Echinococcus granulosus Sensu Stricto and Echinococcus multilocularis in a Gray Wolf (Canis lupus) in Turkey: Further Evidence for Increased Risk of Alveolar Echinococcosis in Urban Areas.

OBJECTIVE: The aim of this study was to identify Echinococcus species by morphological and molecular means. METHODS: A dead gray wolf (Canis lupus) was found near Erzurum province and brought to the parasitology laboratory. Sedimentation and counting technique (SCT) and polymerase chain reaction (PCR) analysis were conducted. RESULTS: The SCT implications indicated that the wolf had a substantial worm burden (62,720 and 49,280 parasites) due to a co-infection of E. granulosus s.l. and E. multilocularis. Genus/species-specific PCR was used to analyze DNA extracted from adult worms and confirmed as E. granulosus s.s. and E. multilocularis, utilizing COI and 12S rRNA gene sequence analysis, respectively. CONCLUSION: This report presents the first co-detection of E. granulosus s.s. and E. multilocularis in a gray wolf found in an urban area in a highly endemic area for human echinococcosis in northeastern Turkey. The results emphasize that AE is not only a problem of rural areas, but also occurs in urban areas, which may pose a threat to public health. Therefore, surveillance in urban areas is crucial. The need to develop new control strategies for domestic and wildlife in the study area is also highlighted.

Cystic echinococcosis in slaughtered sheep in Erzurum province, Turkey.

Cystic echinococcosis (CE) is an important zoonotic infection caused by the larval stages of the genus Echinococcus. Turkey is a highly endemic region for CE and the disease is one of the major public health problems. The study was aimed to assess the situation of the CE in sheep in Turkey and also to provide data on circulating genotypes in the country. A total of 3319 sheep at slaughter were screened during the study. The prevalence of CE in the study area was 31.7% (1052/3319). The lungs were the most frequently CE infected organ (50%, 526/1052). Microscopic examination revealed that overall cyst fertility was 68.1%. Molecular analysis of partial fragments of 12S and COI gene regions were included for 351 selected cyst samples and all of them were identified as E. granulosus sensu stricto. Sequence analysis showed that the predominant genotype in the study areas was G1 (77.1%), and the rest were G3 (22.9%). The prevalence rate of CE in sheep in the study area is lower compared to previous years except for one province. Considering the high cyst fertility rate and the predominance of E. granulosus G1 which is particularly pathogenic to humans, calls for serious control measures like public awareness about the disease, sufficient dog deworming programs, continuity of monitoring the disease should be taken.

Zoonotic Babesia microti infection in wild rodents in Erzurum province, northeastern Turkey.

Wild rodents are natural reservoir hosts of various pathogens, including Babesia microti. This study investigated the presence of B. microti in rodents from Erzurum province in Turkey. A total of 498 rodents and 21 rodent-fed ticks were analysed using the polymerase chain reaction (PCR) technique to test for the presence of B. microti. Babesia spp. were detected in three (0.6%) of the 498 rodent spleen samples. The Babesia-positive rodent species were identified as Microtus socialis by means of molecular analysis. The rodent-fed ticks comprised 15 Ixodes laguri and 6 Rhipicephalus sanguineus, none of which tested positive for Babesia spp. A sequence analysis of the 18S PCR amplicons confirmed the three Babesia-positive samples to be B. microti. The Erzurum isolates were 100% identical to the zoonotic Jena strain. The results of this study indicate the existence of zoonotic B. microti strains that may constitute a potential public health risk in Erzurum province. Future studies should determine the tick vector and other reservoir rodent species of B. microti in Erzurum.

The situation of echinococcosis in stray dogs in Turkey: the first finding of Echinococcus multilocularis and Echinococcus ortleppi.

