Molecular Detection and Characterization of Ehrlichia ruminantium

Ehrlichia ruminantium is an obligate intracellular bacterium that can cause a disease in ruminants known as heartwater. The vertebrate host becomes infected when infected Amblyomma ticks (nymphs or adults) feed on it. When the host survives the infection it becomes a carrier. Ticks become infected with E. ruminantium when they feed on an infected host. The infection is transmitted from one developmental stage to the other except from adult to eggs for as far as is known now. Detection of E. ruminantium is difficult, especially in carrier animals, and is based on either detection of antibodies (MAP1-B ELISA) or detection of bacterial DNA using PCR techniques. The purpose of the research was to molecularly characterize E. ruminantium and to develop new or improved diagnostic tests for the detection of E. ruminantium. For the detection the MAP1-B ELISA was further validated using the TG-ROC method to determine cutoff values with equal sensitivity and specificity, and a new assay based on the reverse line blot (RLB) technique was developed. The developed RLB assays can simultaneously detect and identify 8 Anaplasma and Ehrlichia species. For the characterization several 16S rRNA sequences were determined, which were also used for a reorganization of the taxonomic classification of several rickettsial species. Finally, the transcription of the map1 multigene family was studied in different environments and it was found that map1-1 was transcribed in vitro and in vivo in the vector tick whereas it was not transcribed at a detectable level in vitro in bovine endothelial cells

Development of a polyclonal competitive enzyme-linked immunosorbent assay for detection of antibodies to Ehrlichia ruminantium

A polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) is described for detection of antibodies to Ehrlichia (Cowdria) ruminantium by using a soluble extract of endothelial cell culture-derived E. ruminantium as the antigen and biotin-labeled polyclonal goat immunoglobulins as the competitor. For goats, the diagnostic sensitivity and specificity were both 100% with a cutoff of 80% inhibition (80 PI), with detection of antibodies for 550 days postinfection. For cattle, diagnostic sensitivity and specificity were 86 and 100%, respectively, with a cutoff of 50 PI and 79 and 100% with a cutoff of 70 PI. Cross-reactions with high-titer experimental or field antisera to other Ehrlichia and Anaplasma species were observed at up to 68 PI in cattle and up to 85 PI in sheep, and therefore to exclude these cross-reactions, cutoffs of 70 PI for bovine serology and 85 PI for small-ruminant serology were selected. Application of the PC-ELISA to bovine field sera from South Africa gave a higher proportion of positive results than application of the murine macrophage immunofluorescent antibody test or indirect ELISA, suggesting a better sensitivity for detection of recovered cattle, and results with bovine field sera from Malawi were consistent with the observed endemic state of heartwater and the level of tick control practiced at the sample sites. Reproducibility was high, with average standard deviations intraplate of 1.2 PI and interplate of 0.6 PI. The test format is simple, and the test is economical to perform and has a level of sensitivity for detection of low-titer positive bovine sera that may prove to be of value in epidemiological studies on heartwater

Environmental sampling to assess the bioburden of Mycobacterium avium subspecies paratuberculosis in drylot pens on California dairies.

Mycobacterium avium subspecies paratuberculosis (MAP) is a bacterium that can cause substantial economic losses in infected dairy herds due to reduced milk production and increased cow-replacement costs. In order to control MAP in dairies with drylot pens, a standardized environmental sampling protocol to quantify MAP in fecal slurry was developed based on an existing protocol for freestall pens. Specifically, following a 24 h hold of the flush, a grab sample of approximately 10 ml of fecal slurry was collected every 1 m along the flush lane of the drylot pens, avoiding individual cow fecal pats. To determine the reliability and repatability of the new environmental sampling protocol for estimation of MAP bioburden at the pen level, two collectors simultaneously collected fecal slurry samples every day for 3 days from six drylot cow pens on two Central California dairies. During the study period no cow movement between pens was allowed with the exception of sick cows. The study herds had MAP seroprevalence of 5.8% and 3.2%, respectively, based on whole pen serum ELISA results. Variance components models for quantitative real-time PCR (qPCR) results showed samples collected from different pens on different dairies accounted for greater variablitiy in MAP concentration (65%), while samples collected by different collectors had the least variability (0.1%). In contrast, variability in MAP concentration in environmental samples collected on different days had 25% variability. The intraclass correlation coefficient showed high reliability (93%) of environmental sampling simultaneously by different collectors. In contrast, the reliability of environmental sampling at different days was 65%, which was similar to the reliability for sampling by different collectors on different days. Investigators can expect high reliability when employing the new environmental sampling protocol along with qPCR testing of environmental samples from drylot pens

Alta eficiência diagnóstica do IgM ELISA com o uso de múltiplos antígenos peptídicos (MAP1) de T. gondii ESA (SAG-1, GRA-1 e GRA-7) na toxoplasmose aguda

