Role of invasive carnivores (Procyon lotor and Nyctereutes procyonoides) in epidemiology of vector-borne pathogens: molecular survey from the Czech Republic.

BACKGROUND: Vector-borne pathogens (VBPs) are a major threat to humans, livestock and companion animals worldwide. The combined effect of climatic, socioeconomic and host composition changes favours the spread of the vectors, together with the expansion of invasive carnivores contributing to the spread of the pathogens. In Europe, the most widespread invasive species of carnivores are raccoons (Procyon lotor) and raccoon dogs (Nyctereutes procyonoides). This study focused on the detection of four major groups of VBPs namely Babesia, Hepatozoon, Anaplasma phagocytophilum and Bartonella in invasive and native carnivores in the Czech Republic, with the emphasis on the role of invasive carnivores in the eco-epidemiology of said VBPs. METHODS: Spleen samples of 84 carnivores of eight species (Canis aureus, Canis lupus, Lynx lynx, P. lotor, Martes foina, Lutra lutra, Mustela erminea and N. procyonoides) were screened by combined nested PCR and sequencing for the above-mentioned VBPs targeting 18S rRNA and cytB in hemoprotozoa, groEL in A. phagocytophilum, and using multilocus genotyping in Bartonella spp. The species determination is supported by phylogenetic analysis inferred by the maximum likelihood method. RESULTS: Out of 84 samples, 44% tested positive for at least one pathogen. Five different species of VBPs were detected in P. lotor, namely Bartonella canis, Hepatozoon canis, Hepatozoon martis, A. phagocytophilum and Bartonella sp. related to Bartonella washoensis. All C. lupus tested positive for H. canis and one for B. canis. Three VBPs (Hepatozoon silvestris, A. phagocytophilum and Bartonella taylorii) were detected in L. lynx for the first time. Babesia vulpes and yet undescribed species of Babesia, not previously detected in Europe, were found in N. procyonoides. CONCLUSIONS: Wild carnivores in the Czech Republic are hosts of several VBPs with potential veterinary and public health risks. Among the studied carnivore species, the invasive raccoon is the most competent host. Raccoons are the only species in our study where all the major groups of studied pathogens were detected. None of the detected pathogen species were previously detected in these carnivores in North America, suggesting that raccoons adapted to local VBPs rather than introduced new ones. Babesia vulpes and one new, probably imported species of Babesia, were found in raccoon dogs.

Red fox (Vulpes vulpes) play an important role in the propagation of tick-borne pathogens.

The red fox (Vulpes vulpes) is the most widespread free-living carnivore in the world. Over the years, foxes have been recognized as hosts for a number of tick-borne pathogens. However, their role as reservoirs for zoonotic tick-borne diseases is poorly understood. The aim of our study was to investigate tick-borne pathogens in the red fox population in the Czech Republic. Out of 117 red foxes, 110 (94.02%) individuals tested positive for the presence of at least one pathogen by the combined PCR and sequencing approach. Hepatozoon canis was the most frequently detected pathogen (n = 95; 81.2%), followed by Babesia vulpes (n = 75; 64.1%). Babesia canis was not detected in our study. Four (3.42%) red foxes were positive for Candidatus Neoehrlichia sp., 3 (2.56%) for Anaplasma phagocytophilum, and one red fox (0.85%) tested positive for the presence of Ehrlichia sp. DNA. Overall, DNA of spirochetes from the Borrelia burgdorferi s.l. complex was detected in 8.6% of the foxes and B. miyamotoi in 5.12% of the samples. As a carnivore found in all ecosystems of Central Europe, foxes obviously contribute to transmission of tick-borne pathogens such as A. phagocytophilum, B. burgdorferi s.l., and B. myiamotoi. In addition, foxes apparently harbour a community of pathogens, associated with this host in local ecological context, dominated by H. canis and B. vulpes (possibly also Candidatus Neoehrlichia sp.). These species have the potential to spread to the domestic dog population and should be included in the differential diagnosis of febrile diseases with hematologic abnormalities in dogs.

Further data on the distribution of Dirofilaria spp. in the Czech Republic in dogs.

