Prevalence and Genotyping of Water- and Food-Borne Parasitic Protozoans (Giardia duodenalis and Cryptosporidium spp.) in Hospitalized Patients from Northwestern Romania.

Giardia duodenalis and Cryptosporidium spp. are important zoonotic protozoan pathogens that infect the gastro-intestinal tract of numerous vertebrates, including humans, and both parasites are responsible for water- or food-borne outbreaks of disease worldwide. Although, globally, both parasites are highly prevalent, particularly in developing countries, epidemiological data from Romania are scarce, and genotyping has rarely been performed. The aims of the present study were to investigate the occurrence and genetic diversity of G. duodenalis and Cryptosporidium spp. in patients hospitalized in Northwestern Romania in relation to clinical and paraclinical presentation and to identify the relative frequency of non-specific symptoms and potential risk factors. Between June 2022 and January 2024, 426 fecal samples were screened for gastro-intestinal parasites by rapid tests and microscopical examination, further confirmed by PCR and sequencing. Giardia duodenalis was detected and characterized in 12 samples (2.82%), while Cryptosporidium parvum was confirmed in four samples (0.94%). A majority of positive patients were symptomatic and reported nausea and vomiting with a significantly higher frequency compared to negative ones. This study provides new insights into the epidemiological status and clinical implications of gastro-intestinal parasite species and genospecies in Romania that are necessary for an in-depth understanding of the potential zoonotic transmission and improvement of patient care.

Molecular survey of flea-borne pathogens in fleas associated with carnivores from Algeria and an Artificial Neural Network-based risk analysis of flea-borne diseases.

As ectoparasites and efficient vectors of pathogens fleas constitute a source of nuisance for animals as well as a major issue for public health in Algeria. In this study, a molecular survey has been conducted to investigate the presence of pathogens in fleas infesting domestic and wild carnivores in the central north and eastern north and south of Algeria. The molecular screening that targeted Acanthocheilonema reconditum, Bartonella spp.,and Dipylidium caninum, was supplemented by a comprehensive analysis of risk factors related to flea-borne pathogens, drawing data from all documentation across multiple languages and sources from Morocco, Algeria, and Tunisia. In the current study, several Bartonella spp. 56/430 (13.02%) and Dipylidium caninum 3/430 (0.7%) were identified. The sequencing results revealed 5/23 (21.74%) B. clarridgeiae, 3/23 (13.04%) B. henselae, and 3/23 (13.04%) B. vinsonii. The two haplotypes, H1 and H2, of D. caninum were identified for the first time in North Africa. The results of the Artificial Neural Network risk analyses unveiled that the prevalence of pathogens and the presence of host generalist fleas as well as the vectorial competence are the most determinant risk factors of flea-borne diseases in Maghreb.

Fleas of wild carnivores in Romania: diversity, distribution, and host-associations.

BACKGROUND: Fleas are important hematophagous insects, infesting mammals and birds with a worldwide distribution. Fleas of medical importance have been reported from various carnivores worldwide, such as felids, canids, or mustelids. Romania hosts a wide carnivore diversity, but very little is known about flea species that parasitize these animals in Romania. This study aimed to provide a better understanding of the fleas' diversity and their distribution in a relatively large and diverse number of wild carnivore hosts from Romania. METHODS: From 2013 to 2021, 282 carcasses of wild carnivores from different locations in Romania were collected and examined for the presence of ectoparasites. All collected fleas were morphologically identified using specific keys and descriptions. An analysis of the co-occurrence networks was performed. RESULTS: A total of 11 flea species were identified: Pulex irritans (41.09%), Paraceras melis (20.11%), Ctenocephalides felis (7.33%), Ctenocephalides canis (7.83%), Monopsyllus sciurorum (11.11%), Chaetopsylla trichosa (21.96%), Chaetopsylla homoea (5.5%), Chaetopsylla tuberculaticeps (100%), Chaetopsylla rothschildi (13.33%), Chaetopsylla sp. (14.34%), Chaetopsylla globiceps (5.12%), Echidnophaga gallinacea (10%). The statistical analyses showed a significant difference between the infestation of Martes foina with females being more frequently infected than males (66% versus 33%). Paraceras melis infesting Meles meles had a significantly higher prevalence in female badgers than in males (× 2 = 7.7977, P < 0.01) and higher intensities of infestations in males than in females (t = 1.871, P < 0.05). CONCLUSIONS: This is the first large-scale study investigating the distribution and diversity of flea species infesting wild carnivores in Romania. Three flea species were identified for the first time in Romania (E. gallinacea, C. homoea, and C. tuberculaticeps).

