Culture of Mycobacterium avium subsp. paratuberculosis: challenges, limitations and future prospects.

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants and is suspected to be involved in the development of Crohn's disease and several autoimmune disorders. As such, sensitive and specific MAP detection methods are required to confirm infection in animals and identify potential sources of animal and human exposure. Despite recent developments in immunological and nucleic acid-based detection methods, culture-based detection of MAP remains the 'gold standard' against which the sensitivity and specificity of other detection methods are measured. However, not all culture-based approaches are equivalent in terms of detection capability, which can lead to errors in the evaluation of other detection methods. This review will provide an overview of the chronological development of culture methods for MAP, and will consider the unique growth requirements of MAP, the merits of solid versus liquid culture media, the relative performance of the commonly used MAP culture media, and sample preparation/decontamination protocols for different sample types. The limitations of current MAP culture methods and prospects for improvements are discussed.

Optimization of the composition of a solid culture medium for Mycobacterium avium subsp. paratuberculosis using factorial design and response surface methodology.

AIM: To develop an optimized solid culture medium for improved growth of Mycobacterium avium subsp. paratuberculosis (MAP). METHODS AND RESULTS: Seven medium constituents (factors) were assessed at various concentrations for their ability to positively affect MAP growth. The factors tested were Tween 80, egg yolk, casitone, taurocholic acid, Mycobactin J, agar and either OADC or ADC supplement. After an initial screening of individual factors, a fractional factorial design and a response surface methodology (RSM) central composite design were used to assess the effects of multiple factors simultaneously and design a new solid culture medium. MAP growth became visible on streak plates of the optimized solid medium 2 weeks earlier than on Herrold's egg yolk medium (HEYM). CONCLUSIONS: MAP grew faster on the optimized solid medium than on HEYM. It consisted of Middlebrook 7H9 broth with 1.0% Tween 80, 0.019% casitone, 1.4% bacteriological agar, 10% egg yolk, 10% ADC and 1.65 µg ml-1 Mycobactin J. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to use an RSM approach to optimize the composition of a solid medium for MAP culture. The new medium could improve MAP culture in future by reducing incubation times and increasing MAP colony numbers.

Detection of influenza D virus in bovine respiratory disease samples, UK.

Influenza D is a newly described virus of cattle, pigs and small ruminants first detected in North America during 2011. Cattle have been shown to be the main viral reservoir and mounting evidence indicates that infection with influenza D may contribute to the development of bovine respiratory disease. The virus has been detected across the United States, Europe and Asia. To date, influenza D has not been reported in the UK. During the winter and spring of 2017/2018, we performed molecular testing of cattle submitted for post-mortem examination where respiratory disease signs were present. We detected influenza D virus in 8.7% of cases, often as the sole viral agent and always in conjunction with bacterial co-infection with one or more agents. Viral RNA was present in both the upper and lower respiratory tract and pathological changes in lung tissues were observed alongside signs of concurrent bacterial infections. Sequencing of one UK isolate revealed that it is similar to viruses from the Republic of Ireland and Italy.