Search

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD). One mode of transmission of MAP is through ingestion of contaminated milk and colostrum by susceptible calves. The objective of this study was to determine if the amount of MAP shed into the milk and colostrum of infected cows was affected by severity of infection as well as the number of days in milk (DIM). Milk was collected over the 305-d lactation period from naturally infected cows in the asymptomatic subclinical (n=39) and symptomatic clinical (n=29) stages of disease, as well as 8 noninfected control cows. All milk samples were assayed for MAP by culture on Herrold's egg yolk medium and either BACTEC 12B (Becton Dickinson, Franklin Lakes, NJ) or para-JEM (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH) liquid medium, and by direct PCR for the IS900 target gene. Mycobacterium avium ssp. paratuberculosis was detected in 3.8, 4.1, and 12.6% of milk samples collected from cows with subclinical JD after culture in Herrold's egg yolk medium, liquid medium, and direct PCR, respectively. The frequency of MAP positivity increased to 12.9, 18.4, and 49.2% of milk samples collected from cows with clinical JD by these same methods, respectively. None of the milk samples collected from control cows was positive for MAP by any detection method. Viable MAP was primarily isolated from milk and colostrum of subclinically and clinically infected cows collected in early lactation (DIM 0-60), with negligible positive samples observed in mid (DIM 60-240) and late (DIM 240-305) lactation. This study demonstrates that shedding of MAP into milk is affected by infection status of the cow as well as stage of lactation, providing useful information to producers to help break the cycle of infection within a herd., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Mycobacterium avium ssp. paratuberculosis (MAP) is shed into the milk of cattle affected by Johne's disease and, therefore, is a route of transmission for infection in young stock in dairy herds. The objective of this study was to validate a decontamination and culture protocol for the recovery of MAP from individual bovine milk samples from known infected herds. Decontamination of milk samples (n = 17) with either 0.75% hexadecylpyridinium chloride for 5h or N-acetyl-L-cysteine-1.5% sodium hydroxide (NALC-1.5% NaOH) for 15 min before culture in BACTEC 12 B (Becton Dickinson, Franklin, NJ), para-JEM [Thermo Fisher Scientific (TREK Diagnostic Systems, Inc.), Cleveland, OH], and Herrold's egg yolk (HEY; Becton Dickinson) media was compared. Treatment with NALC-NaOH resulted in a lower percentage (6%) of contaminated samples than did treatment with hexadecylpyridinium chloride (47%), regardless of culture medium. The decontamination protocol (NALC-1.5% NaOH) was then applied to milk samples (n = 144) collected from cows at 7 US dairies. Recovery of viable MAP from the milk samples was low, regardless of culture medium, with recovery from 2 samples cultured in BACTEC 12 B medium, 1 sample cultured in para-JEM medium, and no viable MAP recovered on HEY medium. However, 32 cows were fecal culture positive and 13 milk samples were positive by direct PCR, suggesting that several cows were actively shedding MAP at the time of milk collection. Contamination rates were similar across media, with 39.6, 34.7, and 41.7% of samples contaminated after culture in BACTEC 12 B, para-JEM, and HEY media, respectively. Herd-to-herd variation had a major effect on sample contamination, with the percentage of contaminated samples ranging from 4 to 83%. It was concluded that decontamination of milk with NALC-1.5% NaOH before culture in BACTEC 12 B medium was the most efficacious method for the recovery of viable MAP from milk, although the ability to suppress the growth of contaminating microorganisms varied greatly between herds., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Mycobacterium avium subsp. paratuberculosis is shed into the milk and feces of cows with advanced Johne's disease, allowing the transmission of M. avium subsp. paratuberculosis between animals. The objective of this study was to formulate an optimized protocol for the isolation of M. avium subsp. paratuberculosis in milk. The parameters investigated included chemical decontamination with N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH), alone and in combination with antibiotics (vancomycin, amphotericin B, and nalidixic acid), and the efficacy of solid (Herrold's egg yolk medium [HEY]) and liquid (Bactec 12B and para-JEM) culture media. For each experiment, raw milk samples from a known noninfected cow were inoculated with 10(2) to 10(8) CFU/ml of live M. avium subsp. paratuberculosis organisms. The results indicate that an increased length of exposure to NALC-NaOH from 5 to 30 min and an increased concentration of NaOH from 0.5 to 2.0% did not affect the viability of M. avium subsp. paratuberculosis. Additional treatment of milk samples with the antibiotics following NALC-NaOH treatment decreased the recovery of viable M. avium subsp. paratuberculosis cells more than treatment with NALC-NaOH alone. The Bactec 12B medium was the superior medium of the three evaluated for the isolation of M. avium subsp. paratuberculosis from milk, as it achieved the lowest threshold of detection. The optimal conditions for NALC-NaOH decontamination were determined to be exposure to 1.50% NaOH for 15 min followed by culture in Bactec 12B medium. This study demonstrates that chemical decontamination with NALC-NaOH resulted in a greater recovery of viable M. avium subsp. paratuberculosis cells from milk than from samples treated with hexadecylpyridinium chloride (HPC). Therefore, it is important to optimize milk decontamination protocols to ensure that low concentrations of M. avium subsp. paratuberculosis can be detected.

