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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC AND IMMUNOLOGICAL CHARACTERISTICS OF JOHNE'S DISEASE Title: Pathogenesis of Mycobacterium avium subsp. paratuberculosis in Neonatal Calves after Oral or Intraperitoneal Experimental Infection

Authors
item Stabel, Judith
item Palmer, Mitchell
item Robbe-Austerman, Suelee - USDA-APHIS-NVSL
item Harris, B - USDA-APHIS-NVSL

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 12, 2007
Publication Date: October 28, 2007
Citation: Stabel, J.R., Palmer, M.V., Robbe-Austerman, S., Harris, B.N. 2007. Pathogenesis of Mycobacterium avium subsp. paratuberculosis in Neonatal Calves after Oral or Intraperitoneal Experimental Infection [abstract]. 9th International Colloquium on Paratuberculosis. Abstract No. 30A, p. 115.

Technical Abstract: There is a critical need for a ruminant infection model that can reproduce the progression from subclinical to clinical disease, thereby following a pattern of infection commonly recognized in the field. The current study was designed to compare the effectiveness of different methods of experimental inoculation on the pathogenesis of MAP infection. Twenty neonatal Holstein calves were obtained from status level 4 herds and randomly assigned to 5 treatment groups: 1) control noninfected (C), 2) oral (Oral), 3) oral with dexamethasone pretreatment (Oral/DXM), 4) intraperitoneal (IP), and 5) oral/mucosal (Oral/M). The oral group was fed milk replacer containing 1010 cfu of live MAP, strain K-10, 2x per day for 14 consecutive days. The Oral/DXM group were inoculated in the same manner as the Oral group but the calves were administered 0.25 mg/kg BW dexamethasone IV for 3 consecutive days prior to bacterial challenge, and on days 28 and 56 post-challenge. IP inoculation of calves with 1010 cfu MAP, strain K-10, was performed on days 0, 7, 14, and 21 of the study. The Oral/M calves were inoculated by feeding milk replacer containing live MAP obtained by scraping the ileal mucosa from a clinically infected cow on days 0, 7, and 14. All calves were housed in AAALAC-accredited BSL-2 facilities during the study. Throughout the study, blood and fecal samples were obtained from calves on days -5 and -4 prior to the first inoculation of MAP, and then on days 7, 14, 21, 28, and monthly thereafter for the 12 month term of the study. Fecal culture and PCR data demonstrated that calves in the oral inoculation groups experienced shedding on days 7, 14, 21, and 28, indicative of “pass-through” shedding that is typically observed after large oral boluses of bacteria are administered. Shedding was minimal and infrequent over the course of the study for calves in the Oral, Oral/DXM, and IP treatment groups. Calves in the Oral/M treatment group shed high numbers of bacteria up to 4 months post-inoculation. By 4 months post-infection, shedding was significant only in 1 of the 3 calves (79 cfu/slant), followed by sporadic shedding of few organisms thereafter. Fecal PCR results mirrored the culture results with infrequent positive reactions after the first 4 weeks of infection, regardless of infection group. Colonization was present in a number of intestinal tissues and lymph nodes with the lowest number of affected tissues in the IP calves and the highest for calves in the Oral/M group. Recovery of viable MAP was low in tissues regardless of treatment group with the exception of one calf in the Oral/M whose tissues had very high numbers of MAP. Histopathologic lesions were predominantly found in the ileal and jejunal sections and their associated lymph nodes, as well as the ileocecal valve and node. Lesions were characterized by multifocal small infiltrates in the submucosa of intestinal tissues and small aggregates of macrophages with and without granuloma formation within the lymph nodes. Lesions were most predominant within the tissues from Oral/DXM calves and secondarily for the Oral group. Few lesions were found in the tissues of IP and Oral/M calves. Four SSR loci in the MAP genome (Locus 1, Locus 2, Locus 8 and Locus 9) with the highest indices of diversity were compared across the 2 strains of MAP utilized (K-10, clinical cow isolate) and between the original inoculum of each strain and the output from fecal shedding of infected calves. Analysis of the MAP isolates recovered from the infected calves indicated that there was no variation in the MLSSR types as compared to the original inoculum. However, strain K-10 demonstrated the genotype of >14-10-5-5, consistent with previous reports, but the clinical cow isolate yielded two similar MLSSR types; 7-9-4-3 and 7-10-4-3, suggesting that 2 strains of MAP were present in the ileum of the infected cow. These data suggest that oral inoculation remains the most effective method of experimental infection for MAP and that inoculation with a low passage strain of MAP may induce more clinical signs.

Last Modified: 10/21/2014
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