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In 2022/2023, Europe experienced its third consecutive season of high-pathogenicity avian influenza. During this period, the Czech Republic was again severely affected. For the first time, the number of culled birds approached one million, which was three times higher than in previous seasons. In parallel to the outbreaks in poultry, mass die-offs of gulls were also observed. In the present study, we performed whole-genome sequencing and phylogenetic analysis of 137 H5N1 strains collected in the Czech Republic in 2022/2023 (94.6% of all outbreaks or locations). The analysis revealed four distinct genotypes: AB, CH, BB and AF. Phylogenetic analysis suggested that the AF genotype persisted from the previous H5N1 season without reassortment. In addition, the genotype BB, which was detected mainly in gulls, showed a noticeable strain diversity at the local level. This virus was also responsible for a single outbreak in commercially bred turkeys. Finally, an interesting spatio-temporal cluster with three co-circulating H5N1 genotypes, AB, CH and AF, was identified with no evidence of intrasubtype reassortment. Highly sensitive molecular surveillance and the timely sharing of genomic sequences and associated metadata could greatly assist in tracking the spread and detecting molecular changes associated with the increased virulence of this potentially zoonotic pathogen.

Lyme disease, caused by some strains of bacterial spirochetes Borrelia burgdorferi sensu lato ( Bb sl), affects humans but also domestic animals including horses. The primary pathogens in horses in Europe are B. afzelii , B. garinii and B. burgdorferi sensu stricto. To our knowledge, there are no data available on the seropositivity of B. burgdorferi s.l. in horses from the Czech Republic. In this country, horses are mainly used for sport, breeding, and recreational riding in areas where vectors of B. burgdorferi s.l. are present, which is why they are frequently at risk of infection. The aim of the study was to detect anti-borrelia IgM and IgG antibodies in clinically healthy and sick horses from the Czech Republic and to evaluate the risk factors of infection. In total, sera of 262 horses (247 clinically healthy horses and 15 horses hospitalized due to symptoms of encephalitis/meningoencephalitis) were examined by an indirect sandwich enzyme-linked immunosorbent assay. Positivity of B. burgdorferi was 27% (66/247) in clinically healthy horses (21% IgM, 7% IgG and 3% IgM + IgG antibodies) and 20% (3/15) in horses with clinical signs (20% IgM, 7% IgG and 7% IgM + IgG). In the clinically healthy horses, positivity statistically differed ( p ≤ 0.05) only in Pony and Warmblood breeds, being the most affected at 32% and 30%, respectively, while other characteristics (sex, age, usage and localities) had no effect on positivity. This is the first survey of antibodies to B. burgdorferi s.l. in Czech horses showing that horses are exposed to ticks infected with B. burgdorferi s.l. This should be taken into account when making differential diagnoses in patients with non-specific symptoms to start with adequate therapy.

The aim of this study was to detect antibodies to Toxoplasma gondii and Neospora caninum in exotic animal species kept in three zoos in Slovakia. Antibodies to T. gondii and N. caninum were detected by commercial Enzyme-linked immunosorbent assay (ELISA, ID Screen Toxoplasmosis Indirect Multispecies and ID Screen Neospora caninum Indirect Multispecies, ID Vet, Montpellier, France). Antibodies to T. gondii and N. caninum were detected in 43% (24/56) and 5% (3/55) of animals, respectively. The three animals with N. caninum antibodies: two wolves (Canis lupus) and one Hartmann's mountain Zebra (Equus zebra hartmannae), were clinically healthy, and both wolves simultaneously had antibodies to T. gondii. The results of our study provide a picture of the recent circulation of T. gondii in three Slovakian zoos with the S/P (ratio of antibodies in the sample to antibodies in positive control) value higher than 200%, found in five animals (9%) indicating acute toxoplasmosis. The highest S/P value (296%) was detected in a Roloway monkey (Cercopithecus roloway), which was healthy without clinical signs, presuming that Roloway monkey is a species less susceptible to T. gondii infection. Results of our study showed the presence of T. gondii and N. caninum in Slovakian zoos, confirming recent T. gondii infections according to the high level of antibodies detected in five animals, referring to acute toxoplasmosis., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In 2021/2022, the re-emergence of highly pathogenic avian influenza (HPAI) occurred in Europe. The outbreak was seeded from two sources: resident and reintroduced viruses, which is unprecedented in the recorded history of avian influenza. The dominant subtype was H5N1, which replaced the H5N8 subtype that had predominated in previous seasons. In this study, we present a whole genome sequence and a phylogenetic analysis of 57 H5N1 HPAI and two low pathogenic avian influenza (LPAI) H5N1 strains collected in the Czech Republic during 2021/2022. Phylogenetic analysis revealed close relationships between H5N1 genomes from poultry and wild birds and secondary transmission in commercial geese. The genotyping showed considerable genetic heterogeneity among Czech H5N1 viruses, with six different HPAI genotypes, three of which were apparently unique. In addition, second-order reassortment relationships were observed with the direct involvement of co-circulating H5N1 LPAI strains. The genetic distance between Czech H5N1 HPAI and the closest LPAI segments available in the database illustrates the profound gaps in our knowledge of circulating LPAI strains. The changing dynamics of HPAI in the wild may increase the likelihood of future HPAI outbreaks and present new challenges in poultry management, biosecurity, and surveillance.

