Phagomagnetic separation-quantitative PCR: A rapid,sensitive and specific surveillance tool for viable Mycobacterium avium ssp. paratuberculosis in bulk tank and individual cow milk samples

Bulk tank milk samples from 392 Northern Ireland dairy farms and individual milk from animals (n = 293) on 4 of these farms were tested by a novel phagomagnetic separation (PhMS)-quantitative (q)PCR assay able to detect and quantify viable Mycobacterium avium ssp. paratuberculosis (MAP), to demonstrate its potential utility as a milk surveillance tool. Viable MAP were detected in 26.5% of the bulk tank milks, with MAP contamination levels ranging from 1 to 8,432 MAP/50 mL of milk; less than 2% of farms had MAP contamination levels >100 MAP/50 mL in their bulk tank milk. Follow-up PhMS-qPCR testing of milk from individual animals on 4 farms that had the highest numbers of MAP in their bulk tank milks indicated that 17 to 24% of animals in each herd were shedding viable MAP in their milk. Mean MAP numbers detected ranged between 6.7 and 42.1 MAP/50 mL of milk. No significant correlation was observed between the detection of viable MAP in bulk or individual milks by PhMS-qPCR and parallel milk ELISA results, or between PhMS-qPCR results and any other milk recording results (somatic cell count, total bacterial count, % butterfat, or % protein). Viable MAP was detected by IS900 qPCR in 52 (85.2%) Pozzato broth cultures of 61 PhMS-qPCR-positive individual milks after 12 wk of incubation, suggesting few PhMS-qPCR results were false positives. The mean sensitivities of the PhMS-qPCR assay and milk ELISA applied to individual milks were estimated by Bayesian latent class analysis to be 0.7096 and 0.2665, respectively, and mean specificities were similar (0.9626 and 0.9509). Our findings clearly demonstrate that the novel PhMS-qPCR assay could be a useful milk surveillance tool for dairy processors, or a milk monitoring tool for Johne's disease control or milk quality assurance programs.     

Reduction of viable Mycobacterium avium ssp. paratuberculosis in slurry subjected to anaerobic digestion in biogas plants

Cattle infected with Mycobacterium avium ssp. paratuberculosis (MAP) shed the bacterium in their feces. This may lead to considerable concentrations of MAP in slurry, which has been postulated to contribute to MAP transmission when this slurry is used as fertilizer. For other bacterial species, anaerobic digestion has been shown to reduce bacterial load and to increase the safety of organic waste. Therefore, the objective of this study was to investigate the effects of anaerobic digestion in biogas plants on MAP survival in slurry from 16 dairy farms with a history of MAP infection. Presence of MAP was determined using MAP culture and a commercial MAP IS900 quantitative PCR (qPCR) applied on untreated slurry samples, slurry samples after primary fermentation, and digestate. Unfermented slurry samples from most enrolled farms tested positive for MAP, via both culture and qPCR. After the fermentation process, MAP could no longer be cultured in most samples, with the exception of 2 samples from farms where high numbers of MAP-shedding cows were kept at the time of sampling. A Bayesian binomial model predicted a probability of 93% for a MAP-negative culture result after fermentation. In most samples, MAP DNA was still detectable when using the IS900 qPCR. The probability of a negative result in qPCR was estimated to be 27%. Results of this study indicate that subjecting MAP-positive slurry to anaerobic digestion in biogas plants leads to a reduction of viable MAP below the detection limit; however, MAP DNA remained detectable. It remains undetermined whether MAP DNA detected in fermentation products is a residue of MAP degradation or belongs to viable MAP below the detection limit or in a dormant state. In conclusion, subjecting MAP-positive slurry to anaerobic mesophilic digestion reduces viable MAP concentration below the detection limit. The use of digestion products as fertilizer on pasture and agricultural soils instead of untreated slurry may therefore reduce the risk of MAP transmission.     

