Viable Mycobacterium Avium Subsp. Paratuberculosis Research Articles

Decontamination protocols on bovine fecal and environmental samples for Mycobacterium avium ssp. paratuberculosis growth in solid media

Bacteriological culture of Mycobacterium avium subsp. paratuberculosis (MAP) is considered the gold standard to confirm its presence in several matrices for Johne’s disease diagnosis. Whether it is a liquid or solid culture, a problem with MAP culture is that non-interpretable results arise because of overgrowth by other microorganisms, making MAP growth and identification more difficult or impossible. We systematically reviewed published decontamination protocols and their effects on MAP culture from bovine fecal and environmental samples on solid media. Based on our findings, we suggest a step-by-step decontamination protocol. The OVID®/MEDLINE, PubMed®, SciELO Citation Index®, and Redalyc® platforms as well as the International Colloquium on Paratuberculosis (ICP) proceedings and the reference lists were reviewed to identify relevant studies. The inclusion criteria considered articles published in English, Portuguese, French, German, Spanish, and peer-reviewed journals. The exclusion criteria included unrelated topics, species other than bovines, other than environmental/faecal samples, other than diagnostic techniques of interest, and non-original articles. Definitive studies were obtained through the authors’ consensus regarding their eligibility and quality. In total, 1,004 publications matched the search terms, and 27 articles met the inclusion criteria, of which 45 derived and reported 15 different decontamination protocols. The centrifugation-one-step hexadecylpyridinium chloride protocol, which used over 22,154 fecal samples in three studies, was found to be the most suitable, reporting an average MAP isolation rate of 3.99% (886/22,154) and an average contamination rate of 0.17% (38/22,154). This systematic review highlights the need for further refinement of decontamination protocols to minimize the loss of viable MAP during processing of bovine fecal and environmental samples.

Utilisation of Actiphage in combination with IS900 qPCR as a diagnostic tool for rapid determination of paratuberculosis infection status in small ruminant herds.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a chronic infectious intestinal disease occurring in domestic and wild ruminants. Early diagnosis of infected herds enabling timely adoption of control measures is tremendously important in view of the fact that the disease has a significant economic impact on farmers. The aim of this study was to evaluate the possibility of rapid detection of viable MAP on small ruminant farms based on environmental sample examination using a novel phage-based test named Actiphage. A total of 9 fresh and 28 frozen (8 or 11 years at -70°C) environmental samples originating from paratuberculosis-affected farms were analysed for the presence of MAP by four different diagnostic methods: Actiphage combined with real-time PCR targeting insertion sequence 900 (IS900 qPCR), conventional phage amplification assay, culture (frozen samples only), and direct ĪS900 qPCR. Viable MAP was detected in one fresh environmental sample using Actiphage-IS900 qPCR. None of the frozen samples tested positive using this diagnostic approach, which was consistent with the results of culture examination also providing information on viability. This study describes other possible and innovative uses of phage-based methods in paratuberculosis control strategies. The Actiphage-qPCR was found to be less laborious than culture and provided results within six hours, suggesting that it may be a valuable tool for rapid initial determination of the infectious status of farmed animals based on environmental sample examination.

Viable Mycobacterium avium subsp. paratuberculosis Colonizes Peripheral Blood of Inflammatory Bowel Disease Patients.

Pathobionts, particularly Mycobacterium avium subsp. paratuberculosis (MAP) and Escherichia coli isolates with adherence/invasive ability (AIEC) have been associated with inflammatory bowel disease (IBD), particularly Crohn's disease (CD). This study aimed to evaluate the frequency of viable MAP and AIEC in a cohort of IBD patients. As such, MAP and E. coli cultures were established from faecal and blood samples (with a total n = 62 for each) of patients with CD (n = 18), ulcerative colitis (UC, n = 15), or liver cirrhosis (n = 7), as well as from healthy controls (HC, n = 22). Presumptive positive cultures were tested by polymerase chain reaction (PCR), for a positive confirmation of MAP or E. coli identity. E. coli-confirmed isolates were then tested for AIEC identity using adherence and invasion assays in the epithelial cell line of Caco-2 and survival and replication assays in the macrophage cell line of J774. MAP sub-culture and genome sequencing were also performed. MAP was more frequently cultured from the blood and faecal samples of patients with CD and cirrhosis. E. coli presumptive colonies were isolated from the faecal samples of most individuals, in contrast to what was registered for the blood samples. Additionally, from the confirmed E. coli isolates, only three had an AIEC-like phenotype (i.e., one CD patient and two UC patients). This study confirmed the association between MAP and CD; however, it did not find a strong association between the presence of AIEC and CD. It may be hypothesized that the presence of viable MAP in the bloodstream of CD patients contributes to disease reactivation.