We report an outbreak of SARS-CoV-2 lineage alpha in gorillas and felid species in a zoo in Prague, Czech Republic. The course of illness and clinical signs are described, as are the results of characterization of these particular SARS-CoV-2 variants by next-generation sequencing and phylogenetic analysis. The putative transmission routes are also discussed., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

In 2020-2021, the second massive dissemination of a highly pathogenic avian influenza of the H5Nx subtype occurred in Europe. During this period, the virus caused numerous outbreaks in poultry, including in the Czech Republic. In the present study, we provide an insight into the genetic variability of the Czech/2021 (CZE/2021) H5N8 viruses to determine the relationships between strains from wild and domestic poultry and to infer transmission routes between the affected flocks of commercial poultry. For this purpose, whole genome sequencing and phylogenetic analysis of 70 H5N8 genomes representing 79.7% of the cases were performed. All CZE/2021 H5N8 viruses belonged to the 2.3.4.4b H5 lineage and circulated without reassortment, retaining the A/chicken/Iraq/1/2020 H5N8-like genotype constellation. Phylogenetic analysis suggested the frequent local transmission of H5N8 from wild birds to backyard poultry and extensive spread among commercial poultry farms. In addition, the analysis suggested one cross-border transmission event. Indirect transmission via contaminated materials was considered the most likely source of infection. Improved biosecurity and increased collaboration between field veterinarians and the laboratory are essential to limit the local spread of the virus and to reveal and interrupt critical routes of infection.

Canine distemper is a highly contagious viral disease in carnivores and represents a serious threat for both wild and domestic animals. The aim of our study was to monitor the occurrence of the canine distemper virus in wildlife from the Czech Republic, reveal the H gene heterogeneity in positive samples and perform subsequent phylogenetic analysis. In total, 412 wild animals of 10 species were included in the study: 219 red foxes ( Vulpes vulpes ), 79 European badgers ( Meles meles ), 47 European otters ( Lutra lutra ), 40 stone martens ( Martes foina ), 10 pine martens ( M. martes ), 7 raccoons ( Procyon lotor ), 5 undetermined martens ( Martes sp.), 2 wolves ( Canis lupus ), 1 European polecat ( Mustela putorius ), 1 free-ranging ferret ( Mustela putorius furo ), and 1 free-ranging American mink ( Neovison vison ). Most animals were found dead or were killed by hunters during hunting seasons in the years 2012-2020 and came from all 14 regions of the Czech Republic. In the animals that were hunted, symptoms such as apathy, loss of shyness or disorientation were reported. Canine distemper virus (CDV) was detected by real-time RT-PCR in the tissues of 74 (18%) of the animals, including 62 (28%) red foxes, 4 (10%) stone martens, 3 (43%) raccoons, 2 (20%) pine martens, 2 (2.5%) European badgers and 1 (20%) undetermined marten. There was a statistical difference in positivity among animal species ( p < 0.0001), regions ( p = 0.0057), and the years of sampling ( p = 0.0005). To determine the genetic characteristics of circulating variants of CDV in wildlife, 23 of 74 CDV variants were partially sequenced. Phylogenetic analysis showed that 21 variants belonged to the European lineage and two strains belonged to the European-Wildlife lineage. This study provides the first comprehensive overview of the prevalence and spatial distribution of CDV in wildlife in the Czech Republic, including molecular phylogenetic analysis of currently circulating CDV lineages.

Theileria equi and Babesia caballi are protozoan agents causing equine piroplasmosis, endemic in countries all over the world. The aim of this study was to detect antibodies to T. equi and B. caballi in horses in the Czech Republic and to investigate the origin of the infection. Blood sera from 711 horses were examined with competitive enzyme-linked immunosorbent assay; positive samples were verified with indirect fluorescence immunoassay. Antibodies to T. equi and B. caballi were detected in eight (1.1%) and three (0.4%) horses, respectively. Infection with T. equi was confirmed by PCR and sequencing in the blood of five serologically positive horses. An autochthonous origin of T. equi infection could not be excluded in two (0.3%) horses. Intensive movement of horses across European countries and the expanding occurrence of competent tick vector Dermacentor reticulatus in the Czech Republic create an increasing risk of establishing active foci of equine piroplasmosis in the country., (Copyright © 2021. Published by Elsevier B.V.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kvapil, Račnik, Kastelic, Pittermannová, Avšič-Zupanc, Bártová and Sedlák.)

The protozoan parasites Theileria equi and Babesia caballi, transmitted by ticks, cause equine piroplasmosis, the most prevalent tick-borne disease in equids. Trichinellosis is a worldwide food-borne zoonosis caused by helminth Trichinella spp. that can lead to serious disease in humans, with fatal outcome. Although the infection is rare in horses, it deserves attention due to the increasing use of horse meat as a source of protein for humans. Horse trichinellosis is caused by several Trichinella species, most commonly by T. spiralis. The aim of the study was to determine the prevalence of antibodies to T. equi, B. caballi and Trichinella spp. in equids from three states of Northern Nigeria. Serum samples were collected from 139 clinically healthy animals, comprising 115 horses and 24 donkeys. Antibodies to T. equi and B. caballi were detected in serum by competitive-inhibition enzyme-linked immunosorbent assay (cELISA) and antibodies to Trichinella spp. by ELISA. Antibodies to T. equi were detected in 34% of equids (41% horses and 0% donkeys), antibodies to B. caballi in 9% of equids (8% horses and 13% donkeys), and antibodies to Trichinella spp. in 4% of equids (4% horses and 0% donkeys). There was co-infection of T. equi and B. caballi in 1% of horses and co-infection of T. equi and Trichinella spp. in 2.6% of horses. This is the first report on seroprevalence of Trichinella spp. in equids from Northern Nigeria.

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.