The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis.O principal marcador sorológico para o diagnóstico da toxoplasmose aguda ou recente são os anticorpos IgM específicos, a par de anticorpos IgG de baixa avidez. Entretanto em alguns pacientes, estes anticorpos podem persistir, ultrapassando o período da fase aguda/recente, contribuindo para erro diagnóstico em casos suspeitos de toxoplasmose. No presente estudo, a eficiência diagnóstica de ELISA foi avaliada, com o uso de frações ou peptídeos originados dos antígenos ESA de T. gondii, denominados de SAG-1, GRA-1 e GRA-7. No referido ensaio, estudamos cada uma destas frações isoladamente, como também em quatro diferentes combinações, ou múltiplos peptídeos antigênicos (MAP), visando estabelecer um perfil confiável para a toxoplasmose aguda/recente em amostra única de soro. A melhor eficiência diagnóstica do ensaio foi encontrada com o uso da combinação de peptídeos SAG-1, GRA-1 e GRA-7, denominada MAP1. A detecção de anticorpos IgG e IgM anti-MAP-1 apresentou melhores características diagnósticas para a diferenciação entre a fase aguda/recente da fase não aguda/recente na toxoplasmose. Nossos resultados mostram que anticorpos IgM anti-MAP-1 poderão prestar auxílio como um marcador sorológico, aumentando a eficiência diagnóstica do ensaio para a fase aguda/recente da toxoplasmose

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

Fusarium graminearum gene deletion mutants map1 and tri5 reveal similarities and differences in the pathogenicity requirements to cause disease on Arabidopsis and wheat floral tissue

The Ascomycete pathogen Fusarium graminearum can infect all cereal species and lower grain yield, quality and safety. The fungus can also cause disease on Arabidopsis thaliana. In this study, the disease-causing ability of two F. graminearum mutants was analysed to further explore the parallels between the wheat (Triticum aestivum) and Arabidopsis floral pathosystems. Wild-type F. graminearum (strain PH-1) and two isogenic transformants lacking either the mitogen-activated protein kinase MAP1 gene or the trichodiene synthase TRI5 gene were individually spray- or point-inoculated onto Arabidopsis and wheat floral tissue. Disease development was quantitatively assessed both macroscopically and microscopically and deoxynivalenol (DON) mycotoxin concentrations determined by enzyme-linked immunosorbent assay (ELISA). Wild-type strain inoculations caused high levels of disease in both plant species and significant DON production. The map1 mutant caused minimal disease and DON accumulation in both hosts. The tri5 mutant, which is unable to produce DON, exhibited reduced pathogenicity on wheat ears, causing only discrete eye-shaped lesions on spikelets which failed to infect the rachis. By contrast, the tri5 mutant retained full pathogenicity on Arabidopsis floral tissue. This study reveals that DON mycotoxin production is not required for F. graminearum to colonize Arabidopsis floral tissue

Detección de ocratoxina A en higos secos utilizando el anticuerpo MAP1 y una técnica de ELISA competitivo

Sin resume

The Fusarium graminearum MAP1 gene is essential for pathogenicity and development of perithecia

Fusarium, graminearum is the causal agent of ear blight disease of cereals. Infection occurs at anthesis when ascospores and/or conidia directly penetrate exposed anther and ovary tissue. The hemibiotrophic hyphae colonize floral tissues and developing grains to cause premature ear senescence. During infection, Fusarium hyphae can also produce hazardous trichothecene mycotoxins, thereby posing a threat to human and animal health and safety. The Fusarium MAP1 gene was identified using a PCR approach by its homology to a known pathogenicity gene of Magnaporthe grisea, the mitogen-activated protein kinase gene PMK1. Gene replacement F. graminearum map1 mutants were non-pathogenic on wheat flowers and roots, and also could not infect wounded wheat floral tissue or tomato fruits. Unlike the wild-type strain, map1 mutant inoculations did not compromise grain yield. Map1 mutants lost their ability to form perithecia in vitro, but their rate of asexual conidiation was unaffected. DON mycotoxin production in planta was still detected. Collectively, the observed phenotypes suggest that the Map1 signalling protein controls multiple events in disease establishment and propagation. Novel approaches to control Fusarium ear blight disease by blocking perithecial development are discussed

DIAGNOSIS PENYAKIT TUNGRO PADA PADI

Tungro disebabkan oleh dua jenis virus yaitu virus batang (rice tungro bacilliform virus=RTBV) dan virus bulat (rice tungro spherical virus=RTSV). Kedua jenis virus tersebut hanya dapat ditularkan oleh wereng hijau secara semipersisten. RTBV merupakan virus dependen, sedangkan RTSV sebagai virus pembantu (helper virus). Wereng hijau dapat menularkan RTSV dan RTBV secara bersamaan dari sumber inokulum yang mengandung kedua virus. Penularan RTBV hanya terjadi apabila vektor telah menghisap RTSV terlebih dahulu, sedangkan penularan RTSV dapat terjadi tanpa bantuan RTB