Detection and Molecular Characterization of Canine Babesiosis Causative Agent Babesia canis in Naturally Infected Dogs in the Dobrogea Area (Southeastern Romania).

Canine babesiosis is an emerging tick-borne disease of major veterinary concern in Europe. Its prevalence has increased in the last two decades and is spreading rapidly toward the north. The aim of this study was to investigate the genetic diversity of Babesia spp. strains isolated from naturally infected dogs in a tick-endemic area (Dobrogea) in southeastern Romania. For this purpose, a total of twenty-three samples from dogs diagnosed with various clinical forms of babesiosis, evaluated by means of clinical history, physical examination, and hematological tests, were subjected to a molecular investigation using PCR, sequencing analysis, and genetic characterization. A microscopic examination of thin Diff-quick-stained blood smears revealed large intra-erythrocytic Babesia piroplasms in all dogs. The PCR and sequencing analysis results indicated the presence of Babesia canis in 22 dogs (95.7%) and Babesia vogeli in 1 dog (4.3%). Among the B. canis isolates, two genotypes were distinguished based on two nucleotide substitutions (GA→AG) observed in the 18S rRNA gene sequences (at positions 609 and 610), with the AG genotype predominating (54.5% of samples), while the GA variant was identified in 9.1% of samples. In the remaining isolates (36.4%), both variants were identified. The B. vogeli-positive dog also tested positive for antibodies against Ehrlichia canis and displayed severe disease. This study reports, for the first time, the presence of genetically heterogenic B. canis strains in dogs with clinical babesiosis in Romania. These findings provide a basis for future studies on the relationship between the genetic structure of the causative agents of canine babesiosis in Romania and the course of the disease.

New insights into semen separation techniques in buffaloes.

Male infertility is frequently caused by idiopathic or unexplained reasons, resulting in an increase in demand for assisted reproductive technologies. In buffaloes, more than in other animals due to reproductive hardiness, successful fertilization needs spermatozoa to effectively transit the female reproductive system to reach the oocyte. This mechanism naturally picks high-quality sperm cells for conception, but when artificial reproductive technologies such as in vitro fertilization, intracytoplasmic sperm injection, or intrauterine insemination are utilized, alternative techniques of sperm selection are necessary. Currently, technology allows for sperm sorting based on motility, maturity, the lack of apoptotic components, proper morphology, and even sex. This study provides current knowledge on all known techniques of sperm cell sorting in buffaloes, evaluates their efficiency, and discusses the benefits and drawbacks of each approach.

Serological Evidence of Natural Exposure to Tick-Borne Pathogens in Horses, Romania.

The purpose of this study was to investigate the seroprevalence of selected tick-borne-pathogens (TBPs) among Romanian horses. For this, a total of 223 animals originating from north, central, and southeast Romania, including horses from stud farms (n = 118) and working horses (n = 105), were tested using a commercial rapid ELISA-based test. Overall, 10.3% (95% confidence interval (CI): 6.7-15.1%) of the tested horses were seropositive for antibodies (Ab) against Anaplasma phagocytophilum. Additionally, 18.8% (95% CI: 13.9-24.6%) and 0.5% (95% CI: 0.01-2.5%) of horses were seropositive for Ab against Borrelia burgdorferi sensu lato and Ehrlichia spp., respectively. Among the tested horses, 3.1% were seroreactive to two or three pathogens. These findings show the natural exposure of Romanian horses to zoonotic tick-borne pathogens and emphasize the need for further studies to better understand the epidemiology of equine tick-borne diseases in Romania.

Clinical Occurrence of Canine Babesiosis in the Coastal Area of the Black Sea (Dobrogea) in Southeastern Romania and Associated Epidemiological Implications.