A protocol was optimized for the isolation of Mycobacterium avium subsp. paratuberculosis (MAP) from milk and colostrum, with parameters including chemical decontamination, antibiotics, and different culture media. This study demonstrates that the efficiency of MAP recovery from milk is highly dependent upon the culturing protocol, and such protocols should be optimized to ensure that low concentrations of MAP in milk can be detected.

Mycobacterium avium subsp. paratuberculosis, the causative agent of a chronic enteritis in ruminants (Johne's disease), has been linked to Crohn's disease in humans. This microorganism is shed by infected animals primarily in the feces but is also shed in the milk at much lower levels. Therefore, dairy products from infected animals may be one mode of transmission of this animal pathogen. This study was designed to evaluate the effectiveness of the holder and high-temperature short-time pasteurization standards on the destruction of M. paratuberculosis. One hundred eighty experiments were conducted in this study using a slug-flow pasteurizer unit and a laboratory scale pasteurizer unit. Ultrahigh-temperature milk was inoculated at two concentrations, 10(8) and 10(5) CFU/ml, with three different field strains of M. paratuberculosis. Five different time-temperature combinations were evaluated: 62.7 degrees C for 30 min, 65.5 degrees C for 16 s, 71.7 degrees C for 15 s, 71.7 degrees C for 20 s, and 74.4 degrees C for 15 s. Three replicates of each experiment were run for the pasteurizer unit, time-temperature combination, and strain of M. paratuberculosis. Treatment of milk regardless of bacterial strain or pasteurizer unit resulted in an average 5.0- and 7.7-log kill for the low and high concentrations of inoculum, respectively. Milk treated for cheese production (65.5 degrees C for 16 s) resulted in a much lower and more variable kill. Results from this study indicate that the current U.S. minimum standards for batch and high-temperature short-time pasteurization of grade A milk significantly reduced the survivability of M. paratuberculosis, but some bacteria survived subpasteurization heat treatment of milk used for cheese manufacture.

The 2002 NAHM's Dairy Survey indicated that 87.2% of dairy farms in the United States feed waste milk to their neonatal calves. Although cost-effective, this practice can lead to increased calf morbidity and mortality due to ingestion of pathogenic agents. In an effort to reduce the risk of infection, dairy producers are implementing on-farm pasteurization of the waste milk as a control procedure before feeding the milk to calves. In the present study, the efficacy of a commercial high-temperature, short-time (HTST) on-farm pasteurizer unit to destroy Mycobacterium paratuberculosis, Salmonella enterica spp., and Mycoplasma spp. in raw milk was evaluated. Replicate experiments were run for 3 isolates of M. paratuberculosis, 3 serovars of Salmonella (derby, dublin, typhimurium); and 4 species of Mycoplasma (bovis, californicum, canadense, serogroup 7) at 2 different levels of experimental inoculation. In addition, HTST pasteurization experiments were performed on colostrum experimentally inoculated with M. paratuberculosis. After culture of the pasteurized milk samples, no viable M. paratuberculosis, Salmonella, or Mycoplasma were recovered, regardless of species, strain, or isolate. Pasteurization of colostrum was also effective in the destruction of M. paratuberculosis but resulted in an average 25% reduction in colostral immunoglobulin. These results suggest that HTST pasteurization is effective in generating a safer product to feed to young calves.

Objectives were to estimate percentages of seropositive herds with cows shedding Mycobacterium paratuberculosis in feces and milk, and to estimate sensitivity, specificity, and predictive value of an ELISA relative to fecal culture. Dairy cows (n = 712) were randomly selected from 61 herds previously identified by ELISA as positive for Johne's disease. Fecal and bulk tank milk samples (n = 52 of 61 herds) were obtained from 10 states in the United States. Fecal samples were processed by a double centrifugation, double decontamination culture procedure. Milk samples were processed for both culture and DNA analysis by using polymerase chain reaction (PCR). Of 24 herds with at least three cows that had tested ELISA-positive, 79% were also culture-positive, compared with 18 of 37 herds with one or two ELISA-positive cows. Both fecal-culture and ELISA results were available on 651 cows; only 25% of cows that were fecal-culture positive also tested positive by ELISA and over 6% of cows that were fecal-culture negative tested ELISA-positive. Milk samples all cultured negative, but analysis of milk samples by PCR resulted in 68% of herds positive for M. paratuberculosis DNA including 24 of 31 herds with positive fecal cultures and 11 of 21 herds with negative fecal cultures. Sensitivity and specificity of the ELISA compared with fecal culture is lower than previously reported and perhaps best used in screening herds because of limited efficacy to predict infection in individual cows. In addition, contamination of bulk tank milk samples with M. paratuberculosis does occur in seropositive herds, even in some with negative fecal cultures.

