Investigation On Infection Of Babesia In Host Animals In Some Areas Of Yunnan Province

[objective]To understand Babesia infection among domestic animals and small mammals in Yunnan and provide scientific evidence for developing control measures. Domestic animals(cattles, sheeps, horses, donkeys and dogs) and small mammals were severed as research objects. 18 S rRNA gene of Babesia was amplified by nested PCR and was analyzed. [Method]1. Sample Collection A total of 3,277 domestic animals and small mammals samples were collected. 1,073 domestic animals blood(274 cattle, 395 sheep, 354 dogs, 33 horses and 17 donkeys) were collected. 2204 small mammals(50 species, 26 genera, 9 families and 4 orders) were collected. They contained 346 samples from Deqing, 99 samples from Weixi, 91 samples from Xianggelila, 224 samples from Yulong, 76 samples from Jinggu, 88 samples from Ninger, 39 samples from Tengchong, 215 samples from Yongde, 175 samples from Menghai, 81 samples from Mengla, 68 samples from Yunxian, 128 samples from Shiping, 110 samples from Mile, 94 samples from Yiliang, 22 samples from Mengzi, 134 samples from Fugong, 114 samples from Lushui and 100 samples from Gongshan.2. DNA Extraction Genomic DNA was extracted from blood or spleen of the animals using a TIANamp Genomic DNA Kit.3. PCR amplification(1)As to domestic animals, a near full-length 18 S rRNA gene sequence of Babesia was amplified by using nested PCR.(2)As to small mammals, a near full-length 18 S rRNA gene sequence of Babesia was amplified by using nested PCR. And a partial 18 S rRNA gene sequence of Babesia microti was amplified by using nested PCR.4. agarose gel electrophoresis PCR products was detected by 1.2% agarose gel electrophoresis and was visualized by UV transilluminator.5. Gene sequencing and analysis Gene sequencing of PCR products were carried out. Then gene sequences were compared by Blast program and phylogenetic tree were constructed by software MEGA6.06. [Results]1. 1073 domestic animals blood samples were detected. 50 samples were infected with Babesia, with the infection rate of 4.66%. Among 274 cattle, 11 were infected with Babesia(4.01%). 4 of them were Babesia bovis and 7 of them were Babesia bigemina. Among 395 sheep, 38 were infected with Babesia(9.62%), 37 of them were Babesia odocoilei-like parasites and 1 of them was Babesia capreoli-like parasites. Among 354 dogs, only one was infected with Babesia and it was Babesia vogeli. Horses and donkeys were negative.2. Domestic animals in 6 counties were infected with Babesia. These counties were Xianggelila, Yangbi, Jianchuan, Lanping, Yunlong, Gengma and Mengla.3. Phylogenetic tree of PCR products of blood of domestic animals revealed that among 7 Babesia bigemina sequences, 4 sequences, AY603402 and JQ723014 clustered into one clade, and other 3 sequencs, HQ840960 and JX495402 clustered into one clade. Among 4 Babesia bovis sequences, 3 sequences, EF458213 and H264111 clustered into one clade, the other one, L19077 and FJ426364 clustered into one clade. 37 Babesia odocoilei-like parasites sequences and KC460321 clustered into one clade. Babesia capreoli-like parasites sequences and KM657248 clustered into one clade. Babesia vogeli sequences and HQ148664 clustered into one clade.4. Among 2204 small mammals, 53 were infected with Babesia, with the infection rate of 2.40%. The positive small mammals belonged to 12 species, 9 genera, 4 families and 2 orders. They were, in descending order of infection rate, Neotetracus sinensis(40.00%), Eothenomys eleusis(14.29%), R. brunneusculus(7.53%), Apodemus draco(5.91%), Crocidura russula(5.56%), Eothenomys custos(3.08%), Pitymys leucurus(2.86%), Rattus tanezumi(2.70%), Suneus murinus(1.85%), Mus pahari(1.19%), Apodemus latronum(1.10%), Niviventer confucianus(0.93%). PCR products gene sequences were Babesia microti after BLAST. 10 counties were infected with Babesia. Infection rate in forest area, agricultural area and residential area was 3.37%, 1.79% and 0.93%, respectively. There was no difference in sex of small mammals. Adult small mammals had a higher infection rate than minor ones. As to altitude, small mammals in the altitude of 2000-3000 m had the highest infection rate.5. Phylogenetic tree of PCR products of spleens of small mammals revealed that 12 Babesia microti sequences, AB243679 and AB242176 clusted into one clade. 9 Babesia microti sequences and KC478600 clusted into one clade. [Conclusion]1. Some domestic animals in partial counties in Yunnan were infected with Babesia. These Babesia were Babesia bovis, Babesia bigemina, Babesia odocoilei-like parasites, Babesia capreoli-like parasites and Babesia vogeli. They destroyed domestic animals health and husbandry. Babesia bovis might infect human. Phylogenetic tree revealed that these Babesia were different from Babesia in other places. It was necessary to identify their genomic diversity.2. This paper firstly reported that small mammals were infected with Babesia microti in Yunnan province. 53 positive samples belonged to 12 species, and were from 10 counties. Small mammals in the altitude of 2,000-3,000 m had the highest infection rate. So they threatened workers and travelers who went into forest.3. As was known to all, Babesia microti could infect human. There might be Babesiosis natural focus in Yunnan. Small mammals might be the main host animals. Babesia microti might be the main pathogens. It was necessary to investigate infection rate in human, new Babesia species infecting human and ticks transmitting Babesiosis.