Comparison of PCR Prescreening to Two Cultivation Procedures with PCR Confirmation for Detection of Mycobacterium avium subsp. paratuberculosis in U.S. Department of Agriculture Fecal Check Test Samples

2004 ◽  
Vol 67 (10) ◽  
pp. 2310-2314 ◽  
Author(s):  
JAY L. E. ELLINGSON ◽  
JEFFREY J. KOZICZKOWSKI ◽  
JENNIFER L. ANDERSON
Keyword(s):  
Mycobacterium Avium ◽  
Culture Medium ◽  
Mycobacterium Avium Subsp ◽  
Diagnostic Tools ◽  
Enzyme Linked Immunosorbent Assay ◽  
Culture Methods ◽  
Department Of Agriculture ◽  
Systems Research ◽  
Specificity And Sensitivity ◽  
Mycobacterium Avium Subsp Paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent in Johne's disease in cattle and causes diarrhea, decreased milk production, emaciation, and frequently death. The ability to detect MAP rapidly and accurately is an integral part of herd management. However, detection of this bacterium is complicated due to its slow division time and its ability to enter dormancy. Culture methods are considered the “gold standard,” but they have their limitations. Many enzyme-linked immunosorbent assay methods and conventional PCR methods have been used as diagnostic tools. The present study compares the results of a PCR prescreen to two culture methods of detection paired with confirmatory PCR to determine the most accurate, rapid, and sensitive method using U.S. Department of Agriculture (USDA) fecal check samples. This study involving two laboratories (Marshfield Clinic Laboratories, using solid culture medium [Herrold's egg yolk agar], and TREK Diagnostic Systems Research and Development, using liquid culture medium [ESP Culture System II]) showed that the PCR prescreening method used in this study lacked specificity and sensitivity as a stand-alone test in fecal samples. However, the combination of liquid enrichment culture using the ESP II system, and PCR confirmation with the hspX primer set, was not only 100% sensitive and specific but also correlated with viable MAP and USDA culture results.

scholarly journals Isolation of Mycobacterium avium Subsp. Paratuberculosis in the Feces and Tissue of Small Ruminants Using a Non-Automated Liquid Culture Method

Animals ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 20
Author(s):  
Luigi De Grossi ◽  
Davide Santori ◽  
Antonino Barone ◽  
Silvia Abbruzzese ◽  
Matteo Ricchi ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Liquid Culture ◽  
Culture Media ◽  
Small Ruminants ◽  
Culture Method ◽  
Mycobacterium Avium Subsp ◽  
Type I ◽  
Culture Methods ◽  
Specific Pcr ◽  
Mycobacterium Avium Subsp Paratuberculosis

Paratuberculosis is a chronic disease of ruminants caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Since isolation of MAP type I (S) is rarely reported in Italy, our research was aimed at isolating, by an inexpensive liquid culture manual method, this type of MAP isolates. At first, we used an ELISA to point out to serologically positive samples from five flocks. Secondly, we used a fecal direct IS900-qPCR on the ELISA positive samples, in order to detect shedder animals. Feces from IS900-qPCR positive samples were inoculated in solid and liquid culture media. IS900-qPCR was further used to test the growth of MAP isolates in liquid medium, which were further confirmed by f57-qPCR and submitted to typing by specific PCR in order to identify the MAP type. Twenty-eight samples (24 fecal and four tissutal samples) were processed by culture methods, resulting in the isolation of six type I MAP field isolates. Notably, no isolates were recovered by solid media, underlining the utility of this liquid method. Few data about this type of MAP are currently available in Italy, and further analyses should be carried out in order to study the origin and epidemiology of type I strains circulating in Italy.

Detection of Viable Mycobacterium avium subsp. paratuberculosis in Retail Pasteurized Whole Milk by Two Culture Methods and PCR

2005 ◽  
Vol 68 (5) ◽  
pp. 966-972 ◽  
Author(s):  
JAY L. E. ELLINGSON ◽  
JENNIFER L. ANDERSON ◽  
JEFF J. KOZICZKOWSKI ◽  
ROY P. RADCLIFF ◽  
SALLY J. SLOAN ◽  
...  
Keyword(s):  
United Kingdom ◽  
Mycobacterium Avium ◽  
Culture Medium ◽  
The United States ◽  
Mycobacterium Avium Subsp ◽  
Seasonal Effect ◽  
Pasteurized Milk ◽  
The United Kingdom ◽  
Whole Milk ◽  
Mycobacterium Avium Subsp Paratuberculosis

Cattle with Johne's disease can shed live Mycobacterium avium subsp. paratuberculosis (MAP) in their milk, and MAP can survive under simulated commercial pasteurization conditions. In several studies conducted in the United Kingdom and Canada, MAP DNA has been detected in retail pasteurized milk samples; however, in one study in the United Kingdom viable MAP was identified in commercially pasteurized milk. A double-blind study involving two laboratories was undertaken to evaluate retail pasteurized whole milk in the United States. Marshfield Clinic Laboratories used solid culture medium (Herrold's egg yolk agar slants with mycobactin J and amphotericin B, nalidixic acid, and vancomycin), and TREK Diagnostic Systems, Research and Development used liquid culture medium (ESP culture system). Cultures at both laboratories were confirmed by PCR. A total of 702 pints of retail whole milk were purchased in three of the top five milk-producing states (233 from California, 234 from Minnesota, and 235 from Wisconsin) over a 12-month period and were tested for the presence of viable MAP. The criteria used for identifying samples as positive for viable MAP were similar to those followed by most laboratories (positive culture with PCR confirmation). The combined data from the two laboratories revealed the presence of viable MAP in 2.8% of the retail whole milk pints tested. Although the number of samples containing viable MAP was similar among states (P > 0.05), there was a seasonal effect on the presence of viable MAP in retail milk (P = 0.05). More MAP-positive samples were identified during the third quarter of the year (July through September). Of the 22 brands of retail milk tested, 12 (55%) yielded at least one sample positive for viable MAP.