Canine babesiosis (CB) is a significant tick-borne disease caused by intra-erytrocytic protozoa of the genus Babesia (Apicomplexa: Piroplasmida) and is currently emerging or re-emerging in many European countries. Despite the increasing incidence of clinical CB, little is known of the epidemiology of this disease in Romania. Therefore, the present study was conducted to provide epidemiological data on CB in the coastal area of the Black Sea (Dobrogea), southeastern Romania. For this, 306 owned dogs presented during 2016-2018 at a veterinary clinic in the area and clinically suspected for CB were tested for the presence of intra-erytrocytic piroplasms using Giemsa-stained thin blood smears. Overall, 27.8% (95% CI: 22.83-33.16) of dogs were positive for large piroplasms. The Babesia spp.-positive dogs were 51 males and 34 females aged from 3 mo to 13 yr (mean 4.2 yr; SD = 3.6). A higher rate of infection was registered in Bichon Maltese (n = 13) and mixed-breed (n = 28) dogs. Most cases (76/85; 89.4%) were diagnosed from March to June, with only a few cases registered in November and December (1 case each). Infected dogs displayed different clinical presentations, varying from mild (38.8%) to moderate (29.4%) and severe disease (29.4%). Based on clinical- pathological changes, 38 (44.7%), 34 (40.0%), and 13 (15.3%) dogs were diagnosed with uncomplicated babesiosis, complicated babesiosis with a single organ dysfunction, and complicated babesiosis with multiple-organ-dysfunction syndrome (MODS), respectively. The recovery rate (81.6%, 70.1%, and 38.4%, respectively) was significantly different in these groups. The age of dogs appeared to be a risk factor for both severe disease (mean age of 5.8 yr) and MODS (mean age 6.8 yr). These findings document the common occurrence of CB in Dobrogea, southeastern Romania, and suggest that appropriate control measures need to be taken.

The first seroepidemiological survey for Angiostrongylus vasorum in domestic dogs from Romania.

BACKGROUND: Angiostrongylus vasorum is a metastrongyloid nematode localized in the right heart and the pulmonary arteries of domestic dogs. The number of reports in Europe has recently increased, presumably as a consequence of a growing awareness among clinicians, animal owners and researchers, but also due to a growing incidence and territorial spread. So far, no studies have been conducted to assess the prevalence and distribution of A. vasorum in domestic dogs in Romania, and the awareness among veterinarians is limited or absent. The aim of the present study was to evaluate the countrywide seroprevalence of circulating antigens of A. vasorum and specific antibodies against A. vasorum in domestic dogs from Romania. METHODS: Between November 2016 and July 2017, blood was sampled from a total of 1545 domestic dogs from 23 counties of Romania. Details about their gender, age, breed, housing, use and origin were collected. All serum samples were tested for the presence of A. vasorum circulating antigens (AG) using monoclonal and polyclonal antibodies in a sandwich ELISA. Additionally, a sandwich ELISA using A. vasorum adult somatic antigen purified by monoclonal antibodies was used for specific antibody (AB) detection. RESULTS: A total of 33 dogs (2.14%, 95% CI: 1.82-3.56%) were seropositive for A. vasorum antigen or antibodies against the parasite. Three dogs were positive for antigen only (0.19%, 95% CI: 0.07-0.57%) and 30 dogs (1.94%, 95% CI: 1.36-2.76%) were positive for antibodies only. No dog was positive for both tests. The overall prevalence (AB or AG) and the AB prevalence were significantly higher in pure breed dogs compared to mixed breeds and mongrel dogs (P < 0.05) and in shepherd dogs compared to other groups (P < 0.05). There was no significant difference between males and females, between urban and rural dogs, between dogs with unrestricted access and with restricted access to the environment, and between dogs living outdoors and indoors. CONCLUSIONS: Our data suggest that the disease is present in Romania in dogs, as it was previously demonstrated in foxes. However, so far, no clinical case has been reported in the country and this may be related to a low awareness among vets.

Parasitological and molecular diagnostic of a clinical Babesia caballi outbreak in Southern Romania.

