Identification and Molecular Characterization of Giant Liver Fluke (Fascioloides magna) Infection in European Fallow Deer (Dama dama) in Romania-First Report.

Fascioloidosis is a parasitic disease of primary wild and domestic ruminants, caused by giant liver fluke, Fascioloides magna. The definitive host of the liver fluke in its area of origin (North America) is the white-tailed deer (Odocoileus virginianus). In Europe, the red deer (Cervus elaphus) and European fallow deer (Dama dama) are definitive hosts and the most sensitive hosts to F. magna infection, on which the parasite exerts serious pathogenic effects. In this study, we analyzed fecal samples and livers of 72 D. dama from 11 hunting grounds in Arad County, Romania. Of the 72 fecal samples and livers from D. dama, trematodes of the genus Fascioloides were identified in four (5.56%). Sequencing revealed that the trematodes identified in the samples were similar to the sequence of F. magna (GenBank no. EF534992.1, DQ683545.1, KU232369.1). The sequence obtained from the molecular analysis has been deposited in GenBank® under accession number OQ689976.1. This study describes the first report of giant liver fluke (F. magna) infection in D. dama in Romania.

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.

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.

The First Identification of Trichinella britovi in the Raccoon Dog (Nyctereutes procyonoides) in Romania.

Trichinella spp. are nematodes distributed throughout the world that affect an impressive number of host animals (mammals, birds, and reptiles) involved in the evolution of two cycles, the domestic and the sylvatic. The raccoon dog (Nyctereutes procyonoides) is an omnivorous mammal with great ecological plasticity. The expansion of the raccoon dog in Europe is associated with the risk of the introduction and spread of different pathogens, especially zoonotic ones (Trichinella, Echinococcus). Currently, the raccoon dog's range in Romania is limited to the Danube Delta area, the Lower Danube Meadow, and the Prut Meadow. The aim of this study was to examine the presence of Trichinella larvae isolated from the muscles of raccoon dog from six hunting funds of Giurgeni, Ialomița County, Romania. The muscle samples were examined via artificial digestion, and the obtained larvae were processed via multiplex PCR. The PCR-amplified ESV and ITS1 DNA fragments were then sequenced for species confirmation. The species Trichinella britovi, which is the most common species identified in wild carnivores in temperate zones, was confirmed. Although T. britovi has been reported in several host animals in Romania, this case report confirms its presence in the raccoon dog for the first time.

Research on the Control of Gastrointestinal Strongyles in Sheep by Using Lotus corniculatus or Cichorium intybus in Feed.

The general practice of sheep farmers in gastrointestinal helminth control is based on the use of commercial drugs, making chemoresistance very common. Considering this, our study focused on the biological control of gastrointestinal parasitism using high-tannin plant hay. Three groups of 30 animals each were formed. The control group was additionally fed meadow hay, while the other two groups received chicory (group 2) and bird's foot trefoil hay (group 3). The number of gastrointestinal strongyle eggs, shed through faeces (EPG), was surveyed for 28 days for all animals. The amounts of total tannins for meadow, chicory, and Lotus corniculatus hay supplements were 13.92 mg/g, 78.59 mg/g, and 94.43 mg/g, while their condensed tannin contents were 2.58 mg/g, 29.84 mg/g, and 15.94 mg/g, respectively. Compared to experimental day 0, there was an increase in EPG of 80.83% in the control group, a decrease of 24.72% in group 2, and a 20% decrease in group 3, by day 28. The p-value was <0.05 between group 1 and the other groups, showing significant differences between the control and experimental groups. The decrease in EPG rates in the experimental groups compared to the control group demonstrates an antiparasitic effect of Lotus corniculatus and chicory.

Characterization of the Tongue Worm, Linguatula serrata (Pentastomida), Identified from Hares (Lepus europaeus) in Romania.

Linguatula serrata (Frölich, 1789) is a widespread parasite known as the tongue worm belonging to the family Linguatulidae. The adult form of the parasite is usually located in the upper respiratory tract of domestic and wild carnivores while the larval forms are located in the visceral organs of intermediate hosts (various herbivorous mammals). Twenty-four European brown hares (Lepus europaeus) were examined in this study, of which two were positive with L. serrata nymphs. The collected nymphs were examined morphologically using electron-microscopic analysis and molecularly by amplification of 18S rRNA and COX1 genes. Lung tissue samples were also collected and histopathological examination was performed. Histopathological examination revealed the following lesions: generalized inflammatory oedema, granulomas with necrosis, calcification and fibrosis in the bronchial tree. The results of molecular sequencing for L. serrata specimens collected from the European brown hares are deposited in GenBank. This study presents the first report on Linguatula serrata nymphs collected from L. europaeus in Romania, using molecular and morphological characterization simultaneously.

