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.

The red fox (Vulpes vulpes) plays a minor role in the epidemiology of the domestic cycle of Trichinella in Romania.

Nematode worms of the genus Trichinella are zoonotic parasites with a worldwide distribution. The majority of the biomass of these nematodes circulates among wildlife, but when humans fail in the proper management of domestic animals and wildlife, Trichinella infections are transmitted from the sylvatic to the domestic environment. Such failures occur in Romania, where a high prevalence of Trichinella spiralis has been detected in domestic pigs. The aim of the present study was to provide data about the prevalence of Trichinella spp. infections in red foxes (Vulpes vulpes) that are hunted in Romanian counties, in which the prevalences of Trichinella spp. infection in backyard and free-ranging pigs range from 0.17 to 2.5%, to determine the role played by this carnivore species in the transmission of the parasite to domestic cycle. A total of 121 animals from 45 hunting grounds of three counties were screened to detect Trichinella spp. larvae by the digestion method. Infections were detected in 26 (21.5%) foxes from 18 (40%) hunting grounds of the three counties (13/67 in Arad, 1/3 in Hunedoara, and 12/51 in Timis). The mean larval density was 10.5 larvae per gram. Of the 25 successfully tested samples, the Trichinella larvae from 24 isolates were identified as T. britovi (96%), and the larvae from one isolate were identified as T. spiralis (4%). No mixed infections were recorded. The present results revealed that the red fox should be considered an important T. britovi reservoir in the sylvatic cycle; in contrast, the detection of only a single T. spiralis-positive isolate suggests that red foxes play a minor role in the epidemiology of the domestic cycle in the investigated area of western Romania.

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.

Zoonotic Cryptosporidium parvum in Romanian newborn lambs (Ovis aries).

This study was undertaken to investigate the occurrence and public health significance of Cryptosporidium species/genotypes and subtypes in a newborn lambs. A total of 175 diarrheic fecal samples from lambs (younger than 21 days) were collected in seven sheep flocks located in western Romania, and were microscopically examined for the presence of Cryptosporidium oocysts after staining with modified Ziehl-Neelsen technique. Twenty-four (13.7%) fecal samples were tested Cryptosporidium positive by microscopy and were subjected for molecular characterization. All positive samples were successfully amplified through a nested polymerase chain reaction (PCR) of the small subunit (SSU) rRNA gene (18S). Cryptosporidium species were determined by restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products using the conventional SspI and VspI restriction enzymes. The identified species were: Cryptosporidium parvum (20/24), C. ubiquitum (2/24) and C. xiaoi (2/24), respectively. PCR-RFLP results for C. ubiquitum and C. xiaoi isolates were confirmed by DNA sequencing. Subsequently, subtyping of seven randomly selected C. parvum isolates, based on sequence analysis of the GP60 gene, revealed the presence of five different subtypes (IIaA17G1R1, IIaA16G1R1, IIdA20G1, IIdA24G1 and IIdA22G2R1) belonging in two zoonotic subtype families (IIa and IId). These findings may suggest the potential role of the newborn lambs as a source for human cryptosporidiosis. This is the first published report about the presence of C. ubiquitum and C. xiaoi in lambs from Romania.

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

Serum samples from 376 randomly selected adult cattle, from 25 farms located in 3 counties (Arad, Bihor, and Timis) from western Romania, were sampled for Neospora caninum antibodies using a commercial ELISA-kit. Seroprevalence values and risk factors for neosporosis (cow age, breed, herd size, farming system, previous abortion, and number of farm dogs) were examined using a generalized linear mixed model with a binomial distribution. Overall, the seroprevalence of N. caninum was 27.7% (104/376) with a prevalence of 27.9% (24/86) in Arad, 26.9% (25/93) in Bihor, and 27.9% (55/197) in Timis. Of 25 cattle herds, 23 were seropositive with a prevalence ranging from 10.0 to 52.2%. No correlation was found between N. caninum seropositivity and age, breed, herd size, breeding system, and previous abortion. The number of farm dogs was the only factor (P(Wald)  =  0.03) positively associated with seroprevalence in cows and can be considered the risk factor in the acquiring of infection. The present work is the first regarding serological evidence of N. caninum infection in cattle from western Romania.