| Bovine tuberculosis(bTB)is a chronic consumptive zoonotic disease caused by Mycobacterium bovis(M.bovis).It is thought to be transmitted among herds by inhalation of infected aerosol droplets from infected cattle,the digestive tract exposure also plays an important role,as cattle can become infected following ingestion of feed or water contaminated with nasal secretions,feces,urine,or unpasteurized milk from infected animals.Symptoms vary according to the location of lesions,the lesions often can be observed in lung and lymph nodes,also can be found in other tissues and organs,such as mamma,serosa,reproductive organs and intestine.China is a country with a high burden of bTB,and the prevalence rates are between 0~15%.The data show that after 1950 s,the main manifestation of bTB in China is pulmonary tuberculosis,and the manifestation of bTB in other countries are also mainly pulmonary tuberculosis after generally implementing milk pasteurization.However,in the 21 st century,the national bTB epidemiological investigation found a puzzling common phenomenon: the PPD positive cattle had no respiratory symptoms such as cough,and few pulmonary tuberculosis lesions were found by autopsy.To further understand the prevalence of bTB in some areas of China,to reveal the reasons for the discrepancy between PPD positive cattle and pulmonary tuberculosis examined by autopsy,and to explore the source of infection,route of transmission and pathogen characteristics,an epidemiological survey of bTB was carried out in 14 provinces and Xinjiang Production and Construction Corps.Eight large-scale dairy farms were selected for comprehensive immunological diagnosis,systematic autopsy,histopathological examination,pathogen isolation and identification,whole genome sequencing,phylogenetic analysis,animal pathogenicity test and other biological characteristics.1.From 2015 to 2018,we used comparative intradermal allergy(SICCT)and IFN-? assay to carry out bTB epidemiological investigation in 14 provinces and Xinjiang Production and Construction Corps,and collected disease materials from positive cattle detected by SICCT or IFN-? assay for bacterial isolation and identification.1,594 positive cattle were detected in 6,943 cattle by SICCT,with an average individual positive rate of 22.96%,and 114 positive farms were detected in 185 farms by SICCT,with an average positive rate of 61.62%.1,786 positive cattle were detected in 8,677 cattle by IFN-? assay,with an average individual positive rate of 20.58%.150 positive farms were detected in 236 farms,with an average positive rate of 63.56%.Bacteria were isolated from 64.44% of bTBpositive cattle.The isolates were identified by two-stage multiplex PCR of Mycobacterium tuberculosis complex,of which 72 strains were M.bovis,11 strains were M.avium,and 4 strains were other mycobacteria.The results of epidemiological investigation showed that the positive rate of bTB was still high in some areas of China,and bTB was prevalent in 14 provinces and Xinjiang Production and Construction Corps.However,the positive cattle detected by PPD or IFN-? assay rarely showed typical pulmonary tuberculosis symptoms such as cough,dyspnea and extreme emaciation in field investigation.Moreover,because of the above reasons,there were doubts about the legitimacy of culling of bTB positive cattle and it was difficult to carry out normal quarantine.2.To investigate the current forms of bTB and identify the possible transmission modes in dairy farms of China.13,345 cows from 8 dairy farms in 3 provinces were detected by a comprehensive diagnostic assembly,including SICT,SICCT,IFN-? assay,PPD eye drop reaction and ELISA.The result indicated that 752(5.64%)were determined to have advanced infection of bTB.Examination of 151 positive cattle by necropsy revealed that 131 cattle(86.75%)had typical bTB lesions,of which 119(90.84%)had lesions in liver,spleen,mesenteric lymph nodes,mammary lymph nodes and other organs,whereas only 12 cattle(9.16%)had lesions in lung.HE staining showed that different degrees of pathological changes of bTB could be seen in the tissue sections of 151 cattle.The bacteria from 60.26%(91/151)positive cattle were isolated and characterized by Ziehl-Neelsen acid-fast staining and qPCR,GeneXpert MTB/RIF and Enigma.The results of comprehensive diagnosis showed that the main manifestations of bTB in 8 dairy farms were extrapulmonary tuberculosis(EPTB),and the lesions were mainly distributed in extrapulmonary tissues and organs such as mesenteric lymph nodes,intestinal tract,liver and spleen.Of note,71.26% of EPTB was related to gastrointestinal system.Moreover,the results of field investigation,clinical manifestation,pathological examination,histopathological examination and pathogen isolation and identification all suggest that the transmission route of EPTB in 8 dairy farms may be the digestive tract,and it may be infected by milk or forage contaminated by M.bovis.3.To verify the source of infection and transmission of EPTB in 8 dairy farms,54 milk samples,including 46 from positive cattle and 8 from calving room,were collected.qPCR result revealed bTB nucleic acid positive in all 8 samples from calving room and 39 samples from positive cattle.12 isolates of bTB were further cultured from 54 milk samples.We also tested the 54 feces samples.Similar to milk samples,we found bTB nucleic acid positive in all 8 samples from fecal pool and 34 samples from positive cattle.However,no bTB strain was successfully isolated these 54 feces samples.These results unraveled the possibility of gastrointestinal transmission mode of EPTB in 8 dairy farms.4.To study the pathogenicity of the isolates,the representative isolates(strain 113 and 105)and the positive control strain(M.bovis AN5)were selected for rabbit infection test.The rabbits were divided into groups and infected by intraperitoneal injection,oral administration and nasal drip.The rabbits were subjected to a detailed necropsy after 8 weeks,the diseased materials were collected for histopathological examination,bacterial isolation and culture and plate colony count.The histopathological examination showed that the pathological changes mainly in lungs,hilar lymph nodes,mesenteric lymph nodes and spleen.Typical granulomatous lesions with three-layer structure could be observed,and the Langhans giant cell were also observed sometimes.Also the bacteria were observed in lungs,hilar lymph nodes and mesenteric lymph nodes by Ziehl-Neelsen acid-fast staining.The gross lesions of rabbits in the experimental groups were more extensive and more serious than those in the positive control group at necropsy.Bacteria could be recovered from both the experimental groups and the positive control group,and the CFU of the experimental groups were higher than that of the positive control group,indicating that the isolates of EPTB showed strong pathogenicity and had the potential risk of infecting cattle and spreading among cattle.5.The isolates were genotyped by 24-locus MIRU-VNTR genotyping method,and the whole genome sequencing and phylogenetic analysis of typical strains were carried out.The results of MIRU-VNTR genotyping showed that the isolates were clustered into three large groups,and the isolates from the same region were closely related,which may come from the same evolutionary branch,and M.bovis has unique geographical differences.10 isolates were sequenced,and the homology analysis showed that all the 10 isolates were M.bovis,and the homologies were between 98.80%~99.31% with the M.bovis AF2122/97.Phylogenetic analysis showed that the 10 isolates were divided into two clusters and were closely related to M.bovis AF2122/97 and M.bovis 30,respectively.In conclusion,this is a first detailed investigation on the distribution of lesions of extrapulmonary tissues and organs in bTB in China,and it is confirmed that the high prevalence rate of EPTB transmitted by digestive tract were present in dairy farms in many provinces of China.In this study,the epidemic characteristics of bTB in some provinces of China were determined,the comprehensive diagnosis strategy of EPTB was established,and the pathogenic ability of EPTB isolates was determined.This study provides strong evidence for the difficulty of culling PPD positive cattle in the current industry,diagnostic strategies and prevention and control guidance for dairy farms to rapidly reduce the positive rate of bTB,data support for the analysis of the source and genetic characteristics of bTB epidemic strains in China,and a decision-making basis for the formulation of bTB control and eradication programmes in our country. |
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