The accurate identification of Taylorella equigenitalis strains is essential to improve worldwide prevention and control strategies for contagious equine metritis (CEM). This study compared 367 worldwide equine strains using multilocus sequence typing according to the geographical origin, isolation year and equine breed. The strains were divided into 49 sequence types (STs), including 10 described for the first time. Three major and three minor clonal complexes (CCs), and 11 singletons, were identified. The genetic heterogeneity was low (0.13 STs/strain) despite the wide diversity of geographical origins (n = 16), isolation years (1977-2018) and equine breeds (n = 18). It was highest outside Europe and in the 1977-1997 period; current major STs and CCs already existed before 1998. Previous data associated the major CC1 with the first CEM outbreaks in 1977-1978 in the United Kingdom, Australia and the United States, and revealed its circulation in France. Our study confirms its circulation in France over a longer period of time (1992-2018) and its distribution in Spain and Germany but not throughout Europe. In addition to CC1, relationships between non-European and European countries were observed only through ST4, ST17 and ST30. Within Europe, several STs emerged with cross-border circulation, in particular ST16 and ST46 from the major complexes CC2 and CC8. These results constitute a baseline for monitoring the spread of CEM outbreaks. A retrospective analysis of a higher number of strains isolated worldwide between 1977 and the early 2000s would be helpful to obtain an exhaustive picture of the original CEM situation., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Problems with parasitic infections are common in zoological gardens and circuses. In some animals it can lead to several disorders such as systemic disease, reproductive disorders (abortions and neonatal mortality), and even to death if severe illness is untreated. Thus, the aim of this study was to evaluate the prevalence of three common parasites in 74 animals from three zoos, and four circuses in Southern Italy. Antibodies to Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi were detected in 51%, 12%, and 20% of animals, respectively. Co-infections of T. gondii and N. caninum were reported in seven animals (9%) and co-infection of T. gondii and E. cuniculi in one animal. T. gondii, N. caninum and E. cuniculi seroprevalence differed in type of diet (P ≤ 0.0001; P ≤ 0.037 and P ≤ 0.004, respectively). T. gondii and E. cuniculi seroprevalence also differed in animal families (P ≤ 0.0001) and according to type of housing (P ≤ 0.003), respectively. Statistical differences were not found in other characteristics (gender, age, country of birth, origin, and contact with cats or dogs). This is the first serological study focusing on protozoan and microsporidian parasites in zoo and circus animals from Southern Italy and the first detection of antibodies to E. cuniculi in camels in Europe., (© 2018 International Society of Protistologists.)

We tested sera of 24 free-ranging European brown bears ( Ursus arctos) from six regions of Slovakia for antibodies to 10 viral agents. We tested sera by an indirect fluorescence antibody test for antibodies to canine distemper virus (CDV), canine coronavirus (CCV), canine parvovirus type 2 (CPV-2), canine adenovirus, canine parainfluenza virus type 2 (CPIV-2), and canine herpesvirus type 1 (CHV-1). We used an enzyme-linked immunosorbent assay for detection of antibodies to hepatitis E virus, bluetongue virus, West Nile virus (WNV), and Aujeszky's disease virus (ADV). We detected antibodies to CDV, CHV-1, CPV-2, CPIV-2, CCV, WNV, and ADV in seven (29%), three (12%), two (8%), two (8%), one (4%), one (4%), and one (4%) bear, respectively. Evidence of exposure of free-ranging European brown bears to CCV and ADV has not been reported.

Toxoplasma gondii is protozoan parasite with ability of causing disease in wide-spectrum of animals; many species of animals in captivity died of clinical toxoplasmosis. The monitoring of T. gondii antibodies in zoo animals can be an important indicator of T. gondii circulation in zoo. The aim of this study was to examine sera of animals from eight Czech zoos by latex agglutination test with statistical evaluation and detect T. gondii DNA in stray cats and rodents captured in the zoos. T. gondii antibodies were detected in 33% of 1043 zoo animals without statistical difference between birds (27%, n = 74) and mammals (33%, n = 969). In birds, the chance to be infected with T. gondii was higher in Accipitriformes (71%) compared to Pelecaniformes (6%) (p < 0.0001). In mammals, the chance to be infected with T. gondii was higher in Carnivora (63%) compared to Cetarodactyla (30%), Perissodactyla (26%), Primates (28%) and Rodentia (13%) (p < 0.0001) and higher in Felidae (70%) compared to Bovidae (28%) and Equidae (28%) (p < 0.0001). Mammals with carnivore/scavenger way of feeding were in a higher risk of T. gondii infection compared to herbivores and omnivores (p < 0.0001). T. gondii DNA was detected in tissue of one stray cat while in none of 77 rodents caught in zoo. This study is the first report on toxoplasmosis in zoos from the Czech Republic including seroepidemiology and molecular detection., (Copyright © 2018. Published by Elsevier B.V.)

Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi are important infectious agents, with T. gondii and E. cuniculi having zoonotic potential. There are two main clonal lineages (types I and II) of T. gondii in Europe, but little is known about genotypes of T. gondii in wild animals. The aim of our study was molecular detection of these three pathogens in tissues of wild red foxes ( Vulpes vulpes) from the Czech Republic. Using PCR (B1 gene), we detected T. gondii in 10% of the animals that we tested ( n=100); N. caninum and E. cuniculi were not detected. The T. gondii samples were genotyped by single multiplex PCR assay with 15 microsatellite markers. Five samples were successfully genotyped as genotype II, a unique finding for T. gondii isolated from red foxes from the Czech Republic.

Here, we present a comprehensive analysis of the H5N8/H5N5 highly pathogenic avian influenza (HPAI) virus strains detected in the Czech Republic during an outbreak in 2017. Network analysis of the H5 Hemagglutinin (HA) from 99% of the outbreak localities suggested that the diversity of the Czech H5N8/H5N5 viruses was influenced by two basic forces: local microevolution and independent incursions. The geographical occurrence of the central node H5 HA sequences revealed three eco-regions, which apparently played an important role in the origin and further spread of the local H5N8/HPAI variants across the country. A plausible explanation for the observed pattern of diversity is also provided.

[This corrects the article DOI: 10.1371/journal.pone.0151204.].

