Susan M. Stehman

Development and Application of Quantitative Polymerase Chain Reaction Assay Based on the ABI 7700 System (TaqMan) for Detection and Quantification of Mycobacterium Avium Subsp. Paratuberculosis

Numerous reports have described diagnostic methods based on the polymerase chain reaction (PCR) used to detect Mycobacterium avium subsp. paratuberculosis, the causative agent of Johnes disease. The result of conventional PCR tests has been only qualitative, either positive or negative; it does not present any quantitative information about the number of the agents in the specimen. A quantitative PCR method (IS900 TaqMan) was developed to measure the number of M. a. paratuberculosis organisms present in field and clinical samples. The sensitivity of IS900 TaqMan was 1 colony-forming unit (CFU) for M. a. paratuberculosis ATCC 19698. The specificity of the method was determined by testing 14 mycobacterial species (M abscessus, M. asiaticum, M. avium subsp. avium, M. bovis, M. fortuitum subsp. fortuitum, M. intracellular, M. kansasii, M. marinum, M. phlei, M. scrofulaceum, M. simiae, M. smegmatis, M. terrae, and M. ulcerans) and 9 nonmycobacterial species (Borrelia burgdorferi, Chlamydia psittaci, Ehrlichia canis, E. equi, E. risticii, Escherichia coli, E. coli O157:H7, Streptococcus equi, and S. zooepidemicus). Even at high cell numbers (105 CFU/reaction), most of the organisms tested negative for the IS900 insertion element except M. marinum and M. scrofulaceum. This finding for M. scrofulaceum was consistent with previous reports that several M. scrofulaceum-like isolates were positive for IS900. Those isolates had 71–79% homology with M. a. paratuberculosis in the region of IS900. When used in conjunction with the new liquid medium-based ESP culture system II for bovine clinical fecal samples, IS900 TaqMan confirmed that the ESP II-positive samples contained 105–106 CFU/ml of M. a. paratuberculosis. All of the 222 ESP II-positive and acid-fast bacilli-positive samples tested in this study were positive by IS900 TaqMan. IS900 TaqMan was also useful in the study of growth characteristics of 3 groups of M. a. paratuberculosis strains in bovine fecal samples from 3 shedding levels (heavy, medium, and low) based on cell numbers measured by Herrold egg yolk (HEY) agar culture. When cultured in ESP medium, M. a. paratuberculosis reached 105–106 CFU/ml within 2 weeks for heavy shedders, 3–4 weeks for medium, and 6–8 weeks for low shedders. No significant growth was observed after up to 5 weeks of incubation for some of low shedders. No or extremely slow growth characteristic of low shedders might be a possible explanation for frequent false-negative results by HEY The detection time was dependent on the inoculum size and the growth rate of M. a. paratuberculosis. Generation times were inversely proportional to the shedding level: 1–2 days for medium and heavy shedders and >4 days for low shedders. IS900 TaqMan could be a useful tool for determining viable cell counts by measuring changes in cell numbers over the incubation period.

Paratuberculosis in Small Ruminants, Deer, and South American Camelids

Paratuberculosis in small ruminants is widespread geographically. In some herds and flocks, clinical paratuberculosis, which primarily causes long-term weight loss, can be a significant cause of culling. The effects of subclinical disease are less defined, but they may include decreased milk production in milking sheep and decreased weight gain in deer raised for slaughter. Paratuberculosis also can cause economic losses due to reduced sales of breeding animals from purebred flocks and herds. Diagnosis in goats, deer, and SACs is based primarily on feces or tissue culture and histopathologic examination. Because of the difficulty in growing sheep strains of M. paratuberculosis, diagnosis is based on finding compatible histopathologic lesions with acid-fast organisms. The AGID is a highly specific serologic test for diagnosis of clinical paratuberculosis that correlates with fecal shedding of organisms and degree of severity of lesions; however, it is less sensitive than fecal culture at the herd level for detection of subclinical disease. The CF test detects clinically affected animals but yields poor specificity. The ELISA test is being evaluated for use as a herd screening test in sheep, goats, and SACs. The test seems to be as sensitive as the AGID test but shows a lack of specificity in herds and flocks infected with caseous lymphadenitis. Absorption of sera with Corynebacterium pseudotuberculosis or Mycobacterium phlei may improve specificity, but more studies are needed. Currently, the ELISA test is best used only as a herd or flock screening tool; its use for diagnosis of individual cases requires confirmation with a more specific serologic test or organism detection test such as fecal culture or PCR. Paratuberculosis can be managed and eliminated by combining stringent management with frequent testing and culling or by combining vaccination (where permitted) with management of fecal-oral transmission. Management of fecal-oral transmission is particularly important to prevent exposure of young stock to the infection. Control programs differ with the goals and economics of each farm. The frequency of testing and level of management intervention are determined by each farms abilities, priorities, and finances.

Use of Conventional and Real-Time Polymerase Chain Reaction for Confirmation of Mycobacterium Avium Subsp. Paratuberculosis in a Broth-Based Culture System ESP II:

The ESP II Culture System (ESP II), a broth-based culture system, has been modified and optimized for culturing Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in animal feces since 2000. Conventional and real-time polymerase chain reaction (PCR) assays based on the IS900 sequence were performed as confirmatory tests for M. paratuberculosis in ESP II liquid culture medium. There were no differences between test results of conventional and real-time PCR assays. During the 5-week incubation period, if acid-fast bacilli (AFB) were detected in ESP culture–positive samples, IS900 PCR assays were performed to confirm whether those AFB were M. paratuberculosis. At the end of the 5-week incubation, AF staining was performed on all ESP II–negative cultures to screen any false-negative cultures; IS900 PCR assays were performed on AFB-positive cultures. During a period of 1 year, of a total of 18,499 ESP II cultures, 2,814 (15.2%) PCR confirmation assays were performed. Of those, 2,259 (80%) were both ESP and PCR positive; 104 (4%) were ESP positive and PCR negative; 423 (15%) were ESP negative and PCR positive; 28 (1%) were both ESP and PCR negative. The AF-staining step after the 5-week incubation produced 423 (15%) more PCR-positive cultures. Of a total of 2,814 AFB-positive cultures, 132 (5%) were not confirmed as M. paratuberculosis. Further studies are needed for speciation of non–M. paratuberculosis isolates.

