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

Faburay, Bonto; Geysen, Dirk; Münstermann, S.; Bell‐Sakyi, Lesley; Jongejan, Frans

doi:10.1186/1471-2334-7-85

Cited by 16 publications

(13 citation statements)

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“…Different specific PCR assays were used to detect Anaplasmataceae DNA in blood samples from African buffalo in the Gorongosa National Park, Mozambique. All the 97 blood samples tested were negative in a specific nested PCR for E. ruminantium (based on pCS20 gene) [ 16 ] and in a multiplex real-time PCR for E. chaffeensis (based on vlpt gene) and A. phagocytophilum (based on msp2 gene) [ 15 ]. Seventy African buffalo (72.2 %) were shown to be positive for amplification of fragments of the gene msp1 β in the real-time PCR specific for A. marginale .…”

Section: Resultsmentioning

confidence: 99%

“…DNA samples were screened by different conventional PCR assays: a nested PCR for E. ruminantium [ 16 ]; two nested PCRs for Anaplasma spp. based on partial sequences of the 16S rRNA gene for detection of A. phagocytophilum , A. bovis and A. platys [ 17 ], and for A. centrale and A. marginale [ 18 ]; a nested PCR for the groEL gene [ 19 – 21 ]; a PCR based on the major surface protein 5 (MSP5) gene [ 22 , 23 ].…”

Section: Methodsmentioning

confidence: 99%

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Molecular diagnosis and genetic diversity of tick-borne Anaplasmataceae agents infecting the African buffalo Syncerus caffer from Marromeu Reserve in Mozambique

et al. 2016

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BackgroundTick-borne diseases (TBDs) are very important in relation to domestic ruminants, but their occurrence among wild ruminants, mainly in the African buffalo Syncerus caffer, remains little known.MethodsMolecular diagnostic methods were applied to detect Anaplasma marginale, Anaplasma centrale, Anaplasma phagocytophilum, Ehrlichia ruminantium and Ehrlichia chaffeensis in 97 blood samples of African buffalo captured at the Marromeu Reserve in Mozambique. Molecular detection of agents belonging to the family Anaplasmataceae were based on conventional and qPCR assays based on msp5, groEL, 16S rRNA, msp2, pCS20 and vlpt genes. Phylogenetic reconstruction of new Anaplasma isolates detected in African buffalo was evaluated based on msp5, groEL and 16S rRNA genes.ResultsAll the animals evaluated were negative for specific PCR assays for A. phagocytophilum, E. ruminantium and E. chaffeensis, but 70 animals were positive for A. marginale, showing 2.69 × 100 up to 2.00 × 105msp1β copies/μl. This result overcomes the conventional PCR for A. marginale based on msp5 gene that detected only 65 positive samples. Sequencing and phylogenetic analyses were performed for selected positive samples based on the genes msp5, groEL and 16S rRNA. Trees inferred using different methods separated the 29 msp5 sequences from buffalo in two distinct groups, assigned to A. centrale and A. marginale. The groEL sequences determined for African buffalo samples revealed to be more heterogeneous and inferred trees could not assign them to any species of Anaplasma despite being more related to A. marginale and A. centrale. The highly conserved 16S rRNA gene sequences suggested a close relationship of the new 16 sequences with A. centrale/A. marginale, A. platys and A. phagocytophilum.ConclusionsOur analysis suggests that different species of Anaplasma are simultaneously present in the African buffalo. To the best of our knowledge, this is the first study that diagnosed Anaplasma spp. in the African buffalo and inferred the taxonomic status of new isolates with different gene sequences. The small fragment of msp5 sequences revealed to be a good target for phylogenetic positioning of new Anaplasma spp. isolates.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Molecular diagnosis and genetic diversity of tick-borne Anaplasmataceae agents infecting the African buffalo Syncerus caffer from Marromeu Reserve in Mozambique

et al. 2016

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“…Serological tests for heartwater also exhibit false-negative results, mostly in cattle, as antibody levels are often too low to be detected, even in animals that are under continuous natural challenge by infected ticks (23,24). It has been demonstrated that results from the MAP1B ELISA test do not correlate well with those from a nested pCS20 polymerase chain reaction (PCR) test (25), and it is this latter test that the authors will discuss next.…”

