Kálmán Imre

Zoonotic Cryptosporidium parvum in Romanian newborn lambs (Ovis aries).

This study was undertaken to investigate the occurrence and public health significance of Cryptosporidium species/genotypes and subtypes in a newborn lambs. A total of 175 diarrheic fecal samples from lambs (younger than 21 days) were collected in seven sheep flocks located in western Romania, and were microscopically examined for the presence of Cryptosporidium oocysts after staining with modified Ziehl-Neelsen technique. Twenty-four (13.7%) fecal samples were tested Cryptosporidium positive by microscopy and were subjected for molecular characterization. All positive samples were successfully amplified through a nested polymerase chain reaction (PCR) of the small subunit (SSU) rRNA gene (18S). Cryptosporidium species were determined by restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products using the conventional SspI and VspI restriction enzymes. The identified species were: Cryptosporidium parvum (20/24), C. ubiquitum (2/24) and C. xiaoi (2/24), respectively. PCR-RFLP results for C. ubiquitum and C. xiaoi isolates were confirmed by DNA sequencing. Subsequently, subtyping of seven randomly selected C. parvum isolates, based on sequence analysis of the GP60 gene, revealed the presence of five different subtypes (IIaA17G1R1, IIaA16G1R1, IIdA20G1, IIdA24G1 and IIdA22G2R1) belonging in two zoonotic subtype families (IIa and IId). These findings may suggest the potential role of the newborn lambs as a source for human cryptosporidiosis. This is the first published report about the presence of C. ubiquitum and C. xiaoi in lambs from Romania.

Molecular characterisation of Cryptosporidium isolates from pre-weaned calves in Romania: is there an actual risk of zoonotic infections?

Diarrheic fecal samples from 258 pre-weaned calves (1-30 day-old) from 9 dairy farms located in Banat region, Romania, were microscopically examined for the presence of Cryptosporidium oocysts. Overall, 65 (25%) samples were found positive. A higher percent of infection was recorded in calves aged between 8 and 14 days compared with other age categories (1-7, 8-14, 15-21 and 22-30 days; p<0.05). Genetic characterization was carried out on all Cryptosporidium-positive samples. After DNA extraction, Cryptosporidium species were determined by a nested PCR of the small subunit rRNA gene (18S) followed by RFLP analysis with SspI, VspI and MboII restriction enzymes. The restriction patterns showed that animals were infected with Cryptosporidium parvum. Subsequently, subtyping of 13 C. parvum isolates, based on sequence analysis of the 60 kDa glycoprotein (GP60) gene, showed 2 subtypes (IIaA15G2R1 and IIaA16G1R1) belonging to the subtype family IIa. This is the first molecular study of bovine Cryptosporidium infection in Romania.

Seroprevalence of Babesia canis Infection in Clinically Healthy Dogs From Western Romania

Abstract:u2003 Serum samples from 197 clinically healthy dogs residing in the Banat Region, the western historical part of Romania, were assayed by an indirect fluorescent antibody test for the presence of anti-Babesia canis antibodies. Overall, the seroprevalence was 19.8% (39/197). The percent of seropositive dogs in rural areas (28.4%; 19/67) was significantly higher (P < 0.05) compared to dogs living in urban areas (15.4%; 20/130). Seroprevalence of B. canis infection in hunting dogs was also found to be significantly higher (P < 0.05) compared to canines with other lifestyles, but no significant difference was found between companion and kennel dogs. The statistical analysis showed that no significant differences (P > 0.05) were present between the seroprevalence of infected animals associated with age, gender, or breed. The hunting lifestyle was the only factor (OR = 4.57; 95% CI = 2.1–10.2; P = 0.002) positively associated with seroprevalence in dogs and can be considered the risk factor in the acquisition of infection. Also, the results of the present survey indicate that infection with B. canis in dogs is common in the sampling area and that it is an important pathogen for the local canine population.