Equine piroplasmosis (EP) is a tick-borne disease of equids caused by Babesia caballi and/or Theileria equi, which is endemic in many tropical and temperate areas of the world. However, clinical outbreaks of EP in Romania during the last decades have not been reported Therefore, the aim of this paper is (i) to describe a clinical B. caballi outbreak in horses on several farms in Southern Romania using a diagnostic and therapeutic approach and (ii) the molecular diagnostic of EP in an endemic area of Romania. In the first case, a 10-month-old stallion male was presented with lethargy, anorexia, fever (40.9 °C), pale mucosal/mucous/membranes and a marked anemia. In the subsequent weeks, three horses from other farms located in the same area, displayed similar clinical signs. B. caballi was diagnosed in all the horses based on Giemsa-stained blood smears and the diagnosis was further confirmed by polymerase chain reaction (PCR), using a single-round and multiplex PCR and sequencing. All four horses were treated with imidocarb dipropionate, at a dose rate of 2.2 mg/kg body weight (two injections at 48 h apart), and all horses clinically recovered within 24-48 h, post-treatment. This report presents the first molecularly characterized B. caballi outbreak in Romania in clinically affected horses, confirmed by DNA sequencing.

Differential influence of tacrolimus and sirolimus on mitochondrial-dependent signaling for apoptosis in pancreatic cells.

To examine and compare the mitochondria-related cellular mechanisms by which tacrolimus (TAC) or sirolimus (SIR) immunosuppressive drugs alter the pancreatic exocrine and endocrine ß-cell fate. Human exocrine PANC-1 and rat endocrine insulin-secreting RIN-m5F cells and isolated rat islets were submitted to 1-100 nM TAC or SIR. In cultures, insulin secretion was measured as endocrine cell function marker. Apoptosis was quantified by annexin 5 and propidium iodide staining. Cleaved caspase-3, Bax apoptosis indicators, and p53, p21 cell cycle regulators were detected by Western blot. Cell cycle and mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry and SA-beta-galactosidase (SA-ß-gal) activity by fluorescence microscopy. Only TAC reduced insulin secretion by RIN-m5F after 24 h. TAC and SIR promoted moderate apoptosis in both PANC-1 and RIN-m5F after 24 h. Apoptosis was associated with up-regulated Bax (threefold) and cleaved caspase-3 (fivefold) but only in PANC-1, while p53 and p21 were up-regulated (twofold) in both cell lines. ΔΨm was impaired only in PANC-1 by TAC and SIR. Only SIR prompted cell cycle arrest in both cell lines. The induction of a premature senescence-like phenotype was confirmed in isolated islets by SA-ß-gal activity. TAC and SIR are early inducers of pancreatic cell dysfunction and apoptosis but differentially alter endocrine and exocrine cells via mitochondrial-driven pathways. In rat islets, TAC and SIR prompt a senescence-like phenotype.

New Cases of Thelazia callipaeda Haplotype 1 in Dogs Suggest a Wider Distribution in Romania.

Thelazia callipaeda is an emerging vector-borne zoonotic helminth parasitizing the conjunctival sac of a broad spectrum of definitive hosts, such as dogs, cats, rabbits, wild carnivores, and humans. Its presence is associated with mild to severe ocular disease. Here, we report two new clinical cases in dogs originating from western and southern Romania, with no travel history. On clinical examination, the nematodes were retrieved from the conjunctival sac and identified using morphological keys and molecular tools. Twenty-two adult nematodes (8 males, 14 females) were collected and were identified as T. callipaeda by morphology. The molecular analysis revealed a 100% identity with haplotype h1 of T. callipaeda. This study describes the occurrence of new autochthonous cases of thelaziosis in Romania, reinforcing the spreading trend of this zoonotic eyeworm and highlighting the need for increased awareness among medical and veterinary practitioners. Moreover, we provide additional molecular evidence for the exclusive distribution of haplotype 1 of T. callipaeda in Europe.

Molecular detection of Rickettsia conorii and other zoonotic spotted fever group rickettsiae in ticks, Romania.

