Divergent Antigen-Specific Cellular Immune Responses during Asymptomatic Subclinical and Clinical States of Disease in Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis.

Infection of the host with Mycobacterium avium subsp. paratuberculosis results in chronic and progressive enteritis that traverses both subclinical and clinical stages. The mechanism(s) for the shift from an asymptomatic subclinical disease state to advanced clinical disease is not fully understood. In the present study, naturally infected dairy cattle were divided into subclinical and clinical infection groups, along with noninfected control cows of similar parity, to study host immune responses in different stages of infection. Both infection groups had higher levels of secretion of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) than control cows, whereas only clinical cows had increased secretion of IL-10, IL-12, and IL-18 upon stimulation of peripheral blood mononuclear cells (PBMCs) with antigen. Conversely, secretion of IL-17Α was decreased for clinical cows compared to subclinical and control cows. Proinflammatory cytokine genes were upregulated only for subclinical cows, whereas increased IL-10 and IL-17 gene expression levels were observed for both infection groups. Increased CD4+, CD8+, and γδ T cell receptor-positive (TCR+) T cells were observed for subclinical cows compared to clinical cows. Although clinical cows expressed antigen-specific immune responses, the profile for subclinical cows was one of a dominant proinflammatory response to infection. We reason that a complex coordination of immune responses occurs during M. avium subsp. paratuberculosis infection, with these responses shifting as the host transitions through the different stages of infection and disease (subclinical to clinical). A further understanding of the series of events characterized by Th1/Th2/Th17 responses will provide mechanisms for disease progression and may direct insightful intervention strategies.

Mycobacterium avium subspecies paratuberculosis is able to manipulate host lipid metabolism and accumulate cholesterol within macrophages.

Johne's disease is a chronic wasting disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Closely related pathogenic mycobacteria such as M. tuberculosis are capable of altering host lipid metabolism, highlighting the need to explore the role of lipid metabolism contributing to intracellular survival. This study aimed to identify whether MAP is able to manipulate host lipid metabolic pathways and accumulate host cholesterol during early infection. Macrophages were exposed to four different MAP strains and non-pathogenic M. phlei for up to 72 h, with changes to lipid metabolism examined using fluorescent microscopy and gene expression. MAP-infected macrophages displayed strain-dependent differences to intracellular cholesterol levels during early infection, however showed similarly increased intracellular cholesterol at later timepoints. Gene expression revealed that MAP strains similarly activate the host immune response in a conserved manner compared to M. phlei. MAP significantly upregulated host genes associated with lipid efflux and endocytosis. Moreover, lipid biosynthesis genes were differentially regulated in a strain-dependent manner following MAP infection. Collectively, these results demonstrate that MAP manipulates host lipid metabolism during early infection, however the extent of these modulations are strain-dependent. These findings reflect a conserved pathway contributing to intracellular MAP survival.

Deciphering the virulence of Mycobacterium avium subsp. paratuberculosis isolates in animal macrophages using mathematical models.

Understanding why pathogenic Mycobacterium avium subsp. paratuberculosis (Map) isolates cause disparate disease outcomes with differing magnitudes of severity is important in designing and implementing new control strategies. We applied a suite of mathematical models: i) general linear, ii) and neurofuzzy logic, to explain how the host of origin of several Map isolates, Map genotype, host, macrophage-based in vitro model and time post-infection contributed to the infection. A logistic growth ordinary differential equation (ODE) model was applied to estimate within macrophage growth rates for the different Map isolates. The models revealed different susceptibilities of bovine and ovine macrophages to Map infection and confirmed distinct virulence profiles for the isolates, judged by their ability to grow within macrophages. Ovine macrophages were able to internalize Map isolates more efficiently than bovine macrophages. While bovine macrophages were able to internalize Map isolates from cattle with more efficiency, ovine macrophages were more efficient in internalizing ovine isolates. Overall, Map isolates from goat and sheep grew minimally within macrophages or did not grow but were able to persist by maintaining its initial population. In contrast, the ability of the bovine isolates and the non-domesticated animal isolates to grow to higher CFU numbers within macrophages suggests that these isolates are more virulent than the sheep and goat isolates, or that these isolates are better adapted to infect domestic ruminants. Overall, our study confirms the different virulence levels for the Map isolates and susceptibility profiles of host macrophages, which is crucial in increasing our understanding of Map infection.

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.

Survival of Mycobacterium avium subspecies paratuberculosis in retail pasteurised milk.