A severe case of hyperinfection by Strongyloides stercoralis in a pet dog from Romania.

Strongyloides stercoralis is a zoonotic soil-transmitted nematode affecting mainly humans and dogs but identified also in non-human primates, cats and wild carnivores. It has a cosmopolitan distribution being endemic in tropical and subtropical areas. In Romania, the infection was reported on several occasions in dogs with low prevalence (3.5% -3.8%), assessed by coproscopy and it was confirmed in human patients with no travel history. A 2-year-old male Boston Terrier dog presented to a private clinic due to severe digestive problems, in July 2022. The animal had a long history of health problems. The dog was in a very bad clinical condition with severe abdominal pain, bloody diarrhea, and weight loss. Coproparasitological examinations using the saline flotation method and the modified Baermann's technique were done, both being negative. In addition, an intestinal biopsy was performed during the second endoscopy. Nematodes were collected and identified morphologically and molecularly confirmed. Histology revealed severe inflammation of the duodenal mucosa with areas of edema, necrosis, and hemorrhage, and in the intestinal glands, there were numerous nematodes suggesting a parasitic infection by Strongyloides spp. PCR followed by sequencing confirmed the infection with S. stercoralis. The dog was treated with a combination of oral fenbendazole and milbemycin oxime for 5 months. No relapse was observed 3 months after negativity was attained. This case describes a severe clinical infection by Strongyloides stercoralis in a domestic dog from Romania and the recovery after long-term treatment.

Diversity of Thelazia spp. in domestic cattle from Romania: epidemiology and molecular diagnosis by a novel multiplex PCR.

BACKGROUND: Thelaziosis is a neglected vector-borne disease caused by parasitic nematode worms of the genus Thelazia which affects various hosts. Limited attention has been given to ungulate-associated Thelazia species. Current diagnosis of thelaziosis and the identification/differentiation of species heavily relies on morphological features. Therefore, we conducted an epidemiological study in Romanian cattle, with the aim to obtain morphological and molecular data that can be used for species identification. METHODS: The eyes of 705 slaughtered cattle were sampled and subjected to morphological identification, morphometric analysis, and molecular characterization. PCR amplification and sequence analysis were performed based on the cytochromec oxidase subunit 1 (COI) gene. Statistical tests assessed the correlations between infection parameters and ecological or biogeographical factors. A novel PCR method was developed based on the consensus sequence from each species. Specific forward primers were designed for each of the three species, and a reverse primer (COIintR) was used for all reactions. A consensus thermal profile was established by gradient PCR amplification of each species separately. RESULTS: Of the sampled cattle, 19.3% were infected with Thelazia spp. Prevalence varied significantly with ecogeographical factors. A total of 585 Thelazia nematodes were recovered, with T. rhodesi being the most abundant, followed by T. skrjabini and T. gulosa. Morphometric and molecular analyses supported the morphological identification, yielding unique sequences for each species. From the 59 T. rhodesi specimens sequenced, 29 unique sequences were obtained, with a 99.1-99.85% nucleotide identity to the only other COI sequence present in GenBank®. All nine T. gulosa isolates were unique (99.37-100% nucleotide identity to other sequences), while T. skrjabini specimens displayed 98.47-100% nucleotide identity to the sole available sequence. CONCLUSIONS: Bovine thelaziosis is prevalent in Romania, raising concerns for animal welfare and potential economic impacts. Infected cattle grazing alongside vulnerable wild ruminants, such as the European bison, may affect conservation efforts. Our newly developed multiplex PCR shows promise as a valuable surveillance tool, enabling the detection of occult infections in apparently healthy animals through lachrymal secretion testing.

