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.

Mycobacterium avium subsp. paratuberculosis Virulence: A Review

To propose a solution for control of Mycobacterium avium subsp. paratuberculosis (MAP) infections in animals as well as in humans, and develop effective prevention, diagnostic and treatment strategies, it is essential to understand the molecular mechanisms of MAP pathogenesis. In the present review, we discuss the mechanisms utilised by MAP to overcome the host defense system to achieve the virulence status. Putative MAP virulence genes are mentioned and their probable roles in view of other mycobacteria are discussed. This review provides information on MAP strain diversity, putative MAP virulence factors and highlights the knowledge gaps regarding MAP virulence mechanisms that may be important in control and prevention of paratuberculosis.

Evaluation of real time PCR for the detection of Mycobacterium avium subsp. paratuberculosis in faecal samples of cattle

The efficiency and suitability of a MAP F57 based SYBR Green qPCR assay for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) using a set of known MAP positive (12) and MAP negative (23) DNA samples that were previously identified by conventional IS 900 PCR were assessed. These DNA samples were isolated in our previous study from faecal samples collected from cattle in the livestock farms under government sector with a previous history of Johne’s disease. The MAP F57 qPCR was able to identify all the positive samples accurately and rapidly with Cq values ranging from 20-29. The efficiency of qPCR using recombinant plasmid for standard curve was 0.991 and limit of detection was 10 MAP organisms per microlitre of DNA sample.

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.

Análisis espacial y ambiental de lecherías infectadas con Mycobacterium avium subsp. paratuberculosis en Antioquia (Colombia)

<p>El presente estudio tuvo como objetivo describir la distribución espacial de Mycobacterium avium subsp paratuberculosis (MAP) en hatos lecheros, y detallar variables ambientales tomadas como referencia de los antecedentes físicos del área de estudio, específicamente aquellas relacionadas con los hatos positivos por MAP-qPCR, ubicados en seis municipios de la región norte de la Provincia de Antioquia (Colombia), de acuerdo con el muestreo ambiental y análisis por qPCR. Los hatos del estudio (n = 386) se ubicaron en 63 distritos diferentes de seis municipios. Los hatos participantes fueron visitados una vez entre junio y octubre (2016) para recolectar una muestra ambiental, y la identificación de MAP se logró utilizando un método de PCR cuantitativa dúplex en tiempo real. Las tendencias de lluvia, la temperatura de la superficie diurna y nocturna, y el índice de cobertura vegetal se tomaron como referencias ambientales del entorno físico del área de estudio. Además, se construyeron mapas de distribución de hatos positivos y negativos a MAP-qPCR, así como mapas de variaciones de temperatura y cobertura vegetal. Como resultado, hubo un aumento en los hatos positivos para MAP en el noroeste, sur y sudeste del área de estudio. Se encontró un régimen general de alta precipitación y las temperaturas superficiales diurnas y nocturnas mostraron variaciones importantes durante los meses de muestreo. No se encontró evidencia de manejo de la cubierta vegetal, tanto en pastizales como en áreas con vegetación nativa, excepto en un área de conservación. En conclusión, se reportan las condiciones Recibido: mmmm_AAAA / Aceptado: mmmm_AAAA 11<br />ambientales generales, donde es más probable que ocurra la detección de hatos positivos<br />para MAP, considerando enfoques que utilizan el mismo método de recolección (o uno muy<br />aproximado), el manejo de muestras y el método de detección molecular.</p>