A survey of retail purchased semi-skimmed pasteurised milk (n = 368) for Mycobacterium avium subspecies paratuberculosis (MAP) was conducted between May 2014 and June 2015 across the midlands of England using the Phage-PCR assay. Overall, 10.3% of the total samples collected contained viable MAP cells, confirming that pasteurisation is not capable of fully eliminating human exposure to viable MAP through milk. Comparison of the results gained using the Phage-PCR assay with the results of surveys using either culture or direct PCR suggest that the phage-PCR assay is able to detect lower numbers of cells, resulting in an increase in the number of MAP-positive samples detected. Comparison of viable count and levels of MAP detected in bulk milk samples suggest that MAP is not primarily introduced into the milk by faecal contamination but rather are shed directly into the milk within the udder. In addition results detected an asymmetric distribution of MAP exists in the milk matrix prior to somatic cell lysis, indicating that the bacterial cells in naturally contaminated milk are clustered together and may primarily be located within somatic cells. These latter two results lead to the hypothesis that intracellular MAP within the somatic cells may be protected against heat inactivation during pasteurisation, accounting for the presence of low levels of MAP detected in retail milk.

Environmental sample characteristics and herd size associated with decreased herd-level prevalence of Mycobacterium avium ssp. paratuberculosis.

Environmental sampling is an effective method for estimating regional dairy herd-level prevalence of infection with Mycobacterium avium ssp. paratuberculosis (MAP). However, factors affecting prevalence estimates based on environmental samples are not known. The objective was to determine whether odds of environmental samples collected on farm changed culture status over 2 sampling times and if changes were specific for location and type of housing (freestall, tiestall, or loose housing), the sample collected (i.e., manure of lactating, dry, or sick cows; namely, cow group), and effects of herd size. In 2012-2013 [sampling 1 (S1)] and 2015-2017 [sampling 2 (S2)], 6 environmental samples were collected and cultured for MAP from all 167 (99%) and 160 (95%) farms, respectively, in the province of Saskatchewan, Canada. Only the 148 dairy farms sampled at both sampling periods were included in the analysis. A mixed effects logistic regression was used to determine whether differences between sampling periods were associated with herd size and sample characteristics (cow group contributing to environmental sample, type of housing, and location). In S1 and S2, 55 and 34%, respectively, of farms had at least 1 MAP-positive environmental sample. Correcting for sensitivity of environmental sampling, estimated true prevalence in S1 and S2 was 79 and 48%, respectively. Herds with >200 cows were more often MAP-positive than herds with <51 cows in both S1 and S2. The percentage of positive samples was lower in S2 compared with S1 for all sampled areas, cow groups contributing to samples, types of housing where samples were collected, and herd size categories. However, samples collected from dry cow areas had the largest decrease in MAP-positive samples in S2 compared with all other cow group samples. Herds that were MAP-negative in S1 with a herd size 51 to 100 or 101 to 150 were more likely to stay MAP-negative, whereas MAP-positive herds with >200 cows more frequently stayed MAP-positive. No difference was observed in the odds of a sample being MAP-positive among housing types or location of sample collection in both sample periods. Of all farms sampled, 104 (70%) did not change status from S1 to S2. In conclusion, when herd-level MAP prevalence decreased over the 3-yr interval, the change in prevalence differed among herd size categories and was larger in samples from dry cow areas. It was, however, not specific to other characteristics of environmental samples collected.

In vitro bioassessment of the immunomodulatory activity of Saccharomyces cerevisiae components using bovine macrophages and Mycobacterium avium ssp. paratuberculosis.

The yeast Saccharomyces cerevisiae and its components are used for the prevention and treatment of enteric disease in different species; therefore, they may also be useful for preventing Johne's disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). The objective of this study was to identify potential immunomodulatory S. cerevisiae components using a bovine macrophage cell line (BOMAC). The BOMAC phagocytic activity, reactive oxygen species production, and immune-related gene (IL6, IL10, IL12p40, IL13, IL23), transforming growth factor ß, ARG1, CASP1, and inducible nitric oxide synthase expression were investigated when BOMAC were cocultured with cell wall components from 4 different strains (A, B, C, and D) and 2 forms of dead yeast from strain A. The BOMAC phagocytosis of mCherry-labeled MAP was concentration-dependently attenuated when BOMAC were cocultured with yeast components for 6 h. Each yeast derivative also induced a concentration-dependent increase in BOMAC reactive oxygen species production after a 6-h exposure. In addition, BOMAC mRNA expression of the immune-related genes was investigated after 6 and 24 h of exposure to yeast components. All yeast components were found to regulate the immunomodulatory genes of BOMAC; however, the response varied among components and over time. The in vitro bioassessment studies reported here suggest that dead yeast and its cell wall components may be useful for modulating macrophage function before or during MAP infection.

