Mechanisms of Pathogenic Escherichia coli Attachment to Meat.

Escherichia coli are present in the human and animal microbiome as facultative anaerobes and are viewed as an integral part of the whole gastrointestinal environment. In certain circumstances, some species can also become opportunistic pathogens responsible for severe infections in humans. These infections are caused by the enterotoxinogenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli and the enterohemorrhagic E. coli species, frequently present in food products and on food matrices. Severe human infections can be caused by consumption of meat contaminated upon exposure to animal feces, and as such, farm animals are considered to be a natural reservoir. The mechanisms by which these four major species of E. coli adhere and persist in meat postslaughter are of major interest to public health and food processors given their frequent involvement in foodborne outbreaks. This review aims to structure and provide an update on the mechanistic roles of environmental factors, curli, type I and type IV pili on E. coli adherence/interaction with meat postslaughter. Furthermore, we emphasize on the importance of bacterial surface structures, which can be used in designing interventions to enhance food safety and protect public health by reducing the burden of foodborne illnesses.

Molecular detection of hemotropic mycoplasmas (hemoplasmas) in domestic cats (Felis catus) in Romania.

BACKGROUND: The hemotropic mycoplasmas (hemoplasmas) of the genus Mycoplasma are recognized as important bacteria that parasitize red blood cells, causing hemolytic anemia in many mammalian species, including cats. No information is available concerning the presence of feline hemoplasma infections in cats in Romania. Thus, the objective of the present study was to provide data on the occurrence and molecular characterization of hemotropic mycoplasmas in client-owned cats in Romania. METHODS: Blood samples from 51 unhealthy cats, originating from Timisoara Municipality, Romania, were screened for the presence of hemoplasmas using conventional polymerase chain reaction (PCR) targeting the 16S rRNA gene and sequencing assays. PCR-positive samples were subsequently analyzed by phylogenetic and population genetic analysis. RESULTS: Molecular analysis revealed 11 (21.6%) positive samples, consisting of 8 (72.7%) Candidatus Mycoplasma haemominutum and 3 (27.3%) Mycoplasma haemofelis confirmed positives. Candidatus Mycoplasma turicensis was not detected, and no co-infections were registered. No significant associations (p > 0.05) were found between the hemoplasma infection status and age, gender, breed, presence of ectoparasites, feline leukemia virus/feline immunodeficiency virus positivity of cats, or the sampling season. However, outdoor access was positively associated (p = 0.049) with infection and could be considered a risk factor (OR = 4.1) in acquiring feline hemotropic mycoplasmas. Phylogenetic analysis revealed that our sequences clustered with those selected from the GenBank database in two distinct clades. The registered population genetic indices were strongly supportive of the great variance in sequences between the recorded Mycoplasma species. CONCLUSIONS: The findings support the occurrence of feline hemoplasma infections in previously uninvestigated territories of Europe, providing useful information for small animal practitioners. To our knowledge, the present survey is the first reported molecular evidence of feline hemoplasma infections in Romania.

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.

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%.

Trichinella spiralis Infecting Wild Boars in West, Southwest, and Northwest of Romania: Evidence of an Underrated Risk.

The species of the genus Trichinella are etiological agents distributed all over the world and are able to infect mammals, birds, and reptiles. Trichinella spiralis is the species most adapted to domestic and wild pigs and is also the most important etiological agent of trichinellosis. The wild boar (Sus scrofa) is a nocturnal omnivorous mammal belonging to the Suidae family. S. scrofa has a great appetite and its diet includes a variety of small prey such as mice, rats, and other rodents, as well as carcasses of larger animals. The aim of this study was the identification and the molecular characterization of Trichinella larvae isolated from the muscle tissue of S. scrofa specimens collected in different counties of Romania. The muscle samples were examined by artificial digestion and the larvae identified at the species level by multiplex PCR. T. spiralis, a species that is able to infect a considerable number of different host species including humans, was identified. In Romania, S. scrofa is an important reservoir species for T. spiralis and plays an important role in linking the domestic and the wild cycle of Trichinella, with serious repercussions for human health.