Toxoplasma gondii in small mammals in Romania: the influence of host, season and sampling location.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that infects a large spectrum of warm-blooded animals, including humans. Small rodents and insectivores play an important role in the epidemiology of T. gondii and may serve as a source of infection for both, domestic and wild definitive felid hosts. Factors influencing the occurrence of T. gondii in wild small mammals are unknown, despite the fact that many intermediate host species are identified. We have used small mammals (Rodentia and Lipotyphla) captured over two years in various habitats, both in urbanised and in natural landscapes. We assessed the importance of land-use, season and host ecology on T. gondii infection. RESULTS: We examined 471 individuals belonging to 20 small mammal species, collected at 63 locations spread over wide altitude, habitat and land-use ranges from Romania. Heart tissue samples were individually analysed by PCR targeting the 529 bp repetitive DNA fragment of T. gondii. The overall prevalence of infection was 7.3%, with nine species of rodents and two species of shrews being found to carry T. gondii DNA. Five species showed high frequency of infection, with the highest prevalence found in Myodes glareolus (35.5%), followed by Spermophilus citellus (33.3%), Sorex minutus (23.1%), S. araneus (21.7%) and Micromys minutus (11.1%). Adults seemed more often infected than young, however when controlling for season, the difference was not significant, as in spring both adults and young showed higher infection rates, but more adults were sampled. Contrary to our expectations, urban/rural areas (with their implicit high density of domestic feline presence) had no effect on infection prevalence. In addition, neither habitat, nor land-use at sampling sites was important as only geographical location and host species were contributing factors to the infection risk. CONCLUSIONS: High prevalence of T. gondii infection showed a highly localised, patchy occurrence, with long living and higher mobility host species being the most common carriers, especially during autumn.

Toxoplasmosis Screening during Pregnancy in a Romanian Infectious Diseases Tertiary Center: Results of a 15 Years Follow-Up Program.

Maternal infection with Toxoplasma gondii during pregnancy may have serious consequences for the fetus. In Romania, screening for toxoplasmosis is included in the first antenatal visit. A retrospective study was performed on all toxoplasmosis antenatal screening patients between May 2008 and February 2023. Twenty-seven thousand one hundred sixty-nine (27,169) pregnant women presented for prenatal screening once (22,858) or several times: during the same pregnancy (209) or during multiple pregnancies (4102). Thirty-one thousand six hundred fifty-eight (31,658) tests for IgM and IgG antibodies were performed. Nine thousand eighty-three (9083) tests (28.69%), corresponding to 7911 women (29.12%), were positive for IgG antibodies. The seroprevalence increased with patients' age, decreased in time intervals, and was more frequently associated with rural residence. At risk for acquiring the infection during the pregnancy were women with negative anti-Toxoplasma IgG antibodies (70.88%), but only 0.9% of them presented for rescreening during the same pregnancy. Acute Toxoplasma infection (ATI) was suspected in 44 patients (0.16%) due to IgG seroconversion and/or low or borderline IgG avidity. A questionnaire follow-up interview was performed, and no congenital toxoplasmosis was identified in children born from mothers with probable ATI. Our study demonstrates poor compliance with the screening program in the Romanian population.

Diversity and geographic distribution of haplotypes of Dirofilaria immitis across European endemic countries.

