Equine Piroplasmosis in Asymptomatic Horses of Western Iran: Comparison of Microscopic Examination and Multiplex PCR.

PURPOSE: Piroplasmosis is responsible for anemia, fever, loss of physical activity and even death in equines. In epidemiological studies, accurate diagnostic tests are essential for detecting asymptomatic carriers. This study aimed to investigate the prevalence of infection in asymptomatic horses from Lorestan province, western Iran by developing a multiplex PCR. METHODS AND RESULTS: Blood samples were examined by microscopy and multiplex PCR targeting the SSU rRNA gene of Theileria equi and Babesia caballi. Out of the total of 165 horses, 19 (11.51%) and 31 (18.78%) cases were positive for piroplasms by microscopy and PCR, respectively. The detection rates of both genera were significantly higher in multiplex PCR compared to microscopy (p < 0.0001). Compared with multiplex PCR, the sensitivities of microscopy for the detection of Babesia were only 28.5%. The prevalence of T. equi infection was significantly higher in summer (p = 0.035). The prevalence of B. caballi was significantly higher in males (p = 0.038). CONCLUSION: Findings indicate that the multiplex PCR described here is a sensitive technique for the detection of piroplasm DNA in carriers. Furthermore, asymptomatic carriers must be considered as an important source of infection for equids living in this region.

Molecular Detection and Phylogenetic Analysis of Avian Haemosporidian Parasites in House Sparrows (Passer domesticus) of Western Iran.

House Sparrows (Passer domesticus) are ubiquitous wild birds and can be potential reservoirs of avian haemosporidians (vector-borne blood parasites) in their habitat. The Zagros Mountain range in western Iran is an important habitat of many endangered birds. To investigate the occurrence and molecular characterization of Haemoproteus and Plasmodium spp. in Kuhdasht County in the central western region of Zagros, blood samples from 60 House Sparrows were screened by microscopic examination and PCR. An overall prevalence of 6.6%, with a varied intensity of infection (1-198 parasites per 15,000 erythrocytes) for Haemoproteus spp. and no infection for Plasmodium spp., was observed by microscopy. One of the studied cytochrome b sequences was identical to the lineage PAHIS1, and the other sequence was a new lineage, PADOM41; both were extremely similar to Haemoproteus passeris. This is the first report on the molecular detection and characterization of Haemoproteus in passerines of Kuhdasht.

Exo-erythrocytic stages of Haemoproteus sp. in common buzzard (Buteo buteo): A histopathological and molecular study.

Haemosporidian parasites are responsible for anemia, acute tissue degeneration, and depopulation in wild birds. This study aimed to investigate the prevalence of haemosporidians and also morphologic and molecular evaluation of tissue stages of Haemoproteus sp. in common buzzards (Buteo buteo). Eleven free-living common buzzards were referred to the Avian Clinic of Veterinary School of Lorestan University with lethargy, weight loss, and ataxia. Gametocytes of Leucocytozoon buteonis were found in blood smears of six (54.5 %) birds, while one had simultaneous infection with blood stages of Haemoproteus and Leucocytozoon. During histopathological examinations, exo-erythrocytic stages of the genus Haemoproteus were seen in the lung and kidney of a dead bird. This study is the first report of exo-erythrocytic infection of Haemoproteus in common buzzards. Molecular assays confirmed the infection of Haemoproteus sp. (lineage BUTBUT15) in tissue samples. Phylogenetic analysis using cytochrome b gene suggested that BUTBUT15 was more closely related to the lineages isolated from the family Falconidae in contrast to the Accipitridae.

Molecular evaluation of Toxocara species in stray cats using loop-mediated isothermal amplification (lamp) technique as a rapid, sensitive and simple screening assay.

Toxocara species are parasitic nematodes of dogs and cats with a worldwide distribution. The adult worm lives in the intestine, and horizontal transmission of the infection occurs through eating paratenic host or embryonated eggs. This study aimed to estimate the molecular prevalence of Toxocara species in stray cats using the loop-mediated isothermal amplification (LAMP) technique. A total of 95 stool samples were randomly collected from stray cats in Khorramabad city in western Iran. Microscopic examination was performed after the separation and extraction of supernatants. The LAMP reaction was performed using the internal transcribed spacer 2 (ITS2) gene primers of Toxocara species and the appropriate master mix. The overall prevalence of Toxocara spp. in stray cats was 20% (19/95, CI 95%: 0.2 ± 0.08) by parasitological and molecular assessments. The microscopic examination of stool samples revealed that 19 samples were positive for Toxocara. The same 19 positive samples were also positive by the LAMP technique. Interestingly, based on the results of the LAMP assay, out of 95 studied samples, 18 (18.94%; CI 95%: 0.19 ± 0.08) specimens were Toxocara canis, while only 1 (1.05%; CI 95%: 0.005 ± 0.01) sample was diagnosed as Toxocara cati. The relatively high prevalence of Toxocara species in the studied cats shows the role of this species in spreading the parasite and the role of the cats in transmitting this zoonotic parasite. Preventive measures including the control of stray cat's population by castration and protection of public gardens where children play are recommended. The easy, highly sensitive and specific LAMP method is proposed for the differential detection of Toxocara species in animals and humans.

Bioassay-based detection of Toxoplasma gondii in free-range chickens from Khorramabad, Iran: comparison of direct microscopy and semi-nested PCR.

This study aimed to investigate the occurrence of anti-Toxoplasma gondii antibodies in free-range chickens from Khorramabad, western Iran, and also to compare the performance of direct microscopy and semi-nested PCR in mice bioassayed with tissues from seropositive chickens. We investigated 97 serum samples from free-range chickens, using the modified agglutination test (MAT). Tissues from all seropositive chickens (MAT ≥ 1:10) were bioassayed in mice. All inoculated mice were examined by direct microscopy and a semi-nested PCR targeting the 529 bp repeat element (RE) of the parasite. Anti-T. gondii antibodies were detected in 21.6% of chicken sera. Eighteen of 21 (85.7%) seropositive chickens were positive in mouse bioassay using molecular DNA detection. However, biological forms of the parasite were isolated only from 11 (52.3%) seropositive chickens. Compared with semi-nested PCR, the sensitivity of direct microscopy was 62.1%. It can be concluded that although direct microscopy is a rapid and specific method for the detection of T. gondii, it does not detect the parasite in all experimentally infected mice. The low sensitivity of direct microscopy highlights the need for molecular techniques, such as RE-based semi-nested PCR, to increase the sensitivity of the mouse bioassay.