The diverse tick fauna as well as the abundance of tick populations in Romania represent potential risks for both human and animal health. Spotted fever group (SFG) rickettsiae are recognized as important agents of emerging human tick-borne diseases worldwide. However, the epidemiology of rickettsial diseases has been poorly investigated in Romania. In urban habitats, companion animals which are frequently exposed to tick infestation, play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Therefore, the aim of the present study was to investigate the occurrence of SFG rickettsiae in ticks infesting dogs in a greater urban area in South-eastern Romania. Adult ixodid ticks (n=205), including Rhipicephalus sanguineus sensu lato (n=120), Dermacentor reticulatus (n=76) and Ixodes ricinus (n=9) were collected from naturally infested dogs and were screened for SFG rickettsiae using conventional PCR followed by sequencing. Additionally, ticks were screened for DNA of Babesia spp., Hepatozoon spp., Ehrlichia canis, and Anaplasma platys. Four zoonotic SFG rickettsiae were identified: Rickettsia raoultii (16%) and Rickettsia slovaca (3%) in D. reticulatus, Rickettsia monacensis (11%) in I. ricinus, and Rickettsia conorii (0.8%) in Rh. sanguineus s.l. Moreover, pathogens of veterinary importance, such as B. canis (21%) in D. reticulatus and E. canis (7.5%) in Rh. sanguineus s.l. were identified. The findings expand the knowledge on distribution of SFG rickettsiae as well as canine pathogens in Romania. Additionally, this is the first report describing the molecular detection of R. conorii in ticks from Romania.

New insights into the molecular epidemiology of Trichinella infection in domestic pigs, wild boars, and bears in Romania.

Trichinellosis is a food-borne zoonosis caused by the parasitic nematode Trichinella, characterized by an extremely wide host range and geographical distribution. In Romania, it is recognized as one of the most serious zoonotic diseases. A cross-sectional study, covering all regions of Romania, was conducted in 2014 to investigate and update the prevalence of Trichinella infection among domestic pigs, wild boars, and bears. Additional, molecular identification of Trichinella species circulating among these animals was performed in order to establish the biogeography of Trichinella species within the seven geographical regions of Romania. For this, a total of 113,383 pigs raised in non-controlled housing conditions (backyards), 5596 hunted wild boars and 147 hunted bears were subjected to Trichinella analysis. The highest prevalence of Trichinella infections was found in bears (12.93%), followed by wild boars (1.66%) and domestic pigs (0.20%). Of 294 Trichinella isolates that tested positive by multiplex PCR, 219 (74.49%) were identified as Trichinella spiralis, 66 (22.45%) as Trichinella britovi, and 9 isolates (3.06%) as mixed infections of T. spiralis and T. britovi. T. spiralis was more prevalent in domestic pigs (165/228; 72.37%) than in game (63/228; 27.63%), while T. britovi showed a higher prevalence in game (50/75; 66.66%) than in domestic pigs (25/75; 33.33%). Moreover, the present study revealed a significant host- and area- related distribution of Trichinella species within the seven regions of Romania. Therefore, these findings are of epidemiological relevance, updating data on the prevalence and distribution of Trichinella species circulating among domestic and wild animals in South-Eastern Europe.

Occurrence and genetic characterization of Echinococcus granulosus in naturally infected adult sheep and cattle in Romania.

An epidemiological and molecular study was conducted to investigate the occurrence and genetic diversity of Echinococcus granulosus isolates from adult sheep and cattle in Romania. Overall, 642 sheep (aged over 3 years) and 1878 cattle (aged over 5 years) from 16 counties were examined for hydatid cysts. Of them, 421 (65.6%) sheep and 754 (40.1%) cattle were found infected by cystic echinococcosis (CE). Germinal layers were collected from 98 individual cysts (one cyst per animal; 31 from sheep and 67 from cattle), DNA was extracted and two different mitochondrial DNA genes, namely cytochrome c oxidase subunits 1 (CO1) and 12S ribosomal DNA (12S rDNA) were used as genetic markers. The assessment of the genetic diversity of the Echinococcus strains showed the presence of the E. granulosus sensu stricto complex and disclosed an apparent dominance of the G1 genotype within the G1­G3 complex. Furthermore, several mitochondrial variants were identified for the G1 and G2 genotypes of E. granulosus s.s. complex. Overall, the findings were of epidemiological relevance and highlighted a high potential risk of zoonotic infection.

Comparative study for the detection of antibodies to Neospora caninum in milk and sera in dairy cattle in southern Romania.

