Culture Phenotypes of Genomically and Geographically Diverse Mycobacterium avium subsp. paratuberculosis Isolates from Different Hosts

Abstract

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johne's disease) in ruminants in most countries. Historical data suggest substantial differences in culturability of M. avium subsp. paratuberculosis isolates from small ruminants and cattle; however, a systematic comparison of culture media and isolates from different countries and hosts has not been undertaken. Here, 35 field isolates from the United States, Spain, Northern Ireland, and Australia were propagated in Bactec 12B medium and Middlebrook 7H10 agar, genomically characterized, and subcultured to Lowenstein-Jensen (LJ), Herrold's egg yolk (HEY), modified Middlebrook 7H10, Middlebrook 7H11, and Watson-Reid (WR) agars, all with and without mycobactin J and some with sodium pyruvate. Fourteen genotypes of M. avium subsp. paratuberculosis were represented as determined by BstEII IS900 and IS1311 restriction fragment length polymorphism analysis. There was no correlation between genotype and overall culturability, although most S strains tended to grow poorly on HEY agar. Pyruvate was inhibitory to some isolates. All strains grew on modified Middlebrook 7H10 agar but more slowly and less prolifically on LJ agar. Mycobactin J was required for growth on all media except 7H11 agar, but growth was improved by the addition of mycobactin J to 7H11 agar. WR agar supported the growth of few isolates. The differences in growth of M. avium subsp. paratuberculosis that have historically been reported in diverse settings have been strongly influenced by the type of culture medium used. When an optimal culture medium, such as modified Middlebrook 7H10 agar, is used, very little difference between the growth phenotypes of diverse strains of M. avium subsp. paratuberculosis was observed. This optimal medium is recommended to remove bias in the isolation and cultivation of M. avium subsp. paratuberculosis.Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease of ruminants, which occurs in most countries. Usually it is an enteric infection with a long incubation period and progressively worsening granulomatous infiltration of the small and large intestines leading to weight loss and death. It is economically significant in farmed cattle, goats, sheep, and deer and is known to be capable of infecting a wide range of domesticated and free-living herbivores and occasionally also the carnivores that prey upon them (2, 4, 6, 15). Like many pathogenic mycobacteria, M. avium subsp. paratuberculosis is not host specific, and cross-species transmission is common where there are high contact rates. Although tissue colonization and histological lesions may occur, clinical disease in response to infection is rarely reported for nonruminant species, except for rabbits (2, 4). There is a long-standing debate about the role of M. avium subsp. paratuberculosis in chronic inflammatory bowel disease of humans, specifically Crohn's disease. Consequently, the isolation and cultivation of M. avium subsp. paratuberculosis are attempted in both vet...

Fecal culture and two fecal-PCR methods for the diagnosis of Mycobacterium avium subsp. paratuberculosis in a seropositive herd

“…According to Withlock et al (2000), the disadvantages of some detection test are due mainly because of the intermittent shedding of microorganisms. This means that the sensitivity of direct tests to detect symptomatic animals is high, but low for detection of infected/subclinical animals (Nielsen and Toft, 2008;Schönenbrücher et al, 2008;Whittington et al, 2011).…”

“…Usually, the specificity of fecal culture is considered to be almost 100% if the isolates obtained are confirmed to be MAP by molecular methods such as polymerase chain reaction (PCR; Nielsen and Toft, 2008;Schönenbrücher et al, 2008;Whittington et al, 2011). Fecal culture has been used as an acceptable standard technique for detecting the infection status of animals -related to elimination rate-, for estimating the sensitivity of other diagnostic tests (e.g.…”

Trends and advances in the diagnosis and control of paratuberculosis in domestic livestock

“…One of the important drawbacks of culture is its sluggishness associated with requirement of mycobactin J and high costs. The addition of mycobactin J in the medium is the primary requirement for in vitro culture of MAP enabling iron acquisition and its dependence can be used as discriminatory tests for confirmation of MAP in culture (Whittington et al 2011). Use of automated systems such as BACTEC MGIT 960 shortens the time of detection (4À7 weeks) and can detect as low as 10 CFU/ml .…”

Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

“…Thus, environmental and host factors may potentially influence the phenotype of MAP prior to and during infection. Early studies on this phenomenon have identified a variety of phenotypes between geographically distinct MAP isolates (Whittington et al, 2011), and have illustrated proteomic differences between laboratory culture and mucosal derived isolates (Weigoldt et al, 2011). MAP also develops a more invasive phenotype in response to both the hyperosmolar environment of raw milk and growth within the mammary epithelium (Patel et al, 2006).…”