The Use of Pythium oligandrum in the Biological Control of Roundworm Infection in Dogs and Cats.

Pythium oligandrum is an oomycete used in veterinary medicine to treat dermatophytosis in animals. The ovicidal potential against various types of parasite eggs has not been investigated. Ascarids are very common in young animals and the resistance of eggs in the external environment is very high. A commercial product containing P. oligandrum was used in the present study. Its ovicidal action against Toxocara spp. eggs was investigated. Thus, two categories of media (soil and sandstone) were used, on which the ascarid eggs were dispersed. The commercial product was prepared as a solution and was applied according to the manufacturer. The treatment scheme indicated in case of animals with dermatophytosis was used. Thus, the external natural conditions were recreated in the laboratory, in order to establish whether there is any applicability for this oomycete in the biological control of some parasitic diseases. The results indicated an ovicidal action of Pythium oligandrum, supporting the prospects of its use in the decontamination methods of various surfaces or environments where ascarid eggs from carnivores are found.

PREVALENCE AND INTENSITY OF DIGESTIVE AND PULMONARY PARASITES IN WILD BOARS IN ROMANIA.

The prevalence and intensity of infection with digestive, liver, and pulmonary parasites in wild boars in Romania was determined by examination of 280 cadavers from 26 hunting grounds during the period 2012-2016. Eleven genera of parasites were recovered: nine within the digestive system (Eimeria, Ascaris, Globocephalus, Gongylonema, Hyostrongylus, Oesophagostomum, Physocephalus, Trichuris, and Macracanthorinchus); and two (Dicrocoelium, Metastrongylus ) located in the hepatic and pulmonary systems. The overall prevalence of infection was 80.7% (n = 280). Polyparasitism was found in 82.8% of positive cases. The mean intensity of parasitism was highest for pulmonary parasites (Metastrongylus salmi, 25.95). Regarding gastrointestinal parasites, the highest mean intensity occurred in the case of Oesophagastomum dentatum infections (22.14), whilst the lowest was that of Macracanthorhynchus hirudinaceus (1.66). Wild boars are an important source of infection for domestic pigs in Romania and neighboring countries where extensive breeding systems occur.

The prevalence, abundance and distribution of cyathostomins (small stongyles) in horses from Western Romania.

Forty seven working horses from Romania were post-mortem examined for small strongyles (Cyathostominae) infections. All horses were found infected. The overall cyathostomins intensity ranged from 390 to 13,010 and horses were infected by 8-24 species. The intensity was higher in ventral colon (1531) and dorsal colon (824), the lowest in the caecum (524). Twenty four species were identified. Cyathostomum catinatum, Cylicocyclus insigne, and C. Nassatus had 100% of prevalence. Over 50% of horses were infected by Coronocyclus coronatus, Cylicostephanus calicatus, C. goldi, and C. longibursatus. Other prevalent species (34%-45%) were Cyathostomum tetracanthum, Cylicostephanus minutus and Gyalocephalus capitatus. Coronocyclus labiatus, Parapoteriostomum mettami, Poteriostomum imparidentatum and P. ratzii had the lowest prevalence. Most species showed high organ preference with a niche breadth value between 1 and 1.96 while only 7 species (Coronocyclus labiatus, Cyathostomum tetracanthum, C. brevicapsulatus, Cylicocyclus elongatus, C. insigne, C. leptostomum and C. radiatus) showed a more generalist selection. The niche breadth of 10 species was significantly (p<0.05) influenced by itself intensity (Coronocyclus labratus, Cyathostomum pateratum, C. tetracanthum, Cylicocyclus elongatus, C. radiatus, C. ultrajectinus, C. leptostomum, Cylicodontophorus euproctus, Poteriostomum imparidentatum, P. ratzii). The niche breadth of Cylicocyclus nassatus was positively (p<0.05) influenced by the summed intensity of the other species while that of Cylicocyclus elongatus was negatively (p<0.05) influenced by the intensity of the other species. The cluster analysis of the Cyathostominae community composition showed a major cluster composed by the three dominant species, followed by a cluster composed by Coronocyclus coronatus, while all the other species presented a tree like structure.

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.

Seroprevalence of Toxoplasma gondii in Western Romania.

Toxoplasma gondii is an obligate intracellular parasite that most commonly causes asymptomatic infection in immunocompetent hosts, but can have devastating consequences in congenitally infected infants and immunocompromised patients. We evaluated the seroprevalence of T. gondii in the general population in Western Romania. Sera from 304 individuals were analysed with the Pastorex Toxo test, which allows the simultaneous detection of T. gondii IgG and/or IgM antibodies. T. gondii antibodies were demonstrated in 197 individuals (64.8%) and the prevalence increased with age: 35.0% in those < 20 years versus 76.8% in those ≥ 70 years (p < 0.001). There was a higher prevalence of T. gondii antibodies in rural areas (76.9%) than in urban regions (55.3%) (p < 0.001). Our results suggest a high prevalence of T. gondii antibodies in Western Romania.

First record of autochthonous canine ehrlichiosis caused by Ehrlichia canis in Romania.

This case study describes the first genetically confirmed and clinically manifested autochthonous Ehrlichia canis infection in a 9-year-old female mixed-breed dog from Romania. Health screening of the dog included clinical examination, evaluation of stained peripheral blood smear and hematologic variables, as well as serologic testing and molecular analysis. Clinical signs included fever, apathy, dehydration, pale mucous membranes, and weakness. The microscopic examination of the blood smear and immunologic assays for Borrelia burgdorferi, Anaplasma phagocytophilum, and E canis antibodies, and for Dirofilaria immitis antigen yielded negative results. Hematologic abnormalities included moderate nonregenerative anemia, leucopenia with neutropenia, and moderate thrombocytopenia. The biochemical abnormalities identified were hypoalbuminemia, and mildly increased serum enzyme activities of AST and ALT. In addition, increased urea and creatinine levels associated with low urine specific gravity and proteinuria were also present. Nested PCR amplification of the partial E canis 16S rRNA gene demonstrated the presence of this rickettsial pathogen in the dog's blood, which subsequently was confirmed through sequencing based on the 100% homology with GenBank deposited E canis isolates. After specific treatment with doxycycline (10 mg/kg, orally, SID) for one month, the proteinuria, and hematologic and serum biochemical abnormalities with the exception of mild azotemia resolved. This report supports the geographical expansion of canine ehrlichiosis caused by E canis in nonendemic regions of Europe.

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

Blood samples of 119 red foxes, originating from 44 hunting grounds of 3 western counties (Arad, Hunedoara, and Timis) 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 Timis, 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.

Endoparasites in mammals from seven zoological gardens in Romania.

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%.

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.

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.

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.

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

Serum samples from 376 randomly selected adult cattle, from 25 farms located in 3 counties (Arad, Bihor, and Timis) 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 Timis. 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 (P(Wald)  =  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 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.

The veterinary programs at the Romanian University of Agricultural Sciences and Veterinary Medicine.

Romania has a long tradition in the field of veterinary medicine, interest in which can be traced back to the early nineteenth century. There are four main veterinary educational centers in Romania, one for each province: the Faculty of Veterinary Medicine in Bucharest in the south; the Faculty of Veterinary Medicine in Cluj-Napoca in the northwest; the Faculty of Veterinary Medicine in Iasi in the northeast; and the Faculty of Veterinary Medicine Timisoara in the south west. The authors of this article are faculty members representing each of these schools.