Short communication: Recovery of viable Mycobacterium avium subspecies paratuberculosis from retail pasteurized whole milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic granulomatous enteritis that affects all ruminants worldwide. Some researchers have indicated a possible role of MAP in Crohn's disease. Despite extensive research and large and important advances in the past few decades, the etiology of Crohn's disease remains indefinite. The most probable transmission route of MAP from animals to humans is milk and dairy products. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide, and some studies have reported that MAP is resistant to pasteurization. In Brazil, MAP has been reported in raw milk samples; however, Brazilian retail pasteurized milk has not yet been tested for viable MAP. The aim of this study was to investigate MAP in pasteurized milk in the region of Viçosa (Minas Gerais, Brazil). Thirty-seven samples were collected and processed for culture of MAP. One colony similar to MAP was observed and confirmed by IS900-nested PCR and sequencing. Analysis revealed 97 to 99% identity with the MAP K-10 strain. This study is the first report of the presence of MAP in retail pasteurized whole milk in Brazil.     

Short communication: Viable Mycobacterium avium subspecies paratuberculosis in retail artisanal Coalho cheese from Northeastern Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis and it potentially plays a role in Crohn’s disease. In humans, the main route of transmission of MAP might be the intake of contaminated milk and dairy products. Considering that MAP has already been detected in many types of cheese in different counties, and that Coalho cheese is an important dairy product in northeastern Brazil, the aim of this study was to report the first detection of MAP in retail Coalho cheese in Brazil by PCR and culture. Of 30 retail Coalho cheese samples, 3 (10%) amplified fragments of a similar size to that expected (626 bp) were obtained and viable MAP was recovered by culture from 1 (3.3%) sample. The DNA from the positive culture sample was sequenced and showed 99% identity with the insertion sequence IS900 deposited in GenBank. It was possible to identify the presence of MAP-specific DNA in the analyzed samples for the first time in Brazil, and to recover viable cells from retail Coalho cheese.     

On-farm spread of Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination

A rapid, cheap and sensitive detection method of Mycobacterium avium subsp. paratuberculosis (MAP) in raw milk was needed for routine usage. We developed two duplex real time qPCR systems specific for MAP detection. These real time qPCR assays amplify the multicopy element IS900 for qualitative analysis and the single copy element F57 for quantitative analysis. Both assays incorporate an internal amplification control amplified with the same primers as the targets and the same probes are used in both assays. The specificity of the assays was confirmed by the testing of 6 different MAP isolates, 12 isolates of other mycobacteria or bacterial species and 4 different mammalian DNAs. The sensitivity of the developed assays and isolation efficiency were demonstrated through the analysis of raw milk samples artificially contaminated with MAP cells and with plasmids containing cloned fragments of the targets (IS900 and F57). The developed assays for milk analysis were applied to samples from one farm with two faecal shedding cows. Three hundred and forty five individual milk samples were tested by real time qPCR assays and by cultivation. Hundred and eleven (32.5%) individual milk samples were positive by the real time qPCR, no milk sample was culture positive. The spread of MAP in individual, tank and bulk tank milk samples was also monitored.     

Short communication: Detection of Mycobacterium avium subspecies paratuberculosis by polymerase chain reaction in bovine milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, or Johne's disease, a chronic granulomatous enteritis that affects all ruminants worldwide. Since the isolation of MAP from intestinal tissue of human patients bearing Crohn's disease, there has been a debate on the possibility of this agent playing a role in the etiology of Crohn's disease. Milk could be the potential vehicle for transmission to humans. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide. In Brazil, detection of MAP is uncommon; however, it has already been detected by bacterial isolation and serological test. The aim of this study was to investigate the presence of MAP, by PCR, in raw milk samples in the region of Viçosa, Minas Gerais State, Brazil. Of 222 milk samples evaluated, 8 (3.6%) quarter milk samples amplified fragments of similar size to that expected of 626 bp. These fragments were cloned and sequenced. The genetic analysis revealed a 99% identity match between the sequences obtained in this study and the insertion sequence IS900 deposited in the GenBank. In the analyzed milk samples, MAP DNA was detected, confirming its presence in dairy cattle in the region of Viçosa. This is the first report of MAP presence in raw milk samples in Brazil.     