Neospora spp. and Toxoplasma gondii are considered to be a globally distributed parasites affecting wide range of warm-blooded animals. Neosporosis has caused clinical illness in horses and consumption of horse meat has been epidemiologically linked to clinical toxoplasmosis in humans. This study was conducted to determine Neospora spp. and T. gondii antibodies and risk factors of infection in horses and donkeys from three states of Nigeria. A total of 144 samples were collected from clinically healthy animals (120 horses and 24 donkeys). The sera were tested for antibodies to Neospora spp. and T. gondii by indirect fluorescence antibody test, a titer ≥ 50 was considered positive. Seroprevalence data were statistically analyzed, considering the variables of gender, age, use, state, origin of breed and type of management. Antibodies to Neospora spp. and T. gondii were detected in 8% horses with titers 50 and in 24% horses with titers 50-800, respectively. Co-infection of both parasites was proved in three horses (3%). Statistical differences were found only for T. gondii seroprevalence in horses with different use, locality, origin and management (p-value ≤ 0.05). Antibodies to T. gondii were detected in four (17%) of 24 donkeys with statistical difference (p-value ≤ 0.05) in animals of different use; antibodies to Neospora spp. were not proved in any of the donkeys. This is the first seroprevalence study of Neospora spp. and T. gondii in equids from Nigeria.

Neospora caninum and Toxoplasma gondii are the protozoan parasites with definitive hosts from order Carnivora. Due to vertical transmission, both parasites can cause abortions and neonatal mortality that lead to significant productive and economic losses in the domestic ruminants. The aim of this study was to describe N. caninum and T. gondii seroprevalence in the group of frequently farmed captive exotic ruminants (n = 184) including Bovidae (barbary sheep, bezoar goat, common eland, American bison, water buffalo, and yak) and Camelidae (bactrian camel, guanaco, llama, and alpaca). Antibodies were tested by indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Higher prevalence of T. gondii antibodies (31% in IFAT and 52% in ELISA) was detected compared to N. caninum (24% in IFAT and 17% in cELISA). Mixed infection was found in 18 (10%) and 22 (12%) animals by IFAT and ELISA, respectively. Higher seroprevalence of both N. caninum and T. gondii was found in Camelidae compared to Bovidae. To author knowledge, this is the first detection of T. gondii and N. caninum antibodies in common elands and bezoar goats.

Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates.

Generally, rodents and other small mammals are considered as one of the sources of Toxoplasma gondii or Neospora caninum infection for cats and dogs as the definitive hosts of these two parasites, respectively. The aim of the study was to find out the prevalence of these two parasites in wild small mammals from the Czech Republic and to characterize T. gondii isolates by methods of molecular biology. A total of 621 wild small mammals were caught in the Czech Republic during years 2002-2014. Antibodies to T. gondii were detected by latex agglutination test in six (2.5%) of 240 small mammals (in two A. agrarius and four A. flavicollis). Antibodies to N. caninum were detected by commercially available competitive-inhibition enzyme-linked immunosorbent assay in one A. flavicolis (0.4%). Three of 427 (0.7%) liver samples were positive for T. gondii by PCR while negative for N. caninum. All embryo samples (n=102) were negative for both T. gondii and N. caninum. The three liver samples positive for T. gondii DNA (two from A. flavicollis and one from A. sylvaticus) were genotyped by 15 microsatellite markers and characterized as type II. To our knowledge, this is the first information about genetic characterization of T. gondii isolates in small mammals from Europe and the first detection of N. caninum antibodies in wild rodents from the Czech Republic., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.

Introduction and Objective: Neospora caninum and Toxoplasma gondii are worldwide spread parasites, causing serious illnesses in sensitive animals; toxoplasmosis is also important zoonosis. Although neosporosis is not considered as a zoonosis, it leads to aborted births in cattle, as well as paresis and paralysis in dogs., Objective: The aim of this study was to discover the prevalence of N. caninum and T. gondii antibodies in red foxes (Vulpes vulpes) in the Czech Republic., Materials and Method: Sera of 80 foxes from 8 regions of the Czech Republic were tested for antibodies to N. caninum and T. gondii by competitive enzyme linked immunosorbent assay (cELISA) and indirect ELISA. All samples were simultaneously tested by indirect fluorescent antibody test (IFAT) to detect both N. caninum and T. gondii antibodies., Results: Antibodies to N. caninum were found by IFAT in 3 (3.8%) red foxes with titre 50 and in 2 (2.5%) red foxes with inhibition 42.7% and 30.2 %. Antibodies to T. gondii were found in all tested animals in both IFAT (titres 50-6400) and in ELISA (S/P ranging from 34%-133%)., Conclusion: This is the first prevalence study of N. caninum and T. gondii antibodies in red foxes in the Czech Republic. The results obtained show that red foxes are exposed at different levels to both protozoan infections, and thus could play an important role in the transmission cycle of N. caninum and T. gondii in sylvatic cycle.

Introduction and Objectives: Encephalitozoon cuniculi is an obligate intracellular parasite infecting especially domestic rabbits; however, spontaneous infections have been documented in other mammalian species such as dogs, cats, rabbits, horses, cows and sheep all over the world. Encephalitozoonosis is a chronic and latent disease leading to renal failure, encephalitis, disorders of brain and urinary tract, and may lead to death. There are limited reports on encephalitozoonosis in wildlife, which is why the aim of this study was to detect the prevalence of antibodies to E. cuniculi in European hares., Materials and Methods: Samples of blood sera from 701 wild hares from the Czech Republic (n = 245), the Slovak Republic (n = 211) and Austria (n = 245) were examined by indirect immunofluorescence antibody test (IFAT); samples with titer ≥ 40 were marked as positive., Results: The total seroprevalence of E. cuniculi antibodies was 1.42% with titres in the range 40-640. Antibodies to E. cuniculi were detected in 2.9% (7/245), 0.8% (2/245) and 0.47% (1/211) hares from the Czech Republic, Austria and the Slovak Republic, respectively., Conclusions: This is the first detection of antibodies to E. cuniculi in hares from Europe showing that hares could be exposed to E. cuniculi infection, however with a low rate.