BACKGROUND: Dirofilaria immitis, also known as heartworm, is one of the most important parasitic nematodes of domestic dogs, causing a potentially serious disease, cardiopulmonary dirofilariosis, which can be lethal. This species seems to be less 'expansive' than its sister species Dirofilaria repens, and it is believed that climate change facilitates the spread of this parasite to new non-endemic regions. METHODS: In total, 122 heartworm isolates were analysed from nine endemic countries in Europe (Portugal, Spain, Italy, Greece, Hungary, Romania, Slovakia, and Ukraine) and a single isolate from Bangladesh by amplification and sequencing of two mitochondrial (mt) DNA markers: cytochrome c oxidase subunit 1 (COI) and dehydrogenase subunit 1 (NADH). The main aim of the current study was to determine the genetic diversity of D. immitis and compare it with D. repens haplotype diversity and distribution. DNA was extracted from adult heartworms or microfilariae in blood. Most isolates originated from dogs (Canis lupus familiaris) while 10 isolates originated from wildlife species from Romania, including eight isolates from golden jackals (Canis aureus), one isolate from a Eurasian otter (Lutra lutra) and one isolate from a red fox (Vulpes vulpes). RESULTS: Median spanning network analysis was based on the combined sequence (1721 bp) obtained from two mt markers and successfully delineated nine haplotypes (Di1-Di9). Haplotype Di1 was the dominant haplotype encompassing 91 out of the 122 sequences (75%) from all nine countries and four host species. Haplotype Di2 was the second most common haplotype, formed solely by 13 isolates from Italy. The remaining sequences were assigned to Di3-Di9 haplotypes, differing by 1-4 SNPs from the dominant Di1 haplotype. There was evidence for geographical segregation of haplotypes, with three unique haplotypes associated with Italy and four others associated with certain countries (Di4 and Di7 with Slovakia; Di8 with Greece; Di6 with Hungary). CONCLUSION: Diversity in D. immitis mt haplotypes was lower by half than in D. repens (9 vs. 18 haplotypes in D. immitis and D. repens, respectively), which may be associated with the slower expansion of heartworm in Central and NE Europe. NADH gene appears to be conserved in Dirofilaria sp. by showing lower genetic diversity than the analysed COI gene.

Is the Angiostrongylus vasorum infection in domestic dogs underestimated or misdiagnosed? A comprehensive presentation of four lethal cases.

Introduction: Angiostrongylus vasorum (A. vasorum) is a widely distributed gastropod-borne nematode, causing severe cardio-pulmonary disorders in dogs. In Romania, A. vasorum was detected in foxes and serologically confirmed in domestic dogs, but no clinical cases are currently diagnosed. Methods: Four dogs with clinical history of respiratory distress, originating from different geographical regions of Romania, were included in the study. One dog (case 1) was clinically evaluated using thoracic radiology and cardiac ultrasound; examination of feces and PCR were also performed for the etiologic diagnosis. The postmortem exam was performed in the other three cases, followed by parasitological and molecular analyses. Results: In the first case, parasitic pneumonia was suspected based on the radiographic examination of the thorax and the infection with A. vasorum was confirmed by L1 morphological identification and PCR. The main postmortem changes included large, coalescing, dark red areas of pulmonary consolidation (n = 3) and numerous adult nematodes in the pulmonary arteries (n = 2). The histopathological examination of the lungs showed necrotizing and granulomatous pneumonia with severe hemorrhages and chronic pulmonary arterial changes. Intralesional nematodes were seen in all necropsied cases. Additional inflammatory changes related to A. vasorum infection were identified in the brain and tracheobronchial and mediastinal lymph nodes (n = 2). Identification of larvae, histopathology and PCR confirmed the infection with A. vasorum. Conclusions: This study describes the first cases of canine cardiopulmonary angiostrongylosis in domestic dogs in Romania, and focuses on clinical presentation, pathological findings and molecular analysis. Angiostrongylosis should be included on the list of differential diagnoses of canine cardiopulmonary distress and/or haemorrhagic diathesis in Romania and awareness should be raised among clinicians to avoid post-mortem diagnosis in the future.

Diversity of Crenosoma species in mustelids with the first molecular characterization of C. melesi and C. petrowi.

Species of genus Crenosoma have a wide distribution and are reported in Europe, the Americas, and Asia. Currently, the genus includes 14 nominal species, out of which 9 are parasitic in mustelids. Two species are mostly reported in mustelids from Europe, namely C. melesi and C. petrowi. Up to now, no genetic sequences are deposited in GenBank for any of the two. The aims of this study were to investigate the distribution, prevalence, and diversity of Crenosoma spp. infecting mustelids in Romania and to genetically characterize the species. Mustelids (n = 247) were collected over a period of 7 years from different locations in Romania and the respiratory tract was removed and examined for nematodes. Detected nematodes were morphologically identified and fragments of two genes were sequenced. Sampled mustelids included Eurasian badger, Meles meles (n = 102), Eurasian otter, Lutra lutra (n = 20), beech marten, Martes foina (n = 36), European pine marten, Martes martes (n = 5), steppe polecat, Mustela eversmanii (n = 1), European mink, Mustela lutreola (n = 1), least weasel, Mustela nivalis (n = 2), European polecat, Mustela putorius (n = 78), and marbled polecat, Vormela peregusna (n = 1). Nematodes from Eurasian badgers were morphologically identified as C. melesi (n = 13, 12.74%) and C. petrowi (n = 3, 2.94%). Nematodes from the beech martens were identified as C. petrowi (n = 6, 16.66%), C. vulpis (n = 1, 2.78%) and Crenosoma spp. (n = 3, 8.33%). Co-infections with two Crenosoma species were detected in one beech marten (C. petrowi + C. vulpis, n = 1, 2.77%) and in one European pine marten [C. petrowi + C. vulpis (n = 1, 20%)]. Two genes of Crenosoma melesi and C. petrowi were partly sequenced for the first time. We report new host-parasite associations for M. martes and C. vulpis. However, further studies are needed in order to determine the host-parasite associations and to improve the understanding of the epidemiology of Crenosoma nematodes.