The study aimed to assess the within-herd Neospora caninum exposure in dairy cattle in southern Romania, based on the detection of specific antibodies in milk and serum. A total of 104 paired samples of milk and serum were collected from four dairy farms. Individual samples were analyzed for N. caninum antibodies by ELISA: IDEXX Neospora Ab (Idx) (three farms: A, B, C; n = 60) and ID-VET Lab (Idv) (farm D; n = 44). Additionally, four pooled milk samples, one per each farm (A, B, C) and a composed one (A+B+C), were analyzed with Idx ELISA. Optimized cut-off values for milk samples were determined by receiver operating characteristic (ROC) analysis, with serum results considered as true status. The agreement was expressed by K values. The overall seroprevalence of N. caninum infection was 45% in the farms tested by Idx ELISA and 56.8% in the farm tested by Idv ELISA. A good agreement between serum and milk was obtained for both ELISA kits (K = 0.72 and 0.77, respectively). The specificity and sensitivity at optimized cut-off of S/P>0.704 for Idx and S/P%>7.966% for Idv were 100% and 70.37% for Idx and 89.47% and 88% for Idv. Testing pooled milk samples, there were identified as N. caninum positive the dairy farms with a 15% or higher within-herd seroprevalence at the cut-off value of S/P>0.51. This is the first study in Romania in which milk samples were tested to determine the N. caninum infection status in dairy farms, providing a base for further researches.

Exocrine cell-derived microparticles in response to lipopolysaccharide promote endocrine dysfunction in cystic fibrosis.

BACKGROUND: Diabetes in cystic fibrosis (CF) is a result of exocrine pancreas alteration followed by endocrine dysfunction at a later stage. Microparticles (MPs) are plasma membrane fragments shed from stimulated or damaged cells that act as cellular effectors. Our aim was to identify a new form of interaction between exocrine and endocrine pancreatic cells mediated by exocrine MPs, in the context of recurrent infection in CF. METHODS: MPs from either human exocrine CFTRΔF508-mutated (CFPAC-1) cells or exocrine normal pancreatic (PANC-1) cells were collected after treatment by LPS from Pseudomonas aeruginosa and applied to rat endocrine normal insulin-secreting RIN-m5F cells. MP membrane integration in target cells was established by confocal microscopy and flow cytometry using PKH26 lipid probe. Apoptosis, lysosomal activity, insulin secretion were measured after 18 h. MP-mediated NF-κB activation was measured in HEK-Blue reporter cells by SEAP reporter gene system and in RIN-m5F cells by Western blot. In endocrine normal cells, CFTR inhibition was achieved using Inhibitor-172. RESULTS: Compared to PANC-1, MPs from CFPAC-1 significantly reduced insulin secretion and lysosomal activity in RIN-m5F. MPs induced NF-κB activation by increasing the level of IκB phosphorylation. Moreover, the inhibition of NF-κB activation using specific inhibitors was associated with a restored insulin secretion. Interestingly, CFTR inhibition in normal RIN-m5F cells promoted apoptosis and decreased insulin secretion. CONCLUSIONS: During recurrent infections associated with CF, exocrine MPs may contribute to endocrine cell dysfunction via NF-κB pathways. Membrane CFTR dysfunction is associated with decreased insulin secretion.

Molecular evidence for bacterial and protozoan pathogens in hard ticks from Romania.

The aim of the present study was to provide a preliminary insight into the diversity of tick-borne pathogens circulating at the domestic host-tick interface in Romania. For this, feeding and questing ticks were analyzed by real-time polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, Borrelia burgdorferi sensu latu, and by PCR and subsequent sequencing for Rickettsia spp., Babesia spp. and Theileria spp. A total of 382 ticks, encompassing 5 species from 4 genera, were collected in April-July 2010 from different areas of Romania; of them, 40 were questing ticks and the remainder was collected from naturally infested cattle, sheep, goats, horses or dogs. Tick species analyzed included Ixodes ricinus, Dermacentor marginatus, Hyalomma marginatum, Rhipicephalus bursa, and Rhipicephalus sanguineus. Four rickettsiae of the spotted fever group of zoonotic concern were identified for the first time in Romania: Rickettsia monacensis and Rickettsia helvetica in I. ricinus, and Rickettsia slovaca and Rickettsia raoultii in D. marginatus. Other zoonotic pathogens such as A. phagocytophilum, Borrelia afzelii, and Babesia microti were found in I. ricinus. Pathogens of veterinary importance were also identified, including Theileria equi in H. marginatum, Babesia occultans in D. marginatus and H. marginatum, Theileria orientalis/sergenti/buffeli-group in I. ricinus and in H. marginatum and E. canis in R. sanguineus. These findings show a wide distribution of very diverse bacterial and protozoan pathogens at the domestic host-tick interface in Romania, with the potential of causing both animal and human diseases.