Molecular characterisation of Cryptosporidium (Apicomplexa) in children and cattle in Romania

To investigate the transmission of species of Cryptosporidium Tyzzer, 1907 in Timis County, Romania, 48 isolates of Cryptosporidium coccidia from 11 children, 29 calves and eight pigs were characterised by molecular analysis of two loci (SSU rRNA and 60-kDa glycoprotein gene). Overall, 22 isolates were amplified and sequence analyses revealed that all isolates were Cryptosporidium parvum Tyzzer, 1912. Two subtype families were identified, IIa and IId. Subtype IIdA22G1 (n = 4) was the single C. parvum subtype found in children. Subtypes found in calves included IIdA27G1 (n = 8), a novel subtype, IIdA25G1 (n = 5), IIdA22G1 (n = 2), IIdA21G1a (n = 1), and IIaA16G1R1 (n = 1). Subtype IIdA26G1 was found in a pig. These results were significantly different from previous Romanian reports, as the five subtypes of family IId identified in this study were never identified previously in this country. Thus, cattle may be a source of Cryptosporidium infections for humans and the transmission dynamics of C. parvum in Romania is more complex than previously believed.

Survey of babesiosis in symptomatic dogs from Romania: Occurrence of Babesia gibsoni associated with breed

Blood samples from 49 symptomatic dogs from 5 western and north-western counties of Romania were screened using microscopic examination, polymerase-chain-reaction-restriction-fragment-length-polymorphism procedure (PCR-RFLP), and sequence analysis. Results of the microscopic evaluation of stained blood smears revealed 45 positive samples with the presence of large and small intraerythrocytic piroplasms in 35 and 10 samples, respectively. Babesia canis (35/49, 71.4%) and Babesia gibsoni (14/49, 28.6%) were identified and differentiated by PCR-RFLP targeting the 18S rRNA gene. Results of the sequence analysis of all B. gibsoni and 17 randomly selected B. canis PCR products confirmed the PCR-RFLP-diagnosed species. The distribution of B. gibsoni infection was positively associated (p<0.001) with fighting dog breeds including infection in 12 American Pit Bull Terriers and one American Staffordshire Terrier. This report is the first to present molecular evidence of the occurrence of B. gibsoni in Romania confirmed by sequencing.

Frequency of isolation and antibiotic resistance of staphylococcal flora from external otitis of dogs.

Otitis externa of dogs is a multifactorial aetiology disease, caused by a wide range of bacteria, parasites and fungi (Kiss and others, 1997). Out of these, species and subspecies of the genus Staphylococcus are recognised as important bacteria of the mucous membranes and natural micro flora of the skin including external ear (Cole and others, 1998, Zamankhan-Malayeri and others, 2010). Members of this genus are classified as coagulase-positive (CoPS) and coagulase-negative staphylococci (CoNS), and have been frequently reported as causative agents of the numerous infections in animal and human populations, worldwide (Malik and others, 2005).nnIn veterinary clinical practice, the treatment of dog otitis with infectious origin remain one of the most challenging problems, taking into account the rapid development of antibiotic resistance of bacterial strains associated with frequent usage of prolonged antibiotic treatments (Tejedor and Martin, 2002, Penna and others, 2010). Moreover, the application of empirical systemic or topical treatments by the small animal practitioners, without antimicrobial sensitivity testing, can contribute greatly to undesirable outcomes in a disease control. Therefore, the frequent updating of the sensitivity charts of bacterial strains with zoonotic potential isolated from pets should be one of the most important concerns of clinicians in the treatment of infectious disorders (Lilenbaum and others, 2000).nnIn Romania, the antibiotic susceptibility testing of the bacterial strains isolated from external otitis of dogs was focused on Staphylococcus genus and was carried out especially in veterinary teaching units in the last decade (Degi, 2005, Niculae and others, 2009). Thus, the current study was undertaken to investigate the occurrence of staphylococcal flora in canine patients suffering from otitis externa, and to provide new data about their antibiotic resistance.nnFrom October 2010 to January 2011, 58 dogs with characteristic clinical signs of otitis externa (eg, pruritus, local pain, otorrhoea, xaddesquamation of the ear …

Serological survey of Neospora caninum infection in cattle herds from Western Romania.