Are Reactive Oxygen Species (ROS) the Main Mechanism by Which Copper Ion Treatment Degrades the DNA of Mycobacterium avium subsp. paratuberculosis Suspended in Milk?

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis. This pathogen is able to survive adverse environmental conditions, including the pasteurization process. Copper, a well-studied metal, is considered an important antibacterial tool, since it has been shown to inactivate even MAP in treated milk through unknown mechanisms. The aim of the present study is to show the effect of copper ions, and reactive oxygen species (ROS) generated in response to oxidative stress, on the damage to MAP DNA when exposed to a copper ion challenge in cow's milk. Spiked milk with different MAP bacterial loads was supplemented with blocking agents. These were either the copper chelators ethylenediaminetetraacetic acid (EDTA) and batocuproin (BCS) or the ROS quenchers D-mannitol, gallic acid and quercetin. The DNA protection, MAP viability and ROS production generated after exposure to a copper challenge were then measured. In a bacterial load of 104 cells mL-1, blocking effects by both the copper chelators and all the ROS quenchers offered significant protection to MAP DNA. In a concentration of 102 cells mL-1, only D-mannitol and a mix of quenchers significantly protected the viability of the bacteria, and only at a concentration of 106 cells mL-1 was there a lower production of ROS when supplementing milk with gallic acid, quercetin and the mix of quenchers. Based on these findings, it may be concluded that MAP DNA damage can be attributed to the combined effect of the direct copper ions and ROS generated. Nevertheless, taking into account the antioxidant environment that milk provides, the direct effect of copper could play a prominent role.

Cathelicidin Mediates an Anti-Inflammatory Role of Active Vitamin D (Calcitriol) During M. paratuberculosis Infection

Vitamin D is a key regulator in calcium and phosphorus metabolism which are essential for maintaining bone health. Recent reports also showed a role for vitamin D in immune regulation which may be linked to vitamin D deficiency in autoimmune disorders including inflammatory diseases and Crohn’s disease (CD). This study examines the role of vitamin D deficiency in the regulation of Cathelicidin Antimicrobial Peptide (CAMP) in CD-like macrophages. The latter includes macrophages infected with Mycobacterium avium subsp. paratuberculosis (MAP) isolated from CD patient. Initially, we measured cathelicidin and calcitriol in ex vivo plasma samples from CD patients with or without MAP infection (N=40 per group). We also measured the expression and production of CAMP/LL-37, TNF-α, IL-1β, IL-10, cellular oxidative stress markers, and bacterial viability following treatment of MAP-infected macrophages with four different forms of vitamin D (D2, D3, calcifediol, and calcitriol). From these studies, we determined that LL-37 and calcitriol were significantly lower in CD samples from MAP-positive patients [155.55 ± 49.77 ng/mL and 51.48 ± 31.04 pg/mL, respectively] compared to MAP-negative patients [193.01 ± 78.95 ng/mL and 272.36 ± 94.77 pg/mL, respectively]. Moreover, calcitriol and calcifediol upregulated CAMP expression by nearly 5-fold and 3-fold, respectively. However, following MAP infection, only calcitriol increased CAMP by 3-folds. Both calcitriol and LL-37 reduced intracellular MAP viability by ~3 folds and inhibited TNF-α and IL-1β expression and production in these cells. Treating co-culture of Caco-2 monolayers and MAP-infected macrophages with LL-37 or calcitriol have shown a reduction in NOX-1 expression and DHE signal, in addition to a higher NADPH/NADPt ratio. Notably, calcitriol’s anti-inflammatory effects were lost upon CAMP knockdown by CAMP-siRNA transfection. Altogether, the data indicate that MAP infection and burden is significant in CD by disrupting the conversion of calcifediol to calcitriol and downregulation of CAMP expression leading to vitamin D deficiency.

A viability assay combining palladium compound treatment with quantitative PCR to detect viable Mycobacterium avium subsp. paratuberculosis cells

Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 µM bis(benzonitrile)dichloropalladium(II) or 30 µM palladium(II)acetate at 5 °C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.

