotherapy with Bleomycin of Different types of Cutaneous Tumours in a Ferret (Mustela Putorius Furo).

BACKGROUND: Mast cell tumour, sebaceous gland adenoma, and less common squamous papilloma are skin tumours in ferrets (Mustela putorius furo), and early excisional surgery is usually the treatment of choice. The aim of our study was to investigate the effectiveness of electrochemotherapy (ECT), a new, minimally invasive non-surgical method, as first treatment option of different types of ferret skin tumours located on surgically difficult sites. MATERIALS AND METHODS: A 5-year-old castrated male ferret with two cutaneous masses, presenting 4 months apart and a 7-year-old spayed female ferret with two cutaneous masses, that appeared simultaneously on two locations are presented. In the first patient, both masses were diagnosed as mast cell tumours, and in the second patient, squamous papilloma and sebaceous adenoma were diagnosed. One session of ECT with bleomycin injected intratumourally was applied in all tumours. RESULTS: Complete response (CR) of all tumours was obtained, without recurrence during observation period of 15 months after ECT for first tumour and 11 months after ECT of the tumour located on the right hock in first patient, and 8 months after treatment for the second patient. CONCLUSIONS: In present study, ECT with bleomycin proved to be safe and effective against different cutaneous tumours in ferrets. Due of good results, low cost and relatively easy procedure, ECT could be the treatment of choice instead of surgery for the selected skin tumours in ferrets.

Detection and Phylogenetic Analysis of Herpesviruses Detected in Wild Owls in Slovenia.

Herpesvirus (HV) was detected using PCR in the organs of eight of 55 wild owls (14.5%) from seven species that were found dead in various locations in Slovenia between 1995 and 2015. HV was detected in three species: the Eurasian eagle owl (Bubo bubo), Ural owl (Strix uralensis), and long-eared owl (Asio otus). Phylogenetic analysis of partial DNA polymerase gene nucleotide sequences showed that the detected HVs are similar to the avian and mammal alphaherpesviruses. Two sequences were very similar to known bird HV sequences. One sequence was identical to the columbid herpesvirus 1 (CoHV1) sequence, and the other was very similar to the gallid herpesvirus 2 (GaHV2) sequence. The phylogenetic tree revealed a lower similarity of the other six analyzed Slovenian sequences with the sequences of alphaherpesviruses of birds and mammals. This is the first study to report the detection of different HVs in owls.

Detección y análisis filogenético de herpesvirus detectados en búhos silvestres en Eslovenia Se detectaron virus herpes mediante PCR en los órganos de ocho de 55 búhos silvestres (14.5%) pertenecientes a siete especies y que se encontraron muertos en varios lugares de Eslovenia entre los años 1995 y 2015. Se detectaron virus herpes en tres especies: el búho real (Bubo bubo), cárabo uralense (Strix uralensis) y el búho chico (Asio otus). El análisis filogenético de las secuencias de nucleótidos parciales del gene de la polimerasa de ADN mostró que los virus herpes detectados son similares a los alphaherpesvirus aviares y de mamíferos. Dos secuencias fueron muy similares a las secuencias de virus herpes de aves conocidas. Una secuencia fue idéntica a la secuencia del herpesvirus 1 de palomas (CoHV1) y otra fue muy similar a la secuencia del herpesvirus 2 del pollo (GaHV2). El árbol filogenético reveló menores similitudes entre las otras seis secuencias analizadas de Eslovenia con las secuencias de alphaherpesvirus de aves y de mamíferos. Este es el primer estudio que informa la detección de diferentes virus herpes en búhos.

Uterine heterologous malignant mixed Müllerian tumor in a dwarf rabbit (Oryctolagus cuniculus).

Malignant mixed Müllerian tumor (MMMT) is a rare neoplasm of the female genital system. A case of MMMT in the uterus of an 8-year-old female dwarf rabbit, which died with clinical signs associated with severe acute dyspnea and anorexia, is described. At necropsy, an oval, firm tumor was found in each of the 2 uterine horns. Numerous metastases were scattered throughout the mediastinum, thoracic diaphragm, and all pulmonary lobes. Microscopically, the tumors consisted of a poorly demarcated, unencapsulated neoplasm, composed of closely associated carcinomatous and sarcomatous components and areas of osteosarcomatous differentiation. Metastases were composed entirely of the sarcomatous component with osteosarcomatous differentiation. Immunohistochemically, the neoplastic epithelial component was positive for cytokeratin and negative for α-smooth muscle actin (α-SMA), vimentin, and desmin. The sarcomatous component was diffusely and strongly positive for vimentin, focally positive for α-SMA (<20% of cells positive), and negative for desmin. The neoplasm was diagnosed as a heterologous MMMT with metastases to the lung, mediastinum, and thoracic diaphragm.

Zero prevalence of Clostridium difficile in wild passerine birds in Europe.

Clostridium difficile is an important bacterial pathogen of humans and a variety of animal species, where it can cause significant medical problems. The major public health concern is the possibility of inapparent animal reservoirs of C. difficile and shedding of bacteria to noninfected individuals or populations, as well as being a source of food contamination. Migrating birds can be a key epizootiological factor for transmission and distribution of pathogens over a wide geographic range. Therefore, the purpose of this study was to investigate whether migrating passerine birds can be a source of spread of C. difficile along their migration routes. Cloacal samples were taken from 465 passerine birds during their migration south over the Alps. Selective enrichment was used for detection of C. difficile. Clostridium difficile was not isolated from any of the samples, which indicates that migrating passerine birds are unlikely to serve as a reservoir and a carrier of C. difficile.

Molecular characterization of avian paramyxovirus type 1 (Newcastle disease) viruses isolated from pigeons between 2000 and 2008 in Slovenia.

Fourteen avian paramyxovirus type 1 (APMV-1; Newcastle disease) viruses isolated from dead free-living and domestic pigeons in Slovenia between 2000 and 2008 were analyzed by a molecular characterization of a part of the fusion protein gene, which included the region encoding the fusion protein cleavage site. Phylogenetic analysis indicated that the Slovene pigeon paramyxovirus type 1 (PPMV-1) viruses do not cluster together but instead are divided into two groups--4bi and 4bii--of sublineage 4b. Nine Slovenian strains were placed in group 4bii. Five other strains clustered together with PPMV-1 from group 4bi. The sequence of the fusion protein cleavage site of all Slovenian strains was typical for pathogenic APMV-1. The 112RRQKRF117 motif was present in the strains from group 4bii, whereas strains from group 4bi displayed the 112GRQKRF117 motif.