Section: Ante-mortem Diagnosismentioning

confidence: 97%

Heartwater – Ehrlichia ruminantium infection

Allsopp¹

2015

Rev. Sci. Tech. OIE

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SummaryHeartwater is a notifiable disease that is listed by the World Organisation for Animal Health. It is caused by Ehrlichia ruminantium, an obligately intracellular Gramnegative bacterium in the order Rickettsiales and the family Anaplasmataceae. The disease is borne by ticks in the genus Amblyomma and causes heartwater, or cowdriosis, in wild and domestic ruminants, primarily in Africa, but also in parts of the Caribbean. The disease was recognised in South Africa in the 19th Century and determined to be tick borne in 1900, while the organism was identified in 1925 and first cultured in vitro in 1985. This latter achievement boosted research into the disease at a time when biology was moving into the molecular genetic age. Over the last 20 years, there have been significant improvements in our understanding of E. ruminantium, yielding major advances in diagnosis, epidemiology, genetic characterisation, phylogeny, immunology, and vaccine development. The organism is genetically highly variable; this has important implications for future control measures, and is making it difficult to develop an effective vaccine for protection against tick challenge. Research is continuing into three different types of vaccine, inactivated, attenuated, and recombinant, and the current state of development of each is discussed. KeywordsAmblyomma hebraeum -Amblyomma variegatum -Attenuated vaccine -ControlCowdriosis -Diagnosis -Economic importance -Ehrlichia ruminantium -Heartwater -Inactivated vaccine -Occurrence -Recombinant vaccine -Tick-borne disease.Rev. Sci. Tech. Off. Int. Epiz., 2015, 34 (2), 557-568 AetiologyHeartwater is listed by the World Organisation of Animal Health as a notifiable disease (1). It was known in South Africa for nearly 90 years before the causative organism was identified in 1925 as a rickettsia, originally named Rickettsia ruminantium (2, 3). The name was later changed to Cowdria ruminantium (4), from which arose the term 'cowdriosis'. Molecular phylogenetic studies of the Rickettsiales in the 1990s uncovered the real evolutionary relationships within the order and the organism was reclassified as Ehrlichia ruminantium in the family Anaplasmataceae (5). Ehrlichia ruminantium, which is transmitted by Amblyomma ticks, is obligately intracellular, infects cattle, sheep, goats and some wild ruminants, and is frequently fatal. A comprehensive account of the history and biology of E. ruminantium can be found elsewhere (6). Economic and social importanceHeartwater is a serious economic problem wherever it occurs, in an enormous area covering most of sub-Saharan Africa, its offshore islands, and several islands in the Caribbean. The disease generally prevents livestock farmers from upgrading their herds to modern high-yielding breeds, as these are more susceptible to infection than traditional stock breeds, which often have a measure of resistance (7). Since heartwater is so common in the endemic areas of Africa, farmers are usually unwilling or unable to pay for definitive diagnoses, so it is difficul...

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“…Therefore, to prevent the introduction and spread of Foc races in different new regions of India, a suitable, reliable and rapid detection method is prerequisite. In recent years, PCR-based methods, for instance multiplex and real-time PCR have been developed to detect fungal species and other microorganisms [ 8 - 11 ], however, methods based on PCR can be time-consuming and require the extraction of high-quality DNA due to the effects of inhibitors on PCR sensitivity [ 12 , 13 ]. Loop-mediated isothermal amplification (LAMP) is an alternative amplification technology [ 14 ], is highly sensitive, less time-consuming than conventional PCR-based methods, and less prone to inhibition from DNA preparations [ 15 - 18 ].…”

Section: Introductionmentioning

confidence: 99%

Development of Loop-Mediated Isothermal Amplification (LAMP) assay for rapid detection of Fusarium oxysporum f. sp. ciceris - wilt pathogen of chickpea

et al. 2015

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BackgroundFusarium oxysporum f. sp. ciceris (Foc), the causal agent of Fusarium wilt is a devastating pathogen of chickpea. In chickpea, various soil borne pathogens produce (s) similar symptoms, therefore cannot be distinguished easily at field level. There is real need for a rapid, inexpensive, and easy to operate and maintain genotyping tool to facilitate accurate disease diagnosis and surveillance for better management of Fusarium wilt outbreaks.ResultsIn this study, we developed a loop-mediated isothermal amplification (LAMP) assay targeting the elongation factor 1 alpha gene sequence for visual detection of Foc. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue (HNB) was added before amplification, samples with Foc DNA developed a characteristic sky blue colour but those without DNA or with the DNA of six other plant pathogenic fungi did not. Results obtained with LAMP and HNB were confirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for Foc was 10 fg of genomic DNA per reaction, while that of conventional PCR was 100 pg.ConclusionsIn conclusion, it was found that a LAMP assay combined with HNB is simple, rapid, sensitive, and specific. The LAMP assay does not require specialized equipment, hence can be used in the field for the rapid detection of Foc. This is the first report of the use of LAMP assay for the detection of Foc. The presented LAMP method provides a specific, sensitive and rapid diagnostic tool for the distinction of Foc, with the potential to be standardized as a detection method for Foc in endemic areas and will be very useful for monitoring the disease complex in the field further suggesting the management strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-0997-z) contains supplementary material, which is available to authorized users.

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

Cited by 16 publications

References 35 publications

Molecular diagnosis and genetic diversity of tick-borne Anaplasmataceae agents infecting the African buffalo Syncerus caffer from Marromeu Reserve in Mozambique

Molecular diagnosis and genetic diversity of tick-borne Anaplasmataceae agents infecting the African buffalo Syncerus caffer from Marromeu Reserve in Mozambique

Heartwater – Ehrlichia ruminantium infection

Development of Loop-Mediated Isothermal Amplification (LAMP) assay for rapid detection of Fusarium oxysporum f. sp. ciceris - wilt pathogen of chickpea

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