The isolation and molecular characterization of Mycobacterium avium subsp. paratuberculosis in Shandong province, China

Abstract

BackgroundMycobacterium avium subspecies paratuberculosis (Map) causes Johne’s disease in domestic and wild ruminants. It has been a debate that whether Map can cause Crohn’s disease in human. To our knowledge there is no report about molecular characterization of Map in China, although several Map strains have been reported in other country. The objectives of this study was to know the recent prevalence of Johne’s disease in dairy farms in Shandong province, and have a better understanding of genotypic distribution of Map in China.MethodsJohne’s disease was detected from 1038 individuals in 19 dairy farms by ELISA. Map in fecal and milk specimens was identified by Ziehl-Neelsen staining and confirmed using PCR-REA. In addition, frozen sections of ileum and mesenteric lymph nodes from two Map shedding cows were performed to observe the histopathological changes. Next-generation sequencing technology was performed to get whole genome sequences.ResultA total of 121 (11.7 %) animals were positive for Map antibody from 1038 sera tested, and 11 (57.9 %) dairy herds were positive for Map antibody. Typically histopathologic changes were observed in mesenteric lymph nodes. We have successfully isolated two Map strains, which both were Map-C. The current genome-wide analysis showed that the genome size of our isolates are respectively 4,750,273 and 4,727,050 bp with a same G + C content of 69.3 %, and the numbers of single nucleotide polymorphisms (SNPs) against Map K-10 are respectively 292 and 296.ConclusionMap is a prevalent pathogen among dairy cattle in China. This study successfully isolated two Map strains from one Chinese dairy herd with signs of diarrhoea, and identified that the two isolates were both Map-C. Furthermore, these isolates were most closely related to Map K-10.

Characteristics and Epidemiological Investigation of Paratuberculosis in Dairy Cattle in Tai’an, China

“…In the late stage of the disease, cows became extremely thin and milk production declines sharply. In this study, the identified MAP strain was type C (dairy cattle), consistent with Yue et al's previous research [18]. It indicates that type C is the predominant type in cattle in Shandong Province, China.…”

“…The seroprevalence investigation indicated that 66.7% (6/9) of herds and 14.2% of animals were positive for MAP antibodies, which is higher than the findings from previous studies from Shandong [18] and other provinces in China [41][42][43][44]. These studies recorded MAP antibodies in 2.8%-18% of animals [41][42][43][44].…”

“…One herd consisted of 550 cows and the other had 536 cows. The diagnosis of the case and the isolation and identification of the pathogen were based on the methods reported in a previous study [18]. The five cows were euthanized because they were extremely thin.…”

“…Molecular Examination. The test was performed as per the method of Yue et al [18]. Following euthanasia, the gastrointestinal tracts and mesenteric lymph nodes were collected from two of the five cows for postmortem examination.…”

Examination ofMycobacterium aviumsubspeciesparatuberculosismixed genotype infections in dairy animals using a whole genome sequencing approach

“…There have also been numerous WGS studies that have examined the genetic diversity of Map from dairy animals (Ahlstrom et al, 2015; Bryant et al, 2016; Ahlstrom et al, 2016; Yue et al, 2016; Leão et al, 2016), but none which have analyzed or addressed mixed Map genotype infections using multiple isolates from a single animal. We recently reported that Map with different s hort s equence r epeat (SSR) types could be isolated from individual dairy animals from the island of Newfoundland (Podder et al, 2015).…”