Abstract: Serum samples from 376 randomly selected adult cattle, from 25 farms located in 3 counties (Arad, Bihor, and Timiş) from western Romania, were sampled for Neospora caninum antibodies using a commercial ELISA-kit. Seroprevalence values and risk factors for neosporosis (cow age, breed, herd size, farming system, previous abortion, and number of farm dogs) were examined using a generalized linear mixed model with a binomial distribution. Overall, the seroprevalence of N. caninum was 27.7% (104/376) with a prevalence of 27.9% (24/86) in Arad, 26.9% (25/93) in Bihor, and 27.9% (55/197) in Timiş. Of 25 cattle herds, 23 were seropositive with a prevalence ranging from 10.0 to 52.2%. No correlation was found between N. caninum seropositivity and age, breed, herd size, breeding system, and previous abortion. The number of farm dogs was the only factor (PWald u200a=u200a 0.03) positively associated with seroprevalence in cows and can be considered the risk factor in the acquiring of infection. The present work is the first regarding serological evidence of N. caninum infection in cattle from western Romania.

Molecular Survey of Hepatozoon canis in Red Foxes (Vulpes vulpes) from Romania

Abstract:u2003 Blood samples of 119 red foxes, originating from 44 hunting grounds of 3 western counties (Arad, Hunedoara, and Timiş) of Romania, have been examined for the presence of Hepatozoon canis infection using the conventional polymerase chain reaction (PCR) of the fragment of 18S rRNA gene. Overall, 15 (12.6%) samples were found to be PCR-positive. Of the sampled hunting grounds, 29.5% (13/44) were found positive. Positive samples were recorded in all screened counties with the prevalence of 14.8% (9/61) in Arad, 9.8% (5/51) in Timiş, and 14.3% (1/7) in Hunedoara, respectively. No correlation was found (P > 0.05) between H. canis positivity and gender or territorial distribution of the infection. To confirm PCR results, 9 randomly selected amplicons were sequenced. The obtained sequences were identical to each other, confirmed the results of the conventional PCR, and showed 98–100% homology to other H. canis sequences. The results of the current survey support the role of red foxes as sylvatic reservoirs of H. canis in Romania.

Environmental Occurrence and Antibiotic Susceptibility Profile of Listeria monocytogenes at a Slaughterhouse Raw Processing Plant in Romania

This survey was conducted to investigate the occurrence and antimicrobial susceptibility of Listeria monocytogenes isolates in the environment of a pig slaughterhouse raw processing plant complex in western Romania. A total of 97 environmental samples from food contact (n = 60) and nonfood contact (n = 37) surfaces were examined with standard methods. The susceptibility of the isolates to 17 antimicrobial agents was determined with the VITEK 2 automated system. Overall, 25 (25.8%) samples were positive for L. monocytogenes , with recovery rates of 23.3% (n = 14) and 29.7% (n = 11) for food contact and nonfood contact surfaces, respectively. All 25 tested isolates were resistant to benzylpenicillin, imipenem, and fusidic acid. Resistance was observed to oxacillin (23 isolates; 92%), fosfomycin (23 isolates; 92%), clindamycin (22 isolates; 88%), rifampin (14 isolates; 56%), trimethoprim-sulfamethoxazole (12 isolates; 48%), tetracycline (11 isolates; 44%), and ciprofloxacin (1 isolate; 4%). No resistance was found to seven tested agents: gentamicin, moxifloxacin, erythromycin, linezolid, teicoplanin, vancomycin, and tigecycline. All tested L. monocytogenes isolates were resistant to four to nine antimicrobial agents. These results indicate a high prevalence of multidrug-resistant L. monocytogenes isolates in this pork-processing environment, providing baseline information for hygienists and public health specialists.

Review of Cryptosporidium and Giardia in the eastern part of Europe, 2016

Introduction This paper reviews the current knowledge and understanding of Cryptosporidium spp. and Giardia spp. in humans, animals and the environment in 10 countries in the eastern part of Europe: Bosnia and Herzegovina, Croatia, Czech Republic, Estonia, Hungary, Latvia, Poland, Romania, Serbia and Slovenia. Methods: Published scientific papers and conference proceedings from the international and local literature, official national health service reports, national databases and doctoral theses in local languages were reviewed to provide an extensive overview on the epidemiology, diagnostics and research on these pathogens, as well as analyse knowledge gaps and areas for further research. Results: Cryptosporidium spp. and Giardia spp. were found to be common in eastern Europe, but the results from different countries are difficult to compare because of variations in reporting practices and detection methodologies used. Conclusion: Upgrading and making the diagnosis/detection procedures more uniform is recommended throughout the region. Public health authorities should actively work towards increasing reporting and standardising reporting practices as these prerequisites for the reported data to be valid and therefore necessary for appropriate control plans.