William P. Shulaw

Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis: Evidence for Limited Strain Diversity, Strain Sharing, and Identification of Unique Targets for Diagnosis

ABSTRACT The objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium avium subsp. paratuberculosis isolated in the United States and to identify M. avium subsp. paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900 integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. avium subsp. paratuberculosis isolates recovered from animals (n = 203) and patients with Crohns disease (n = 7) from diverse geographic localities. Six hundred bacterial cultures, including M. avium subsp. paratuberculosis (n = 303), non-M. avium subsp. paratuberculosis mycobacteria (n = 129), and other nonmycobacterial species (n = 168), were analyzed to evaluate the specificity of two IS900 integration loci and a newly described M. avium subsp. paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. avium subsp. paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. avium subsp. paratuberculosis isolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. avium subsp. paratuberculosis isolates from ovine and bovine sources from the same state were more closely associated than were isolates from different geographic regions, suggesting that some of the strains are shared between these ruminant species. AFLP fingerprinting revealed a similar pattern, with most isolates from bovine sources clustering into two major nodes, while those recovered from sheep or humans were clustered on distinct branches. Overall, this study identified a high degree of genetic similarity between M. avium subsp. paratuberculosis strains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. avium subsp. paratuberculosis isolates recovered from human and ovine sources.

Rapid Detection and Typing of Strains of Mycobacterium avium subsp. paratuberculosis from Broth Cultures

ABSTRACT A liquid culture followed by molecular confirmation was evaluated for potential to improve sensitivity and reduce time to diagnosis of Mycobacterium avium subsp. paratuberculosis infection. Fecal samples from 240 animals from Ohio farms were assessed for presence of M. avium subsp. paratuberculosis using four different protocols: (i) sedimentation processing followed by inoculation on Herrolds Egg Yolk media (HEYM) slants (monitored biweekly up to 16 weeks), (ii) double centrifugation processing followed by inoculation on HEYM slants (monitored biweekly up to 16 weeks), (iii) liquid-solid double culture method using modified 7H9 broth (8 weeks) followed by subculture on HEYM slants (monitored up to 8 weeks), and (iv) liquid culture using modified 7H9 broth (8 weeks) followed by molecular assays for the presence of two M. avium subsp. paratuberculosis-specific targets. The number of positive samples detected by each protocol was 37, 53, 65, and 76, respectively. Twenty-seven samples were positive by all four methods. Based on samples positive by at least one method (n = 81), the sensitivities for sedimentation processing, double centrifugation processing, liquid-solid double culture, and liquid culture followed by molecular confirmation were 46%, 65%, 80%, and 94%, respectively. Fingerprinting of the positive samples using two polymorphic (G and GGT) short sequence repeat regions identified varying levels of within-farm and between-farm diversity. Our data indicate that liquid culture followed by molecular confirmation can significantly improve sensitivity and reduce time-to-diagnosis (from 16 to 8 weeks) of M. avium subsp. paratuberculosis infection and can also be efficiently employed for the systematic differentiation of M. avium subsp. paratuberculosis strains to understand the epidemiology of Johnes disease.

Field trials evaluating the safety and serologic reactions of reduced-dose Brucella melitensis Rev 1 vaccination in adult sheep

Abstract Field trials of two reduced doses of Brucella melitensis Rev 1 vaccine were conducted in Portuguese Merino crossbred ewes. All ewes had been bred 73–100 days prior to vaccination. Doses of 106 and 107 Rev 1 live organisms were given subcutaneously in treatment groups of 43 ewes and 42 ewes respectively, with an additional 25 ewes maintained as non-vaccinated controls. Ewes were monitored for 20 weeks post-vaccination by clinical observation, vaginal culture to detect B. melitensis Rev 1 excretion, and repeated serologic testing by Rose Bengal, serum agglutination, Complement fixation, and rivanol tests. No difference in live lambing rates was observed between the two treatment groups or the control. No shedding of vaccine organism was detected in vaccinated ewes. Serologic reactions were stronger and persisted longer in the higher-dose treatment group. Titers fell rapidly in the vaccinated ewes, with only one ewe testing positive beyond the eighth week.