Piroplasms in farmed American bison, Bison bison from Romania.

The American bison (Bison bison) is the largest terrestrial mammal of North America, with around 350,000 individuals in the wild and in private herds but the knowledge regarding the presence of different vector-borne pathigens in these mammals is very poor. Babesia and Theileria spp. are tick-borne apicomplexan parasites which are considered to be among the most commonly found blood parasites of large ruminants, often with a high economic importance. However, the knowledge on piroplasms of bisons is extremely scarce. The aim of our study was to evaluate the presence of apicomplexan parasites in blood and tissues of farmed American bison from Romania. Overall, we tested 222 blood samples and 11 tissues samples (heart, liver, and spleen) from farmed B. bison raised for meat in Romania. All the samples were analyzed by nPCR targeting the 18SrRNA gene for piroplasmids. All positive samples were sequenced and analyzed phylogenetically. The overall prevalence of infection with piroplasmids in American bison was 1.65%, with Babesia divergens and Theileria sp. identified following sequencing. To our knowledge, this is the first report of piroplasms detected in blood and tissues of farmed B. bison from Europe. Further studies are necessary in order to obtain a better overview on the epidemiological status and clinical relevance of piroplasms in farmed American bisons.

First report of Thelazia callipaeda Railliet & Henry, 1910 in a zoo lion (Panthera leo) (Linnaeus, 1758) from Romania.

Thelazia callipaeda, the zoonotic "oriental eye worm", is an emerging nematode known to infect a wide range of hosts, particularly carnivores (wild and domestic canids and felids, mustelids, and ursids), but also other mammal groups (suids, lagomorphs, monkeys, and humans), across a wide geographical area. New host-parasite associations and human cases have mostly been reported in endemic areas. A less studied group of hosts is represented by zoo animals that may harbor T. callipaeda.A 23-year old male lion (Panthera leo) that was born in a public zoo in Reșița (Caraș-Severin County, Romania) was submitted to the University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca for investigations after death from unknown reasons in November 2022. Four nematodes were collected from the right eye during the necropsy and submitted to morphological and molecular characterization.Collected nematodes were morphologically identified as three females and one male T. callipaeda. The BLAST analysis revealed 100% nucleotide identity to numerous isolates of T. callipaeda haplotype 1.The present paper presents a new host-parasite association between T. callipaeda and P. leo from a zoo in Romania.

Retrospective Survey of Human Trichinellosis in a Romanian Infectious Diseases Hospital over a Thirty-Year Interval-The Never-Ending Story.

Trichinellosis remains a food-safety risk in Romania due to cultural traditions and food behavior. The aim of the present study was to evaluate the epidemiological, clinical and therapeutical data of all human trichinellosis cases in patients admitted to an Infectious Diseases Hospital from northwestern Romania during a thirty-year interval. Between 1 January 1988 and 31 December 2018, a total of 558 patients were hospitalized with the diagnosis of trichinellosis. The number of cases/year varied between 1 and 86. The source of infection was known for 524 patients, represented by domestic pig meat (n = 484; 92.37%) and wild boar (n = 40; 7.63%). Most patients (410; 73.48%) presented were part of family or group outbreaks. Demographical and clinical data of patients will be presented. Antiparasitic therapy was prescribed in 99.46% of cases, and corticosteroids were prescribed in 77.06% of patients. In total, 48 patients (8.6%) presented complications of trichinellosis: 44 for a single complication (neurological, cardiovascular or respiratory); the others multiple complications. Pregnancy was documented in five patients. No fatalities occurred during the study period. Although the number of hospitalized patients has decreased in the last years, trichinellosis still remains an important public health problem in northwestern Romania.

