First detection of Toxoplasma gondii Africa 4 lineage in a population of carnivores from South Africa.

Introduction: There have only been a few molecular studies conducted on the detection of T. gondii in tissues of carnivores in South Africa, with no data on the genetic diversity of this parasite. That is why the aim of this study was to detect and genotype T. gondii DNA in tissues of selected wild and domestic carnivores in South Africa. Methods: Samples were collected from 80 animals of 20 species (mainly road-killed) in the four provinces of Limpopo (n=57), Mpumalanga (n=21), Gauteng (n=1) and Free State (n=1) during the period 2014-2018. Samples of brain (n=31), heart (n=4), liver (n=40), spleen (n=2) and lung (n=3) were used to detect T. gondii by real-time PCR targeting a 529 bp repeating fragment of T. gondii DNA. Samples that were positive in real-time PCR were genotyped using 15 microsatellite markers. Results: T. gondii DNA was detected in 4 (5 %) samples: in the brain from a Black-backed Jackal (Canis mesomelas), in the liver from a African Wildcat (Felis silvestris lybica) and in the liver and heart of two Rusty-spotted Genets (Genetta maculata) respectively. The DNA sample from Black-backed Jackal was genotyped and characterized as belonging to the type Africa 4 lineage (equivalent to RFLP genotype ToxoDB#20), that is a widespread lineage in Africa. Discussion: This is the first genetic characterization of T. gondii isolated from a wild carnivore on the African continent and the first report of T. gondii in Black-backed Jackal. The Africa 4 lineage was also confirmed in the region of Southern Africa for the first time.

Molecular Detection of Toxoplasma gondii, Neospora caninum and Encephalitozoon spp. in Vespertilionid Bats from Central Europe.

Bats may carry various viruses and bacteria which can be harmful to humans, but little is known about their role as a parasitic source with zoonotic potential. The aim of this study was to test wild bats for the presence of selected parasites: Toxoplasma gondii, Neospora caninum and microsporidia Encephalitozoon spp. In total, brain and small intestine tissues of 100 bats (52 Myotis myotis, 43 Nyctalus noctula and 5 Vespertilio murinus) were used for the DNA isolation and PCR detection of the abovementioned agents. Toxoplasma gondii DNA was detected by real-time PCR in 1% of bats (in one male of M. myotis), while all bats were negative for N. caninum DNA. Encephalitozoon spp. DNA was detected by nested PCR in 25% of bats, including three species (twenty-two M. myotis, two N. noctula and one V. murinus). Positive samples were sequenced and showed homology with the genotypes Encephalitozoon cuniculi II and Encephalitozoon hellem 2C. This is the first study on wild vespertilionid bats from Central Europe and worldwide, with a relatively high positivity of Encephalitozoon spp. detected in bats.

Detection of antibodies to Toxoplasma gondii and Neospora caninum in animals from three zoos in Slovakia.

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.

The Amount of Food Ingested and Its Impact on the Level of Uric Acid in the Blood Plasma of Snakes.

In mammals, lipemic blood from sampling too soon after an animal feeds can have substantial effects on biochemical values. Plasma biochemical values in reptiles may be affected by species, age, season, and nutritional state. However, fasting status is not routinely considered when sampling reptile blood. Assessing uric acid levels in snakes is an important part of the diagnosis of the renal disease. However, the use of this biochemical indicator is undervalued without knowledge of natural uric acid fluctuations and the lack of differentiation from pathological changes. This study aimed to look at the relationship between snake feeding and uric acid concentrations. The investigation aims to better understand the feed-induced changes that occur and render the analysis of this biochemical parameter a more potent diagnostic tool. The study used ten snakes belonging to seven species, and basal uric acid values were evaluated by blood biochemical analysis before feeding. The snakes were fed in two rounds, with successive blood sampling and monitoring of uric acid changes carried out for each. The snakes were fed approximately 50% more with the second round of feeding to investigate the relationship between food supply and uric acid level. The findings show feeding led to substantial elevations in uric acid values, whereby postprandial concentrations were significantly elevated for up to 8 days after feeding. The findings show the significant changes in uric acid levels that occur after feeding and the similarities between postprandial rises in uric acid and those reported in snakes with renal disease. To minimize misdiagnosis and differentiate transient postprandial hyperuricemia from pathological increases, it is recommended that sufficient anamnestic data on time since the last feeding be collected, as well as repeated samples following weeks of fasting. This knowledge is crucial because the amount of feed in terms of intensity and volume has a significant effect on uric acid levels in the blood of snakes.