Based on previously published data, the Czech Republic is regarded an endemic country of the onchocercid nematodes Dirofilaria immitis (Leidy, 1856) and Dirofilaria repens Railliet et Henry, 1911. Nevertheless, while cases of D. repens are commonly reported from dogs in South Moravia, no recent records of D. immitis are available. Therefore, the present study was performed to clarify the occurrence of both species of Dirofilaria Railliet et Henry, 1910. Blood samples of 551 dogs sampled during 2015 and 2016 were analysed microscopically for presence of microfilariae and blood sera were examined by IDEXX SNAP® 4Dx® test (IDEXX, USA). DNA from blood of microscopically positive dogs was extracted and PCR protocol amplifying fragment of cytochrome c oxidase I (COI) gene was performed; PCR products were then sequenced. All dogs from the Bohemian part of the Czech Republic were negative. The prevalence of D. repens in the Moravian region was 5.7 % (27/476). BLAST analyses of obtained sequences confirmed the presence of D. repens (99-100% identical to KX265049). All sampled animals showed a negative result for D. immitis antigen in IDEXX SNAP® 4Dx® test. Our study confirmed the previously reported occurrence of D. repens in South Moravia and revealed its spreading from the epicentre to the north and west. PCR with subsequent sequencing together with negative results for D. immitis antigen in IDEXX SNAP® 4Dx® test revealed only D. repens infection. A previously published autochthonous infection of dogs with D. immitis in South Moravia was not confirmed.

Baylisascaris transfuga (Ascaridoidea, Nematoda) from European brown bear (Ursus arctos) causing larva migrans in laboratory mice with clinical manifestation.

Due to the recent recovery of brown bear populations in Central Europe, information about their ascarid parasite, Baylisascaris transfuga is necessary as the parasite represents a part of natural ecological networks. B. transfuga can lead to larva migrans syndrome in accidental hosts, but its zoonotic potential has not been confirmed. The resent study compares development of larva migrans in infected mice inoculated with two infectious doses (ID 200 and ID 2000) of B. transfuga embryonated eggs, and the clinical manifestation to evaluate the pathogenicity of the larvae. Histopathology revealed that the liver was the most severely infected organ. The moderately infected organs included lung, brain, skeletal muscles and jejunum and the less infected ones were the eyes, heart, kidneys and spleen. The high pathogenicity of B. transfuga to mice was reflected in high mortality (33,3%) after infection, with mortality increasing with higher infectious dose. The results extend the knowledge of the interaction of B. transfuga and its aberrant hosts and contribute to the understanding of the epidemiology and transmission of this bears roundworm.

Identification of Tapeworm Species in Genetically Characterised Grey Wolves Recolonising Central Europe.

PURPOSE: Restored role of the grey wolf in ecological networks of newly recolonized areas can be studied via surveys of parasite communities of this predator. As helminths circulating in multi-host systems, the tapeworms directly reflect wolves' diet, while some species are also important from the One Health perspective. The Czech experienced centuries of wolves' absence, however, now it is situated on the crossroad of recolonising wolves' populations, which is opening questions of their role in ecological networks in this area and thus in sylvatic cycles of heteroxenous parasites. METHODS: Five wolf carcasses from this area were obtained and genetic affinity to a particular population was inspected. Tapeworms isolated from wolves' intestines during necropsies were molecularly identified based on sequences of COI marker. RESULTS: Three wolf haplotypes (w1, w2, w14) correspond with the dominance of haplogroup 1 (w1, w2) within Central European lowland population and haplogroup 2 (w14) within the Carpathian population. Two Taenia spp. were revealed: T. krabbei in Central European population wolves and T. hydatigena in an individual from Carpathian population. CONCLUSIONS: The results serve as a base for future monitoring and studies of the recolonising wolf population and its impact on ecosystems in the studied area to contribute to the hypothesis about differentiation of parasite communities in particular wolf population and higher parasite diversity and richness in established populations in comparison to newly settled ones.

Three new species of Cytauxzoon in European wild felids.

Protists of the genus Cytauxzoon infect a wide variety of wild and domestic felids worldwide. While the American Cytauxzoon felis has been well described, data on the European isolates of Cytauxzoon are still scant. The aim of the current study was to determine the genetic diversity of European Cytauxzoon spp. in wild felids across Europe by analyzing one nuclear and two mitochondrial genes, along with representative complete mitochondrial genomes. Overall, 106 biological samples from wild felids (92 from Felis silvestris and 14 from Lynx lynx) from Germany, Romania, Czech Republic, and Luxembourg were collected and screened for the presence of Cytauxzoon spp. using nested PCR protocols, targeting the highly conserved 18S rDNA, mitochondrial cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) genes. Furthermore, 18 previously confirmed wild felid biological samples from Europe, and comparative material from USA positive for C. felis, were included in the study. In 18S rDNA sequences analyses, Cytauxzoon spp. from felids formed two separate clades of New World and Old World isolates, with a low inner diversity of the European clade. In contrast to 18S rDNA, the phylogenetic analyses of CytB and COI genes affirmatively revealed three highly supported clades, resulting in three defined genotypes. Similar intra- and interspecific variability of CytB and COI genes was observed in the case of different Babesia spp. Considering geography, host species and analyses of three genes, we conclude that the three detected genotypes of Cytauxzoon in European wild felids represent three new species, which we herein describe.