An optimised milk testing protocol to ensure accurate enumeration of viable Mycobacterium avium subsp. paratuberculosis by the PMS-phage assay

There is interest in determining levels of Mycobacterium avium subsp. paratuberculosis (MAP) contamination in milk. The optimal sample preparation for raw cows' milk to ensure accurate enumeration of viable MAP by the peptide-mediated magnetic separation (PMS)-phage assay was determined. Results indicated that milk samples should be refrigerated at 4 °C after collection and MAP testing should commence within 24 h, or samples can be frozen at −70 °C for up to one month without loss of MAP viability. Use of Bronopol is not advised as MAP viability is affected. The vast majority (>95%) of MAP in raw milk sedimented to the pellet upon centrifugation at 2500 × g for 15 min, so this milk fraction should be tested. De-clumping of MAP cells was most effectively achieved by ultrasonication of the resuspended milk pellet on ice in a sonicator bath at 37 kHz for 4 min in ‘Pulse’ mode.     

Short communication: Passive shedding of Mycobacterium avium ssp. paratuberculosis in commercial dairy goats in Brazil

Goat farming is a low-cost alternative to dairy production in developing countries. In Brazil, goat production has increased in recent years due in part to the implementation of programs encouraging this activity. Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a disease that causes chronic granulomatous enteritis in ruminants, but MAP transmission dynamics are still poorly understood in goats. In a previously published study of our research group, 10 dairy goat farms (467 animals) from Minas Gerais state were analyzed for MAP detection; 2 fecal cultures and 11 milk samples tested positive for MAP by conventional PCR and were confirmed by sequencing. Because no clinical signs were observed over 1 yr of monitoring, we hypothesized that these MAP-positive goats could be passive shedders. Thus, in the present study, 4 positive goats (4/13) from the previous study were purchased and feces and milk samples were collected for evaluation (twice, with an interval of 3 mo between tests) by culture of MAP, IS900 PCR, or both. All analyses were negative for MAP. At the last time point, blood samples were collected for ELISA, the animals were killed, and tissues collected for tissue culture and histopathology. At necropsy, no macroscopic lesions related to paratuberculosis were observed. Similarly, no histological changes were observed and MAP in samples stained by Ziehl–Neelsen was not detected. These animals were characterized as potential passive shedders with upward contamination of the teat canal by MAP. This is the first report of the passive shedding phenomenon in goats in Brazil and it highlights the importance of identifying these animals for control programs and to ensure the quality of dairy products.     

Short communication: Investigation into Mycobacterium avium ssp. paratuberculosis in pasteurized milk in Italy

This study investigated the presence of viable Mycobacterium avium ssp. paratuberculosis (MAP) in pasteurized milk produced by Italian industrial dairy plants to verify the prediction of a previously performed risk assessment. The study analyzed 160 one-liter bottles of pasteurized milk from 2 dairy plants located in 2 different regions. Traditional cultural protocols were applied to 500 mL of pasteurized milk for each sample. The investigation focused also on the pasteurization parameters and data on the microbiological characteristics of raw milk (total bacterial count) and pasteurized milk (Enterobacteriaceae and Listeria monocytogenes). No sample was positive for MAP, the pasteurization parameters complied with European Union legislation, and the microbiological analysis of raw and pasteurized milk showed good microbiological quality. The results show that a 7-log (or >7) reduction could be a plausible value for commercial pasteurization. The combination of hygiene practices at farm level and commercial pasteurization yield very low or absent levels of MAP contamination in pasteurized milk, suggesting that pasteurized milk is not a significant source of human exposure to MAP in the dairies investigated.     