A rapid phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis in milk

Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phage-bead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively.

A Novel Approach to the Viability Determination of Mycobacterium avium subsp. paratuberculosis Using Platinum Compounds in Combination With Quantitative PCR

Mycobacterium avium subsp. paratuberculosis (MAP) represents a slow-growing bacterium causing paratuberculosis, especially in domestic and wild ruminants. Until recently, the assessment of MAP viability relied mainly on cultivation, which is very time consuming and is unable to detect viable but non-culturable cells. Subsequently, viability PCR, a method combining sample treatment with the DNA-modifying agent ethidium monoazide (EMA) or propidium monoazide (PMA) and quantitative PCR (qPCR), was developed, enabling the selective detection of MAP cells with an intact cell membrane. However, this technology requires a laborious procedure involving the need to work in the dark and on ice. In our study, a method based on a combination of platinum compound treatment and qPCR, which does not require such a demanding procedure, was investigated to determine mycobacterial cell viability. The conditions of platinum compound treatment were optimized for the fast-growing mycobacterium M. smegmatis using live and heat-killed cells. The optimal conditions consisting of a single treatment with 100 μM cis-dichlorodiammine platinum(II) for 60 min at 5°C resulted in a difference in quantification cycle (Cq) values between live and dead membrane-compromised mycobacterial cells of about 6 Cq corresponding to about 2 log10 units. This optimized viability assay was eventually applied to MAP cells and demonstrated a better ability to distinguish between live and heat-killed mycobacteria as compared to PMA. The viability assay combining the Pt treatment with qPCR thereby proved to be a promising method for the enumeration of viable MAP cells in foodstuffs, environmental, and clinical samples which could replace the time-consuming cultivation or laborious procedures required when using PMA.

Application of the Actiphage® Assay to Detect Viable Mycobacterium avium subsp. paratuberculosis Cells in Fresh Sheep and Goat Milk and Previously Frozen Milk and In-Line Milk Filters.

Mycobacterium avium subsp. paratuberculosis (MAP) is a well-known causative agent of paratuberculosis, a chronic infectious granulomatous enteritis of ruminants contributing to significant economic losses worldwide. Current conventional diagnostic tools are far from being sufficient to manage and control this disease. Therefore, increased attention has been paid to alternative approaches including phage-based assays employing lytic bacteriophage D29 to detect MAP cells. The aim of the present study was to assess the applicability and efficiency of the recently developed phage-based kit termed Actiphage® combined with IS900 real-time PCR (qPCR) for rapid detection and quantification of viable MAP in milk samples. We demonstrated that Actiphage® in combination with IS900 qPCR allows for rapid and sensitive detection and identification of viable MAP in milk samples with a limit of detection of 1 MAP per 50 ml milk. Using this method, the presence of viable MAP cells was successfully determined in 30.77% of fresh goat, sheep and cow milk samples originating from paratuberculosis-affected herds. We further used Actiphage assay to define the time-lapse aspect of testing naturally contaminated milk and milk filters frozen for various lengths of time by phage-based techniques. Viable MAP was detected in 13.04% of frozen milk samples and 28.57% of frozen milk filters using Actiphage-qPCR. The results suggest the ability to detect viable MAP in these samples following freezing for more than 1 year. The obtained results support the views of the beneficial role of this technology in the control or monitoring of paratuberculosis.

Attempted Control of Paratuberculosis in Dairy Calves by Only Changing the Quality of Milk Fed to Calves.