Neospora caninum infection in dogs from Southern Romania: coproparasitological study and serological follow-up.

Neospora caninum is an important cause of bovine abortion worldwide for which dogs are the definitive host. The present study was aimed at investigating the exposure to N. caninum infection based on lifestyle categories of dogs from southern Romania. For this purpose, randomly selected rural and urban dogs were examined for fecal N. caninum -like oocysts and were serologically tested for the presence of anti- N. caninum IgG antibodies. Of the 386 dog fecal samples, N. caninum -like oocysts were found in 19 (4.9%; 95% CI = 2.89-7.59) as follows: rural guard dogs (4/41; 9.8%), cattle farm dogs (6/118; 5.1%), and stray dogs (9/192; 4.7%) (P > 0.05). None of the 35 urban guard dogs was positive. Serum samples (n = 84) from all of the 19 N. caninum -like oocysts-positive dogs and another 65 randomly selected canines (15 cattle farm dogs, 21 rural guard dogs, and 29 strays) were tested by indirect fluorescent antibody test (IFAT). None of these dogs exhibited any symptoms of clinical neosporosis. However, IgG antibodies against N. caninum were detected in 17/84 (20.2%) (P < 0.05) serum samples. The highest prevalence was registered in cattle farm dogs (38.1%) followed by strays (18.4%) and rural guard dogs (8.0%). The seropositivity to N. caninum increased significantly with age (P < 0.05), reaching 66.7% in dogs >10 yr of age, suggesting post-natal exposure to N. caninum is the predominant mechanism of N. caninum recruitment.

Cystic echinococcosis in Romania: an epidemiological survey of livestock demonstrates the persistence of hyperendemicity.

An epidemiological survey of cystic echinococcosis (CE) in cattle and sheep was conducted from October 2009 to October 2011 in endemic areas of north-eastern and southern Romania. A total of 8569 animals (3043 cattle and 5526 sheep) were examined, and hydatid cysts were found in 984 (32.34%) cattle and 2756 (49.87%) sheep, respectively. The average number of cysts per animal was 11.3 (range, 1-47) in cattle and 4.2 (range, 1-16) in sheep. Of the CE-positive animals, 177 (17.9%) of the cattle and 358 (12.98%) of the sheep had hydatid cysts only in the lungs, 62 (6.3%) and 803 (29.13%) had only in the liver, and 723 (73.47%) and 1572 (57.04%), respectively, had hydatid cysts both in the liver and in the lungs. Very few animals had cysts in spleen (19 cattle and 23 sheep) and kidney (three cattle). From a sample of 422 cysts undergoing closer examination (258 from cattle, 164 from sheep), the highest fertility rate (38.41%) was found in sheep cysts, while from the cattle only four cysts (1.55%) were found to be fertile. Species identification of 13 animal isolates (nine from sheep, four from cattle) and one human isolate were done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the mitochondrial nad1 gene and confirmed by partial sequencing of the cox1 gene. All showed the same RFLP band pattern (Echinococcus granulosus sensu stricto) and were identified as the "sheep strain" G1 by sequencing. The study emphasizes the hyperendemic presence of E. granulosus in Romania and outlines the necessity for the urgent development of sustainable surveillance and control strategies both in animals and humans.

Seroprevalence of Neospora caninum infection on dairy cattle in farms from southern Romania.

