Growth of Mycobacterium avium subsp. paratuberculosis in the Presence of Hexadecylpyridinium Chloride, Natamycin, and Vancomycin

Johansen, Kristine A.; Hugen, Erin E.; Payeur, Janet B.

doi:10.4315/0362-028x-69.4.878

Cited by 10 publications

(5 citation statements)

References 15 publications

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“…One explanation for this may be that many of the cultures observed in this study had fewer than 10 cfu/g. Although, HEYA culture is the long-standing method, often used as a gold standard for comparison, it has been observed that the decontamination procedure is detrimental to MAP growth (Johansen et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Detection of Mycobacterium avium Subspecies paratuberculosis: Comparing Fecal Culture Versus Serum Enzyme-Linked Immunosorbent Assay and Direct Fecal Polymerase Chain Reaction

Clark

Koziczkowski

Radcliff

et al. 2008

Journal of Dairy Science

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Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle. The disease causes diarrhea, reduced milk production, poor reproductivity, emaciation, and eventually death. Culture on Herrold's egg yolk agar is considered to be the definitive test for diagnosis of Johne's in cattle. This method has moderate sensitivity (30 to 50%) and is 100% specific; however, it can take up to 16 wk due to the slow growth of MAP. Currently, serum ELISA is used to screen herds for Johne's disease, but positive tests must be confirmed culturally or by PCR. The current research sought to evaluate an in-house direct fecal PCR procedure and directly compare it to ELISA using culture as the gold standard. Serum and fecal samples were collected from cows (n = 250) with unknown Johne's status. Fecal samples were processed for culture on Herrold's egg yolk agar and direct PCR. Serum samples were tested using the Parachek serum ELISA. Overall, 67/250 [26.8%, 95% confidence interval (CI) 21.4 to 32.8] animals were culturally confirmed to be shedding MAP. The PCR and ELISA detected 74/250 (29.6%, 95% CI 24 to 35.7) and 25/250 (10%, 95% CI 6.6 to 14.4), respectively. Culture and PCR were able to detect more positive animals than ELISA. Overall, direct fecal PCR was 70.2% sensitive and 85.3% specific when using culture as the gold standard. The ELISA method was 31.3% sensitive and 97.8% specific. When culture reported <10 cfu, the sensitivity and specificity of PCR and ELISA were 57.1 and 85.3%, and 4.8 and 97.8%, respectively. When culture reported 10 to <40 cfu, the sensitivity of PCR and ELISA were 75 and 50%, respectively. When culture reported > or =40 cfu, the sensitivity of PCR and ELISA were 100 and 88.2%, respectively. Specificity could not be calculated at these levels because there were no negative samples. The direct PCR outperformed the ELISA in detecting animals potentially infected with MAP and was not significantly different when compared with culture. The direct fecal PCR method described here provides faster results than traditional culture and is more sensitive than ELISA at detecting animals suspected of Johne's disease. These data support the use of PCR as an alternative method for screening herds for prevalence and diagnosis of Johne's disease.

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Section: Discussionmentioning

confidence: 99%

Detection of Mycobacterium avium Subspecies paratuberculosis: Comparing Fecal Culture Versus Serum Enzyme-Linked Immunosorbent Assay and Direct Fecal Polymerase Chain Reaction

Clark

Koziczkowski

Radcliff

et al. 2008

Journal of Dairy Science

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“…These authors attribute this phenomenon to a decrease of viable MAP bacteria due to the decontamination protocol for fecal samples. This especially concerns the reduction of cultivated bacteria in fecal samples from cows shedding the pathogen in low numbers (Reddacliff et al, 2003;Johansen et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Field study on bovine paratuberculosis using real-time PCR and liquid culture for testing environmental and individual fecal samples implemented in dairy cow management

Schwalm

Metzger-Boddien²,

Seemann³

et al. 2019

Journal of Dairy Science

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fecal samples using the liquid culture. Using fecal PCR, we revealed 43 positive samples of which 39 also tested positive in the liquid culture, revealing MAP-positive cows in all 3 herds. The fecal PCR procedure allows rapid detection of MAP-specific DNA with 74% of the sensitivity of liquid culture. For the purpose of testing with maximal sensitivity, cultivation in liquid medium is recommended. Cultivation of MAP in liquid medium M7H9C means a significant time gain in comparison to cultivation on solid media, which requires twice as much time. Thus, this testing fits within the 6-to 8-wk dry period of gravid cows and provides test results before calving, a prerequisite to prevent fecal-oral transmission to newborn calves.