Echinococcosis, caused by larval stage of the genus Echinococcus, is one of the most important zoonotic diseases worldwide. The purpose of this study was to determine the presence and prevalence of Echinococcus species in stray dogs of Erzurum, a highly endemic region for cystic echinococcosis (CE) and alveolar echinococcosis (AE) in Turkey. The study samples consisted of 446 stray dog faecal specimens collected from an animal shelter in Erzurum, Turkey, between October 2015 and February 2016. The faecal samples were collected from individual dogs for the isolation of taeniid eggs using the sequential sieving and flotation method (SSFM). Molecular analyses and sequencing revealed the prevalence of Echinococcus spp. as 14.13% (63/446) in faecal samples. The stray dogs harboured five different Echinococcus spp.: E. granulosus s.s. (G1/G3) (n = 41), E. equinus (G4) (n = 3), E. ortleppi (G5) (n = 1), E. canadensis (G6/G7) (n = 3) and E. multilocularis (n = 16). E. granulosus s.s. was the most abundant species. Surprisingly, the occurrence of E. multilocularis in dogs was revealed for the first time in Turkey. E. ortleppi was also reported for the first time in Turkey. These findings highlight a significant public health risk for human AE and CE, presenting useful baseline data on Echinococcus spp. infection in dogs for designing control strategies.

Unravelling the genetic diversity and relatedness of Echinococcus multilocularis isolates in Eurasia using the EmsB microsatellite nuclear marker.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe helminthic zoonotic disease distributed in the Northern Hemisphere. The lifecycle of the parasite is mainly sylvatic, involving canid and rodent hosts. The absence of genetic data from most eastern European countries is a major knowledge gap, affecting the study of associations with parasite populations in Western Europe. In this study, EmsB microsatellite genotyping of E. multilocularis was performed to describe the genetic diversity and relatedness of 785 E. multilocularis isolates from four western and nine eastern European countries, as well as from Armenia and the Asian parts of Russia and Turkey. The presence of the same E. multilocularis populations in the Benelux resulting from expansion from the historical Alpine focus can be deduced from the main profiles shared between these countries. All 33 EmsB profiles obtained from 528 samples from the nine eastern European countries belonged to the European clade, except one Asian profile form Ryazan Oblast, Russia. The expansion of E. multilocularis seems to have progressed from the historical Alpine focus through Hungary, Slovakia, the Czech Republic and southern Poland towards Latvia and Estonia. Most of the samples from Asia belong to the Asian clade, with one EmsB profile shared between Armenia and Turkey, and two between Turkey and Russia. However, two European profiles were described from two foxes in Turkey, including one harboring worms from both European and Asian clades. Three EmsB profiles from three Russian samples were associated with the Arctic clade. Two E. multilocularis profiles from rodents from Lake Baikal belonged to the Mongolian clade, described for the first time here using EmsB. Further worldwide studies on the genetic diversity of E. multilocularis using both mitochondrial sequencing and EmsB genotyping are needed to understand the distribution and expansion of the various clades.

Echinococcus multilocularis in Red Foxes in Turkey: Increasing risk in urban.

This study was conducted to determine the occurrence of E. multilocularis in foxes and environmental fecal contamination by E. multilocularis in Erzurum, the most highly endemic region for AE in Turkey. The study materials consisted of 50 red fox carcasses collected from 20 counties of Erzurum, Turkey, between October 2015 and February 2016. After the application of the sedimentation and counting technique (SCT), E. multilocularis was identified through the identification of typical morphological structures. Fox fecal samples (n = 600) were also collected from these counties for the isolation of taeniid eggs using the sequential sieving and flotation method (SSFM). Then, the collected adult worms and taeniid eggs were subjected to molecular and sequence analyses. Mature E. multilocularis parasites were found in 42% (21/50) of the fox intestines, with a mean number of 7,806 (150-31,644). The severity of infection was higher in carcasses obtained from the central district (48.6%, 17/35) than in those obtained from the peripheral district (26.7%, 4/15). The prevalence of environmental fecal contamination with E. multilocularis was 10.5% (63/600) in fecal samples collected from all counties of Erzurum. This infection rate was higher in the central district (32.1%, 36/112) than in the peripheral district (5.5%, 27/488; P < 0.0001). In conclusion, contrary to expectation, the prevalence of E. multilocularis positivity was high in urban areas. This could have been due to alterations in the dietary habitats of definitive and intermediate hosts. Therefore, new control strategies are essential to eliminate human AE cases in the future as urbanization advances.

Molecular Characterization of Echinococcus multilocularis and Echinococcus granulosus from Cysts and Formalin-Fixed Paraffin-Embedded Tissue Samples of Human Isolates in Northeastern Turkey.

