Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Ehrlichia ruminantium

<p>Abstract</p> <p>Background</p> <p>The rickettsial bacterium <it>Ehrlichia ruminantium </it>is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus <it>Amblyomma</it>. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of <it>E. ruminantium</it>.</p> <p>Results</p> <p>Two sets of LAMP primers were designed from the pCS20 and <it>sodB </it>genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for <it>sodB</it>, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of <it>E. ruminantium </it>from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic <it>Ehrlichia </it>species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries.</p> <p>Conclusions</p> <p>Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of <it>E. ruminantium</it> in both heartwater-endemic areas and regions that are at risk of contracting the disease.</p

Longitudinal monitoring of Ehrlichia ruminantium infection in Gambian lambs and kids by pCS20 PCR and MAP1-B ELISA

<p>Abstract</p> <p>Background</p> <p>The epidemiology of <it>E. ruminantium </it>infection in extensively managed young animals is not adequately understood. Thus in this study, we monitored the onset (age at first infection) and kinetics of <it>E. ruminantium </it>infection and antibody response in extensively managed newborn lambs and kids at three sites in The Gambia.</p> <p>Methods</p> <p>We used a nested pCS20 PCR and MAP1-B ELISA in a longitudinal study to monitor the onset (age at first infection) and kinetics of <it>E. ruminantium </it>infection and antibody response respectively, in 77 newborn lambs and kids under a traditional husbandry system at three sites (Kerr Seringe, Keneba, Bansang) in The Gambia where heartwater is known to occur. The animals were monitored for field tick infestation and the comparative performance of the two assays in detecting <it>E. ruminantium </it>infection was also assessed.</p> <p>Results</p> <p>The infection rate detected by pCS20 PCR varied between 8.6% and 54.8% over the 162-day study period. Nineteen per cent of the animals in week 1 post-partum tested positive by pCS20 PCR with half of these infections (7/14) detected in the first 3 days after birth, suggesting that transmission other than by tick feeding had played a role. The earliest detectable <it>A. variegatum </it>infestation in the animals occurred in week 16 after birth. Antibodies detected by MAP1-B ELISA also varied, between 11.5% and 90%. Although there is considerable evidence that this assay can detect false positives and due to this and other reasons serology is not a reliable predictor of infection at least for heartwater. In contrast to the pCS20 PCR, the serological assay detected the highest proportion of positive animals in week 1 with a gradual decline in seropositivity with increasing age. The pCS20 PCR detected higher <it>E. ruminantium </it>prevalence in the animals with increasing age and both the Spearman's rank test (<it>r</it>_{<it>s </it>}= -0.1512; P = 0.003) and <it>kappa </it>statistic (-0.091 to 0.223) showed a low degree of agreement between the two assays.</p> <p>Conclusion</p> <p>The use of pCS20 PCR supported by transmission studies and clinical data could provide more accurate information on heartwater epidemiology in endemic areas and single-occasion testing of an animal may not reveal its true infection status. The view is supported because both the vector and vertical transmission may play a vital role in the epidemiology of heartwater in young small ruminants; the age range of 4 and 12 weeks corresponds to the period of increased susceptibility to heartwater in traditionally managed small ruminants.</p

Determination and expression analysis of functional genes in Lactobacillus plantarum

In this study, the bacteriocin production of two Lactobacillus plantarum strains was investigated and their effectiveness as protective cultures for the biopreservation of turkey meat was assessed. Especially, the genetic loci for bacteriocin production of Lactobacillus plantarum strains BFE 5092 and PCS20 were completely analysed and the protective Lactobacillus plantarum BFE 5092 grew and produced bacteriocin at 8 or 10°C as well as during sessile growth on turkey meat

Protective cultures against foodborne pathogens in a nitrite reduced fermented meat product

In the present work, a combined hurdle approach for fermented meat preservation was investigated. Challenge tests were performed in Chorizo sausage model using the maximum allowed NaNO2 amount (150 mg/kg), a reduced amount (75 mg/kg) and no nitrite, with and without protective cultures inoculation. Cocktail strains of L. monocytogenes and Salmonella spp. were used as indicator strains. In a nitrite reduced sausage model, L. monocytogenes growing trend did not significantly change (p &gt; 0.05) when compared with that containing higher nitrite concentration (150 mg/kg NaNO2). The addition of L. plantarum PSC20 significantly lowered L. monocytogenes growth when compared with control batches without PCS20 (p &lt; 0.05), obtaining 3.84 log cfu/g and 2.62 log cfu/g lower counts in the batches with 150 mg/kg NaNO2 and 75 mg/kg NaNO2 respectively. None of the protective cultures demonstrated in situ antagonistic activity against Salmonella spp. This work pointed out that the reduction of nitrites with the combined use of a protective culture could be a feasible approach to control L. monocytogenes growth in fermented meat foods

Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium

<p>Abstract</p> <p>Background</p> <p>Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model <it>Ehrlichia ruminantium (ER</it>), the causative agent of heartwater, is transmitted by tick <it>Amblyomma variegatum</it>. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood.</p> <p>Results</p> <p>We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales <it>ER </it>to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of <it>ER</it>'s cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome <it>ER </it>microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from <it>ER </it>microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R²= 0.7). Moreover, SCOTS method is crucial for microarrays analysis of <it>ER</it>, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively.</p> <p>Conclusions</p> <p>We conclude that this SCOTS method has a key importance for the transcriptomic analysis of <it>ER </it>and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both <it>ER </it>pathogenicity and the adaptation of obligate intracellular bacteria to their environment.</p

Ehrlichia ruminantium variants which do not cause heartwater found in South Africa

In 1994 a batch of apparently healthy goats was selected for intended export to the USA from a heartwater-free and vector tick-free region of South Africa. The animals were tested serologically for heartwater, using either or both an IFA and an ELISA test, and 52% were found to be serologically positive. A PCR assay based on Ehrlichia ruminantium 16S gene sequences gave positive results for 54% of the animals, suggesting that apparently non-pathogenic E. ruminantium variants existed in this heartwater-free area. To identify and characterise the agents responsible for the positive serological and PCR results, ticks and animal blood samples were collected from two of the three farms involved in the original survey during two successive seasons of expected peak tick activity. Ticks were kept alive for a minimum of 3 weeks to allow digestion of any blood meal before being processed. Over the two seasons, 28% of the livestock and 15% of the ticks sampled were found to be carrying E. ruminantium. E. ruminantium 16S and pCS20 sequences were detected in all of the four tick species collected from the livestock (Rhipicephalus evertsi evertsi, Rhipicephalus evertsi mimeticus, Hyalomma truncatum, Hyalomma marginatum rufipes), suggesting that some of the species may act as vectors. Animals generally carried multiple E. ruminantium 16S genotypes, whereas ticks rarely carried more than one. Infection levels in both animals and ticks were too low to generate a marked response when a blood stabilate was sub-passaged in a clean sheep, preventing the subsequent establishment of any of the organisms in culture.This research was funded by the Red Meat Research and Development Trust (RMRDT) of South Africa. We thank Prof. Ivan Horak for advice and critical reading of the manuscript