Characterization of a Cryptosporidium parvum Gene Encoding a Protein with Homology to Long Chain Fatty Acid Synthetase

ABSTRACT: We describe here the cloning, sequencing, and characterization of a novel Cryptosporidium parvum gene, encoding a protein with significant homology to the long‐chain fatty acyl‐CoA synthetase (LCFA, EC 6.2.13). The gene has an open reading frame of 2,301 bp, coding for a 766 amino acid polypeptide, and with an estimated MW of 86.1 kDa. By indirect immunofluorescence assay, monoclonal antibodies C3CE7 and ESD labeled the anterior pole of fixed C. parvum sporozoites and developmental stages in C. parvum‐infected cultures at 24, 48, and 72 h post‐infection. These monoclonal antibodies inhibited more than 3.5% of parasite growth in vitro. The effect of triacsin C, a potent selective inhibitor of LCFA synthetase, on parasite growth was assessed in cell culture; complete inhibition of parasite growth at 2.5 ug/inl was obtained with little evidence of drug‐associated cytotoxicity. These results suggest that the fatty acyl‐CoA synthetase may be a useful target in the development of selective inhibitors and immunologic interventions against C. parvum

A testing scheme for the detection of Mycobacterium avium subsp. paratuberculosis in bovine feces utilizing the ESP para-JEM liquid culture system

A testing scheme for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in broth cultures of bovine fecal samples carried out in ESP para-JEM System was evaluated. The scheme included acid-fast staining (on signal-positive and signal-negative samples), and confirmation by PCR for 2 MAP-specific targets and subculture of all acid-fast positive PCR-negative samples. Two hundred and fifty bovine fecal samples were evaluated for the presence of MAP using this scheme. Thirty-seven (15%) of 250 fecal samples had a positive culture result when the proposed testing scheme was used, compared to 14 (6%) positive results when using the standard ESP para-JEM protocol (requiring samples to have a positive signal from the system, a positive acid-fast stain, and a positive IS900 PCR result), and 20 (8%) positives when conventional culture was performed on Herrold egg yolk (HEY) media. A preliminary comparison of real-time and conventional PCR on DNA extracted from 15 MAP-positive broth cultures by 3 different protocols suggested that conventional PCR may be a better choice for the confirmation of the presence of MAP in the liquid cultures than real-time PCR.

Calculation Method for Likelihood Ratios Dictates Interpretation

Recently, likelihood ratios were published for use as an aid in the clinical interpretation of test results obtained from a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in cattle (2). As described by Dr. M. T. Collins, likelihood ratios can provide diagnostic information for multiple levels of a diagnostic test result when results are reported on a continuous scale rather than as a dichotomous (positive and negative) outcome. Unfortunately, the author did not take advantage of this characteristic of likelihood ratios. Because the formula used to calculate likelihood ratios presented in this article incorporates sensitivity and specificity estimates for the ELISA at several arbitrary cutoff points, the resulting comparisons remain dichotomous in nature. Further, the interpretation of these likelihood ratios as calculated is incorrect. For example, the likelihood ratio calculated at the 0.10 level indicates that cows with S/P ratios of ≥0.10 are five times more likely to be infected than noninfected. With this approach, all cows with ELISA S/P ratios of ≥0.10 are included in the comparison. Multiple likelihood ratios that allow for comparisons between different levels of S/P ratios were not calculated. Because the ELISA S/P ratios were not stratified into multiple categories, the likelihood ratios presented in Table ​Table11 of Collinss article (2) are overestimates resulting from heterogeneity within the defined groups of interest. TABLE 1. Calculation of likelihood ratios using a multilevel approach Table ​Table11 provides likelihood ratios based on the multiple-level approach proposed by Sackett et al. (3) and calculated from the data presented by Dr. Collins in his Table 1 (2). The likelihood ratios calculated in this manner differ considerably from those presented by Dr. Collins, and differences in their subsequent interpretations may have significant clinical and economic consequences. For example, cows with ELISA S/P ratios of 0.10 to <0.25 are 0.5 times as likely (i.e., half as likely) to be infected than noninfected, not “5 to 15 times” as likely to be infected, as Dr. Collins indicated in his Table 3 (2). Likelihood ratios demonstrating less-than-eightfold differences between comparison groups have been suggested to provide weak statistical evidence that a test result is indicative of a defined outcome (1). Additionally, the exclusion of ELISA S/P ratios of <0.00 from this analysis is a matter for concern. ELISA results from 1,097 animals, over one-third of the available results, were not considered. As this report indicates, ELISA S/P ratios of <0.00 may constitute a significant proportion of the results obtained during herd screening. These results could have been incorporated into the analysis had the multiple-level approach for likelihood ratio calculations been utilized. Likelihood ratios can provide additional diagnostic information for use in the clinical interpretation of the ELISA for M. avium subsp. paratuberculosis in cattle. However, care must be taken in order to interpret these values correctly and to make correct management recommendations based on them. These data do not support the interpretation of ELISA S/P ratios presented by Dr. Collins.