First report of Crenosoma vulpis in Africa and Eucoleus aerophilus in Algeria.

Introduction: Crenosoma vulpis and Eucoleus aerophilus are widely distributed lungworms infecting carnivores, mainly red foxes, and are localized in the trachea, bronchi and bronchioles. There are no reports from Africa regarding the presence of C. vulpis. The aim of the present study was to report a co-infection with C. vulpis and E. aerophilus in a red fox from Algeria. Materials and methods: In January 2022, a road-killed male juvenile red fox (Vulpes vulpes) was collected from Bouhadjar-Tarf locality and was submitted for a complete parasitological necropsy. Detected nematodes were collected and preserved in ethanol for morphological and molecular identification. Tissue samples were also collected and analyzed by histopathological methods. Results: Collected nematodes were identified as a male C. vulpis and several Eucoleus aerophilus. The histological techniques of the lung tissue did not reveal the presence of any larvae, or lung inflammation. Conclusion: To the best of our knowledge, this is the first report of C. vulpis infecting a carnivore in this continent, highlighting the importance of further studies to update the geographical distribution of C. vulpis. E. aerophilus was first reported in Algeria. Red foxes are important spreaders of parasitic diseases. Further studies are required for a better understanding of its epidemiology across North Africa and other areas overlapping the range of the red fox.

Haplotypes of Dirofilaria repens from Poland and selected countries of Central, North-Eastern Europe and the Middle East: An evaluation on the relation between the genetic diversity and the geographic distribution of the fast-spreading parasite.

Subcutaneous dirofilariosis is a fast-spreading infection of dogs, and occasionally of other carnivores and humans. Several factors contribute to its spread, including climate change, which facilitates development and survival of Dirofilaria repens in the mosquito vector. Movement/relocation of infected definitive hosts (dogs) from endemic regions to non-endemic regions is another possible cause of local emergence and the presence of a wide variety of wild reservoirs of the parasite may also contribute to its spread. The main aim of this study was to evaluate the genetic diversity of D. repens from different regions of Europe and to evaluate the spread of identified haplotypes and their geographic origin. A total of 95 D. repens isolates were obtained from Central and Eastern Europe (Poland, Belarus, Ukraine, Austria, Romania), NE Europe (Lithuania, Latvia, Estonia), Italy and Israel. All but two positive samples were obtained from the blood of dogs while one positive sample was obtained from an adult worm from a human case from the Lublin area in SE Poland and one sample was obtained from Anopheles plumbeus mosquito from Austria. Genetic diversity in D. repens isolates was evaluated by PCR amplification and sequencing of three genetic markers, including two mitochondrial genes (mtDNA): the cytochrome c oxidase subunit I (COI) and dehydrogenase subunit I (NADH). Additionally, the genomic marker, internal transcribed spacer 1 (ITS-1) was amplified and sequenced. Haplotypes were differentiated based on sequence alignments by identifying Single Nucleotide Polymorphism (SNPs) using DnaSP and Mega X. PopArt was used to construct a haplotype network including all identified haplotypes. Both mtDNA sequences (COI and NADH) were combined together for phylogenetic and network analyses. Altogether 18 haplotypes (DR1-DR18) were identified in combined mtDNA markers among 95 analysed samples. Haplotype DR1 was the most common encompassing 66 isolates: 42 isolates from Poland (41 from dogs and one from a human), 13 from Lithuania, 4 from Latvia, 2 from Ukraine and 5 from Romania. All other haplotypes grouped around haplotype DR1 separated by 1-5 SNPs, forming a star-like shape. Haplotype DR2 was the second most common haplotype, formed by six isolates from Romania. Interestingly, haplotype DR3 was represented only by four isolates from Israel. The remaining 15 haplotypes were represented by 1-4 isolates of different origins. Our study showed that only minor genetic diversity was found in D. repens since all isolates appear to have clustered in or branched out from haplotype DR1 with 1-5 SNP differences. The genetic diversity appears to be governed by geographic origin since isolates from neighbouring populations (countries) appear to share unique haplotypes while other populations that are geographically distant from individual haplotypes.