Bovine peripheral blood WC1+ and WC1neg γδ T lymphocytes modulate monocyte-derived macrophage effector functions during in vitro Mycobacterium avium subspecies paratuberculosis infection.

The importance of bovine γδ T lymphocytes during anti-mycobacterial immunity is recognized; however, the role of major subsets of γδ T lymphocytes (WC1+ and WC1neg) in this process remains unclear. We investigated how WC1+ and WC1neg γδ T lymphocyte subsets of calves modulate monocyte-derived macrophage (MDM) functions during Map infection in vitro. To achieve this, Map-infected or uninfected MDMs from young calves were co-cultured with autologous WC1+ or WC1neg γδ T lymphocytes. Our data indicate that WC1+ and WC1neg γδ T lymphocytes of young calves modulate effector functions of MDMs with respect to Map killing, CD11b and MHC-II expression. We observed differences in IFN-γ production and CD25 expression on γδ T lymphocyte subsets, as well as MDM expression of CD1b when in contact with WC1neg γδ T lymphocytes.

Genetic diversity of Mycobacterium avium subspecies paratuberculosis and the influence of strain type on infection and pathogenesis: a review.

Mycobacterium avium subspecies paratuberculosis (Map) is an important pathogen that causes a chronic, progressive granulomatous enteritis known as Johne's disease or paratuberculosis. The disease is endemic in many parts of the world and responsible for considerable losses to the livestock and associated industries. Diagnosis and control are problematic, due mostly to the long incubation period of the disease when infected animals show no clinical signs and are difficult to detect, and the ability of the organism to survive and persist in the environment. The existence of phenotypically distinct strains of Map has been known since the 1930s but the genetic differentiation of Map strain types has been challenging and only recent technologies have proven sufficiently discriminative for strain comparisons, tracing the sources of infection and epidemiological studies. It is important to understand the differences that exist between Map strains and how they influence both development and transmission of disease. This information is required to develop improved diagnostics and effective vaccines for controlling Johne's disease. Here I review the current classification of Map strain types, the sources of the genetic variability within strains, growth characteristics and epidemiological traits associated with strain type and the influence of strain type on infection and pathogenicity.

Two state model for a constant disease hazard in paratuberculosis (and other bovine diseases).

Many diseases are characterized by a long and varying sub-clinical period. Two main mechanisms can explain such periods: a slow progress toward disease or a sudden transition from a healthy state to a disease state induced by internal or external events. We here survey epidemiological features of the amount of bacteria shed during Mycobacterium Avium Paratuberculosis (MAP) infection to test which of these two models, slow progression or sudden transition (or a combination of the two), better explains the transition from intermittent and low shedding to high shedding. Often, but not always, high shedding is associated with the occurrence of clinical signs. In the case of MAP, the clinical signs include diarrhea, low milk production, poor fertility and eventually emaciation and death. We propose a generic model containing bacterial growth, immune control and fluctuations. This proposed generic model can represent the two hypothesized types of transitions in different parameter regimes. The results show that the sudden transition model provides a simpler explanation of the data, but also suffers from some limitations. We discuss the different immunological mechanism that can explain and support the sudden transition model and the interpretation of each term in the studied model. These conclusions are applicable to a wide variety of diseases, and MAP serves as a good test case based on the large scale measurements of single cow longitudinal profiles in this disease.

Synthesis and Biological Evaluation of N-Alkoxyphenyl-3-hydroxynaphthalene-2-carboxanilides.

A series of fifteen new N-alkoxyphenylanilides of 3-hydroxynaphthalene-2-carboxylic acid was prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Staphylococcus aureus, three methicillin-resistant S. aureus strains, Mycobacterium tuberculosis H37Ra and M. avium subsp. paratuberculosis. Some of the tested compounds showed antibacterial and antimycobacterial activity against the tested strains comparable with or higher than that of the standards ampicillin or rifampicin. 3-Hydroxy-N-(2-propoxyphenyl)naphthalene-2-carboxamide and N-[2-(but-2-yloxy)-phenyl]-3-hydroxynaphthalene-2-carboxamide had MIC = 12 µM against all methicillin-resistant S. aureus strains; thus their activity is 4-fold higher than that of ampicillin. The second mentioned compound as well as 3-hydroxy-N-[3-(prop-2-yloxy)phenyl]-naphthalene-2-carboxamide had MICs = 23 µM and 24 µM against M. tuberculosis respectively. N-[2-(But-2-yloxy)phenyl]-3-hydroxynaphthalene-2-carboxamide demonstrated higher activity against M. avium subsp. paratuberculosis than rifampicin. Screening of the cytotoxicity of the most effective antimycobacterial compounds was performed using THP-1 cells, and no significant lethal effect was observed for the most potent compounds. The compounds were additionally tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. N-(3-Ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide (IC50 = 4.5 µM) was the most active PET inhibitor. The structure-activity relationships are discussed.