Erzurum province of Turkey is known to be highly endemic for alveolar echinococcosis (AE) and cystic echinococcosis (CE). In this study, we confirmed Echinococcus multilocularis cases, searched genetic variations of the isolates, and-for the first time-determined the genotypes of Echinococcus granulosus s.l. infecting humans in the province. A total of 5 alveolar and 106 hydatid cysts as well as 23 formalin-fixed paraffin-embedded (FFPE) samples that were diagnosed as AE were collected from hospitals between 2015 and 2017. Partial sequences of two mitochondrial genes were amplified to detect E. multilocularis and E. granulosus sensu lato with conventional polymerase chain reactions (PCRs) and genotypes confirmed by sequencing. PCR amplification of a partial 12S rRNA gene on an alveolar cyst and FFPE tissue samples yielded the expected bp in 5 cysts and 19 of 23 FFPE samples; all Erzurum E. multilocularis isolates were confirmed by sequencing. Phylogenetic analysis of the isolates indicated that some of them were identical to European isolates, whereas some of them were identical to Asian isolates. Off all hydatid cyst samples, 101 (95.2%) yielded the expected bp (94 with 12S rRNA-PCR and 7 with COI-PCR). Sequence analysis showed that 98 (97%) of them corresponded to the G1 genotype, whereas 3 (3%) corresponded to the G3 genotype. Results of the study emphasize that E. multilocularis isolates of Erzurum, based on short sequencing, are similar to both European and Asian isolates, and the G1 genotype of E. granulosus is the main causative agent of human CE in Erzurum.

Echinococcus multilocularis in a Eurasian lynx (Lynx lynx) in Turkey.

Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most threatening zoonoses in Eurasia. Human AE is widespread in the Erzurum region of Turkey, but the situation of the disease in intermediate and definitive hosts is unknown. A Eurasian lynx (Lynx lynx) was killed in a traffic accident in the north of Erzurum, and was taken to our laboratory. Sedimentation and counting technique (SCT), DNA isolation and polymerase chain reaction (PCR) analysis were performed. The SCT results showed that the lynx was infected with E. multilocularis with a medium (745 worms) worm burden. The DNA of adult worms obtained from the lynx was analyzed with a species-specific PCR, and the worms were confirmed to be E. multilocularis by 12S rRNA gene sequence analysis. This is the first report of E. multilocularis from Eurasian lynx in Turkey.

First detection of Echinococcus multilocularis in rodent intermediate hosts in Turkey.

Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), a potentially fatal zoonotic disease. Large parts of Turkey are considered as endemic for E. multilocularis. The aim of this study was to determine the occurrence of metacestode of E. multilocularis in wild rodents in Erzurum, an endemic region for human AE in Turkey. During the sampling period, a total of 498 rodents were trapped in twenty counties of Erzurum Province. Suspected lesions were observed on the livers of 48 rodents, and then partial fragment of mitochondrial 12S rRNA gene was PCR-amplified. Five liver samples exhibited E. multilocularis infection. The prevalence of E. multilocularis for Microtus spp. was 1·3%. All of the infected rodents had fertile metacestodes. Infected rodents were morphologically and molecularly analysed and were confirmed to be Microtus irani by the mitochondrial cytochrome b gene sequence analysis. This is the first report of the presence of E. multilocularis in rodent intermediate hosts in Turkey. Our findings of infected M. irani with protoscoleces show that this rodent can act as suitable intermediate host for E. multilocularis' life cycle in Turkey. However, there was a complete lack of data on the infection of carnivores from the country. An extensive survey is recommended to determine the prevalence of E. multilocularis in definitive hosts in this endemic region.

Vector-Borne Pathogens in Stray Dogs in Northeastern Turkey.

This experiment was carried out to attain prevalence and molecular characterization of pathogens causing canine vector-borne diseases (CVBDs) including babesiosis, hepatozoonosis, leishmaniasis, filariosis (Dirofilaria immitis, Dirofilaria repens, and Acanthocheilonema reconditum), ehrlichiosis (Ehrlichia canis), and anaplasmosis (Anaplasma platys) in stray dogs. The study material consisted of 133 asymptomatic female (n = 96) and male (n = 37) stray dogs (≤1 year old, n = 16 and 1-6 years old, n = 117) housed in the Animal Care and Rehabilitation Center, Erzurum, Northeastern Turkey. Conventional and nested PCR were performed on blood samples to detect Babesia spp., Leishmania spp., Hepatozoon spp., D. immitis, D. repens, A. reconditum, E. canis, and A. platys. Sex and age association with the pathogen prevalence was determined using X2 statistics. The positivity rate for at least one CVBD pathogen was 48.9% (65/133). DNA of B. canis, Hepatozoon spp., H. canis, D. immitis, and E. canis were detected in 5.3% (7/133), 27.1% (36/133), 5.3% (7/133), 1.5% (2/133), and 9.8% (13/133) of the dogs, respectively. Leishmania spp., D. repens, A. reconditum, and A. platys DNA were not detected. Mixed pathogens were determined in seven (10.8%) of the infected dogs, with predominant involvement of Hepatozoon spp. or H. canis. The pathogen prevalence did not vary by sex or age. Nucleotide blast analysis of Erzurum isolates showed 99.8-100% identities with the corresponding reference isolates. This study indicates presence of five CVB pathogens, including the first report of E. canis, in stray dogs in Erzurum, Turkey.