Apicomplexan haemoparasites in domestic cats in Romania.

BACKGROUND: Apicomplexan haemoparasites are protozoans that infect a variety of domestic and wild animal species, as well as humans. Data regarding haemoprotozoans in domestic cats are limited; therefore, the aim of this study was to assess the occurrence of Babesia spp., Cytauxzoon spp., and Hepatozoon spp. in domestic cats in Romania using molecular tools. METHODS: Blood samples from 371 domestic cats were screened for the presence of piroplasmids. All samples that yielded a visible band in agarose gels were subsequently tested by specific assays targeting the 18S rDNA of Babesia spp., Cytauxzoon spp., and Hepatozoon spp. Moreover, nested PCR assays targeting mitochondrial genes of Babesia spp. were used for screening of all Babesia spp. 18S rDNA-positive samples. RESULTS: From the total number of sampled cats, 19.4% were positive in the PCR assay targeting piroplasmids. Babesia spp. were identified in 15.1% of cats, while 0.5% were positive for Hepatozoon spp. Molecular analyses confirmed the presence of Babesia canis. No samples were positive for Cytauxzoon spp. CONCLUSIONS: The high infection rates of domestic cats with Babesia spp. and the need for species differentiation highlight the importance of mitochondrial genes as targets for molecular protocols.

Thelazia lacrymalis in horses from Romania: epidemiology, morphology and phylogenetic analysis.

BACKGROUND: Equine thelaziosis is a neglected vector-borne parasitic disease in modern veterinary medicine, lacking recent reports. It is transmitted by Musca autumnalis, and potentially other Muscidae species, by ingesting the lachrymal secretions of its equine host. The distribution of both Thelazia lacrymalis and its intermediate hosts remains largely unknown throughout Europe, with most studies dating back 20 years. The aim of this study was to assess the presence, prevalence and distribution of T. lacrymalis in horses from Romania. METHODS: The eyes of 273 horses, slaughtered at two abattoirs from the Northwestern and Western regions of Romania, were examined for the presence of T. lacrymalis between March and November 2021. Upon detection, the nematodes were collected and morphologically identified using the keys from literature. Following identification, one specimen from each animal was selected for molecular analysis while the rest underwent detailed morphometric measurements. Mapping and distribution, according to ecoregions, was done using the QGis 3.20 software, while sequences obtained were compared to those available in GenBank through BLAST analysis using the MEGA X software. RESULTS: Of the 273 animals sampled, 12 (4.39%) were positive for Thelazia spp. INFECTION: Eighty-seven nematodes were recovered, all morphologically identified as T. lacrymalis. The intensity of infestation varied between one and 33 nematodes/animal while five animals presented a bilateral infestation and seven a unilateral one. The highest prevalence was encountered in Pannonian ecoregion (12.12%) while the lowest was in the Alpine ecoregion (0%). Seventy-five intact specimens underwent detailed morphometric analysis, of the 18-20 parameters, resulting in notable differences in striation lengths compared to the data available in other reports. BLAST analysis identified a 96.46-98.60% similarity to the only other COI gene sequence available for T. lacrymalis. CONCLUSIONS: The current study represents the first report of T. lacrymalis in horses in Romania. The low prevalence rates are probably linked to the wide use of macrocyclic lactones.

European Hares, Lepus europaeus, Represent a Reservoir Host for Thelazia callipaeda in Romania.