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.

Survey of Toxoplasma gondii and Trichinella spp. in hedgehogs living in proximity to urban areas in the Czech Republic.

Hedgehogs (Mammalia: Erinaceidae) are omnivorous nocturnal animals typically living in anthropogenic areas. They may be suitable as sentinels for a wide range of zoonotic infections. Only a few studies have investigated hedgehogs (and then as representative wildlife species) to establish their role in the life cycle of such tissue parasites with zoonotic potential as Toxoplasma gondii or Trichinella spp. Working with frozen hedgehog cadavers, we tested for these parasites using T. gondii DNA-specific magnetic capture isolation plus polymerase chain reaction and Trichinella spp. digestion assay. All of 50 examined hedgehogs were negative for Trichinella spp. larvae in their muscles, but brain tissue from 5 out of 26 Erinaceus europaeus (19.2%) and 4 out of 24 E. roumanicus (16.6%) tested positive for T. gondii DNA. Frequency of T. gondii for both hedgehog species was equal, as was distribution between males and females and across age categories. Although a few studies have suggested the possibility of Trichinella spp. infection in hedgehogs, the zero prevalence in the tested hedgehogs is not surprising in view of the generally low prevalence of Trichinella spp. in Central Europe. Our results show that hedgehogs are susceptible to infection by T. gondii and can be used as indicator wildlife animal species in anthropogenic ecosystems.

A new case of the enigmatic Candidatus Neoehrlichia sp. (FU98) in a fox from the Czech Republic.

This study reports a new case of Candidatus Neoehrlichia sp. (FU98) infection in a fox from the Czech Republic, and provides confirmatory evidence on the occurrence of this newly identified sequence type. However, further studies are needed to investigate the distribution, host range and possible vector(s) for this bacterium, as well as its impact on animals and humans.

Role of golden jackals (Canis aureus) as natural reservoirs of Dirofilaria spp. in Romania.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are mosquito-transmitted zoonotic nematodes, causing heartworm disease and skin lesions, respectively, in carnivores. In Europe, the domestic dog is apparently the main definitive host, but patent infections occur also in other species of carnivores. The rapid spread of the golden jackals (Canis aureus) throughout Europe opens a question of involvement of this species in the sylvatic cycle of pathogens in the colonised territories, including Dirofilaria spp. METHODS: Between January 2014 and May 2015, 54 golden jackals from 18 localities in Romania were examined by full necropsy for the presence of adult filarioid nematodes and blood samples from all animals were screened for the presence of microfilariae of D. immitis, D. repens and Acanthocheilonema reconditum by multiplex PCR DNA amplification. RESULTS: Nematodes morphologically identified as D. immitis were found in 18.52% of the animals, originating from the southern part of Romania. No D. repens or A. reconditum were found at necropsy. The molecular prevalence in blood samples from the same animals was 9.26% for D. immitis and 1.85% for D. repens. All samples were negative by PCR for A. reconditum. CONCLUSION: The relatively high prevalence of Dirofilaria spp. infections in golden jackals from Romania together with the increasing density of the jackal populations highlight their potential role in the transmission of these zoonotic parasites and in the maintenance of natural disease foci.

Predilection sites for Toxoplasma gondii in sheep tissues revealed by magnetic capture and real-time PCR detection.

Undercooked lamb and mutton are common sources of Toxoplasma gondii infection for humans. A sequence specific magnetic capture technique in combination with quantitative real-time PCR targeting the 529 bp repeat element of T. gondii was used for estimation of the parasite burdens in various sheep tissues (n = 6) three months after peroral experimental inoculation with 10,000 T. gondii oocysts. Brain was the most frequently affected organ (positive in all 6 sheep) and showed the highest estimated parasite loads (0.5-30,913 parasites/g tissue). Lung samples were positive in three sheep, with load estimates of 36.3 to <1 parasite/g tissue. Heart tissue was positive in three sheep and kidney only in one animal with low parasite loads (<1 parasite/g tissue). Only few skeletal muscle samples in 2 animals showed positive results, with very low parasite burdens, while samples from further internal organs (i.e. liver and spleen) were negative in all animals. This study identified the brain as the most important predilection site and therefore the most appropriate tissue for T. gondii detection.

