Molecular Epidemiological Investigation And Genetic Identification Of Anaplasmas In Sheep And Goats’ Blood And Milk

ABSTRACT:Anaplasmosis, a widespread vector-borne disease, is caused by species of the genus Anaplasma that parasitized in animals’ and humans’ blood. Sheep and goats are susceptible to this disease. After the infection, sick animals show symptoms such as fever, anemia, weight loss, aurigo, malnutrition and death in serious condition. For this reason, establishing a fast and simple diagnostic method of anaplasmosis is extremely urgent both in domestic and overseas scholars now. In recent years, a large amount of diagnostic approaches for the diagonose of anaplamosis in ruminant animals especially in sheep and cattle have been developed. However, there is no report about the detection method of Anaplasma in milk. The purpose of this study is to Identify the situation of Anaplasma infection and developed a method for directly detecting the Anaplasma from the sheep and goat’s milk. This will provide a scientific basis for diagnosis and prevention anaplasmosis in sheep industry, and is significant to development of sheep industry and human health.1. In order to get a general knowledge of the prevalence of the anaplasmosis in sheep and goats in China, and provide the scientific reference for prevention and control of this disease, and promote the development of sheep industry, the present study was carried out to reflect the epidemiology of Anaplasma in sheep and goats’blood in partial regions of China. From March 2013 to January 2014, a total of 605 blood samples were collected from Luoyang, Zhengzhou, Kaifeng, Nanyang, Jiaozuo, Pingdingshan, Shangqiu, Xinxiang and Jiyuan in Henan province, some regions of Yunnan province and Qinghai province. All blood samples were tested by the nested PCR. The results showed that three Anaplasma pathogens A.phagocytophilum, A.bovis and A.ovis were detected in some blood samples, with varying infection rate as A.phagocytophilum 4.96%(30/605), A.bovis 13.06% (79/605),A.ovis 30.41%(184/605). What’s more, after data analysis, the results revealed that the infection rates were different between two feeding patterns, between sheep and goats, and the infection rates also had discrepancy in different ages of animals. The cases of mixed infection were also found in infected animals. Coinfection of A. ovis and A. bovis was dominant, with an infection rate of 3.47% (21/605). Coinfection rate of A. ovis and A. phagocytophilum was 0.17%(1/605) and that of A. bovis and A. phagocytophilum was 2.81%(17/605). Coinfection rate of all three pathogens was 0.50%(3/605).2. In order to have a global understanding of the prevalence of anaplasmosis in milk, a fast and simple method for directly detecting the Anaplasma from the sheep and goats’milk was established. From March 2013 to January 2014, a total of 105 blood samples were collected from Luoyang, Zhengzhou, Kaifeng, Nanyang, Pingdingshan, Shangqiu and Jiyuan in Henan province and some regions of Yunnan province of China. All milk samples were tested by the nested PCR method. The results showed that three Anaplasma pathogens A.phagocytophilum, A. bovis and A.ovis were deteced in some milk samples, with varying infection rate as A.phagocytophilum 1.90%(2/105),A.bovis 1.90%(2/105),A.ovis% 8.51%(9/105) and the tatal infection rate was 11.43% in milk. What’s more, after data analysis, the results indicated that the infection rates had discrepency between two feeding patterns, between sheep and goats. Anaplasmas infection rate (31.25%) was significantly higher in the sheep in grazing than those of stabling feeding (2.74%). The infection rate was significantly higher in the goats’ milk (14.86%) than in sheep’s milk (3.23%)3. In order to identify species or genotypes of detected Anaplasma isolates,111 isolates of positive samples were selected randomly and sequenced. The obtained sequences of 16S rRNA and MSP4 gene were put into NCBI using Blast tool to search for similar sequences. Finally, gene sequences of 19 representative isolates were picked out and then these sequences were spliced and aligned by Clustal X 2.11 and the homology analysis and phylogenetic tree were made using DNAStar 7.5 and MEGA 5.0, respectively. The results showed that two isolates of A. phagocytophilum from Jiyuan and Yuanyang have been identified with one and three nucleotides differences as compared to the reported strain (KC916731.1) respectively. All the isolates of A.bovis were the same to the reported strain (KF465986.1).63 isolates of A. ovis were compard with the reported strain (JN572936.1), among them,9 isolates had been identified to have 3-5 nucleotides differences with the reference sequence.In this study, we detected the Anaplasma in blood and milk samples of sheep and goats by PCR method. It was found to infect with A. bovis, A. ovis and A.phagocytophilum. The result shows that the situation of infected Anaplasma is common. However, A.phagocytophilum can also infected the Human. It is the first to detected the Anaplasma in milk samples by PCR method.2 isolates of A.phagocytophilum and 9 isolates of A. ovis had been identified. This will provide a scientific basis for diagnosis and prevention anaplasmosis, and will also significantly reduce the threat of anaplasmosis to human health.