Vaccination with recombinant Mycobacterium avium subsp. paratuberculosis proteins induces differential immune responses and protects calves against infection by oral challenge

We previously reported the in vitro cellular immune responses to recombinant antigens (rAgs) of Mycobacterium avium subsp. paratuberculosis (MAP). Here we report the differential immune responses and protective efficacy of four rAgs of MAP (85A, 85B, 85C, and superoxide dismutase (SOD)) used with two adjuvants (monophosphoryl lipid A (MPLA) containing synthetic trehalose dicorynomycolate, cell wall skeleton (MPLA) and bovine IL-12), against MAP challenge in calves. Group I was administered the four rAgs with MPLA and IL-12. Group II was administered the four rAgs and MPLA. Group III received MPLA and IL-12, and Group IV MPLA. rAgs induced significant lymphoproliferative responses in vaccinated animals (Groups I and II). All the rAgs induced significant IFN-gamma production from 11 to 23 wk after primary vaccination (APV), except for SOD. Significant increases were noted in CD3(+), CD4(+), CD8(+), CD21(+), CD25(+), and gammadelta(+) cells against all four rAgs in vaccinated animals. rAg-specific expression of IL-2, IL-12p40, IFN-gamma and TNF-alpha was significantly higher in the two vaccinated groups. Culture results found 4/8 animals in Group I, 3/8 animals in Group II, and 3/4 animals in Groups III and IV were positive for MAP in one or more tissues. Among the seven positive animals in Groups I and II, all but one had had <10CFU. Isolation was confined to one tissue in these animals, except in one animal in which MAP was isolated from two tissues. In the control groups (III and IV), MAP was cultured from up to five different tissues with >250CFU. Preliminary data from this study indicates that all four rAgs induced a good Th1 response and conferred protection against MAP infection in calves.

Development of a Polymerase Chain Reaction Test to Confirm Mycobacterium Avium Subsp. Paratuberculosis in Culture

A polymerase chain reaction (PCR) assay for confirmation of Mycobacterium avium subsp. paratuberculosis was developed using the primer set derived from ISMav2. The PCR product was 494 base pairs (bp) and could be digested with ClaI, which produced 311- and 183-bp fragments. No amplification of 494-bp DNA fragment was detected from DNA of other Mycobacterium spp., including Mycobacterium avium complex, other bacteria, including Escherichia coli, Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae, Leptospira interrogans serovar pomona, Corynebacterium pseudotuberculosis, Salmonella typhimurium, Borrelia burgdorferi, and Staphylococcus aureus, and the Scedosporium sp. This PCR assay could detect 5–8 genome equivalents.

Immune responses in mice to Mycobacterium avium subsp. paratuberculosis following vaccination with a novel 74F recombinant polyprotein.

Johnes disease (JD) is a chronic infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Here, we report the cloning and expression of a 74kDa recombinant polyprotein (Map74F) and its protective efficacy against MAP infection in mice. Map74F was generated by the sequential linkage of the ORFs of the approximately 17.6-kDa C-terminal fragment of Map3527 to the full-length ORF of Map1519, followed at the C-terminus with approximately 14.6-kDa N-terminal portion of Map3527. Mice immunized with Map74F had a significant IgG1 response but not IgG2a. In immunized animals, the IgG1/IgG2a ratio increased until 4 weeks after MAP challenge. The ratio decreased from 8 weeks indicating a shift to a Th1 response. Antigen specific IFN-gamma response, CD3+ and CD4+ T cells increased significantly in immunized mice. Following challenge, MAP burden was significantly lower in liver, spleen and mesenteric lymph nodes of immunized animals compared to control animals indicating protection against MAP infection. This was further evident by the improved liver and spleen pathology of the immunized animals, which had fewer granulomas and lower numbers of acid-fast bacilli. Results of this study indicated that immunization of mice with Map74F protected mice against MAP infection.

Cloning and Characterization of the Genes Coding for Antigen 85A, 85B and 85C of Mycobacterium avium subsp. paratuberculosis

Three genes encoding the secreted proteins (antigen 85-A, B, and C) of Mycobacterium avium subsp. paratuberculosis were cloned, sequenced and studied. The complete sequences of these three 85-complex proteins revealed their similarity with 85-complex proteins of other mycobacterial species. Specifically, these sequences showed 99% homology with M. avium 85-complex protein sequences. The multiple homology analysis of these sequences revealed that variations occur at only certain amino acid positions and this is true with all other 85-complex protein sequences of mycobacteria. However, the proposed three conserved regions involved in fibronectin binding in other mycobacteria were observed in N-terminal regions 85A, B and C of M. avium subsp. paratuberculosis.

Diagnosis of Enteric Disease in Small Ruminants

Diagnosis of gastrointestinal disease in small ruminants requires integration of information obtained in the signalment, history, physical or necropsy examination, and ancillary diagnostic tests. The purpose of this article is to provide the practitioner with a review of the clinical features of several common gastrointestinal diseases of sheep and goats. Rumen acidosis, enterotoxemia, gastrointestinal parasitism, neonatal diarrhea, and salmonellosis are discussed, and where appropriate, reviews of the pathophysiology, prevention, and control of these diseases are cited for further reading.