Thelaziosis caused by Thelazia callipaeda is an emerging disease in Europe. Only two reports of naturally infected lagomorphs have been published so far. The aim of this study was to evaluate the status of the Romanian populations of European brown hares, Lepus europaeus as reservoir hosts for T. callipaeda. Between November 2019 and November 2021, the eyes of 326 L. europaeus carcasses were examined for the presence of ocular parasites. Nematodes were stored in plastic vials with physiological saline, followed by morphological and molecular identification. QGis 3.20 and EpiInfoTM 7 were used for mapping and statistical analysis. Four (1.23%) hares harbored T. callipaeda infection, with a total of 84 nematodes collected (mean intensity 21 nematodes/host), with 45 males, 39 females (two sexually immature, seven with only eggs, and 30 with eggs and larvae). One specimen from each host was successfully sequenced resulting in a 100% similarity with several other sequences of T. callipaeda haplotype 1. Statistical analysis revealed no significant results. The current study represents a first report of T. callipaeda in the European brown hare in Romania, and the second in Europe, also reiterating the role of lagomorphs as reservoir hosts for this zoonotic ocular nematode.

Epidemiological Survey of the Main Tick-Borne Pathogens Infecting Dogs from the Republic of Moldova.

Despite the significant burden of tick-borne diseases (TBDs), epidemiologic studies are missing, and TBD awareness is low in the Republic of Moldova. Our study is the first to assess the prevalence of the main tick-borne pathogens (TBPs) infecting dogs in this country and associated risk factors. In this cross-sectional, multi-centre study (June 2018-July 2019), blood samples were collected from dogs presenting in veterinary clinics (Chisinau: N = 30) and hosted in public dog shelters (Cahul: N = 42; Chisinau: N = 48). TBPs were assessed by molecular techniques and risk factors by the logistic regression model. Hepatozoon canis was the most prevalent TBP (15.8% [19/120]), followed by Babesia canis (11.7% [14/120]), Anaplasma phagocytophilum (5.8% [7/120]), and Bartonella spp. (0.8% [1/120]). Blood samples tested negative for Borrelia spp., Rickettsia spp., Francisella tularensis, Anaplasma platys, and Ehrlichia canis. Dogs originating from the veterinary clinics had a higher prevalence of A. phagocytophilum infection than those from the shelters (16.6% versus 2.2%, respectively, p = 0.0292; OR: 27.0 [95%CI: 1.4-521.9]). Dogs from Chișinau had a higher prevalence of Hepatozoon canis infection versus those from Cahul (19.2% versus 9.5%, respectively, p = 0.0295; OR: 3.9 [95%CI: 1.1-13.4]). We recommend routine use of acaricides and deworming of dogs to prevent or/and limit TBD spread. Further TBD surveillance studies are needed.

Intestinal parasites of buffalo calves from Romania: molecular characterisation of Cryptosporidium spp. and Giardia duodenalis, and the first report of Eimeria bareillyi.

Buffaloes represent an important economic resource for several regions of the world including Romania. In the present study, we examined 104 faecal samples collected from 38 buffalo calves (2-11 weeks old) from household rearing systems in Romania for gastrointestinal parasites. All samples were tested using the saturated salt flotation, McMaster and modified Ziehl-Nielsen staining methods. PCR coupled with sequencing isolates were used to identify assemblages of Giardia lamblia (Kunstler, 1882) and species of Cryptosporidium Tyzzer, 1907. Overall, 33 out of 38 examined buffalo calves were infected with different gastrointestinal parasites: 16 had single infections and 17 had mixed infections with two or three parasites. Species of Eimeria Schneider, 1875 (32/38; 84%) were the most prevalent parasites; eight species were identified according to the oocyst morphology, including the pathogenic E. bareillyi (Gill, Chhabra et Lall, 1963) which was detected for the first time in buffaloes from Romania. The nematodes Toxocara vitulorum (Goeze, 1782) (11/38; 37%) and Strongyloides papillosus (Wedl, 1856) (6/38; 16%) were also detected. Cryptosporidium spp. were found in four (11%) buffalo calves; two of them were molecularly identified as C. ryanae Fayer, Santin et Trout, 2008, and another one clustered in the same clade with C. ryanae, C. bovis Fayer, Santin et Xiao, 2005, and C. xiaoi Fayer et Santin, 2009. Giardia duodenalis assemblage E was also molecularly detected in a single (2.6%) buffalo calf. The presence of other buffaloes in the same barn was identified as a risk factor for infection with T. vitulorum. Our results indicate extensive parasitic infections in buffalo calves from northwestern Romania and underline the necessity of prophylactic treatments for T. vitulorum and E. bareillyi.