Prevalence and molecular characterization of Theileria equi and Babesia caballi in jereed horses in Erzurum, Turkey.

Equine piroplasmosis (EP) is a hemoprotozoan tick-borne disease with worldwide distribution that is caused by Theileria equi and Babesia caballi. There are studies reporting the presence of equine piroplasmosis in Turkey but the situation in Erzurum is unknown. The aim of the current study was to determine the situation of equine piroplasmosis in jeered horses in Erzurum. Between April and August 2015, a total of 125 Arabian horse were examined and blood samples were collected. At the time of sampling, animals were also examined for tick infestations and clinical signs. Besides microscopic examination of Giemsastained blood smears, multiplex PCR performed with species specific primers partially amplifying the 18S rRNA gene of B. caballi and T. equi. During the microscopic examination of blood smears, T. equi piroplasms were found in 6 (4.8%) samples. In total, 11 (8.8%) T. equi DNA were detected with multiplex PCR. B. caballi piroplasms or DNA were not obtained. BLAST analysis of the sequenced T. equi samples (GenBank: KU921661-KU921667) indicated 98.8-100% identity to each other, and 100% similarity to T. equi isolates in South Africa, Iran, China, Sudan, India, Mongolia, Trinidad, Kenya, Spain, Serbia, Bosnia and Herzegovina and Turkey (Bursa). The results of our study indicate that T. equi occurs more frequently than B. caballi in the study area. To the authors' knowledge, this is the first report of the molecular detection of equine piroplasmosis in jeered horses in Erzurum, Turkey.

Parasites Determined by Fecal Examination in Horses in Erzurum.

OBJECTIVE: This study aimed to determine the parasites present in horses belonging to the Erzurum Province. METHODS: Fecal samples were collected from 76 horses of different ages, genders and breeds in Erzurum. Individual fecal samples were collected and examined by flotation and sedimentation methods. RESULTS: The following species were detected: strongylid egg (57.89%), Parascaris equorum (10.52%), Dicrocoelium dendriticum (2.63%), Fasciola spp. (2.63%) eggs, and Eimeria spp. oocysts (5.26%). CONCLUSION: Equine animals are significantly infected with Strongylosis in the Erzurum Province, and effective parasite control measures should initiated.

First Molecular Characterization of Echinococcus multilocularis in Turkey.

This study aimed to find out the occurrence of Echinococcus multilocularis in foxes in Erzurum province, the highest endemic region for human alveolar echinococcosis in Turkey. The sedimentation and counting technique was used to reveal adult Echinococcus spp. in the intestines of foxes. One out of the 10 foxes was infected with E. multilocularis. The adult worms were analyzed morphologically and molecularly and were confirmed to be E. multilocularis by species-specific PCR. Pairwise comparisons between the 12S rRNA sequences of the E. multilocularis isolate from Erzurum and other E. multilocularis isolates showed 100% similarity of the Erzurum isolate with European isolates. With this study, the presence of E. multilocularis in a fox in Erzurum was confirmed by PCR, and molecular identification of E. multilocularis is reported for the first time in Turkey.

Molecular determination of Tritrichomonas spp. in aborted bovine foetuses in Eastern Anatolian Region of Turkey.