[When should empirical antibiotic therapy be indicated in acute community-onset diarrhea of suspected bacterial etiology]. / Kdy indikovat empirickou antibiotickou lécbu u akutní komunitní gastroenyerokolitidy suspektní bakteriální etiologie?

BACKGROUND: The aims of this study were to describe the basic parameters of adult patients with acute community-onset salmonellosis or campylobacteriosis responsible for more than 90 % of all cases of community-onset diarrhea in the Czech Republic, and, according to the results of this analysis, to update the diagnostic and therapeutic algorithms. MATERIALS AND METHODS: The data were collected retrospectively between January 1, 2011 and December 31, 2013. Patients with systemic signs of infection having at least 2 signs of systemic inflammatory response syndrome (SIRS) or with elevated serum procalcitonin levels (more than 0.5 ng/ml) were classified as being at risk for the invasive form of the disease. The remaining patients were classified as having the simple intestinal form of the disease. Patients with community-onset post-antibiotic diarrhea were excluded. The data were statistically processed. RESULTS: The following clinical factors were statistically significantly correlated with the high-risk form of the disease: duration of illness of less than 3 days before admission and any of the following (some of them are part of the SIRS classification), even after being adjusted for age: body temperature above 38 °C, peripheral blood white cell count (WBC) above 12 × 109/l, neutrophil count above 9 × 109/l and CRP level above 150 mg/l. The risk form of the disease occurred in 60 cases (18.7 %). The mean WBC was 9.4 × 109/l (median, 8.4; range, 1.7-89.0). The WBC within the normal range was seen in 194 cases (60.4 %). The mean CRP level was 92.9 mg/l (median, 77.0; range, 1.0-342.0). An elevated procalcitonin level was seen in 21 patients; the marker was not routinely measured. Positive blood culture results were obtained in 2 persons with salmonellosis; the examination was not routinely performed. There were 34 patients (10.6 %) with documented immune system dysfunction; the risk form of salmonellosis or campylobacteriosis was seen in only 11 of them (3.4 %). A total of 306 patients (95.3 %) were treated with antibiotics; the mean duration of antibiotic therapy was 8.7 days (median, 7; range, 2-31). CONCLUSIONS: Antibiotic treatment in salmonellosis/campylobacteriosis should not be indicated only due to elevated CRP levels but rather after comprehensive evaluation of the duration of symptoms, individual risk factors and dynamic changes in markers of inflammation. Blood culture tests should be carried out more frequently.

Different clinical utility of oropharyngeal bacterial screening prior to percutaneous endoscopic gastrostomy in oncological and neurological patients.

BACKGROUND: The aim of this study was to monitor oropharyngeal bacterial colonization in patients indicated for percutaneous endoscopic gastronomy (PEG). METHODS: Oropharyngeal swabs were obtained from patients prior to PEG placement. A development of peristomal infection was evaluated. The analysis of oropharyngeal and peristomal site pathogens was done. RESULTS: Consecutive 274 patients referred for PEG due to neurological disorder or cancer completed the study. Oropharyngeal colonization with pathogens was observed in 69% (190/274), dominantly in the neurologic subgroup of patients (P < 0.001). Peristomal infection occurred in 30 (10.9%) of patients and in 57% of them the correlation between oropharyngeal and peristomal agents was present. The presence of oropharyngeal pathogens was assessed as an important risk factor for the development of peristomal infection only in oncological patients (OR = 8.33, 95% CI: 1.66-41.76). Despite a high prevalence of pathogens in neurological patients, it did not influence the risk of peristomal infection with the exception for methicillin resistant Staphylococcus aureus (MRSA) carriers (OR 4.5, 95% CI: 1.08-18.76). CONCLUSION: During oropharyngeal microbial screening prior to the PEG insertion, the detection of pathogens may be a marker of the increased risk of peristomal infection in cancer patients only. In neurological patients the benefit of the screening is limited to the detection of MRSA carriers.

[Campylobacteriosis]. / Kampylobakterioza

Campylobacteriosis belongs to the most frequent bacterial gastrointestinal infections worldwide. In the past several years, an increasing trend in the prevalence of campylobacteriosis has been observed in many countries. The rapid spread of antibiotic resistance in Campylobacter spp. also poses a major challenge. The authors review current knowledge on the microbiology of Campylobacter spp., complex pathogenetic as well as pathophysiological mechanisms in the development and course of campylobacteriosis and related complications such as Guillain-Barré syndrome.

