Molecular Detection and Genotyping of Babesia species in cattle in Baghdad city.

In cross-sectional study carried in areas surrounding


Introduction:
Bovine Babesiosis, also called red water disease or Piroplasmosis of cattle are protozoan parasitic diseases caused by Genus Babesia Order Piroplasmida Phylum Apicomplexa. (1). Babesia bigemina and Babesia bovis are the most widely distributed species affecting cattle in most part of the world except some part of Europe were Babesia divergens are the most prevalent .
(2). Diagnosis of Babesiosis is same as for other blood protozoan infections were the conventional diagnostic methods are still depend on the demonstration of infective stages in the blood , by Giemsa stained blood smear were it consider the gold standard for diagnosis, beside detection of circulating antibodies in the serum of infected animals by serological test like Enzyme Linked Immunosorbent Assay ( ELISA ) (3), (4).More over Polymerase chain reaction(PCR) had been widely used for detection of Babesia parasites owing to their high specificity and sensitivity (5), (6). Because of shortage in studies concerning molecular and genotyping of bovine babesiosis in Iraq and specially in Baghdad province , beside the current study aimed to sight a light on Babesia species genotype of cattle of Baghdad province  The blood sample had mixed for 10 minutes at room temperature.  A volume of 20 µl of Proteinase K (PK) had dispensed into a 1.5 ml micro-centrifuge tube.  From each sample, 250 µl of blood was added to the tube containing the Proteinase K (PK) solution, and briefly mixed.  Cell lysis Buffer (CLD) had added in avolume of 200 µl into a tube. This tube has capped and mixed by vortexing for 10 seconds  In water bath, tubes had incubated at 56 c˚ (optimal for Proteinase activity) for 10 minutes .  While the blood sample had incubated , a ReliaPrep™ Binding Column had placed into empty collection tube.  After tube removing from water bath incubation, a volume of 250 µl of binding Buffer (BBA) had added for each tubes. These tubes had capped and mixed by vortexing for 10 minutes.  Tubes contents had added to the labeled ReliaPrep ™ Binding Column, there for the column has capped and placed in microcentrifuge.  Centrifugation had conducted for 1 minute at maximum speed.  Collection tubes containing the flow through had removed, and the liquid has discarded as hazardous waste.  Binding columns had placed into a new collection tubes A volume of 500 µl of column wash solution (CWD) has added to the column and centrifuged for 3 minutes at maximum speed through that the flow has excluded.  Washing steps had repeated twice for three washes.  Columns had placed in a clean 1.5 ml microcentrifuge tubes.  Volume of 200 µl of nuclease-free water had added to the column and centrifuged for 1 minute at maximum speed.  Binding Column has discarded and the eluate has saved at -30 c˚. (7).

3-Determination of DNA Concentration and Purity:
Quantusflorometer used to detect the concentration of the extracted DNA in order to detect the quality of samples for downstream application. For 1 µl of DNA , 199 µl of diluted Quanty flour dye has mixed. There for, after incubation at room temperature , DNA concentration values had detected.(8).

4-Detection of Presence of B.bigemina and B.bovis :
Purified DNA samples had used to assess the specificity of Group 1 and Group 11 primers (Table 1), that described by (5). Detection the presence of B.bigemina and B.bovis , from study blood samples , using Grpup 1 primer , PCR product has added into the second (nested) PCR mixture compromising similar composition of reagents as the first-round PCR except that the external primers had replaced with the nested PCR primers.  Phylogenetic analysis for the sequenced genes was performed using ClustalW in MEGA 6, the neighbor-joining method to identify the genetic similarity and build phylogenetic tree for 8 Babesia bovis isolates and the two reference strains.

Computational Studies:
Several computerized programs and databases were used through this study for different purposes, as shown in table (3).

5-Statistical Analysis:
Results statistical analysis were carried using SPSS -version 14,using t-test and chi-square test according to (9).

Results and Discussions:
Among PCR, results of this study revealed that 10% of samples were infected with B.bovis without recorded any infection with B.bigemina as shown in table (4) . Figure (1).

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Results were not agreed with most of studies concerning molecular prevalence of cattle Babesiosis , like (10) in Iraq , and(111) in Pakistan who recorded the presence of both B.bovis and B.bigemena in their studies, this is may due to difference in study area and study sample or using different genes and different primers design may play important role in this differences.
Among age group of cattle examined by PCR , age group (4-6) years showed the highest rate of infection(73.33%) and the lowest infection rate (6.67%) were recorded in age group(7-9) years with significant difference p=0.016 , table (5). These results were in agreement with (13 ) who recorded (83.3%) in animals more than 3 years old, while our results disagree with (14) who declared that were no significantly difference between infection rate in young and older cattle infected with Babesia bovis . Distribution of cases by PCR according to study areas showed that Al Tajy and AbuGraib recorded the highest rate of infection (40%) for both areas , followed by Al Doura (13.33%) , and the lowest rate of infection was recorded in AlNahrawan(6.67%) with these results were shown in table (6).These results indicated that Al Tajy and AbuGraib considered as endemic areas with Babesiosis for the availability of the tick vectors.  .,2013). Phylogenetic tree was constructed using the neighbor-joining method. Figure  (1.1) show the genetic similarity between reference strains Babesia bovis isolate vaccine rhoptry associated protein 1 (rap-1) gene, partial cds (KM504166.1) and Babesia bovis isolate T21 rhoptry associated protein 1 (rap-1) gene, partial cds (KM504165.1)] and 8 Babesia bovis isolates according (rap-1) gene. The variability percentage was (0.01), this meaning there is variability in one nucleotide for each 100 nucleotides. Figures(2), showed neighbor-joining trees based on rap-1 gene sequences. (KM504166.1) and (KM504165.1). As reference strains.(Mega v.6).