Short communication: Mycolicibacterium smegmatis,basonym Mycobacterium smegmatis,causing pyogranulomatous mastitis and its cross-reactivity in bovine (para)tuberculosis testing

Different mycobacterial species are encountered in bovine medicine. The fastidiously growing mycobacteria (Mycobacterium bovis as the cause of bovine tuberculosis, and Mycobacterium avium ssp. paratuberculosis, MAP, as the cause of paratuberculosis) are well known and targeted in eradication/control or monitoring programs in different countries, whereas the rapidly growing species is only rarely identified from bovine disease. The latter have occasionally been reported as the cause of bovine clinical mastitis, but recent reports are scarce. In this study, Mycolicibacterium smegmatis (basonym Mycobacterium smegmatis) was identified as cause of granulomatous, relapsing clinical mastitis in 2 cows from one Belgian dairy herd. Milk, blood, and fecal samples were collected, as well as tissue samples after the cows were culled. Serological analysis conducted on milk and serum samples resulted in positive reactions for MAP, but negative for Mycobacterium bovis. Production of IFN-γ showed sensitization with mycobacteria or similar organisms, other than M. bovis, in one cow. Detection of MAP by bacteriological culture and IS900-based quantitative PCR on milk and feces remained negative. In conclusion, this paper describes M. smegmatis as a cause of bovine clinical mastitis in Belgium and suggests cross-reactivity of the intramammary M. smegmatis infection with routinely used serological tests for MAP.     

Are we closer to understanding why viable cells of Mycobacterium avium subsp. paratuberculosis are still being reported in pasteurised milk?

Mycobacterium avium subsp. paratuberculosis (MAP) continues to be associated with Crohn’s disease. Following work in the 1990s that suggested that statutory pasteurisation of milk (72 °C, 15 s) was insufficient to destroy MAP, the UK Dairy Industry increased the holding time to 25 s. Since then, some plants have increased the lethality of pasteurisation further with a number using 78 °C for 27 s. Despite the increase in lethality, a recent survey of pasteurised milk in England found that 10.3% of pasteurised milk samples tested positive for viable MAP. This article discusses the significance of MAP and why viable MAP might be found in pasteurised milk.     

Prevalence of Mycobacterium avium subspecies paratuberculosis in Swiss raw milk cheeses collected at the retail level

A total of 143 raw milk cheese samples (soft cheese, n = 9; semihard cheese, n = 133; hard cheese, n = 1), collected at the retail level throughout Switzerland, were tested for Mycobacterium avium ssp. paratuberculosis (MAP) by immunomagnetic capture plus culture on 7H10-PANTA medium and in supplemented BAC-TEC 12B medium, as well as by an F57-based real-time PCR system. Furthermore, pH and water activity values were determined for each sample. Although no viable MAP cells could be cultured, 4.2% of the raw milk cheese samples tested positive with the F57-based real-time PCR system, providing evidence for the presence of MAP in the raw material. As long as the link between MAP and Crohn's disease in humans remains unclear, measures designed to minimize public exposure should also include a focus on milk products.     

Short communication: Application of an N-acetyl-l-cysteine-NaOH decontamination method for the recovery of viable Mycobacterium avium subspecies paratuberculosis from milk of naturally infected cows

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 5 h or N-acetyl-l-cysteine-1.5% sodium hydroxide (NALC-1.5% NaOH) for 15 min before culture in BACTEC 12B (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 12B 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 12B, 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 12B 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.     

Detection of viable Mycobacterium avium subsp. paratuberculosis in retail pasteurized whole milk by two culture methods and PCR

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 eonfirmed 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.     

Clinical disease and stage of lactation influence shedding of Mycobacterium avium subspecies paratuberculosis into milk and colostrum of naturally infected dairy cows

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.     

