Reserch Of Several Correlation Factors Of Diarrhea And Epidemiology Investigate Of Two Respiratory Diseases In Yaks

Yaks have been the most important production of consumer for local herders because of the great adaptability to cold, hypoxia, lack of grass and other harsh conditions in Qinghai-Tibet plateau region, which made it an irreplaceable role in ecotope, society and economic status. However, grassland degradation, serious desertification, unreasonable structure of the herd and degradation of yak breeds have been appeared due to the extensive predatory management carried by farmers and severe inbreeding among yaks. So morbidity of yaks has been rising in recent years, especially for calves. However, diarrhea of perinatal yak is the most common disease and also one of the main factors impacting yaks breed. Diarrhea of perinatal yak is mainly related to physiological functions, nutrition, stress and invasion of pathogenic microorganisms of calves and so on, though the factors are more complex. Diarrhea has seriously affected the growth and production performance of yaks, resulting in a extremely serious economic loss for yak breeding industries in Plateau region.Therefore, the relationship between trace elements, diarrhea and intestinal flora, the main pathogens causing diarrhea of perinatal yak and the major respiratory disease serology were investigated in this experiment and obtained the following results:1. The relationship between trace elements in the serum of normal and diarrheic yaksThe objective of this study was to investigate the blood serum for copper(Cu), iron(Fe), magnesium(Mg), manganese(Mn), selenium(Se) and zinc(Zn) concentrations in yak serum samples by inductively coupled plasma mass spectrometry(ICP-MS) from 35 yaks consisting of 10 healthy and 25 diarrheic ones. Our observations indicated that the concentrations of Mn, Zn and Fe in yak affected with diarrhea had statistically significant reduced levels as compared to the control group(P<0.05). The mean concentration of Cu in blood serum of diarrheic yaks was found significantly higher than the normal ones(P<0.05). The Mn and Zn in serum of diarrheic groups were observed significantly different(P<0.05) with different ages.These findings can provide for some valuable information for investigation on relationship between trace elements in the serum of normal and diarrheic yaks.2. Diversity analysis of intestinal bacterial microbiota in diarrthea and healthy in Perinatal Yaks through High-Throughput sequencingTo investigate the impact of diarrhea on the number and structure of intestinal flora in perinatal yak, healthy and diarrhea fresh feces were chosed respectively as objects in this study, and the changes in number and structure of intestinal flora were also compared by16 S r DNA V3-V4 region high-throughput sequencing. The result revealed that total of 272071 optimized sequences were acquired from bacterias, which were identified from 9 gates, 13 classes, 17 orders, 36 families and 72 genera; The number of bacteria species among diarrhea intestinal flora was lower than healthy group, but there were no significant differentces between the two groups(P>0.05); what’s more,significant differentces were presented in phylum, class, order, family and genus level between the two groups(P <0.05). This study showed that diarrhea of perinatal yak was related to intestinal flora structural changes.3. Isolation, Identification, Molecular Typing and Virulence Genes Detection of Pathogenic Escherichia coli in perinatal yaksTo investigate Escherichia coli(E. coli) of perinatal yaks serotype distribution and virulence genes, during June to August 2014, rectal swabs from 50 diarrhea calves were collected aseptically from Hongyuan area for the further isolation and identification of E. coli. Besides, O serotype determination, mouse pathogenicity experiments, 16 Sr RNA sequencing and virulence gene detection were also operated for the isolates.The result revealed that total of 47 E. coli were identified by culture and biochemical tests; 12 kinds of serotypes O were identified from 37 isolates by slide agglutination and another 10 have not been finalized, all of which covered the main serum O125, O145 and O91; all isolates were showed pathogenic by intraperitoneal injection in mice, and all mice had obvious lesions; 12 isolates randomly selected were 16 Sr RNA sequenced and analyzed by phylogenetic analysis, and showed a homology from 99.7% to 100%, which has a homology from 94% to 98% with other strains in Genbank; virulence gene iss, fim H,Omp A, pap C, tra Ta, tsh, iuc D and ETT2 were found 65.2%, 21.7%, 91.3%, 34.8%, 8.7%, 34.8%, 30.4% and 65.2% by PCR respectively, both of KspⅡand cva were 4.3%, and STa and irp2 were not detected. This study showed that not only erotypes of E. coli in Perinatal Yaks were more complex but virulence genes were more various, which provided a theoretical foundation for prevention of E. coli and development of vaccine for perinatal yak.4. Whole Genome Sequencing analysis of Pathogenic E.coli from diarrhea perinatal yakThe result revealed the 6.92 G raw sequences and 6.89 G filtered sequences, representing a 1384-fold and 1378-fold coverage of the genome respectively, which were satisfied with the further analysis; different kmer genomes about 76 contigs n50 were assembled into 52203 bp by using velvet; contig n50 and scaffold n50 reached 130308 bp and 354007 bp after scaffold was constructed by SSPACE_Standard and assembled by Gap Closer with showing a genome of 5.3 M and 5438 expected gene models, at last, which were in accordance with the genome of 5M of E. coli in magnitude. By refering the genomic sequence of E. coli available at NCBI, the location of scaffold assembled by velvet was performed by software of Mauve Contig Mover. Scaffold was connected into a complete linear chromosome by using of 200 n after obtained the order information between each two scaffold. A total 5543 Protein-coding genes, 10 r RNA and 92 t RNA were discovered by gene prediction of mitochondrial chromosome. Repetitive sequence, Genome Island, Prophage and structure of CRISPR prediction were performed,respectively. And NR, Swiss-prot 、 COG 、 KEGG and GO function annotation of Protein-coding gene were preformed respectively..5. Seroprevalence of two respiratory diseases in yaks(Bos grunniens) on the Qinghai-Tibetan Plateau of ChinaThe seroprevalence of Bovine Tuberculosis infection and Bovine Rhinotracheitis in yaks was investigated in Qinghai-Tibet Plateau of China in 2011. A total of 1124(Tibet 938, Qinghai 206)and 1840(Tibet 988, Qinghai 475,Sichuan 377) serum samples was examined by ELISA. The results showed that 24(2.56 %)of Tibetan samples and 3(0.98 %)of Qinghai’s samples were positive for Bovine Tuberculosis. And 381(38.6 %)of Tibetan samples, 212(44.6 %)of Qinghai’s samples and 105(27.9%)of Sichuan’s samples were positive for Bovine Rhinotracheitis. The observations of the present investigation indicate that Bovine Tuberculosis is common in yaks in Qinghai and Tibet and Bovine Rhinotracheitis is common in yaks in Tibet, Qinghai and Sichuan. The objective of this study was to survey the prevalence of Bovine Tuberculosis infection and Bovine Rhinotracheitis infection in yaks in Qinghai-Tibetan Plateau.In conclusion, the present reserch reveals that a comparatively high prevalence two respiratory diseases of yaks; Changes of trace element and intestinal flora disturbance causing of diarrhea in yak calves; demonstrates that the pathogeny of diarrhea in Hongyuan is E. coli and uncovers its biological characteristic by Whole Genome Sequencing analysis.