Study Aim: The West Nile virus (WNV) is an important mosquito-borne flavivirus occurring around the world. Occasionally found in Central Europe, the virus spread massively through whole Hungary between 2008 and 2009. The aim of our study was to determine the recent prevalence of the WNV infection in horses in the Czech Republic., Material and Methods: Overall, 2349 serum samples, collected from healthy unvaccinated adult horses in the Czech Republic between 2011 and 2013, were tested. A commercially available competitive ELISA kit (cELISA) was used for this purpose and positive samples were confirmed by virus neutralisation tests using WNV and tick-borne encephalitis virus (TBEV)., Results: Altogether 271 of 2348 samples (11.5%) were positive by cELISA. Confirmatory VNT revealed 16 WNV positive samples, 11 of which had titres from 8 to 1024; VNTs with TBEV were negative. Three samples had antibodies against both viruses and the WNV antibody titres were less than or equal to the TBEV antibody titres. A cross reactivity of flaviviruses might have had an impact on the results, but in samples with similar WNV and TBEV titres, co-infection with both pathogens cannot be ruled out either. VNT antibody titres in two horses were inconclusive (cut-off titre 4). The place of birth and transfers (if any) were checked for each WNV seropositive horse. Five WNV positive/TBEV negative samples (0.2%) came from five administrative regions (South Bohemian, Karlovy Vary, Central Bohemian, South Moravian, and Moravian-Silesian) and the respective animals were never moved to a foreign country. Four of these horses never left the farm. Other six WNV positive/TBEV negative horses were imported to the Czech Republic from North America or Central and West Europe and therefore, it is not possible to tell unambiguously whether their infection is autochthonous or imported., Conclusion: The results of the present study confirm that WNV antibodies occur sporadically in horses in the Czech Republic. WNV was found to circulate in different parts of the Czech Republic and not only in the South of Moravia.

Sera of 414 cats coming from different parts of the Czech Republic were tested for N. caninum antibodies. Sera samples were collected during years 2002-2011. N. caninum antibodies were detected by a commercial competitive-inhibition enzyme-linked immunosorbent assay (cELISA) with cut off ≥30% inhibition. Samples positive in cELISA were confirmed by an indirect fluorescence antibody test (IFAT); titre ≥50 was considered positive. In total, 137 (33%) cats reacted positively in cELISA; N. caninum antibodies in IFAT were detected in 16 (3.86%) cats with titres 50 and 100. In 6 cats, positive for N. caninum antibodies, T. gondii antibodies were also detected by IFAT. It is the first report of N. caninum antibodies in domestic cats from the Czech Republic and third report in Europe.

In some European countries there is an increasing interest on donkey. Despite there are few data regarding the donkey's parasitic diseases especially those with a protozoal etiology as neosporosis. Samples used in the study were collected from 238 domestic donkeys during year 2010 in Southern Italy from 207 females and 31 males of five breeds (Martina-Franca, Amiata, Sicilian-Grey, Ragusano, Sardinian) and crossbreeds with the average age 9 years (1 month - 24 year). Sera were tested by a competitive-inhibition enzyme-linked immunosorbent assay for antibodies against Neospora caninum; the sera were marked positive, if more than 30% inhibition was found. Out of a total 238 donkeys, 28 (11.8%) were found positive for Neospora antibodies with 12% in females and 6% in males. Different seroprevalence 15.4%, 16%, 12% and 8.8% were found in age categories <1 year, 1-4 years, 5-9 years and ≥10 years, respectively. The seroprevalence ranged in different breeds from 36% (Sicilian-Grey) to 0% (Sardinian) and in different use from 17% (for breeding) to 0% (for meat production). Logistic regression analysis demonstrated evidence of a significant (P<0.05) association between crossbreed origin of samples and risk of protozoan infection; age of donkeys was also significant risk factor for protozoan infection. No statistical significant difference (P>0.05) was found among genders and use of donkeys and risk of N. caninum infection. This is the first serological survey for Neospora spp. performed in donkeys., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We have performed a survey of Babesia canis prevalence within group of dogs living in Southern and Western Slovakia. Blood samples and sera from 217 dogs, including individuals suspected of having babesiosis, were examined by nested PCR-RFLP, light microscopy and indirect fluorescence antibody test (IFAT). The detection of B. canis DNA revealed the highest number of infected dogs in the region of Nové Zámky, with 23 B. canis-positive blood samples (35.4%, n=65), followed by an area close to Komárno (both areas of Southern Slovakia), where 1 dog out of 52 collected (1.9%) had detectible B. canis DNA in the blood stream. The serological method revealed an opposing pattern, with only 3 dogs (4.8%, n=63) sampled at Nové Zámky presenting IgG antibodies against B. canis, while in Komárno region such antibodies were detected in 15 dogs (28.8%, n=52). This discrepancy may be because the majority of samples from Nové Zámky were dogs suspected of an acute phase of canine babesiosis, whereas dogs at Komárno were sampled during a vaccination campaign, and thus were without any clinical signs of the disease. The latter group contains evidently recovered carriers of IgG against B. canis. Hence, the combination of PCR-based and serological methods enabled us to discover both recently infected as well as recovered dogs, thus obtaining a more realistic view on the epidemiological situation. Remarkably, we did not find any positive samples in the vicinity of Stupava (district Malacky, Western Slovakia), either by PCR-RFLP, microscopy or IFAT (n=100). Considering the numerous falsely diagnosed cases of canine babesiosis, we suggest that light microscopy as the simplest and most accessible diagnostic test. Southern Slovakia was confirmed as an area of high risk of canine babesiosis, whereas conclusions about B. canis spreading over Western Slovakia should be considered with wariness., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Samples of blood (serum, smears and blood preserved with ethanol) were collected from dogs during a vaccination campaign in northern Kenya in the years 2006 and 2007. Blood was screened for filarial parasites using molecular and microscopy methods and sera were tested for antibodies against canine distemper virus (CDV). Parasitological examination revealed the presence of two species of canine filariae: Acanthocheilonema dracunculoides and A. reconditum. The DNA from the former species was detected in 58% dogs sampled in 2006 and 36% dogs sampled in 2007, whereas the latter was found only in 4.2% samples collected in 2007. Microfilariae were found in 33.8% blood smears collected in 2006 and 10.6% blood smears collected in 2007. The seroprevalence of CDV was 33.4% in 2006 and 11.2% in 2007. The effect of sex, age and CDV-seropositivity/seronegativity on the occurrence of A. dracunculoides was evaluated. Infection by A. dracunculoides was more common in males and in dogs with a positive antibody titer for canine distemper, but evenly distributed among different age groups. The difference in the prevalence of A. dracunculoides in two isolated mountain ranges was not statistically significant. Methodologies available for detection and determination of canine filariae are compared, underlining methodical pitfalls arising through the determination of less common filarial species. The role of single epidemiological factors and possible association between canine distemper and filariasis are discussed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the Czech Republic, sera from 551 clinically healthy adult slaughtered pigs (females, 6-8 months old) were collected during the first half of June in 2010. Sera were tested for Toxoplasma gondii-specific IgG antibodies by an enzyme-linked immunosorbent assay; samples with more than 50% S/P were considered as positive. The same samples were also analysed for Neospora caninum antibodies using a commercial competitive-inhibition enzyme-linked immunosorbent assay; samples with more than 30% inhibition were considered as positive. Antibodies against T. gondii were found in 198 pigs (36%) in all districts with prevalences ranging from 18% to 75%. Antibodies against N. caninum were found in 16 pigs (3%); positive animals were found in 4 districts with prevalences ranging from 1% to 20%. Indication of mixed infections (concurrent presence of both N. caninum and T. gondii antibodies) was found in 8 (1·5%) pigs. The results of our study indicate that pigs in the Czech Republic have a relatively high seroprevalence for T. gondii, while they have only a low seroprevalence for N. caninum. Therefore, natural infection with T. gondii seems to be very common in Czech pigs. It is the first evidence of N. caninum antibodies in pigs in the Czech Republic. These results complete data about N. caninum infection in pigs in Europe.

