African swine fever virus (ASFV) is the causative agent of a deadly disease in pigs and is spread rapidly across borders. Samples collected from suspected cases must be sent to the reference laboratory for diagnosis using polymerase chain reaction (PCR). In this study, we aimed to develop a simple DNA isolation step and real-time recombinase polymerase amplification (RPA) assay for rapid detection of ASFV. RPA assay based on the p72 encoding B646L gene of ASFV was established. The assays limit of detection and cross-reactivity were investigated. Diagnostic performance was examined using 73 blood and serum samples. Two extraction approaches were tested: silica-column-based extraction method and simple non-purification DNA isolation (lysis buffer and heating, 70 °C for 20 min). All results were compared with well-established real-time PCR. In a field deployment during a disease outbreak event in Uganda, 20 whole blood samples were tested. The assay’s analytical sensitivity was 3.5 DNA copies of molecular standard per µL as determined by probit analysis on eight independent assay runs. The ASFV RPA assay only detected ASFV genotypes. Compared to real-time PCR, RPA diagnostic sensitivity and specificity were 100%. Using the heating/lysis buffer extraction procedure, ASFV-RPA revealed better tolerance to inhibitors than real-time PCR (97% and 38% positivity rate, respectively). In Uganda, infected animals were identified before the appearance of fever. The ASFV-RPA assay is shown to be as sensitive and specific as real-time PCR. Moreover, the combination of the simple extraction protocol allows its use at the point of need to improve control measures.

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.

Knowledge of Mycobacterium avium subsp. paratuberculosis (MAP) herd infection status is important to plan appropriate control and prevention strategies for Paratuberculosis (PTB); however, in Uganda MAP infection status of most herds is unknown. This study aimed at determining the MAP infection status of cattle herds and the associated risk factors for MAP infection in six western districts of Uganda. The survey covered a total of 93 herds where faecal and blood samples were collected from 1814 cattle. A Recombinase Polymerase Amplification (RPA) and an antibody-based (ELISA) assays were used to test for the presence of MAP DNA in faeces and MAP antibodies in serum, respectively. The apparent cow-level prevalence of MAP infection was 3.2 and 2.7% using ELISA and RPA respectively and the true cow-level prevalence using ELISA and RPA was 4.9 and 3% respectively. A herd-level prevalence of 43% (ELISA) and 40.8% (RPA) and a within-herd prevalence of 3.8 ± 2.1% based on ELISA were obtained. Among the risk factors investigated, long dry spells were significantly associated with high MAP infection (p < 0.05). These results indicate that MAP is actively present in most areas where surveillance was carried out. This poses a serious threat to the livestock industry and potentially to public health as MAP is highly suspected to play a role in the pathogenesis of several diseases in humans. Other areas of the country are to be surveyed as well in order to establish full data on MAP infection status to enable interventions for the control and prevention of the disease.