Fatal toxoplasmosis in wild European brown hares (Lepus europaeus) in tularaemia endemic areas of the Czech Republic: Poses risk of infection for humans?

Toxoplasma gondii may cause fatal infection in European brown hares (Lepus europaeus). However, the role of this parasite in terms of mortality rate in tularaemia endemic areas, amount of parasites in affected organs and circulating genotypes, is still unknown. In total, 36 hares (killed or found dead) were submitted for pathomorphological examination as a part of the national tularaemia and brucellosis monitoring. Tissue samples (lung, heart, liver, spleen and kidney) were tested by quantitative real-time PCR targeting 529 bp region of T. gondii. Genotyping was performed by a 15 microsatellite markers method in a single multiplex PCR assay. The same tissues of hares were simultaneously used for the bacteriological cultivation. Toxoplasma gondii was detected by qPCR in the tissues of two hares. Spleen and lungs of one infected hare have been found harbouring up to ~7 millions of T. gondii parasites per gram of tissue. Both positive samples were characterized as T. gondii type II, one archetypal clonal type II and the other one a type II variant (W35 = 244). Bacteria Francisella tularensis was proved in pooled samples of three hares but without coinfection with T. gondii; all hares were negative for Brucella suis. Toxoplasma gondii has significant impact on mortality of European brown hares in tularaemia endemic areas and parasite load within the animal tissues may present high risk of human infection.

DETECTION OF ZOONOTIC PATHOGENS IN WILD BIRDS IN THE CROSS-BORDER REGION AUSTRIA - CZECH REPUBLIC.

To assess the importance of wild birds as a reservoir of zoonotic pathogens in Austria and the Czech Republic, we sampled 1,325 wild birds representing 13 orders, 32 families, and 81 species. The majority belonged to orders Columbiformes (43%), Passeriformes (25%), and to birds of prey: Accipitriformes, Strigiformes, and Falconiformes (15%). We collected cloacal swabs from 1,191 birds for bacterial culture and 1,214 triple swabs (conjunctiva, choana, cloaca) for DNA and RNA isolation. The cloacal swabs were processed by classical bacteriologic methods for isolation of Escherichia coli , Salmonella spp., methicillin-resistant Staphylococcus aureus (MRSA), and thermophilic Campylobacter spp. Nucleic acids isolated from triple swabs were investigated by PCR for West Nile virus, avian influenza viruses, and Chlamydia spp. We also tested tissue samples from 110 fresh carcasses for Mycobacterium spp. by PCR and we cultured fresh droppings from 114 birds for Cryptococcus spp. The most-frequently detected zoonotic bacteria were thermophilic Campylobacter spp. (12.5%) and Chlamydia spp. (10.3%). From 79.2% of the sampled birds we isolated E. coli , while 8.7% and 0.2% of E. coli isolates possessed the virulence genes for intimin (eaeA) and Shiga toxins (stx1 and stx2), respectively. Salmonella spp. were rarely found in the sampled birds (2.2%), similar to findings of MRSA (0.3%). None of the samples were positive for Cryptococcus neoformans , Mycobacterium spp., avian influenza viruses, or West Nile virus.

Seasonal Dynamics, Parity Rate, and Composition of Culicoides (Diptera: Ceratopogonidae) Occurring in the Vicinity of Wild and Domestic Ruminants in the Czech Republic.

In the light of the emergence of bluetongue and Schmallenberg viruses in northern and central Europe, an extensive entomological survey within the framework of a bluetongue control program was undertaken from 2008 to 2013 in the Czech Republic to investigate Culicoides biting midges (Diptera: Ceratopogonidae) collected in close proximity of domestic livestock and semiwild ruminants. Insects were sampled using CDC black-light suction traps placed overnight near ruminants in farms or in forest game preserves to provide data on Culicoides fauna collected near these two groups of hosts inhabiting different environments. From almost a half million biting midge specimens collected at 41 sampling sites, 34 species were identified including three species newly recorded for the Czech Republic: Culicoides (Oecacta) clastrieri Callot, Kremer & Deduit, Culicoides (Oecacta) odiatus Austen, and Culicoides (Pontoculicoides) saevus Kieffer. The Culicoides obsoletus species group, incriminated as a bluetongue virus vector, was predominant in both domestic livestock (91%) and semiwild game (52%). A relatively high proportion (around 30%) of C. obsoletus Meigen females with pigmented abdomen (= more likely parous) was observed from spring till autumn. In contrast, adult biting midges were found to be largely absent during at least three winter months, approximately December till March, which could be considered as the biting midge vector-free period.

