Preliminary validation of Mycobacterium avium subspecies paratuberculosis detection in ovine pooled faecal culture and species verification by PCR

Abstract

Paratuberculosis interchangeably referred to as Johne’s disease (JD), is a condition in ruminants due to infection with Mycobacterium avium subspecies paratuberculosis (MAP). The infection is acquired by animals feeding on food contaminated with faeces of an excreting infected animal. The term “wasting” describes the condition with which is marked by progressive emaciation and weakening of the infected animal. The disease is incurable and culling is currently the only solution. The infected flocks are worthless to the national and international trade and cause a strain to the agricultural sector. Paratuberculosis is a controlled disease in South Africa, yet remains an unsolved and increasing problem. MAP is currently only diagnosed serologically, but the tests are ineffective due to low sensitivity. Asymptomatically infectious animals are the major stumbling block to manage the disease. It is equally difficult to diagnose it and to date no one method gives a 100% detection rate. Culture is the gold standard method not withstanding its lengthy (16 weeks or longer) turnaround time, but liquid culture medium can reduce this time. This study was designed to evaluate an internationally recognised method (automated liquid culture) that is used as a tool to combat the disease in a South African setting. The ability of the liquid culture method to detect SA MAP strain was assessed by collecting 284 samples of either tissue, faecal or both samples from herds that are known to be infected with MAP. While the ability to distinguish faecal samples not infected with MAP was assessed by collecting 50 faecal samples from herds that have not had occurrence of MAP infection. The samples were processed and cultured in the Versa Trek liquid culture system for a maximum of 70 days, growth signals or no growth signals were further assessed with molecular method to confirm the signal. The detection limit of the method was evaluated using the spiking and serial dilution method. The reproducibility of the liquid culture method was assessed through proficiency testing and precision. Finally, the sensitivity and specificity of the liquid culture method was determined by comparison with the standard reference method, histopathology. In this study 16 MAP isolates (7 faecal and 9 tissue samples) were isolated and confirmed of the 284 cultured samples from flocks with an existing MAP infection. Thirty-five growth signals were detected from flocks that have not had an occurrence of MAP infection and none of these were MAP DNA positive. The detection limit in individual faecal samples and pooled faecal samples was 1.08x104(2) MAP/ml and pool of 10 faecal samples respectively. The threshold for the detection limit is reduced when the recommended antibiotic supplement is excluded. Reproducibility of the method on average was 79.1%% and precision was inconclusive due to the shortage of original samples. Lastly, the sensitivity & specificity of the liquid culture method against the standard reference method was determined to be 33% & 25%, 97% & 98% for faecal and tissue culture, respectively. On the other hand, for tissue culture, the sensitivity increased to 100% when we excluded the antibiotic supplement. In addition, the study demonstrated that there might be a strong inhibitory effect posed by the antibiotic supplement on the SA MAP faecal isolated strain. This needs further assessment using larger sample size and antibiotic profiling. In conclusion, the study did demonstrate that the method can detect SA MAP strain but this is limited for the faecal isolates. By this, the study has emphasised the importance of validating a diagnostic method from different geographic regions. When dealing with bacteria, the behaviour of type species may differ from one location to another and we have demonstrated that this can possibly fall short as a method in one region while it may succeed in another, therefore, validation of a method for its intended use is imperative. The determined pool size will be valuable in a screening process part of a control program, offer reduced cost associated with individual sample processing. Validation is a continuous process and the information attained in this study will form a foundation for this and the validated method will be applied routinely as a diagnostic service offered by the Tuberculosis laboratory.