Seroprevalence of Toxoplasma gondii and Encephalitozoon cuniculi in rabbits from different farming systems.

The breeding of domestic rabbit (Oryctolagus cuniculus) for human consumption has a long tradition mainly in European and Asian countries. Infections that can affect the production of meat or even be transmitted from animals to humans are important to monitor, especially for public health reasons as well as for their impact on animals health. This study aimed to collect sera from rabbits bred in different conditions and test the presence of Toxoplasma gondii and Encephalitozoon cuniculi antibodies. Whether infections were active or latent was assessed by determining the occurrence of IgM or IgM together with IgG antibodies which indicated active infection whereas latent infection was characterized by finding IgG antibodies only. An ELISA test was performed with 1883 sera samples collected throughout the Czech and Slovak Republics. The seroprevalence of T. gondii in 902 samples from 6 commercial farms (CF) was very low with only 4 rabbits (0.4%) being positive. In total 99 (10.1%) individuals out of 981 samples from 29 household farms (HF) were positive for T. gondii antibodies. Only 2 (50%) of the T. gondii positive CF rabbits had active infections while the rest were latently infected. The serological results showed that 35 (35.4%) rabbits from the T. gondii positive HF group suffered from active infection. Out of CF samples 185 (20.5%) were positive for E. cuniculi. Antibodies of E. cuniculi were detected in 497 (50.7%) HF rabbits. Active E. cuniculi infections were determined in 85.9% of CF and 56.3% of HF rabbits; respectively. Interestingly, the E. cuniculi positive rabbits were significantly more often positive for anti-T. gondii antibodies in comparison to E. cuniculi negative individuals. Prevalence of T. gondii in CF rabbits was negligible. According to our results meat of HF rabbits still poses a risk of T. gondii infection. Nevertheless, the risk is on its lowest level in 20 years which is apparently caused due to changes in feeding practices. The occurrence of E. cuniculi antibodies was significantly lower in rabbits from commercial farms, apparently because of better hygiene conditions.

Survival of experimentally induced Toxoplasma gondii tissue cysts in vacuum packed goat meat and dry fermented goat meat sausages.

Ingestion of raw or undercooked meat is a potential source of human toxoplasmosis. The aim of this study was to determine the viability of Toxoplasma gondii cysts in vacuum packed (VP) goat meat and in dry fermented sausages (DFS), and evaluate certain physical and chemical parameters, like water activity (aw), pH value, content of salt, dry matter and fat. A portion of muscle tissue from experimentally infected animals was used for production of VP meat with or without addition of 2.5% curing salt, and stored at 4 °C or at -20 °C. Results of bioassay showed that, samples of vacuum packed Toxoplasma positive meat without salt addition were alive after six weeks at 4 °C. Incubation at -20 °C supported the viability after 3 h, but not after 4 h. After 7 days in 2.5% of curing salt, samples of T. gondii VP goat meat were still viable, but not after 14 days at 4 °C. All the DFS samples were not positive for infective cysts which mean that, they do not pose a risk of T. gondii transmission. These data suggest that vacuum packaging increases the survival of T. gondii cysts.

Efficacy of magnetic capture in comparison with conventional DNA isolation in a survey of Toxoplasma gondii in wild house mice.

Toxoplasma gondii is a zoonotic parasite with a world-wide distribution. House mice (Mus musculus) play an important role as a reservoir host in the parasite life cycle. However, their detection in mouse brain is limited because the host potentially harbours only a few tissue cysts. In order to improve the diagnosis, we tested a novel protocol for T. gondii detection in mice and compared this technique to a standard PCR-based protocol using a commercial kit for DNA isolation. Efficacy of magnetic capture for isolation of T. gondii DNA from whole host brains was tested in brain samples of laboratory mice spiked with 1 up to 10(4) tachyzoites. Real-time PCR revealed that even 1-5 tachyzoites can be detected after magnetic capture. Also this method is suitable to quantify parasite numbers in mouse brains with more than 10 tachyzoite equivalents. To assess the two techniques in wild mice, we employed a dataset consisting of 243 individuals. The prevalence of T. gondii detected by magnetic capture and qPCR and by commercial isolation and PCR was 1.2% and 0%, respectively. The magnetic capture and quantitative PCR seems to be a highly sensitive and specific diagnostic method for both laboratory research and wild population surveys.