Association between Mycobacterium avium subspecies paratuberculosis infection and milk production in two California dairies

The association between Mycobacterium avium ssp. paratuberculosis (MAP) and milk production was estimated on 2 California dairies using longitudinal data from 5,926 cows. Both study herds had moderate MAP seroprevalence, housed cows in freestalls, and had Johne's disease control programs. Cow MAP status was determined using both serum ELISA and fecal culture results from cows tested at dry-off and from whole-herd tests. Potential confounders were evaluated based on a causal diagram. Mixed models with 2 functions (splines) for days in milk (DIM) representing milk production pre- and postpeak used in similar studies were further modified to use each cow's observed DIM at peak and lactation length. Cows that were seropositive produced 2.5 kg less 4% fat-corrected milk (FCM) per day than their seronegative herdmates. In addition, cows that were fecal-culture positive by liquid culture and confirmed by PCR produced 2.2 kg less 4% FCM per day than their fecal-culture negative herdmates. The decrease in milk production in MAP test-positive compared with test-negative cows started in the second lactation. A switch in MAP status in either ELISA or fecal culture results from positive to negative had no significant association with milk production. Modified DIM functions that used the observed DIM at peak had better model fit than another function that assumed a fixed peak at 60 DIM. Cows that tested positive for MAP on serum ELISA or fecal culture produced less milk than cows that tested negative, and the association between MAP and milk production was not confounded by mastitis, elevated somatic cell counts, or uterine or metabolic cow conditions.     

Detection and enumeration of Staphylococcus aureus from bovine milk samples by real-time polymerase chain reaction

The aim of this study was to develop and evaluate a real-time quantitative PCR (qPCR)-based method to detect and quantify Staphylococcus aureus in bronopol-preserved milk samples from subclinical intramammary infections (IMI). Serial dilutions of milk artificially inoculated with Staph. aureus ATCC 29213 were used to establish a standard curve (cfu/mL) of the qPCR assay targeting the Staph. aureus thermonuclease-encoding gene nuc according to the strain plate count. The analytical sensitivity, specificity, and repeatability of the qPCR assay were determined. A total of 60 milk samples, collected from mammary quarters without abnormal appearance and with positive isolation of Staph. aureus, were submitted to both the qPCR protocol and Staph. aureus plate counting and results from both methods were compared. Staphylococcus aureus from bronopol-preserved, subclinical IMI milk samples were not accurately enumerated by qPCR compared with plate counting of the nonpreserved, raw milk sample. The detection limit of the qPCR protocol of inoculated Staph. aureus ATCC 29213 in bronopol-preserved milk samples was 1.04 × 10¹ cfu/mL. The qPCR protocol can be a high-throughput and rapid diagnostic assay to accurately detect Staph. aureus IMI from bronopol-preserved milk samples compared with a traditional culturing method. However, the proposed qPCR protocol is not accurate for counting of Staph. aureus in bronopol-preserved milk samples from naturally infected mammary glands.     

Evaluation of milk ELISA fordetection of Mycobacterium avium subspecies paratuberculosis indairy herds and association with within-herd prevalence

Cow-level milk ELISA results can be used to determine herd Mycobacterium avium ssp. paratuberculosis (MAP) status. Milk sample collection is minimally invasive and ELISA results can be obtained quickly and economically. The objectives were to evaluate the herd-level test characteristics of 3 commercial milk ELISA, and to determine the impact of within-herd MAP prevalence on the performance of the milk ELISA herd test. A total of 32 purposively selected herds with a median herd size of 66 milking cows were used in this 2-yr project. Fecal and milk samples were collected from all milking cows at 6-mo intervals. Fecal samples were pooled by cow age, with 5 cow samples per pool; individual fecal culture was completed on cow samples from positive pools. Herd MAP status was defined as MAP positive if, at any point during the longitudinal study, a pooled fecal culture from the herd was positive. Milk samples were analyzed using each of 3 commercial milk ELISA kits; a cow-level result from each ELISA was classified as positive following the respective manufacturer’s recommended threshold for a positive result. Herd-level milk ELISA test characteristics were estimated using generalized estimating equations logistic models, which accounted for repeated measurements. Using a cutoff of 2% milk ELISA-positive cows, milk ELISA herd sensitivity relative to a herd MAP status based on all pooled fecal culture results collected during the study was as follows: ELISA A: 59% [95% confidence interval (CI): 36–78%), ELISA B: 56% (95% CI: 32–77%), and ELISA C: 63% (95% CI: 41–81%). Herd specificity for ELISA A, B, and C was 80% (95% CI: 71–88%), 96% (95% CI: 89–98%), and 92% (95% CI: 86–96%), respectively. The remainder of the analyses focused on results from ELISA B. Herd sensitivity of ELISA B increased as MAP prevalence increased. In herds with a mean MAP prevalence ≤5%, the herd sensitivity of the milk ELISA was low, ranging from 11% when MAP prevalence was 1%, to 62% when MAP prevalence was 5%. Categorical likelihood ratios based on milk ELISA within-herd prevalence predicted that herds with milk ELISA prevalence above 0 but <2% had a similar likelihood to be MAP positive or MAP negative, whereas herds with a milk ELISA prevalence between 2 and 4% were 3.7 times more likely to be MAP positive than MAP negative. All herds with a milk ELISA prevalence >4% were MAP positive. Although milk ELISA B worked well to establish herd MAP status in high-prevalence herds, interpretation was unreliable in MAP-negative and low-prevalence herds.     