Simple SummaryMilk is an important transmission route of Mycobacterium avium subsp. paratuberculosis (MAP) for dairy calves. The aim of the present study was to assess the efficacy of milk treatments to prevent MAP infection transmission to calves. A one-year field longitudinal study was carried out. Newborn calves were assigned to one of four experimental groups. MAP numbers were estimated for treated and untreated milk. Infection progression was monitored monthly. After one year, calves were euthanized, and tissue samples examined grossly and by histopathology. Milk treatments were highly effective though MAP shedding was also observed in all groups. It was not enough to try to block MAP infection transmission controlling only the quality of milk consumed by calves.One of the important routes of Mycobacterium avium subsp. paratuberculosis (MAP) transmission in dairy calves is milk. The aim of the present study was to assess the efficacy of milk treatments to prevent MAP infection transmission to calves. A one-year longitudinal study was carried out. Newborn calves were assigned to one of four experimental groups: 5 calves received naturally MAP-contaminated milk, 5 calves received copper treated milk, 4 calves were fed calf milk replacer, and 3 were fed UHT pasteurized milk. MAP load in milk was estimated. Infection progression was monitored monthly. After one year, calves were euthanized, and tissue samples were cultured and visually examined. MAP was undetectable in milk replacer and UHT milk. Copper ion treatment significantly reduced the number of viable MAP in naturally contaminated milk. Fecal shedding of MAP was observed in all study groups but began earlier in calves fed naturally contaminated milk. Paratuberculosis control programs must place multiple hurdles between the infection source, MAP-infected adult cows, and the most susceptible animals on the farm, young calves. As our study shows, strict dependence on a single intervention to block infection transmission, no matter how important, fails to control this insidious infection on dairy farms.

Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR

Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, − 20 °C and − 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.

Is the effectivity of copper ions treatment of milk enough to block Mycobacterium avium subsp. paratuberculosis infection in calves?

Milk is an important transmission route of Mycobacterium avium subsp. paratuberculosis (MAP) for dairy calves. Given its resistance to pasteurization, alternative milk treatments are needed to control MAP transmission via milk. The present study reports the evaluation of a novel milk decontamination treatment based on copper ions as a means of preventing infection in dairy calves. Ten newborn calves were assigned to one of two experimental groups (n=5) which were studied for 1 year. The first group was fed milk naturally contaminated with MAP and the second one received the same milk but after being treated with copper ions. In both groups, milk MAP load was estimated. The progression of the infection was monitored monthly and at the end of the study, calves were euthanised, and tissue samples were examined both grossly and by histopathology. The treatment of milk with copper ions significantly reduced the number of viable MAP. Faecal shedding of MAP was observed in both study groups, but the calves fed naturally contaminated milk began to shed MAP earlier. Only calves fed copper-treated milk showed histopathological evidence consistent with MAP infection. The latter offers more questions than answers, and maybe the presence of a more tolerant and virulent MAP strain could be the final answer to this situation.

Effectiveness of copper ions against Mycobacterium avium subsp. paratuberculosis and bacterial communities in naturally contaminated raw cow's milk.

The focus of the present study was to evaluate the copper ions treatment on the viability of Mycobacterium avium subsp. paratuberculosis (MAP) and other bacterial communities in cow's milk. A copper ions treatment was evaluated in naturally contaminated cow's milk to assay MAP load and/or viability, and relative abundance of other bacterial communities. In addition, physical-chemical analyses of the milk were also performed. All analyses were carried out before and after a copper ions treatment. After copper ions treatment, pH and copper concentration markedly increased in milk; the numbers of viable MAP significantly decreased. The relative abundance of the four target phyla decreased, with the phyla Bacteroidetes and Firmicutes surviving treatment in higher proportions (4 and 2·1% of original populations, respectively). A progressively higher percentage of dead bacterial cells after 5 and 20min copper ions treatments was found (12 and 35%, respectively). With the exception of some MAP-tolerant strains, we have once again demonstrated that copper ions have a significant inactivating effect on MAP as well as certain other bacterial communities found in naturally contaminated cow's milk. This study showed a significant inactivation of both MAP and other bacteria by copper ions in raw cow's milk, information that could be useful as a tool for MAP control.

Interaction of Mycobacterium avium subsp. paratuberculosis with bovine sperm

Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Paratuberculosis mainly affecting domestic ruminants. The interaction between MAP and sperm and/or germ cells has not yet been established, however the adherence between MAP and the host cell surface is associated to the 85 complex proteins that bind to the host cell’s fibronectin. Therefore, this study aimed to evaluate the binding of MAP to bovine sperm and to verify changes in these cells by the presence of MAP before and after sperm cryopreservation. Polyclonal antibodies to MAP 85 complex proteins were produced and utilized in the analyzes. Two Nelore bulls were used for semen collection and MAP dilutions (103–108 CFU/mL) were inoculated in the samples; sperm motility and vigor were evaluated using light microscopy at different times before and after cryopreservation and in the presence and absence of the antibodies 85A and 85B. Interaction of MAP and sperm, interaction of MAP and sperm in the presence of Ab 85A and in the presence of Ab 85B were analyzed by scanning electron microscopy. The viability of MAP after sperm cryopreservation were evaluated by plating the samples after thawing. It was observed that sperm in the presence of MAP shows a decrease in motility and vigor, and that the higher the MAP concentration, the lower the sperm performance. It was possible to determine the viability of MAP after cryopreservation in samples of higher concentrations, which demonstrates the potential of transmission of this pathogen through artificial insemination. The interaction of MAP with bovine sperm occurs mainly in the midpiece and may be linked to the proteins 85A and 85B present in the MAP membrane.