Isolation and characterization of a novel bacteriophage against Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, has a doubling time of 24 hours, making rapid detection very difficult. Mycobacteriophages can be used in the detection of disease-causing mycobacteria such as MAP. Isolation and sequencing the genomes of lytic MAP bacteriophages are important preliminary steps towards designing phage-based rapid detection assays for this bacterium. A simple optimized protocol was developed to allow reproducible production of confluent growth of MAP on plates within four to six weeks of incubation at 30 °C. This protocol was applied to the screening of environmental and fecal samples for bacteriophages inhibiting the growth of MAP. As a result, a lytic phage, vB_MapS_FF47, was isolated from bovine feces. FF47 contains a double-stranded DNA genome ~48 kb in length with 73 protein coding sequences. It does not carry temperate or known virulence genes. This phage was shown to be most closely related to Mycobacterium phage Muddy, isolated in South Africa, and Gordonia phage GTE2; however, it could not infect any of the tested Gordonia, Rhodococcus, or Nocardia spp. that GTE2 could. The protocols that were developed for growth and phage isolation have potential applications in a high-throughput screening for compounds inhibiting the growth of MAP. This work describes the first time that a phage was isolated against M. paratuberculosis.

Optimization of hexadecylpyridinium chloride decontamination for culture of Mycobacterium avium subsp. paratuberculosis from milk.

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.

Development and validation of a liquid medium (M7H9C) for routine culture of Mycobacterium avium subsp. paratuberculosis to replace modified Bactec 12B medium.

Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johne's disease in ruminants. In Australia, New Zealand, the United States, and some other countries, the Bactec 460 system with modified Bactec 12B medium (Becton, Dickinson) has been the most commonly used liquid culture system, but it was discontinued in 2012. In this study, a new liquid culture medium, M7H9C, was developed. It consists of a Middlebrook 7H9 medium base with added Casitone, albumin, dextrose, catalase, egg yolk, mycobactin J, and a cocktail of antibiotics. We found that polyoxyethylene stearate (POES) was not essential for the cultivation of M. avium subsp. paratuberculosis in either the Bactec 12B or the M7H9C medium. The limit of detection determined using pure cultures of the C and S strains of M. avium subsp. paratuberculosis was 7 bacilli per 50 µl inoculum in the two media. The new medium was validated using 784 fecal and tissue samples from sheep and cattle, >25% of which contained viable M. avium subsp. paratuberculosis. Discrepant results for the clinical samples between the two media were mostly associated with samples that contained <10 viable bacilli per gram, but these results were relatively uncommon, and the performances of the two media were not significantly different. M7H9C medium was less than half the cost of the Bactec 12B medium and did not require regular examination during incubation, but a confirmatory IS900 PCR test had to be performed on every culture after the predetermined incubation period.

Sensitivity of Mycobacterium avium subsp paratuberculosis, Escherichia coli and Salmonella enterica serotype Typhimurium to low pH, high organic acids and ensiling.

AIMS: To evaluate the persistence of Mycobacterium avium subsp paratuberculosis (Myco. paratuberculosis), Salmonella enterica serotype Typhimurium (Salm.Typhimurium) and a commensal Escherichia coli (E. coli) isolate under the low pH and high organic acid (OA) conditions of ensiling of forages. METHODS AND RESULTS: Decay rates and the time required to obtain a 90% reduction in cell concentration were calculated following (i) exposure to buffered OA (pH 4.0, 5.0, 6.0 or 7.0) (ii) exposure to silage exudates and (iii) survival through ensiling of forage materials. Salm. Typhimurium had higher decay rates in silage exudates (-0.5601 day(-1) ) than did E. coli (-0.1265 day(-1) ), but both exhibited lower decay rates in silage than in OA or silage exudates. Myco. paratuberculosis showed no decrease in silage and decay rates in silage exudates were significantly lower (2-12 times) than for the other two organisms. CONCLUSIONS: Escherichia coli, Salm. Typhimurium and Myco. paratuberculosis exhibit marked differences in response to acidity. All three organisms show acid resistance, but Myco. paratuberculosis in particular, if present in manure and applied to forage grasses, may survive the low pH and high OA of the ensilaging process; silage may therefore be a potential route of infection if ingested by a susceptible animal. SIGNIFICANCE AND IMPACT OF STUDY: This information contributes to the understanding of potential risks associated with silage preservation and contamination of livestock feed with manure-borne pathogens.