Blends of Organic Acids Are Weaponizing the Host iNOS and Nitric Oxide to Reduce Infection of Piscirickettsia salmonis in vitro.

For the last 30 years, Piscirickettsia salmonis has caused major economic losses to the aquaculture industry as the aetiological agent for the piscirickettsiosis disease. Replacing the current interventions, based on antibiotics, with natural alternatives (e.g., organic acids) represents a priority. With this study, we aimed to better understand their biological mechanism of action in an in vitro model of infection with salmon epithelial cells (CHSE-214). Our first observation revealed that at the sub-inhibitory concentration of 0.5%, the organic acid blend (Aq) protected epithelial cell integrity and significantly reduced P. salmonis invasion. The MIC was established at 1% Aq and the MBC at 2% against P. salmonis. The sub-inhibitory concentration significantly increased the expression of the antimicrobial peptides Cath2 and Hepcidin1, and stimulated the activity of the innate immune effector iNOS. The increase in iNOS activity also led to higher levels of nitric oxide (NO) being released in the extracellular space. The exposure of P. salmonis to the endogenous NO caused an increase in bacterial lipid peroxidation levels, a damaging effect which can ultimately reduce the pathogen's ability to attach or multiply intracellularly. We also demonstrate that the increased NO release by the host CHSE-214 cells is a consequence of direct exposure to Aq and is not dependent on P. salmonis infection. Additionally, the presence of Aq during P. salmonis infection of CHSE-214 cells significantly mitigated the expression of the pro-inflammatory cytokines IL-1ß, IL-8, IL-12, and IFNγ. Taken together, these results indicate that, unlike antibiotics, natural antimicrobials can weaponize the iNOS pathway and secreted nitric oxide to reduce infection and inflammation in a Piscirickettsia salmonis in vitro model of infection.

Natural Antimicrobial Mixtures Disrupt Attachment and Survival of E. coli and C. jejuni to Non-Organic and Organic Surfaces.

The contact and adherence of bacteria to various surfaces has significant consequences on biofilm formation through changes in bacterial surface structures or gene expression with potential ramifications on plant and animal health. Therefore, this study aimed to investigate the effect of organic acid-based mixtures (Ac) on the ability Campylobacter jejuni and Escherichia coli to attach and form biofilm on various surfaces, including plastic, chicken carcass skins, straw bedding, and eggshells. Moreover, we aimed to explore the effect of Ac on the expression of E. coli (luxS, fimC, csgD) and C. jejuni (luxS, flaA, flaB) bacterial genes involved in the attachment and biofilm formation via changes in bacterial surface polysaccharidic structures. Our results show that Ac had a significant effect on the expression of these genes in bacteria either attached to these surfaces or in planktonic cells. Moreover, the significant decrease in bacterial adhesion was coupled with structural changes in bacterial surface polysaccharide profiles, impacting their adhesion and biofilm-forming ability. Essentially, our findings accentuate the potential of natural antimicrobials, such as Ac, in reducing bacterial attachment and biofilm formation across various environments, suggesting promising potential applications in sectors like poultry production and healthcare.

Epidemiological Surveillance of Hypodermosis in Cattle from Romania.

Hypodermosis, or warble fly, is an endemic parasitic disease, common in countries from the northern hemisphere. The use of effective insecticides has decreased the frequency of this parasitic disease, with untreated cattle remaining to act as reservoirs. This study focused on assessing the status of hypodermosis in northwestern Romania by means of clinical examination (skin inspection and palpation performed in order to identify nodules) conducted on a number of 11.741 cattle. The study was carried out from March until June 2021. The identified larvae were subject to molecular assays for species identification and genotyping, followed by comparison with data available in the GenBank database. The average prevalence rate of parasitism caused by Hypoderma spp. was 0.31%, with values ranging from a minimum of 0.11% to a maximum of 1.32%. The dominant age group among positive animals was the 1-3 years old category and in terms of breed distribution, most positive cases were seen in cows belonging to an indigenous breed: Bruna de Maramures. April was the most prolific month in terms of nodule count/animal. The species identified in our study by means of molecular assays was H. bovis with two haplotypes: HB3 and HB8.