Tritrichomonas foetus is the causative agent of venereal trichomonosis in cattle causing infertility, pyometra and abortions. The objectives of this study were to determine the positivity rate of Tritrichomonas spp. in abomasal content of aborted foetuses from Eastern Anatolian Region of Turkey, using staining, culture and PCR methods and to present the isolates found in the region. A total of 246 abomasal content of aborted foetuses were tested and 14 of 246 (5.7%) were Tritrichomonas spp. positive only by polymerase chain reaction (PCR). Positivity was not attained by staining or culture method. Four of the positive samples in PCR were confirmed to be T. foetus by sequencing of the amplified 5.8S rRNA gene and flanking ITS regions. Nucleotide sequences of TR-Erzurum T. foetus isolates have been entered into the GenBank sequence database under accession numbers KC236423 through KC236426. This preliminary study suggests that future studies are needed on the systematic relationships and epidemiology of T. foetus isolates in the region.

A comparison of the efficacy of subcutaneously administered ivermectin, doramectin, and moxidectin against naturally infected Toxocara vitulorum in calves.

This study was conducted to investigate the efficacy of ivermectin (IVM), doramectin (DRM), and moxidectin (MXD) against Toxocara vitulorum in calves. In the study, 20 calves naturally infected with T. vitulorum were divided into four groups: three different treatment groups (n = 5) and one positive control (n = 5). The animals in each group received either IVM (Baymec®, Bayer), DRM (Dectomax®, Pfizer), or MXD (Cydectin®, Fort Dodge) by subcutaneous injection at a single dose of 0.2 mg/kg. Fecal egg counts were performed on all animals on days 0, 2, 4, 8, 12, and 16 post-treatment. In conclusion, IVM, DRM, and MXD significantly reduced the fecal egg counts on day 8 post-treatment (99.90%, 98.77%, and 99.57%, respectively). After the 12th day, IVM, DRM, and MXD were found to be 100% effective. There was no significant difference in efficacy between the three treatment groups at any of the sampling dates (P > 0.05). No side effects associated with nervous, respiratory, and gastrointestinal systems were observed. This is the first study to evaluate the comparative efficacy of subcutaneous administration of ivermectin, doramectin, and moxidectin against naturally infected T. vitulorum in calves.

Efficacy of eprinomectin against Toxocara vitulorum in calves.

This study was made to investigate efficacy of eprinomectin pour-on against to Toxocara vitulorum in calves. In the study, 16 calves naturally infected with T. vitulorum were divided into two groups as treatment (eight calves) and control (eight calves). Eprinomectin (0.5 mg/kg, Eprinex, Merial) was given to treatment group calves, and eggs per gramme were determined in the faeces on the day of pre-treatment and the second, third, fourth, fifth, sixth, 14th and 28th days of post-treatment. No side effects associated with nervous, respiratory and gastrointestinal systems were observed. In conclusion, eprinomectin was determined to be 100% effective against T. vitulorum. This is the first study to evaluate the efficacy of eprinomectin against a natural T. vitulorum infection in calves.

The relationship of public park accessibility to dogs to the presence of Toxocara species ova in the soil.

This survey was conducted to determine prevalence of Toxocara spp. ova in public parks in Erzurum, Turkey. A total of 214 soil samples were collected from July 2007 to June 2008 in 36 public parks, of which 28 were unfenced and 8 were fenced. Prevalence of Toxocara spp. was 64.28% in unfenced public parks, while no contamination was observed fenced public parks (p<0.001). Average number of Toxocara spp. ova was 1.43 per 50 g sand ranging from 1 to 7. Moreover, soils from unfenced public parks were contaminated with Taeniid spp. ova (3.12%). In conclusion, public parks and/or playgrounds should be fenced to prevent fecal contamination, suggesting that a more frequent surveillance should be performed and preventive measures should be taken and enforced by local governments to reduce likelihood of zoonoses in children.

Control of helminth contamination of raw vegetables by washing.

This study was conducted to determine the control of helminth egg contamination of raw vegetables by washing. A total of 199 unwashed and 199 washed lettuce, parsley, carrots, dill, rocket, and green-peppers, provided by a catering service in Bursa, Turkey, between March and June 2009, were subjected to helminth egg count under light microscopy. Helminth eggs were detected in six (3.0%) unwashed samples and not in any washed samples (p<0.01). Ascaris lumbricoides and Toxocara spp. were detected in four (2.0%) and two (1.0%) unwashed vegetables, respectively, mostly among leafy vegetables such as lettuce and parsley. Our data confirm that washing procedures before consumption of raw vegetables regardless of the providers' sanitation should be performed to avoid transmission of helminths.