During January 2007, blood samples were collected from 552 healthy horses from nine different regions of the Czech Republic. Sera were tested for serum antibodies to Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay and confirmed by an indirect fluorescent antibody test. The same samples were tested for serum antibodies against Toxoplasma gondii by a latex agglutination test. In total, 131 of 552 (24%) horses reacted positively for Neospora antibodies in competitive-inhibition enzyme-linked immunosorbent assay; seven of them had > or =50% of inhibition. Samples were confirmed in indirect fluorescence test, and only two samples were positive with final titres 50 and 100, while others were negative. Antibodies against T. gondii were found in 125 (23%) horses. This is the first serologic survey for Neospora spp. antibodies performed on horses in the Czech Republic.

European brown hares (Lepus europaeus) coming from the Czech Republic, Slovakia and Austria were tested for serum antibodies against Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay and for serum antibodies against Toxoplasma gondii by an indirect fluorescent antibody test. In a total of 925 samples, 280 (30%) and 132 (14%) reacted positively for N. caninum and T. gondii antibodies, respectively. Out of 333 hares in the Czech Republic, 129 (39%) and 71 (21%) reacted positively for N. caninum and T. gondii antibodies, respectively. Out of 209 hares in Slovakia, 8 (4%) and 13 (6%) reacted positively for N. caninum and T. gondii antibodies, respectively. Out of 383 hares in Austria, 143 (37%) and 48 (13%) reacted positively for N. caninum and T. gondii antibodies, respectively. Mixed infection (concurrent presence of both N. caninum and T. gondii antibodies) was found in 25 (8%) hares in the Czech Republic, in 14 (4%) hares in Austria and in none hare in Slovakia. It is the first report of antibodies against Neospora caninum in hares in the Czech Republic and Austria., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The present study was carried out on domestic fowls in the Czech Republic in the years 2003 and 2004. A total of 1108 samples obtained from 538 water birds (geese and ducks) and 570 gallinaceous birds (chickens and turkeys) were tested for specific antibodies against Toxoplasma gondii using the indirect fluorescent antibody test. In geese, 43% prevalence (77 positive out of 178 examined) was found with titres of 40, 320 and 2560 in 40, 29 and eight geese, respectively. In ducks, 14% prevalence (52 out of 360) was found with titres of 40 and 320 in 46 and six ducks, respectively. In gallinaceous birds, only one of 293 broiler chickens was positive with a titre of 40; sera of 217 hens and 60 turkeys were negative. We found extremely high antibody prevalence in geese and ducks compared with gallinaceous birds. This is the first seroprevalence study in domestic birds by indirect fluorescent antibody test in Europe.

In the Czech Republic, serum from 547 sheep coming from nine farms was examined for antibodies against parasites Toxoplasma gondii and Neospora caninum by screening ELISA. Antibodies against T. gondii were found in 325 sheep (59%) with prevalence ranging from 11% to 96% in different farms. Antibodies against N. caninum were found in 63 sheep (12%) with prevalence ranging from 4% to 21% in different farms. Mixed infections were found in 53 sheep (10%). It was the first evidence of N. caninum antibodies in sheep from the Czech Republic.

The main aim of the present study was to establish the prevalence of antibodies against Neospora caninum dogs from the Czech Republic and to examine the dynamics of antibody titers during a long-term period. For this purpose, sera of 858 dogs were examined for the presence of anti-N. caninum antibodies using an indirect immunofluorescent antibody test (IFAT). Four groups of dogs of various origins were included in the survey: the first group (A, n=470) comprised dogs purchased by the Czech Army from the civilian sector throughout the Czech Republic, with 22 (4.7%) N. caninum-positive dogs, second group (B, n=115) represented police dogs with no seropositive animal, third group (C, n=195) were pet dog sera collected for veterinary clinic with 5 (2.6%) anti-N. caninum sera and the fourth group (D, n=78) of canine shelter dogs with the seroprevalence of 19.2%. The differences in seroprevalence were significant (P< or =0.01) between groups B and A, and between D and A. None of the serologically positive animals had clinical signs of neurological disorders. Coprological examination did not reveal any dog shedding N. caninum oocysts. The seropositivity rates for N. caninum were analyzed in relation to other data, such as age, breed and gender. Increased prevalence rates of anti-N. caninum antibodies were found in the older age strata of the dog population sample tested in the present study. We found significantly higher (P=0.02) prevalence in 3-3.5-year-old dogs (11.1% of 36), as compared to 1-1.5-years-old dogs (2% of 98). A longitudinal study of antibody dynamics was carried out in 19 initially seropositive dogs over a period of 4 years. The second and third examinations revealed that antibody titers decreased in majority of positive dogs (10, 52.6%), of which in seven cases (36.8%) the titers fell to levels that are currently considered as being seronegative (titer <1:50), or even became undetectable (titer <1:25).