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“…The pellet was then weighed, using the conversion where 1 mg is equivalent to 1 × 10 7 CFU. CFU counts were then confirmed via serial dilution on 7H11 plates, where 7H11 plates were supplemented with 2 mg/L mycobactin J (Allied Monitor), 10% OADC, 10µg/mL vancomycin (V2002, Sigma) 20µg/mL amphotericin B(A2942-20ML, Sigma-Aldrich), and 30µg/mL nalidixic acid (N8878, Sigma) to prevent culture contamination [74,75]. GFP-MAP cultures used in all experiments were plated on blood agar plates to ensure the absence of other bacteria.…”

Section: Map Culturementioning

confidence: 99%

Mycobacterium aviumsubspeciesparatuberculosistargets M cells in enteroid-derived monolayers through interactions with β1 integrins

Baruta,

Flannigan,

Alston

et al. 2024

Preprint

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Paratuberculosis is a global infectious disease caused by the bacterium, Mycobacterium avium subspecies paratuberculosis (MAP). MAP infection of ruminants triggers progressive wasting disease characterized by granulomatous lymphadenitis, enteritis, and severe intestinal pathology that often requires early culling of the animal. The resulting economic burden is significant and MAP exposure in the workplace constitutes a significant zoonotic risk. While it has been established the MAP propagates within resident intestinal immune cells, including macrophages and dendritic cells, significantly less is known about how it attaches, enters and traverses the epithelium. The current paradigm suggests MAP infects the small intestinal epithelium by targeting both enterocytes and M cells, with a potential tropism for the latter. In the current study, we employed emerging enteroid technology to identify the target cells for MAP's entry into the small intestinal epithelium. We generated mouse enteroid-derived monolayers with functional M cells capable of transcytosis. Upon exposure to MAP, the bacteria were detected within both enterocytes and M cells. Following quantification, it was apparent that MAP exhibited tropism for M cells. Complementary studies using the Caco-2/Raji-B co-culture system provided similar results, wherein MAP was found primarily in cells expressing functional M cell markers. Since other mycobacteria have been shown to initiate cell attachment and entry by using a fibronectin-bridging process, we tested whether these interactions were involved in MAP's targeting of M cells. We found that MAP's M cell tropism was significantly enhanced in the presence of fibronectin and that this effect was abolished when monolayers were pretreated with an integrin-blocking peptide. Taken together, our data indicate the MAP preferentially targets M cells and that this process involves a fibronectin-bridging process. Furthermore, our data suggest that targeting M cell-associated integrins could provide a mechanism to reduce MAP infection and transmission within livestock herds.

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Growth of Mycobacterium avium subsp. paratuberculosis in the Presence of Hexadecylpyridinium Chloride, Natamycin, and Vancomycin

Cited by 10 publications

References 15 publications

Detection of Mycobacterium avium Subspecies paratuberculosis: Comparing Fecal Culture Versus Serum Enzyme-Linked Immunosorbent Assay and Direct Fecal Polymerase Chain Reaction

Detection of Mycobacterium avium Subspecies paratuberculosis: Comparing Fecal Culture Versus Serum Enzyme-Linked Immunosorbent Assay and Direct Fecal Polymerase Chain Reaction

Field study on bovine paratuberculosis using real-time PCR and liquid culture for testing environmental and individual fecal samples implemented in dairy cow management

Mycobacterium aviumsubspeciesparatuberculosistargets M cells in enteroid-derived monolayers through interactions with β1 integrins

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