First Morphological and Molecular Identification of Demodex injai in Golden Jackal (Canis aureus Linnaeus, 1758) in Romania.

Demodicosis is one of the most important external parasitic diseases found in carnivores. Three species of the Demodex mite inhabit the skin of dogs and related species, D. canis being the most prevalent. This paper describes the first case of infestation with D. injai in a golden jackal in Romania. An emaciated golden jackal female body found in Timiș County, western Romania, was examined at Parasitology Department of Faculty of Veterinary Medicine, Timișoara. The gross lesions were present on different regions of the body: feet, tail, axillary and inguinal areas, and skin folds as well, consisting of erythema, extensive severe alopecia with lichenification, seborrhea, and scaling. In order to establish diagnosis, microscopic examination of skin scrapes, trichogram (hair plucking), acetate tape test (impression), fungal culture, and PCR were performed. Both microscopic measurements and PCR analysis have confirmed the presence of D. injai.

Epidemiology of Cryptosporidium Infection in Romania: A Review.

Since 1983, when the first report of a human Cryptosporidium spp. infection was published in Romania, and until now, many studies on cryptosporidiosis have been published in our country, but most of them are in the Romanian language and in national journals less accessible to international scientific databases. Although the infection was first recognized as a problem in children or immunocompromised people or more of a problem in low-income or underdeveloped global countries, we have shown in this review that it can also occur in people with normal immunological function and that the epidemiology of our country can provide a theoretical basis for the formulation of a Cryptosporidium spp. prevention strategy. In addition, 9.1% of healthy children and 73% of immunocompromised children were observed to have Cryptosporidium spp. infections. Higher rates have also been reported in immunocompromised adults (1.8-50%). Analyzing the prevalence of Cryptosporidium spp. infection in animals, we found values of 28.52% in cattle, 18% in buffalo calves, between 27.8 and 60.4% in pigs, 52.7% in dogs, and 29.4% in cats. Furthermore, in Romania, the burden of cryptosporidiosis, including acute infections and long-term sequelae, is currently unknown.

First Molecular Identification of Calicophoron daubneyi (Dinnik, 1962) and Paramphistomum leydeni (Nasmark, 1937) in Wild Ruminants from Romania.

Rumen flukes are geographically widespread trematodes affecting wild and domestic ruminants. The juvenile forms, which are found in the small intestine, are more pathogenic compared to the adults. Severe diarrhoea and weight loss are the major clinical signs, and the disease might be fatal in severely infested individuals. In the last decade, paramphistomosis has been described as an emerging parasitic disease in Europe. This study aimed to identify the rumen fluke species in wild ruminants from western Romania. Fifty-two pre-stomachs obtained from roe deer (Capreolus capreolus) that were hunted on 14 hunting grounds from Timiș and Arad counties were examined for the presence of paramphistomes. Three (9.09%) out of 33 samples were positive in Timiș County, and one (5.26%) out of 19 samples was positive in Arad County. Subsequent PCR testing revealed that three samples were positive for Calicophoron daubneyi and one for Paramphistomum leydeni. The presence of C. daubneyi and P. leydeni in roe deer has not been previously reported in Romania. Two Paramphistomum species-C. daubneyi and P. leydeni-were revealed as the main species of rumen flukes in roe deer from forests in Romania.

Investigation on Descriptive Epidemiology, Geographical Distribution, and Genotyping of Echinococcus granulosus s.l. in Bovine from Romania.