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.

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.

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.

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.

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.

Survey of the Occurrence and Human Infective Potential of Giardia duodenalis and Cryptosporidium spp. in Wastewater and Different Surface Water Sources of Western Romania.

From the group of parasitic protozoa, Giardia and Cryptosporidium are the most common pathogens spread in surface water sources, representing a continuous threat to public health and water authorities. The aim of this survey was to assess the occurrence and human infective potential of these pathogens in treated wastewaters and different surface water sources. A total of 76 western Romanian water bodies in four counties (Arad, Bihor, Caraș-Severin and Timiș) were investigated, including the effluents of wastewater treatment plants (n = 11) and brooks (n = 19), irrigation channels (n = 8), lakes (n = 16), and ponds (n = 22). Water samples were collected through polyester microfiber filtration. Giardia cysts and Cryptosporidium oocysts were isolated using immunomagnetic separation, according to the US EPA 1623 method, followed by their identification and counting by immunofluorescence (IF) microscopy. All samples were screened through PCR-based techniques targeting the gdh gene for Giardia spp. and the 18S rRNA gene for Cryptosporidium spp., followed by sequencing of the positive results. Cryptosporidium-positive samples were subtyped based on sequence analysis of the GP60 gene. Giardia spp. was found in all tested water types with a cumulative detection rate of 90.1% in wastewaters, 26.3% in brooks, 37.5% in irrigation channels, 31.2% in lakes, and 36.4% in ponds. Except for ponds, all monitored water bodies harbored the Giardia duodenalis AII subassemblage with human infective potential. In addition, the ruminant origin assemblage E was widely distributed, and the domestic/wild canid-specific assemblage D was also recorded in a pond. Three (27.3%) wastewater samples were Cryptosporidium positive, and the identified species was the zoonotic Cryptosporidium parvum, with IIaA15G2R1 (n = 2) and IIdA18G1 subtypes. The results highlight that this threat to the public health must be brought to the attention of epidemiologists, health officials, and water authorities.

Giardia duodenalis and Cryptosporidium spp. as contaminant protozoa of the main rivers of western Romania: genetic characterization and public health potential of the isolates.

The objective of this study was to establish the prevalence, contamination level, and public health significance of Giardia duodenalis and Cryptosporidium spp. in the primary rivers of western Romania. A total of 53 sampling points in the 24 most important western Romanian rivers in four counties (Arad, Bihor, Caraș-Severin, and Timiș) were investigated from March to September 2016. Surface water samples were collected by microfiber filtration. Cryptosporidium and Giardia (oo)cysts were isolated using immunomagnetic separation (IMS) according to the USEPA 1623 method and, after staining with fluorescently labeled (FITC) monoclonal antibodies, were identified and counted under a microscope. The Cryptosporidium and Giardia (oo)cysts were identified to species and assemblage/sub-assemblage level through the nested PCR-RFLP procedure targeting the 18S ribosomal RNA and gdh genes, respectively. PCR-based techniques were utilized for all water samples. Overall, 22 samples (41.5%) were determined to be positive for Giardia cysts (ranging from 0.05 to 300 cysts per liter), and four samples (7.5%) tested positive for Cryptosporidium oocysts (0.17-48 oocysts/l). G. duodenalis was molecularly identified in 13 water samples (24.5%), indicating the presence of the sub-assemblage A-II (n = 12) and assemblage E (n = 1). PCR-RFLP showed that two samples (3.8%) contained Cryptosporidium DNA, and the identified species were Cryptosporidium parvum and Cryptosporidium canis. All positive results were successfully confirmed by DNA sequencing. Subtyping of the zoonotic C. parvum isolate based on sequence analysis of the GP60 gene revealed the occurrence of the IIaA16G1R1 subtype. The results of this study highlight considerable contamination of river waters with pathogenic Giardia spp. and Cryptosporidium spp., suggesting a potential risk for the public and animal health. This report presents the first extended published description of the presence of Giardia spp. and Cryptosporidium spp. in the aquatic environment in Romania.

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

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