Sera collected from hunter-killed wild boars (Sus scrofa) during 1999-2005 from seven different regions of the Czech Republic were assayed for antibodies to Toxoplasma gondii by indirect fluorescence antibody test and to Neospora caninum by competitive-inhibition enzyme-linked immunosorbent assay and by indirect fluorescence antibody test. Antibodies to T. gondii were detected in 148 (26.2%) of 565 wild boars with serum dilutions of 1:40 in 40, 1:80 in 40, 1:160 in 27, 1:320 in 19, 1:640 in 18 and 1:1280 in 4 wild boars. Antibodies to N. caninum were detected in 102 (18.1%) of 565 wild boars with 30.1-94.6% inhibition in ELISA; statistical significant differences were observed between sampling regions, ranging from 0% to 31.8%. Sera, positive in ELISA, were examined in IFAT; 58 of 102 (56.9%) were positive with titres 1:40-1:160. Mixed infection (concurrent presence of both T. gondii and N. caninum antibodies) was found in 38 wild boars. It is the first report of antibodies to N. caninum in wild boar. Serological results indicate a common exposure to T. gondii and to N. caninum among wild boars in the Czech Republic.

Neospora caninum is an apicomplexan parasite that causes neuromuscular disease in dogs and abortions in cattle. Little is known about the prevalence of antibodies to this parasite in zoo animals. Sera from 556 animals, from 13 Czech and Slovak zoos were tested for antibodies to N. caninum and Toxoplasma gondii by indirect fluorescent antibody test. Antibodies to N. caninum were found in 31 of 556 zoo animals (5.6%), representing 18 of 114 species tested: Eurasian wolf (Canis lupus lupus), Maned wolf (Chrysocyon brachyurus), fennec (Vulpes zerda), cheetah (Acinonyx jubatus), jaguarundi (Herpailurus yaguarondi), Eurasian lynx (Lynx lynx), Indian lion (Panthera leo goojratensis), fisher (Martes pennanti), blackbuck (Antilope cervicapra), European bison (Bison bonasus), lechwe (Kobus leche), African buffalo (Syncerus caffer caffer), eland (Taurotragus oryx), sitatunga (Tragelaphus spekei gratus), Thorold's deer (Cervus albirostris), Eastern elk (C. elaphus canadensis), Vietnam sika deer (C. nippon pseudaxis) and Père David's deer (Elaphurus davidianus). Titres ranged from 1:40 to 1:2560. The highest prevalence 50% was found in family mustelidae of the order carnivora. Antibodies to T. gondii were detected in 193 of 556 zoo animals (34.7%) representing 72 of 114 species tested, with titres ranging from 1:40 to 1:40960. The highest prevalence 100% was found in families: hyaenidae, mustelidae, ursidae and viveridae of the order carnivora. The results of this study indicate that zoo animals have more exposure to T. gondii than to N. caninum. It is the first report of seroprevalence of antibodies to N. caninum in European zoo animals.

Three captive female nilgais (Boselaphus tragocamelus) either showed late-term abortion or their newborn calves died within 2 days of birth. Toxoplasma gondii DNA was demonstrated in the brain and liver of each fetus and in one of the two neonates by single-stage polymerase chain reaction (PCR) with TGR1E and by seminested PCR with B1 gene. Retrospectively, antibodies titers > or =640 to T. gondii by indirect fluorescence were found in the sera of all three female and one male nilgais. No other cause of abortion was detected. Fatal toxoplasmosis was also diagnosed in one captive, adult female saiga antelope (Saiga tatarica), which died suddenly. Toxoplasma gondii was detected in the liver, lung, spleen, kidney, and intestine. An unusual finding was the presence of numerous tissue cysts in the liver of this animal. Toxoplasmosis was confirmed by PCR with TGR1E and immunohistochemically. Toxoplasmic hepatitis and pneumonia were considered to be the primary causes of death.

A total of 28 domestic ducks were divided into seven groups of four ducks. Six groups were inoculated per os with 10(1), 10(2), 10(3), 10(4), 10(5) and 10(5.7) oocysts Toxoplasma gondii oocysts (K21 strain, which is avirulent for mice), and the remaining group was used as a control. Antibodies to T. gondii were detected in all ducks by the indirect fluorescence antibody test first on day 7 post-inoculation (p.i.). Antibody titres were found in the range of 1:20 to 1:640 depending on the infectious dose of the oocysts. From day 14 p.i. antibody titres increased to 1:80 to 1:20 480. Between days 14 and 28 p.i. (end of the experiment), antibody titres decreased in 14 ducks, remained the same in seven ducks, and continued to increase in three ducks. Bioassay in mice revealed T. gondii in the breast and leg muscles and the heart (100%, n=47), brain (91%, n=22), liver (54%, n=13) and stomach (46%, n=24). The infected ducks showed no clinical signs; however, the results of bioassay indicate that, compared with some gallinaceous birds, domestic ducks were relatively susceptible to T. gondii infection.

In total 53 budgerigars (Melopsittacus undulatus) were divided into six groups and orally infected with a suspension of oocysts of low virulent Toxoplasma gondii K21 strain in the doses of 10(2), 10(3), 10(4), 10(5) and 10(6), respectively. Blood was collected from the birds prior to the inoculation and then on days 10, 20 and 30 post infection. Latex-agglutination test (LAT) was used for the detection of antibodies in the inoculated birds. The infected birds showed no apparent signs of disease. The antibodies were found in all but two birds inoculated a dose of 10(2) oocysts. Haematological values remained unchanged after infection. T. gondii was isolated by bioassay in mice from all 37 birds fed 10(3) or more oocysts and 6 of 9 fed 10(2) oocysts. The results demonstrate that budgerigars are resistant to T. gondii infection.