A recent dairy survey conducted in 1996 by the National Animal Health Monitoring System suggests between 20 and 40% of dairy herds in the United States have some level of Johne's disease. This figure will continue to increase unless producers implement management regimes that will help control the spread of this disease within their herds. The neonatal calf is the target for infection with Mycobacterium paratuberculosis, the causative agent of Johne's disease. Calves become infected via exposure to the bacterium through contaminated feces, bedding, colostrum, and milk. Shedding of viable M. paratuberculosis has been documented in the colostrum and milk of infected dams. This study evaluated the efficacy of on-farm pasteurization to destroy M. paratuberculosis in waste milk fed to calves to circumvent this mode of transmission. In three replicate experiments, waste milk was experimentally inoculated with M. paratuberculosis and heated at 65.5 degrees C for 30 min. No viable bacteria were recovered after 28 wk of incubation. These results suggest that batch pasteurization of waste milk contaminated with M. paratuberculosis was effective at generating a clean product to feed to young calves.

Mycobacterium paratuberculosis, an acid-fast bacillus that causes enteritis in ruminants, has been suggested as an etiological agent of Crohn's disease in humans. The mode of transmission is unclear; however, some evidence suggests that humans may become infected via contaminated milk. Currently, it is not known whether commercial pasteurization effectively kills M. paratuberculosis in contaminated raw milk. Using a laboratory-scale pasteurizer unit designed to simulate the high-temperature, short-time method (72 degrees C, 15 sec) currently used by commercial dairies, we previously demonstrated that treatment of raw milk inoculated with 10(4) to 10(6) cfu of M. paratuberculosis/ml reduced numbers to an undetectable level. However, M. paratuberculosis is an intracellular pathogen that resides within the macrophages of the host and evades destruction. We subsequently performed further experiments examining heat treatment of milk inoculated with mammary gland macrophages containing ingested M. paratuberculosis. Heat treatment of these samples under high-temperature, short-time conditions demonstrated that the macrophage does not protect the organism because we were unable to recover any viable M. paratuberculosis from the samples. Conversely, other researchers have demonstrated that a residual population of M. paratuberculosis may survive heat treatment of milk. In addition, a recent news report stated that viable M. paratuberculosis organisms have been cultured from retail-ready milk in Ireland. A summary of past and current studies concerning this issue along with a discussion of methodologies used to recover M. paratuberculosis from experimentally inoculated milk will be presented in this paper.

Recent evidence in cattle and mice has suggested that 1,25-dihydroxyvitamin D3 may stimulate T-lymphocyte differentiation pathways responsible for humoral immunity. The use of 1,25-dihydroxyvitamin D3 as an adjuvant for an Escherichia coli J5 vaccine was tested. Ten midlactation cows received J5 vaccine and were revaccinated 6 wk after the first vaccine administration. Five of these cows were given 200 micrograms of 1,25-dihydroxyvitamin D3, in addition to the primary vaccination, and they received an additional 200 micrograms of 1,25-dihydroxyvitamin D3 1 wk after the primary vaccination. No 1,25-dihydroxyvitamin D3 was administered with the wk 6 J5 vaccine booster. Milk immunoglobin (Ig)M, IgG, and IgA antibodies to E. coli J5 were significantly increased in cows receiving 1,25-dihydroxyvitamin D3 + E. coli J5 vaccine compared with cows that received only E. coli J5 vaccine. Serum IgG and IgG1 antibodies to E. coli J5 were also significantly enhanced by 1,25-dihydroxyvitamin D3 treatment compared with cows receiving vaccine alone. In contrast, serum IgG2 titers tended to decline in cows receiving 1,25-dihydroxyvitamin D3. Taken together, these data suggest that 1,25-dihydroxyvitamin D3 has potential usefulness in boosting humoral responses to vaccines such as J5 and may further enhance the protective qualities of vaccines.

Currently, it is not known whether commercial pasteurization effectively kills Mycobacterium paratuberculosis in contaminated raw milk. Results from holder test tube experiments indicated that a residual population of viable bacteria remained after treatment at 65, 72, 74, or 76 degrees C for 0 to 30 min. Use of a laboratory-scale pasteurizer unit demonstrated that treatment of raw milk at 72 degrees C for 15 s effectively killed all M. paratuberculosis.

A brief history of the development of milk pasteurisation is presented and updated. Concerns about the margin of safety provided by current pasteurisation standards in terms of milk-borne pathogens such as mycobacteria (in particular Mycobacterium paratuberculosis) and other emerging pathogens such as Listeria monocytogenes and Escherichia coli O157:H7 are discussed. With the exception of the endospores of Bacillus cereus, current standards appear to be adequate for public health assurance of milk safety provided good manufacturing practices are followed.