A study on prevalence, molecular identification and characterization of blood and tissue protozoa of domestic ruminants in Chittagong, Bangladesh

Abstract

Blood and tissue parasites infect both the small and large ruminants causing significant economic losses all over the world including Bangladesh. The actual extent of these parasitic diseases and their epidemiologic features is not yet comprehensively studied in Bangladesh. Accurate identification and characterization are essential for any epidemiologic investigation. With the availability of modern DNA based tools and resources, the capacity for accurate and reliable detection of any disease agents are now possible which was not available before. The present study was therefore designed to investigate the prevalence followed by molecular identification of selected hemoparasites (Babesia, Anaplasma, Trypanosoma) of cattle in different geographic areas of the country. In addition, an attempt was made towards the molecular characterization of tissue protozoan (Toxoplasma gondii) in small ruminants in the Chittagong Metropolitan area located in southern part of Bangladesh. Further analyses of the associated risk factors were also done to help to develop necessary prevention and control options against these important protozoa. Blood samples collected from cattle were preserved in the laboratory for further examination with conventional microscopy and modern molecular tools like PCR and DNA sequencing. Sample size varied among different protozoa species and initial screening was aided by Giemsa stained thin smear examinations. A variable numbers of samples were assessed to detect hemoprotozoa including Anaplasma (n=1680), Babesia (n=1680) and Trypanosoma (n=480) where 8.21%, 1.43% and 0.63% prevalence was recorded respectively from different study areas inside Bangladesh. Bovine anaplasmosis was found as an important rickettsial disease with limited reports related to its prevalence inside Bangladesh. Comparative prevalence of this hemoparasitewas recorded as 9.33% in the hilly areas, 9.00% in coastal areas and 5.83% in plain lands inside the country. Further analyses indicated a higher prevalence (P>0.05) in crossbred cattle compared to local or indigenous breeds. Variations in seasonal incidence were also observed with high case numbers recorded in winter season of the year. Age of the animals was also found an important epidemiologic factor where animals aged 18-30 months were more vulnerable to anaplasmosis than other age groups. Housing and floor types also found to be associated with variable prevalence and animals living in non-cemented or soil-type floors were more prone to suffer from anaplasmosis. Molecular characterization through PCR amplification of 16S rRNA gene was achieved where an 80% cases (40 out of 50 samples) were found positive for A. marginale infections. Further amplification of MSP4 gene fragments successfully identified all forty positive cases in 16S rRNA screening. Further DNA sequencing and phylogenetic studies revealed that all the isolates of this present study were closely related to the isolates already reported from countries like Argentina, Australia and China. Bovine babesiosis is an economic important protozoan disease with sporadic reports on its prevalence and incidence in Bangladesh. During this study, prevalence of babesiosis was found as 1.17%, 1.67% and 1.46% in the selected hilly, coastal and xviii plain areas respectively in the study areas. Further data analysis showed crossbred and female animals more vulnerable than local or indigenous cattle and their male counterparts. High number of babesiosis was recorded in summer season compared to other time of the year. Animals aged 6-18 months were more susceptible to babesiosis than other age groups. Molecular identification of the organism were achieved through PCR amplification of 18SrRNA gene fragments where all positive cases (n=24) screened by conventional microscopy were successfully amplified. Further DNA sequencing and phylogenetic analysis revealed that two distinct isolates of Babesia, Babesia bigemina and Babesia ovata were present in the country. Trypanosomiasis is one of the least studied blood parasites of cattle in Bangladesh and thought to be distributed in different parts where relevant vectors are available. During this study out of 480 blood samples, only three were found positive in a single dairy farm located in Chittagong Metropolitan area. All positive cases were recorded in female and Holstein Frisian crossbred cattle and in one farm where previous incidence and disease record was not available. The time of the year when these cases were found was in rainy season and all the animals were aged 6 years or more and none of the younger animals were found positive. Molecular identification was achieved through PCR amplification of 18SrRNA gene fragment following previously published protocols. Sequencing of amplified DNA and further phylogenetic analysis revealed that Trypanosoma evansi were circulating in Bangladesh which is closely related to the isolates reported from countries like Taiwan, Thailand and Japan. Toxoplasmosis is an important zoonotic tissue protozoan disease of small ruminants in Bangladesh and has been documented by a number of publications. Several studies reported variable prevalence rate of toxoplasmosis in different parts of the country. The present study revealed an overall prevalence of toxoplasmosis in sheep and goats as 30.43% and 41.30% respectively. Further statistical analysis of the data indicated that animals aged two years or more were highly seropositive to Toxoplasma gondii. History of abortion and sources of drinking water were found to be significantly associated with toxoplasmosis. Animals with previous history of abortion were highly seropositive compared to the other groups. High seroprevalence was recorded in farms where ponds and municipality supply water are used as water sources for animals. Apart from serodiagnosis, acute and chronic cases of toxoplasmosis are always troublesome to detect hindering their prevention and control efforts. Combined histopathological and molecular approaches were applied during this study to detect the Toxoplasma gondii from several aborted tissues of sheep and goats. Prevalence of T. gondii was detected as high as 25% in sheep (n=4) and 36.84% (n=19) in goats. All the positive cases were detected in animals that aborted at 3.5 months of gestation. Further sequencing of amplified DNA and phylogenetic analysis of the sequences confirms the presence of T. gondii in a single cluster with minimum genetic variations among the four isolates which are closely related to the isolates recorded from countries like India and Iran.