Detection of Mycobacterium avium Sub sp. paratuberculosis in Pasteurized Milk Samples in Northeast of Iran by Culture, Direct Nested PCR and PCR Methods

Mycobacterium avium Subsp. paratuberculosis (MAP) is a gram-positive, small, acid-fast bacillus with high environmental resistance. In animals, especially ruminants, it leads to Paratuberculosis (PTB) or Johne's disease, which is chronic granulomatous enteritis. This bacterium as the main causative agent of Crohn's disease can be a serious threat to human health. This study aimed to detect MAP in pasteurized milk samples produced in Khorasan Razavi province, Iran, using Direct Nested PCR, PCR, and culture methods. In this study, 544 milk samples from Pasteurized Milk Production Companies were selected randomly during the 3-month period. DNA was extracted from milk fat after centrifugation. In order to identify the bacteria, Direct Nested PCR and PCR tests were applied using IS900 and f57, respectively. Furthermore, to detect viable MAP, positive samples resulted from Direct Nested PCR assays were cultured on Herrold's egg medium. For identification of mycobacterial isolates, all colonies were processed by PCR based on f57. A total of 544 pasteurized milk samples were assayed, and Mycobacterium paratuberculosis was detected in 39% of them by IS900 Nested PCR, and only 4.9% of samples were positive in the culture method. All the colonies were positive for the f57using PCR. The results of this study indirectly indicated a high level of contamination of pasteurized milk to Mycobacterium paratuberculosis which is due to the large number of affected animals in livestock farms in Khorasan Razavi province. However, in comparison with the other researches, the low percentage of viable bacteria in pasteurized milk can be due to changes in temperature and time in pasteurizing systems of milk production companies in Khorasan Razavi province, Northeast of Iran.

A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows\u2019 milk

Bacteriophage-based methods for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis (MAP) in veterinary specimens are a recent addition to the Johne’s disease diagnostic toolbox. Here, we report the use of D29 mycobacteriophage-coated tosylactivated paramagnetic beads to capture and concentrate MAP cells from samples (termed phagomagnetic separation, PhMS) and then naturally lyse viable MAP cells (from the inside out) to provide DNA for IS900 qPCR purposes. Transmission electron microscopy confirmed that D29 phages had bound to beads in the correct orientation and that the phage-coated beads captured MAP cells from a suspension. During test optimization, conventional IS900 PCR results were used to subjectively assess the effect of different phage:bead coating ratios, differing amounts of coated beads during PhMS, optimal incubation time post-PhMS to obtain maximal MAP DNA, and the potential benefit of a brief heat shock (55 °C/1 min) prior to IS900 TaqMan qPCR. The limit of detection 50% (LOD50%) of the optimised PhMS-qPCR assay was 10.00 MAP cells/50 ml milk (95% CI 1.20–82.83). Finally, in order to demonstrate the new assay’s ability to detect viable MAP in naturally contaminated milk, bulk tank milk samples from 100 dairy farms were tested. Forty-nine (49%) of these tested PhMS-qPCR-positive, with viable MAP numbers detected ranging from 3–126 MAP/50 ml. The novel PhMS-qPCR assay is a sensitive, specific and easy-to-apply phage-based assay for viable MAP, with potential application for milk surveillance or diagnosis of Johne’s disease.Key points• Phage-coated magnetic beads could capture, concentrate and lyse MAP cells from milk.• PhMS-qPCR assay proved to be a rapid, sensitive and specific test for viable MAP.• A potential application of PhMS-qPCR assay for milk surveillance was demonstrated.