Bovine echinococcosis is a zoonotic disease with worldwide distribution, causing significant economic losses in the affected animals and important public health concerns. The aim of the current study was to investigate the presence and geographical distribution of cystic echinococcosis, and molecular characterization of Echinococcus granulosus s.l. from slaughtered cattle in Romania. In the period 2020−2021, a total of 2693 cattle (aged 2−21 years) slaughtered from the breeds Bruna de Maramureș, Balțata Româneasca, and a mixed breed were examined to identify hydatid cysts. Cysts were identified in 66 cattle (2.45%). The predominant location of hydatid cysts was the lungs. Most cysts were non-fertile. By age categories, hydatidosis was reported in the age categories 2−12 years (6/2341; 0.25%), 13−16 years (11/244; 4.5%), 17−20 years (41/85; 48.23%), and over 21 years (8/23; 34.78%). Following the PCR analysis of 10 samples from the lungs (protoscoleces/proliferative membrane/hydatid liquid), DNA fragments were identified in 5 samples. Following sequencing, the identified species was Echinococcus granulosus sensu stricto (s.s.). The study results indicate that the disease is still present in several of the investigated regions of Romania, highlighting a potential public health risk.

Seroprevalence of Anti-Theileria equi Antibodies in Horses from Three Geographically Distinct Areas of Romania.

Equine piroplasmosis (EP) is an endemic tick-borne disease found in most countries around the world. It affects all species of Equidae, and it is caused by Theileria equi, Babesia caballi and T. haneyi. The research herein is the second study on the prevalence of piroplasms in Romania conducted in the past two decades. The aim of this study was to assess the seroprevalence of anti-Theileria equi antibodies and the geographical distribution of this disease in the southwest, west, and northwest regions of Romania in order to obtain a more thorough understanding of the parasitological status of horses in this country. This study included 522 apparently healthy, mixed-breed horses from three different counties. The serum samples were analysed using the cELISA Theileria equi Antibody Test Kit. The overall seroprevalence rate was 12.84%. From the total number of positive horses, 13.96% were females and 11.21% were males. Based on the distribution of positive cases into age groups, the following values were obtained: 0−60 months: 16.26%, 60−180 months: 10.03%, and >180 months: 15.83%. There was no statistically significant difference between samples, based on age or gender. The positivity percentage in the localities included in the study ranged from 8.33 to 100%. In the population under study, the seroprevalence rate was high, indicating a possible exposure risk in this area of Romania, which could have severe effects on equids in the case of clinical manifestations of the disease. EP represents a serious threat for equine health in Romania; therefore, close and continuous monitoring of the situation is required.

The Impact of COVID-19 Pandemic During Lockdown on the Veterinary Profession in Romania: A Questionnaire-Based Survey.

The lockdown period in Romania lasted for 60 days and had the purpose of limiting the spread of SARS-CoV-2 virus outbreak and manage its consequences through emergency measures on many important areas of activity. This study aimed to gather, assess, analyze and disseminate relevant social, economic, and medical aspects on the impact of COVID-19 pandemic during lockdown on the veterinary profession in Romania. A survey was created using an online questionnaire platform, and disseminated. The survey was completed by a total of 409 individuals. A high number of respondents (71.64%; n = 293) felt exposed to medium or high risk of infection with SARS-CoV-2 at their workplace and many (56.97%; n = 233) felt that their professional environment was more stressful than usual during lockdown. Almost all respondents (89.73%; n = 367) declared the implementation of several control measures recommended by FECAVA and FVE (e.g., social distancing, wearing protective equipment, hand washing), but few mentioned the opportunity of remote work or visiting restrictions. Overall, the results show that the impact of lockdown lies directly on four main categories of importance on veterinarian professional's life-human resource, activity management, relationship between veterinarian and authorities, and continuing education.

Feline Demodicosis Case Report-First Molecular Characterization of Demodex Mites in Romania.