The virulence of the oocysts of 7 Czech Toxoplasma gondii isolates was tested. The oocysts were obtained by experimental infection of cats with the tissue cysts of T. gondii isolates from dogs, cats, and rabbits. The cats shed the oocysts in feces, with prepatent periods of 3-5 days postinfection (PI); the patent period was 7-18 days. The number of oocysts shed varied between 0.94 million and 47 million, with 0.66 million-39 million oocysts found in the daily samples of excrement. The cats ceased oocyst production at 11-22 days PI. Sporulated oocysts were used to prepare infective doses of 1 to 10(5) oocysts for oral infection of 10 mice. Deoxyribonucleic acid isolated from 4 T. gondii isolates was used in polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for amplification of the ROP1 gene and restriction of the product of amplification by restriction endonuclease DdeI. On the basis of their biological characteristics, all 7 isolates belonged to the group of "avirulent" strains. In the PCR-RFLP tests, 2 isolates, K9 and K19, showed an "avirulent" strain pattern.

A serological survey for antibodies against Neospora caninum in aborting cattle was carried out in the Czech Republic. Serum samples from 463 aborting dairy cows originated from 137 farms from different parts of the Czech Republic were tested for presence of N. caninum antibodies by use of an enzyme-linked immunosorbent assay (ELISA) and an indirect fluorescent antibody test (IFAT). Antibodies (> or = 1:640) to N. caninum were found in 18 (3.9%) of 463 aborting cows. Farm prevalence in aborting cows was 12.4% (17/137). The antibody titres of cows were 1:200 (9 cows), 1:640 (7 cows), 1:1280 (3 cows), 1:2560 (3 cows), 1:5120 (3 cows), 1:10,240 (2 cows) and 1:20,480 (0 cow). A case-control study was conducted to estimate the association of N. caninum infection and abortion. For this 407 serum samples were collected from cows on five dairy farms with repeated occurrence of endemic and sporadic abortion of unidentified etiology. These samples were obtained from aborting cattle (n=44) and normally calving cattle (control group; n=363) and tested for N. caninum antibodies by an immunofluorescent antibody test (IFAT). Overall, 3.19% (13/407) of cows sampled had positive N. caninum fluorescence with a cut-off titre of 1:200. The prevalence of N. caninum was significantly higher (P<0.05) in the aborting group (13.64%; 95% confidence interval (CI): 5.2, 27.4) than in the control group (1.93%; 95% CI: 0.8, 3.9). A strong association between seropositivity and abortion was found, with seropositive cows being eight times more likely to abort than seronegative cows (odds ratio=8; 95% CI: 2.6, 25.1). This first report on the serological prevalence of N. caninum in cows in the Czech Republic verified a strong association between N. caninum infection and abortions in five dairy farms. Thus, the neosporosis should be considered in differential diagnosis of bovine abortion.

Common voles (Microtus arvalis) in groups of nine to 10 animals were inoculated per os with a dose of 1, 10, 1x10(2), 1x10(3), and of the K1 strain of Toxoplasma gondii. All the common voles inoculated with 1 to 1 x 10(3) oocysts remained subclinical and survived. Three of the 10 voles inoculated with 1 x 10(4) oocysts died between days 7 and 12 post inoculation (p.i.). Antibodies were demonstrated in all the infected voles killed on day 60 p.i. The highest antibody titres in voles detected by the dye test (DT) and latex agglutination test (LAT) were 1,024 and 1,280, respectively.

Brown hares (Lepus europaeus) trapped in the countryside and domestic rabbits were experimentally infected with Toxoplasma gondii (K7 strain) oocysts. Hares (n=12) were divided into groups of 4 and infected with 10, 10(3) and 10(5) oocysts. Rabbits (n=12) were infected in the same way. The experimentally infected animals were monitored for 33 days after infection (p.i.). Most of the infected hares demonstrated behavioural changes, and all of them died between 8 and 19 days p.i. Three of the rabbits demonstrated only clinical changes related to the concurrent pasteurellosis. The typical pathological finding in the hares were haemorrhagic enteritis, enlargement and hyperaemia of mesenteric lymph nodes, splenomegaly and multiple miliary necrotic lesions in the parenchyma of the liver and other organs. Pathological changes in the rabbits were less pronounced than in the hares. In rabbit brains, tissue cysts of the T. gondii were found. The incidence of T. gondii antibodies both in the hares and the rabbits was first ascertained on day 7 p.i. On day 12 p.i., antibodies were already found in all the animals infected. Antibody titres in indirect fluorescence antibody test (IFAT) using the anti-rabbit conjugate were markedly higher in rabbits than in hares. In all hares, T. gondii was isolated post mortem from the liver, brain, spleen, kidney, lung, heart and skeletal muscles. Although T. gondii was also isolated in all rabbits, it was not always isolated in all their organs. In all hares, parasitemia was demonstrated on days 7 and 12 p.i. The percentage of rabbits with detected parasitemia was lower. In hares, a decrease in the numbers of leukocytes during the infection was observed. No such decrease was observed in the rabbits. The lymphocyte activity after the stimulation with non-specific mitogens showed significant differences between the hares and the rabbits even before the infection. After the infection, the hares infected with 10(3) and 10(5) doses and in rabbits infected with a 10(5) dose showed a decrease of lymphocyte activity. Rabbits infected with a 10(3) dose showed an increase of the lymphocyte activity. While in hares toxoplasmosis was an acute and fatal disease, the infection in rabbits had subclinical manifestations only and easily passed to a latent stage. The different courses of toxoplasmosis in the hare and the rabbit may be due to the differences in the natural sensitivity of the two species to the T. gondii infection or a negative impact of stress to the immune status of hares.