Assessing efficacy of N-Acetyl-l-Cysteine–Sodium Hydroxide on bacterial viability and enhanced recovery of Mycobacterium avium subsp. paratuberculosis from bovine colostrum

The standard procedure for the improved cultural recovery of viable Mycobacterium spp. from diverse samples mainly depends on reducing the viability of background microbiota using different chemical compounds. This study was designed to i) evaluate the efficacy and comparison between N-Acetyl-l-Cysteine–Sodium hydroxide (NALC-2% NaOH) and hexadecylpyridinium chloride (0.75% HPC) treatment and exposure time on reducing the viability of undesirable microorganisms with minimal impact on colostrum consistency; and ii) assess the impact of NALC-2% NaOH on improved and enhanced recovery of Mycobacterium avium subsp. paratuberculosis (MAP) in spiked postpartum colostrum samples and consistency of colostrum. A total of 40 samples, each treated with NALC-2% NaOH for 15 min or 0.75% HPC for 5 h, were investigated for total mesophilic aerobic bacteria (MAB) and enterobacteria (EB) (CFU mL−1). The results showed that treatment of colostrum samples with NALC-2% NaOH completely eliminated EB and significantly reduced MAB (3.6 log10 CFU mL−1). Conversely, samples treated with 0.75% HPC produced a complex mixture following interaction with the colostrum protein and showed non-significant and variable results. In addition, the spiked colostrum treated with NALC-2% NaOH for 15 min revealed recovery of viable MAP cells with a minimum limit of detection of 1.36 log10 CFU 10 mL−1 where no change in the consistency of colostrum was observed. In conclusion, 15-min NALC-2% NaOH treatment of colostrum may significantly reduce the viability of undesirable microorganisms and help to enhance the efficient recovery of MAP without impacting the consistency of high quality postpartum colostrum. This rapid procedure is suitable for efficient recovery and early detection of MAP as well as preventing its transmission to neonates and young calves in MAP infected herds.

The specificity of phage testing for MAP — where might it fit into the diagnostic armoury?

The current individual tools available for the diagnosis of Johne's disease are far from suitable to tackle this endemic disease. Culture, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) tests, when used together can be useful in managing the disease in the later stages of infection at a herd level. They are, however, ill-suited to detecting the causative agent Mycobacterium avium subsp. paratuberculosis (MAP) at the early stages of infection and at an individual level. Phage technology offers another tool in the attempt to better manage and control this disease. Phage-technology has been demonstrated to rapidly and sensitively detect and specifically identify viable MAP in the milk and blood of cattle. Although in relatively-early stages of development phage technology offers a strong addition to the armoury of tests used to detect MAP in blood and milk, and may go on to be part of ongoing control measures to reduce the burden of disease to farmers and veterinarians.

Fate of Mycobacterium avium subsp. paratuberculosis and changes in bacterial diversity populations in dairy slurry after chemical treatments.

A major drawback of using dairy slurry as fertilizer is that it may contains pathogens such as Mycobacterium avium subsp. paratuberculosis (MAP), and it could represent a risk to animal and public health. Thus, the aim of this study was to evaluate the fate of MAP and bacterial communities in dairy slurry after chemical treatments. Cattle slurry, naturally contaminated with MAP, was collected from a dairy herd and divided into 32 glass bottles which were assigned to eight different treatments (control, 3·0% CaO, 0·5% NaOH; 0·087%, 0·11% and 0·14% H2 SO4 ; and 1·0 and 2·5% KMnO4 ). Treated dairy slurry samples were evaluated at 0, 1, 3, 7, 15, 30 and 60-days following treatment application for viable MAP and dairy slurry pH, and in addition temperature in this material was monitored continuously. Bacterial counts were estimated at each sampling time. A Bayesian zero-inflated Poisson mixed model was fitted to assess the effect of each treatment on the count of MAP cells. Model results indicated that only the 3·0% CaO treatment had a statistically important negative effect on MAP counts during the study period. For most treatments, MAP was undetectable immediately after chemical treatment but re-appeared over time, in some replicates at low concentrations. However, in those cases MAP counts were not statistically different than the control treatment. Regarding the fate of the other bacterial populations, the Firmicutes phylum was the dominant population in the un-treated slurry while Clostridia class members were among the most prevalent bacteria after the application of most chemical treatments. Only 3% CaO treatment had a statistically important negative effect on MAP viability in cattle slurry. This study provides evidence of MAP partial control in dairy slurry. This information should be considered as a best management practice to reduce MAP and other pathogens for slurry management on dairy farms.

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.