Microscopic And Molecular Identification of Anaplasma Ovis in Small Ruminants in Duhok Province in Kurdistan Region of Iraq

In last ten years, there has been a developing enthusiasm for microscopic organisms from the genus Anaplasma, particularly the species A. ovis. It is associated with the pathogenic action of these microscopic organisms in livestock. Anaplasma ovis is a tick-borne obligate intracellular rickettsial bacterium that causes anaplasmosis in domestic and wild small ruminants. The samples of the present study were collected from small ruminants from inside seven distinct regions (Akre, Simele, Zummar, Feshchapoor, Deraboon, Bajed Kandal,Karoda)of Duhok province, 389 (goats 75 and sheep 314) during the period of April and May 2018, blood sample were taken and thin smear was formed, after Giemsa’s staining the slide is observed under microscope. In this study used Giemsa stain for microscopic examination out of 389 animals 250 were found positive for Anaplasma ovis infection with a prevalence rate of 64.26 % and 139 of them were negative with a prevalence rate of 35.73 %. According to the species of animals, the highest prevalence of A. ovis infection in animals by using microscopic examination was 67.83 %, 213 positive sample from total 314 blood samples from sheep and lowest prevalence was 49.33 %, 37 positive sample from total 75 blood samples from goats. PCR analysis of 100 blood samples obtained from total 250 positive blood samples after DNA extraction and measure of concentration and purity we used 2 primers that target major surface protein 4 (MSP4) in A. ovis genomic DNA. The results of PCR test with major surface protein 4 primer was 83 samples positive from total 100 samples, According to the species of animals, the highest prevalence of A. ovis was 83.7 %, 72 positive sample from total 86 blood samples from sheep and lowest prevalence was 78.5 %, 11 positive sample from total 14 blood samples from goats.

Development and evaluation of a double-antigen sandwich ELISA to identify Anaplasma marginale–infected and A. centrale–vaccinated cattle

Bovine anaplasmosis is a worldwide infectious disease caused by the intraerythrocytic bacterium Anaplasma marginale, which is transmitted by ticks and fomites. A. centrale is a less virulent subspecies used as a live vaccine in cohorts of 8- to 10-mo-old calves that did not naturally reach enzootic stability. We developed 3 variants of a double-antigen sandwich ELISA (dasELISA) using a recombinant major surface protein 5 (MSP5) from A. marginale (dasELISAm) or from A. centrale (dasELISAc) or using MSP5 from both organisms (dasELISAmc). Each dasELISA was tested for the detection of antibodies against A. marginale and A. centrale. The tests were validated using serum samples from cattle not infected with Anaplasma spp. ( n = 388), infected with A. marginale ( n = 436), and vaccinated with A. centrale ( n = 358), confirmed by nested PCR. A total of 462 samples were compared with a commercial competitive ELISA (cELISA). For dasELISAm, dasELISAc, and dasELISAmc, specificities were 98.7%, 98.7%, and 97.4%, and overall sensitivities were 92.6%, 85.7%, and 97.4%, respectively. For A. marginale–infected and A. centrale–vaccinated cattle, sensitivities were 97.7% and 86.3% for dasELISAm, and 77.7% and 95.5% for dasELISAc, respectively. Sensitivity of dasELISAmc was similar for both groups (>96%). The agreement rate between dasELISAmc and cELISA was 96.3% (κ = 0.92); the former test allowed earlier detection of seroconversion of vaccinated cattle than did cELISA. Based on these results, the test could be used to 1) determine the enzootic stability or instability of anaplasmosis in calves, 2) conduct epidemiologic studies, and 3) evaluate the immunogenicity of A. centrale live vaccine.