Neospora caninum, a coccidian parasite closely related to Toxoplasma gondii, is one of the major causes of abortion in cattle worldwide. Conventional serological techniques, such as the indirect fluorescent antibody test and enzyme-linked immunosorbent assay (ELISA), are routinely used in adult animals and aborted fetuses for the detection of anti- N. caninum antibodies. In Romania, infection with N. caninum in cattle has been reported recently, but only in limited areas from the north and central parts of the country. Therefore, the aim of this study was to obtain additional seroepidemiological data on infection with N. caninum on dairy farms from the south of Romania. A total of 258 blood samples was analyzed from 230 dairy cows and 28 calves from 9 dairy farms in southern Romania; the presence of specific IgG antibodies against N. caninum was determined using an indirect ELISA test. The average seroprevalence was 40.3%, but the within-herd prevalence ranged between 11.5 and 80.0%; the seroprevalence in dairy cows was 41.7%, while in calves it was 28.6%. Of the positive samples, 74.0% (77/104) had a high positive reaction (S/P ratio more than 1.0), while 26.0% (27/104) had a low positive reaction (S/P ratio between 0.5 and 1.0). This study indicates that N. caninum infection is widespread in the south of Romania, which could explain the causes of abortions registered in some herds in the studied area. However, a serological screening across the country is planned in order to assess the actual national prevalence of N. caninum infection, followed by implementation of a prevention and control program.

Canine babesiosis in Romania due to Babesia canis and Babesia vogeli: a molecular approach.

Canine babesiosis is a tick-borne disease caused by the protozoa Babesia spp. that affects dogs worldwide. In Romania, canine babesiosis has become quite frequent in the last few years, with a wide variety of clinical signs, ranging from mild, nonspecific illness to peracute collapse, and even death. Traditionally, a Babesia infection in dogs is diagnosed based on the morphologic appearance of the intraerythrocytic piroplasms observed in peripheral blood smears. To date, no data on genetic characterization of Babesia species in dogs has been documented for Romania. Therefore, a molecular survey on natural Babesia infections of dogs in Romania using polymerase chain reaction and genetic sequence analysis of a fragment of the ssRNA gene was performed. A total number of 16 blood samples were tested for the presence of Babesia DNA. Blood samples were collected from 11 dogs with symptoms of babesiosis and microscopically proven positive for Babesia and from a group of five asymptomatic dogs, not tested microscopically for Babesia, which were included in the study for comparative analysis. The piroplasm-specific PCR amplifying the partial 18S rRNA gene confirmed Babesia spp. infection in all 11 samples from dogs with clinical babesiosis, and in one of the clinically normal dogs. Sequence analysis revealed the presence of Babesia canis in all clinically affected dogs and Babesia vogeli in one clinically normal dog. This is the first molecular evidence of B. canis and B. vogeli in dogs from Romania. The results of the study provide basic information toward a better understanding of the epidemiology of canine babesiosis in Romania and will help to promote an effective control program.

Use of a reverse line blot assay to survey small strongyle (Strongylida: Cyathostominae) populations in horses before and after treatment with ivermectin.

A sensitive and specific PCR hybridization assay was applied for species-specific monitoring of the small strongyle (Strongylida: Cyathostominae) populations in horses in a herd before and after treatment with the anthelmintic drug ivermectin. Fecal samples were collected pre- and post-treatment weekly from eight individual horses (four foals and four yearlings) for 6 weeks to determine counts of strongyle eggs per gram of feces (EPGs). Additionally, one foal and one yearling were nontreated controls. Also, one horse, from another herd known to be infected with Strongylus spp., was a positive control for these parasites. Genomic DNA was obtained from eggs in groups of approximately 6000-7000 eggs except for two samples containing low EPGs in which 450 eggs were used. Amplification of the intergenic spacers (IGSs) of ribosomal DNA (rDNA) of small and large strongyles followed by reverse line blot (RLB) assay were performed to identify the presence of the 12 most common equine small strongyle species and to discriminate them from Strongylus spp. Overall, 11 small strongyle species were identified in pretreatment samples. In the samples collected at 4 weeks after ivermectin treatment, eight small strongyle species were identified and four of them were predominant (Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus calicatus and Cylicostephanus minutus). At 5 and 6 weeks post-treatment, the RLB assay analysis showed almost the same composition in the small strongyle population as before treatment. Strongylus spp. were identified only in samples collected from the positive control horse for these parasites. These data confirm the ability of the PCR-RLB technique for simultaneous species-specific differentiation of equine strongyle eggs, indicating a valuable way of furthering drug-resistance studies.