دراسة تشخيصية جزيئية ومناعية لأنواع الأكريات Eimeria sp. المخمجة للدجاج في محافظتي كربلاء المقدسة وبابل

Identification of Chickens Coccidian species by Molecular and Immunological Methods in Kerbala and Babylon Provinces

This study was conducted to demonstrate the prevalence and determined the species of parasites that responsible of Poultry Coccidiosis in each of provinces of Babel and Kerbala , A different diagnostic methods are used during this study including the traditional methods ,which including all of the clinical examination , Macroscopic and microscopic examination to determine the positive samples in the suspected chickens.
The present study Observations were revealed increasing and decreasing the percentages of infection with coccidiosis during different months of a year and that due to the large change in temperature and humidity in Iraq,so the present study showed that the highest rate of infection in both of Kerbala and Babylon provinces were in December (23.12%) and the lowest rate in July (1.87%).
A lot off 200 samples of feces and intestines were collected from chickens that suspected infection with coccidiosis during the period from August 2013 to |July 2014 and the results revealed that 160 samples were positive depending on the macroscopic and microscopic examination which in turn have undergone the second stage of the examination for the purpose of qualitative accurate diagnosis of the species of Eimeria that causing coccidia in chickens using of molecular methods for the target region ITS1 nucleic acid DNA encoded ribosomal RNA , these techniques include the qRT- PCR technique and Conventional PCR, Which diagnosed three species of Eimeria includes E.tenella which formed the largest proportion of the total positive samples (66.8%) then the species E.necatrix which accounted for (30%) and finally the species E.maxima which form (3.13%).
A Fifteen DNA samples (five samples for each of three species) of Eimeria has been sequenced and analyzed in Korean Bioneer corporation in which multiple sequence alignment online based analysis for the ITS1 region that previously amplified by polymerase chain reaction, A comparison between the sequences of bases of local isolates of Eimeria with global isolates that recorded in Gen Bank and the comparative molecular screening of the present study results revealed the Validity and accuracy of diagnosis of three Eimerian species.
Phylogenetic tree analysis using the program (MEGA 6) were adopted to determine genetic tree of the species analysis ( Test UPGMA tree) (Unweighted Pair Group Method with Arithmetic) to compare the three of local species with global strains of Eimeria and found the Homology sequence identity of Eimeria tenella local isolates in comparison with NCBI-Gen bank Eimeria tenella (JX853830 ) . using NCBI-BLAST the results showed 98% & 99% , while the Homology sequence identity of Eimeria necatrix of local isolates in comparison with NCBI-Genbank Eimeria necatrix (JX853832.1) were 91% & 100 % and the Homology sequence identity of Eimeria maxima of local isolates to NCBI-Genbank Eimeria maxima (JX853828.1) was 98%.
Immunological examination of Indirect ELISA Technique was adopted for diagnosis of IgY in chickens sera that experimentally infected with E .tenella and E .necatrix , positive samples were determined after calculating the value of the detection level ( Cut -off level ) which equal to 0.313, where the average value of absorbance ( Optical density ) in chickens which experimentally infected with E.tenella was (0.96 ± 0.029), while the average value of absorbance in chicken sera that infected with the species E .necatrix was (0.95 ± 0.029).
To measure the possibility of Cross Reactions between the two species of Eimeria that used in experimental infection. The present study results showed the possibility of cross – reaction as the use of chicken sera that infected with species E.tenella species with antigens prepared from sporulated oocysts of E.necatrix species showed a positive result and the value of the average absorbance (0.75 ± 0.096), either in the case of the use of chicken sera infected with E.necatrix species with antigens prepared from sporulated oocysts of E .tenella species also showed a positive result and the average absorbance value was ( 0.75 ± 0.097).