Mirela 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.

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

Abstract:  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.

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.

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  =  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:  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.

The red fox (Vulpes vulpes) plays a minor role in the epidemiology of the domestic cycle of Trichinella in Romania.

Nematode worms of the genus Trichinella are zoonotic parasites with a worldwide distribution. The majority of the biomass of these nematodes circulates among wildlife, but when humans fail in the proper management of domestic animals and wildlife, Trichinella infections are transmitted from the sylvatic to the domestic environment. Such failures occur in Romania, where a high prevalence of Trichinella spiralis has been detected in domestic pigs. The aim of the present study was to provide data about the prevalence of Trichinella spp. infections in red foxes (Vulpes vulpes) that are hunted in Romanian counties, in which the prevalences of Trichinella spp. infection in backyard and free-ranging pigs range from 0.17 to 2.5%, to determine the role played by this carnivore species in the transmission of the parasite to domestic cycle. A total of 121 animals from 45 hunting grounds of three counties were screened to detect Trichinella spp. larvae by the digestion method. Infections were detected in 26 (21.5%) foxes from 18 (40%) hunting grounds of the three counties (13/67 in Arad, 1/3 in Hunedoara, and 12/51 in Timiş). The mean larval density was 10.5 larvae per gram. Of the 25 successfully tested samples, the Trichinella larvae from 24 isolates were identified as T. britovi (96%), and the larvae from one isolate were identified as T. spiralis (4%). No mixed infections were recorded. The present results revealed that the red fox should be considered an important T. britovi reservoir in the sylvatic cycle; in contrast, the detection of only a single T. spiralis-positive isolate suggests that red foxes play a minor role in the epidemiology of the domestic cycle in the investigated area of western Romania.

ENDOPARASITES IN MAMMALS FROM SEVEN ZOOLOGICAL GARDENS IN ROMANIA

Abstract: Animals from seven zoological gardens located in Romania, including 18 species of herbivores, 10 species of carnivores, and 13 species of omnivores, were screened for the presence of parasites. Overall, the prevalences of parasites identified in the sampled population were 54.2% (58/107) for herbivores, 54.5% (24/44) for carnivores, and 32.6% (17/52) for omnivores. In herbivores, Eimeria spp., Dicrocelium lanceolatum, and pulmonary and digestive strongyles were detected. In carnivores, the genera Eimeria and Cystoisospora and nematodes from Ancylostomatidae, Strongyloidae, Ascaridae, Capillariidae and Trichocephalidae were identified. Of 13 omnivore species included in the study, parasites from Eimeridae, Ascaridae, Strongyloidae, and Trichocephalidae were identified in seven species. Toxoplasma antibodies were identified using enzyme-linked immunosorbent assay in all definitive hosts (lions and wild cats) examined. In intermediate hosts (herbivores and omnivores), antibodies to Toxoplasma gondii had a prevalence of 58.8%, except in wild boars (Sus scrofa), in which the prevalence was 100%.

Occurrence and first molecular characterization of Sarcocystis spp. in wild boars (Sus scrofa) and domestic pigs (Sus scrofa domesticus) in Romania: Public health significance of the isolates

Domestic and wild pigs, as intermediate hosts, can harbor tissue cysts of three Sarcocystis species namely S. miescheriana, S. suihominis and S. porcifelis. Out of them, S. suihominis is zoonotic. Romania is a country with high consumption of raw and/or undercooked traditional pork products. This fact may greatly favor the acquiring of the zoonotic Sarcocystis infections by humans, as definitive host. Based on this consideration and in order to investigate the occurrence and public health significance of Sarcocystis spp. in two western counties (Caraş-Severin and Timiş) of Romania, a total of 165 heart samples from hunted wild boars (Sus scrofa, n=101) and home slaughtered domestic pigs (Sus scrofa domesticus, n=64) were screened using microscopic fresh examination and molecular methods. Microscopic examination revealed the presence of sarcocysts in 60.4% of wild boars, and 23.4% of domestic pigs. Genetic characterization of isolates through the PCR-RFLP procedure, targeting the 18S rRNA gene, was successfully achieved for all microscopically positive samples, indicating the presence of a single species, S. miescheriana, in both hosts. The identity of 13 selected S. miescheriana isolates was also confirmed through sequencing. The tested hosts older than 27 months were found to be significantly higher infected (p<0.05) with Sarcocystis than the 6 to ≤27months age group. Although the human infective S. suihominis has not been registered, for a more reliable epidemiological picture, further molecular studies enrolling a larger number of animals and diagnosis on human intestinal Sarcocystis infections are still necessary.