Cat demodicosis is uncommon to rare, and is caused by Demodex cati, Demodex gatoi and another unnamed species. The investigated patient was a mix-breed, 10-year-old feline with no dermatological history. Alopecia, erythema, minor erosions and ulcerations and crusts, associated with pruritus and self-trauma, were observed on the head. Dark, agglutinated cerumen was also present in the external ear canal. The agent causing the skin condition in the feline patient was identified as being a Demodex genus mite, based on the specific, morphological characteristics noticed upon the microscopic examination of deep skin scrapes. Biological samples were collected from the patient with to perform a PCR assay for clear species-determination and morphological assessment. PCR amplification of DNA extracted from the Demodex mites produced a single band of ~330 bp, indicating the presence of the D. cati species. The acaricidal treatment consisted of topical treatment using a fluralaner and moxidectin-based spot-on. Upon follow-up appointments, scheduled three times at a monthly interval, the patient failed to provide a positive result upon deep skin scrapes. The negative scrapes were also accompanied by the complete resolution of the existing lesions. In conclusion, this is the first molecular study to highlight the presence of Demodex cati within the feline population of Romania, and the fluralaner-moxidectin spot-on therapy has led to a complete recovery of the feline patient affected by feline demodicosis.

Identity of Microfilariae Circulating in Dogs from Western and South-Western Romania in the Last Decade.

Dirofilaria infections in dogs are recognized as widespread mosquito-borne diseases with zoonotic potential, that are caused by the filarioid nematode (Onchocercidae) species Dirofilaria immitis and Dirofilaria repens. The long-term survey conducted in western and south-western Romania was undertaken in order to provide valuable data on the occurrence of Dirofilaria infections in dogs. Blood samples from 1088 dogs, originating from 73 localities of 11 western and south-western counties in Romania, were collected and examined using the modified Knott test. Subsequently, all of the microscopically positive samples were subjected to molecular analysis for confirmation. Altogether, the data obtained showed the percentage of dogs with circulating microfilariae to be 21.42% (233/1088) of dogs tested in the investigated region. The identified species, in cases of monoinfections, were D. immitis, D. repens, and Acanthocheilonema reconditum in 106 (9.74%) samples, 102 (9.38%) samples, and 1 (0.09%) sample, respectively. Twenty-four (2.21%) samples were simultaneously positive for D. immitis and D. repens. There was no association (p > 0.05) between infection status and breed; however, sex and lifestyle were positively associated (p < 0.05) with the percentage of dogs with circulating microfilariae and might be regarded as risk factors for infection. The results of the present investigation indicate potential zoonotic risks for humans living in the screened area and support the imperative to increase awareness among both veterinarians and physicians, regarding the continuous spread of these zoonotic filariae.

Sarcocystis spp. in Romanian Slaughtered Cattle: Molecular Characterization and Epidemiological Significance of the Findings.

Species of the genus Sarcocystis are recognized as protozoan parasites infecting a wide range of animals, including humans. This study aimed to provide data on the occurrence, genetic characterization, and epidemiological significance of Sarcocystis spp. in cattle destined for human consumption in Romania. A total of 117 heart samples from slaughtered cattle in three southwestern Romanian counties (Dolj, Timiș, and Gorj) were analyzed in order to detect sarcocysts, using fresh examination microscopic techniques. Subsequently, the isolated sarcocysts and/or cyst fragments (5-15 per sample) from each infected animal were molecularly characterized. Overall, 17.9% (21/117) of the tested animals were found to be Sarcocystis spp. positive by microscopy. Genetic characterization of Sarcocystis spp. isolates, based on sequence analysis of the 18S rRNA gene, showed the presence of a single species, namely S. cruzi. No correlation was found (p > 0.05) between S. cruzi infection and the origin, age, breed, and gender of cattle, but the grazing farming system was positively associated (p=0.031) with the pathogen prevalence and can be considered a risk factor (OR = 3.6) in acquiring infection. To evaluate the possible public health risk, further investigation focused on the processing of other Sarcocystis-specific tissue matrices and evidence of human infections is recommended. This is the first study of bovine Sarcocystis infection in Romania.

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.

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 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.