Survey of Toxoplasma gondii antibodies in meat juice of wild boar (Sus scrofa) in several districts of the Czech Republic.

INTRODUCTION AND OBJECTIVE: The aims of the study were: 1) to detect antibodies against Toxoplasma gondii from wild boar meat; 1) establish seroprevalence of toxoplasmosis in the wild boar population; 3) establish risk factors concerned in higher possible seroprevalence; 4) to estimate the usefulness of meat juice for detection of T. gondii antibodies in wild boar. MATERIAL AND METHODS: Diaphragm meat juice samples from 656 wild boar (Sus scrofa) were collected during the hunting seasons between September 2008 - October 2010 from 9 districts of the Czech Republic. The samples were stratified per age category into 2 groups: piglets (n = 279) and yearlings together with adults (n = 377). The in-house ELISA test was used for the detection of antibodies against T. gondii from the meat juice samples. RESULTS: Antibodies against T. gondii were detected by in-house ELISA in 260 of 656 wild boars (40%) with 26% prevalence in piglets (72/279) and 50% prevalence in yearlings and adults (188/377). The district total seroprevalences ranged between 32% - 59%, with a significantly higher prevalence in the district of Havlíckuv Brod (59%). Statistically significant differences (p-value < 0.05) were found between 2 age categories, and between 9 districts, with a significant variability in the district of Havlíckuv Brod. Seroprevalence correlated positively with farm density, but without any statistical significance. CONCLUSION: The obtained results indicate that consumption of raw or undercooked meat from wild boars can carry an important risk of toxoplasma infection. Post mortem detection of antibodies in meat juice samples using ELISA is a useful alternative to blood serum examination. In addition, a diaphragm sample has been well-proven as a matrix sample for the contemporaneous diagnostics of trichinellosis and toxoplasmosis.

Morphological and molecular characterization of Karyolysus--a neglected but common parasite infecting some European lizards.

BACKGROUND: Blood parasites of the genus Karyolysus Labbé, 1894 (Apicomplexa: Adeleida: Karyolysidae) represent the protozoan haemogregarines found in various genera of lizards, including Lacerta, Podarcis, Darevskia (Lacertidae) and Mabouia (Scincidae). The vectors of parasites are gamasid mites from the genus Ophionyssus. METHODS: A total of 557 individuals of lacertid lizards were captured in four different localities in Europe (Hungary, Poland, Romania and Slovakia) and blood was collected. Samples were examined using both microscopic and molecular methods, and phylogenetic relationships of all isolates of Karyolysus sp. were assessed for the first time. Karyolysus sp. 18S rRNA isolates were evaluated using Bayesian and Maximum Likelihood analyses. RESULTS: A total of 520 blood smears were examined microscopically and unicellular protozoan parasites were found in 116 samples (22.3% prevalence). The presence of two Karyolysus species, K. latus and K. lacazei was identified. In total, of 210 samples tested by polymerase chain reaction (PCR), the presence of parasites was observed in 64 individuals (prevalence 30.5%). Results of phylogenetic analyses revealed the existence of four haplotypes, all part of the same lineage, with other parasites identified as belonging to the genus Hepatozoon. CONCLUSIONS: Classification of these parasites using current taxonomy is complex - they were identified in both mites and ticks that typically are considered to host Karyolysus and Hepatozoon respectively. Furthermore although distortions to the intermediate host erythrocyte nuclei were observed, the defining characteristic of Karyolysus, the haplotypes were nearly identical to those reported from lizards in the Iberian Peninsula, where such distortions were not reported and which were thus identified as Hepatozoon. Based on the phylogenetic analyses, neither vertebrate host, nor geographical patterns of the studied blood parasites could be established.