Brain is the predilection site of Toxoplasma gondii in experimentally inoculated pigs as revealed by magnetic capture and real-time PCR.

Pigs represent an important source of food in many countries, and undercooked pork containing tissue cysts is one of the most common sources of Toxoplasma gondii infection for humans. A magnetic capture method for the isolation of T. gondii DNA and quantitative real-time PCR targeting the 529 bp TOXO repeat element were used to estimate the parasite burden in different tissues of pigs experimentally infected with T. gondii oocysts, and to determine the predilection sites of T. gondii in this host species. The highest concentration of T. gondii DNA was found in brain tissues, equivalent to [median] 553.7 (range 3857.7-121.9) parasites per gram, followed by lungs, heart and dorsal muscles with median values corresponding to 0.3 (range 61.3-0.02); 2.6 (range 7.34-0.37) and 0.6 (range 2.81-0.31) parasites per gram of tissue, respectively. Skeletal muscles from fore and hindlimb, liver and kidney presented very low infection burdens equivalent to [median] ≤0.2 parasites per gram of tissues, and no parasite DNA could be detected in the spleen. This study contributes to understanding the value of different pig tissues as a source of T. gondii infection for humans and shows that the brain, while not being of major importance as human food source, may represent a first-line selection tissue when performing non-serological surveys (e.g. bioassays, histopathological, immunohistochemical or molecular studies) to detect T. gondii infections in pigs.

Quantification of Toxoplasma gondii in tissue samples of experimentally infected goats by magnetic capture and real-time PCR.

Undercooked meat containing tissue cysts is one of the most common sources of Toxoplasma gondii infection in humans. Goats are very susceptible to clinical toxoplasmosis, and especially kids are common food animals, thereby representing a risk for human infection. A sequence-specific magnetic capture method was used for isolation of T. gondii DNA from tissue samples from experimentally infected goat-kids and real-time PCR for the 529 bp repeat element allowed quantification of T. gondii DNA. The contamination level in different types of tissue and in two groups of goats euthanized 30 and 90 dpi was compared. The highest concentration of T. gondii DNA in both groups of goats was found in lung tissue, but only the higher parasite count in lung tissue compared to other organs in group A (euthanized 30 dpi) was statistically significant. T. gondii concentrations were higher in liver and dorsal muscle samples from goats euthanized 90 dpi than in goats euthanized at 30 dpi, while the T. gondii concentration in hearts decreased. This study describes for the first time distribution of T. gondii parasites in post-weaned goat kids. New information about T. gondii predilection sites in goats and about the progression of infection between 30 and 90 dpi was achieved.

Dissemination of IncFII(K)-type plasmids in multiresistant CTX-M-15-producing Enterobacteriaceae isolates from children in hospital paediatric oncology wards.

In this study, extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae isolates in children with malignancies hospitalised at a paediatric oncology department in the Czech Republic were investigated. From June 2009 to January 2010, a total of 50 ESBL-producing faecal isolates of Enterobacteriaceae were obtained from 28 patients. These isolates were characterised with regard to ESBL enzymes, plasmid-mediated quinolone resistance genes, multilocus sequence typing (MLST) and plasmids conferring resistance to cephalosporins and fluoroquinolones. ESBL-producing isolates included Klebsiella pneumoniae (n=36), Escherichia coli (n=7), Klebsiella oxytoca (n=3), Enterobacter cloacae (n=2) and Citrobacter freundii (n=2). Klebsiella pneumoniae isolates belonged to 7 MLST types, including sequence types ST280, ST321, ST323 and ST416 as well as the novel types ST626, ST627 and ST628. The multiresistant epidemic clone E. coli B2-O25b-ST131 was detected in one patient. The gene bla(CTX-M-15) was found on large conjugative IncFII(K) plasmids along with bla(TEM-1), bla(OXA-1), qnrB1, aac(6')-Ib-cr, strA, sul2, aac(3')-II and tet(A) genes in most isolates. Dissemination of IncFII(K) plasmids among various Enterobacteriaceae isolates was considered an important aspect of nosocomial colonisation in the wards by Enterobacteriaceae species producing ESBLs. This is the first study documenting multiple antibiotic resistance elements, including qnr genes, in IncFII(K) plasmids in various bacterial species isolated in a single hospital department. The results highlight the evolution of IncFII(K) plasmids into new variants containing novel antibiotic resistance elements and their important role in spreading ESBL-producing bacteria among hospitalised patients.