Association between the presence of antibodies to Mycobacterium avium subspecies paratuberculosis and somatic cell count

Somatic cell counts (SCC) in bulk tank milk delivered for human consumption are one of the indicators of milk quality and are used for milk pricing. Consequently, milk from cows with high SCC is frequently used by farmers for feeding of calves to lower the SCC in bulk tank milk. Young calves are more susceptible to Mycobacterium avium ssp. paratuberculosis (MAP) and may acquire the infection early in life through ingestion of MAP-contaminated milk. The occurrence of MAP antibodies can be an indicator of MAP shedding. Because MAP can be shed in milk from infected cows, and antibodies to MAP can be an indicator of the infectious status, an association between antibodies to MAP and high SCC can result in high-SCC milk being at risk of containing MAP. Feeding milk containing high SCC to susceptible calves may result in MAP infections. Somatic cell counts and MAP antibodies in milk were measured repeatedly in 7,251 cows from 26 Danish dairy herds to investigate the association between the occurrence of MAP antibodies and high SCC. The results of robust regression showed a log-linear relationship between the age at first positive ELISA and the age at first high SCC sample (R² = 0.51). Of the 1,733 cows positive for MAP antibodies and with high SCC, high SCC was detected prior to MAP antibodies in 46% of the cows. Still, in 40% of the cows, MAP antibodies were detected before a high SCC. Therefore, the findings do not point to a causal relationship between high SCC and antibodies to MAP, but suggest a strong association and highlight a potentially increased risk of MAP transmission when milk with high SCC is fed to calves.     

Short communication: Examination of milk filters by real-time PCR as a herd-level indicator of the presence of Mycobacterium avium subspecies paratuberculosis in dairy herds

The aim of this study was to assess the suitability of real-time quantitative PCR (qPCR) for the detection of Mycobacterium avium ssp. paratuberculosis (MAP) in milk filters as a herd level indicator of paratuberculosis infection. Seventy-nine samples from textile or metal milk filters from 15 herds with defined MAP prevalence (infection status = noninfected, 0-5%, 5-10%, or >10% of animals with clinically confirmed paratuberculosis) were analyzed. The MAP DNA was isolated by a modified commercially available protocol for feces, and detection and quantification of the pathogen was performed by the IS900 qPCR. Mycobacterium avium ssp. paratuberculosis DNA was detected in 63 (79.7%) samples. Determination of MAP infection established by fecal and tissue culture was correctly confirmed by the analysis of milk filters on 11 of 12 infected farms; MAP was not detected in filters from 3 farms where paratuberculosis was never diagnosed. Statistical analysis of the data supports the evidence that milk filters can be used as a template for the direct detection of MAP on the herd level. The probability of successful MAP detection in milk filters in a herd with MAP-infected cows is at least 94.3%. Absolute numbers of MAP detected on the milk filter can be used for a rough estimation of paratuberculosis prevalence >10% in the herd. Analysis of milk filters for the presence of MAP can be a useful tool for the detection of paratuberculosis on the herd level before any individual control strategies.