scholarly journals Seroprevalence of Immunoglobulin G Antibodies Against Mycobacterium avium subsp. paratuberculosis in Dogs Bred in Japan

2020 ◽  
Vol 7 (3) ◽  
pp. 93
Author(s):  
Takashi Kuribayashi ◽  
Davide Cossu ◽  
Eiichi Momotani
Keyword(s):  
Mycobacterium Avium ◽  
Immunoglobulin G ◽  
Mycobacterium Avium Subsp ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
The Difference ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Veterinary Hospital

In this study, the seroprevalence of immunoglobulin G (IgG) antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) in dogs bred in Japan was evaluated. Ninety-two non-clinical samples were obtained from three institutes and fifty-seven clinical samples were obtained from a veterinary hospital in Japan. Serum titers of total IgG, IgG1 and IgG2 isotype antibodies against MAP were measured using an indirect enzyme-linked immunosorbent assay (ELISA). The IgG antibodies against MAP in non-clinical serum obtained from three institutes was observed to be 2.4%, 20% and 9.0%. Similarly, the IgG1 antibodies titers against MAP were observed to be 7%, 20% and 0%. Lastly, the IgG2 antibodies against MAP were observed to be 7%, 20% and 4.4%. No significance differences in these titers were observed among the three institutes. The IgG, IgG1 and IgG2 antibodies in serum obtained from a veterinary hospital were observed to be 55.3%, 42% and 42%, respectively. Significant differences were found between the non-clinical and clinical samples. The titers in the clinical samples showed a high degree of variance, whereas low variance was found in the non-clinical samples. The IgG antibody levels were thought to be induced following exposure to MAP-contaminated feed. The difference in titers between the clinical and non-clinical samples is likely to be related to the amount of MAP antigen contamination in dog foods.

scholarly journals The collection of lymphatic fluid from the bovine udder and its use for the detection of Mycobacterium avium subsp. paratuberculosis in the cow

2011 ◽  
Vol 24 (1) ◽  
pp. 23-31 ◽  
Author(s):  
Johannes L. Khol ◽  
Pablo J. Pinedo ◽  
Claus D. Buergelt ◽  
Laura M. Neumann ◽  
Walter Baumgartner ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Fluid Collection ◽  
Johne's Disease ◽  
Johne’S Disease ◽  
Mycobacterium Avium Subsp ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lymph Fluid ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Lymphatic Fluid ◽  
Elisa Result

The objective of the current study was to evaluate the feasibility of lymph collection from the bovine udder and to investigate if the lymphatic fluid might be of diagnostic value in cows infected with Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis. Lymph fluid collection was attempted from 58 cows, and the reactions of the cows as well as the level of difficulty of the procedure were recorded in 56 animals. Lymph samples (51 in total) were tested for the presence of MAP by nested polymerase chain reaction. Collection of the lymphatic fluid caused no or mild signs of discomfort in 94.6% of the cows; in 51.8% of cows, lymphatic fluid was attained on the first attempt, while sample collection was unsuccessful in 12.1%. Mycobacterium avium subsp. paratuberculosis was detected in 43.1% of all lymph samples. The bacterium was present in 66.7% of cows with clinical Johne’s disease, in 42.8% of asymptomatic cows with a positive or suspicious enzyme-linked immunosorbent assay (ELISA) result in blood, and in 38.7% of cows with a negative ELISA result in blood. The present study shows that the procedure was well tolerated by most cows and can easily be performed on farm. The current report of the isolation of MAP from lymph fluid suggests that the present approach could be used for the early detection of Johne’s disease in cattle.

scholarly journals Effect of Egg Yolk on the Detection of Mycobacterium Avium Subsp. Paratuberculosis Using the ESP II Liquid Culture System

2005 ◽  
Vol 17 (6) ◽  
pp. 554-560 ◽  
Author(s):  
N. Beth Harris ◽  
Suelee Robbe-Austerman ◽  
Janet B. Payeur
Keyword(s):  
Mycobacterium Avium ◽  
Culture System ◽  
Liquid Culture ◽  
Egg Yolk ◽  
Mycobacterium Avium Subsp ◽  
Culture Methods ◽  
Solid Media ◽  
Liquid Culture System ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Time To Detection

Rapid diagnosis of paratuberculosis in infected cattle is important for the successful control of Johne disease within herds. Thus, improving culture methods for Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) will aid in the identification of asymptomatic animals. Egg yolk is a component of the media used for growing M. paratuberculosis, but its requirement as a supplement has not been reported. Using the ESP II liquid culture system, 2 different sources and 5 concentrations (3.3%, 1.6%, 0.8%, 0.4%, and 0%) of egg yolk were analyzed. Egg yolk source did not affect either recovery rate or time to detection, but both parameters were significantly improved when the 3.3% egg yolk concentrations (final volume) were used over media containing no egg yolk. This study also assessed the recovery of M. paratuberculosis from fecal samples that were cultured multiple times using Herrold egg yolk agar (HEY). Specimens containing greater than 70 cfu/g feces could routinely be identified as positive for M. paratuberculosis after only 1 culture attempt, whereas specimens with fewer bacteria were only intermittently positive, even after 5 replicate cultures. Therefore, this study indicates that the sensitivity of the Trek Diagnostic ESP II liquid culture system for M. paratuberculosis is affected by egg yolk concentration and that single culture attempts using HEY solid media may not identify specimens containing low numbers of bacteria.

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