To assess the genetic diversity in Cryptosporidium parvum, we have sequenced the small subunit (SSU) rRNA gene of seven Cryptosporidium spp., various isolates of C. parvum from eight hosts, and a Cryptosporidiumisolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genusCryptosporidium, with at least four distinct species (C. parvum, C. baileyi, C. muris, and C. serpentis). Other species previously defined by biologic characteristics, including C. wrairi, C. meleagridis, and C. felis, and the desert monitor isolate, clustered together or within C. parvum. Extensive genetic diversities were present among C. parvum isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate mostCryptosporidium spp. and C. parvum genotypes, but sequence analysis of the PCR product was needed to differentiateC. wrairi and C. meleagridis from some of theC. parvum genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal C. parvum isolates.

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 Crohn's 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.Paratuberculosis or Johne's disease (JD) is a chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (48). It is estimated that 35% of the U.S. herds are infected with M. avium subsp. paratuberculosis, resulting in annual losses of $200 million (7). Crohn's disease (CD) is also a chronic inflammation of distal intestines exhibiting a pathology similar to that of JD in ruminants. The prevalence of CD is estimated to be 0.15% among the U.S. population resulting in substantial morbidity and medical costs (1). M. avium subsp. paratuberculosis has been implicated as a cause of CD in humans (13,21,36). Currently, the evidence for a link remains inconclusive since strain sharing or a causal role of M. avium subsp. paratuberculosis has not been demonstrated.Comprehensive analysis of the molecular diversity and comparative molecular pathology of M. avium subsp. paratuberculosis will help establish the degree of heterog...

Results obtained with use of the new ELISA agreed favorably with those of a previous ELISA. Practitioners must be aware of variability in the sensitivity of the test, which depends on the clinical and shedding status of the cattle, because this may affect interpretation of test results.

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 Herrold's 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 Johne's disease.

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