Culture- and quantitative IS900 real-time PCR-based analysis of the persistence of Mycobacterium avium subsp. paratuberculosis in a controlled dairy cow farm environment.

The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 10(3) were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 10(2) after 24 months. M. avium subsp. paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable.

Genetic parameters for paratuberculosis infection and effect of infection on production traits in Israeli Holsteins.

Paratuberculosis (Johne's disease) is an infectious enteric disease in dairy cattle and other species that causes substantial economic loss worldwide. In this study, two recursive Gaussian-threshold models were employed in order to infer the effects of Johne's disease on milk yield, fat yield, and protein yield while simultaneously estimating genetic parameters (i.e. heritability and genetic correlation) in an Israeli Holstein population. Disease diagnosis was based on ELISA serum antibody tests. Data were available for 4694 daughters of 361 sires; 3.5% were positive; and 1.6% were suspect for the disease test. Disease status was coded either as a binary character (negative vs. positive) or as an ordered categorical trait (negative, suspect, and positive) in the two recursive models and as a binary trait in a linear model. Among sires with ≥ 50 daughters, predicted probability of Mycobacterium avium ssp. paratuberculosis (MAP) infection in future daughters ranged from <1% to 16.5%. Heritability estimates for Johne's disease were near 0.15, confirming a genetic contribution to disease susceptibility. Genetic correlation estimates for Johne's disease with the three yield traits were 0.15-0.22. Residual correlations for Johne's disease with the yield traits were between -0.01 and -0.10. For the linear regression model, yield losses associated with a positive disease diagnosis during 305 days of lactation were 300 kg milk and around 10 kg for fat and protein. Yield loss estimates from the recursive models were 25-50% less than linear model estimates. Recursive modeling has theoretical advantages over linear models for these phenotypes, but the estimated genetic parameters in this study did not differ significantly between the two types of models.

Bovine paratuberculosis: a review of the advantages and disadvantages of different diagnostic tests.

Paratuberculosis (PTB), or Johne's disease, is a chronic infectious granulomatous enteritis of ruminants, caused by Mycobacterium avium subspecies paratuberculosis (Map). It is characterized by diarrhea and progressive cachexia, which may cause the death of the animal. Calves are the most susceptible to infection. Infected animals excrete Map mainly by the feces. PTB is endemic worldwide, with high prevalence levels, strong economic impact and public health relevance because of its possible association with Crohn's disease. Although the current reference diagnostic test is identification of Map in the bacterial culture, there are different diagnostic tests to identify infected individuals and/or herds. The sensitivity and specificity of these tests vary according to the stage of the disease in the animals to be evaluated. The correct choice and application of each of these diagnostic tests will ensure their success and may allow to establish a control program. The aim of this work is to review and discuss the different diagnostic tests used in the detection of Map-infected animals, focusing on their advantages and disadvantages.

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.

Estimation of Mycobacterium avium subsp. paratuberculosis growth parameters: strain characterization and comparison of methods.

The growth rate of Mycobacterium avium subsp. paratuberculosis was assessed by different methods in 7H9 medium supplemented with OADC (oleic acid, albumin, dextrose, catalase), Tween 80, and mycobactin J. Generation times and maximum specific growth rates were determined by wet weight, turbidometric measurement, viable count, and quantitative PCR (ParaTB-Kuanti; F57 gene) for 8 M. avium subsp. paratuberculosis strains (K10, 2E, 316F, 81, 445, 764, 22G, and OVICAP 49). Strain-to-strain differences were observed in growth curves and calculated parameters. The quantification methods gave different results for each strain at specific time points. Generation times ranged from an average of 1.4 days for viable count and qPCR to approximately 10 days for wet weight and turbidometry. The wet-weight, turbidometry, and ParaTB-Kuanti qPCR methods correlated best with each other. Generally, viability has been assessed by viable count as a reference method; however, due to M. avium subsp. paratuberculosis clumping problems and the presence of noncultivable M. avium subsp. paratuberculosis cells, we conclude that qPCR of a single-copy gene may be used reliably for rapid estimation of M. avium subsp. paratuberculosis bacterial numbers in a sample.