Henriette van Heerden

Phylogenetic Relationships among Ehrlichia ruminantium Isolates

Abstract: Ehrlichia ruminantium, the causative agent of heartwater, is a tick‐borne pathogen infecting ruminants throughout sub‐Saharan Africa and on some Caribbean islands. The most reliable test for E. ruminantium is PCR‐based, but this gives positive results in some areas free of clinical heartwater and of the known Amblyomma spp. tick vectors. To investigate the molecular basis for this finding we have sequenced and carried out phylogenetic analysis of a range of genes from a number of E. ruminantium isolates. The genes include ribonuclease III and cytochrome c oxidase assembly protein genes (the pCS20 region), groESL, citrate synthase (gltA), and 16S ribosomal RNA. Relationships among major antigenic protein (map1) genes have been exhaustively investigated in a previous study that showed that the genes are variable in length, have non‐synonymous mutations, and show no geographical specificity among isolates. The 16S sequences are highly conserved, except in the V1 loop region. The pCS20, groESL, and gltA genes show only single nucleotide polymorphisms (SNPs) dispersed throughout the sequenced regions. Phylogenetic analysis using pCS20 data differentiates the western African isolates into a single clade, which also includes a southern African isolate. All other southern African isolates and a Caribbean isolate fall into a further clade, which is subdivided into two groups. Sequence variation within this clade is greater than that within the western African clade, suggesting that E. ruminantium originated in southern Africa.

Molecular characterisation and regulation of a Nicotiana tabacum S-domain receptor-like kinase gene induced during an early rapid response to lipopolysaccharides.

The isolation, characterization and regulation of the first lipopolysaccharide (LPS)-responsive S-domain receptor-like kinase (RLK) in Nicotiana tabacum are reported. The gene, corresponding to a differentially expressed LPS-responsive EST, was fully characterised to investigate its involvement in LPS-induced responses. The full genomic sequence, designated Nt-Sd-RLK, encodes for a S-domain RLK protein containing conserved modules (B-lectin-, S- and PAN-domains) reported to function in mediating protein-protein and protein-carbohydrate interactions in its extracellular domain, as well as the molecular architecture to transduce signals intracellularly through a Ser/Thr kinase domain. Phylogenetic analysis clustered Nt-Sd-RLK with S-domain RLKs induced by bacteria, wounding and salicylic acid. Perception of LPS induced a rapid, bi-phasic response in Nt-Sd-RLK expression with a 17-fold up-regulation at 3 and 9h. A defence-related W-box cis element was found in the promoter region of Nt-Sd-RLK and the transient induction of Nt-Sd-RLK in cultured cells by LPS exhibited a pattern typical of early response defence genes. Nt-Sd-RLK was also responsive to salicylic acid induction and was expressed in differentiated leaf tissue, where LPS elicited local as well as systemic up-regulation. The results contribute new knowledge about the potential role that S-domain RLKs may play within interactive signal transduction pathways associated with immunity and defence.

Identification and Molecular Characterisation of a Lectin Receptor-like Kinase (GhLecRK-2) from Cotton

Verticillium dahliae, the causative agent of Verticillium wilt, results in significant cotton (Gossypium hirsutum) crop losses worldwide. Identification of host genes involved in mounting an effective defence response may suggest targets for alternative control methods for this devastating fungus. In this study, the identification, cloning and characterisation of a V. dahliae elicitor-responsive lectin receptor-like kinase (GhLecRK-2) is described. Bioinformatic analysis of its deduced amino acid sequences revealed that GhLecRK-2 consists of an external legume-like lectin (L-lectin) domain, a single transmembrane spanning domain and a C-terminal Ser/Thr-specific protein kinase domain. In silico analysis of the promoter sequence revealed that it contains several defence-responsive regulatory elements. Expression analysis revealed that transcription of GhLecRK-2 is significantly up-regulated in response to treatment with a cell wall-derived V. dahliae fraction. The nature of the L-lectin domain suggests that GhLecRK-2 may be capable of interacting with the glycoconjugate-based fraction, and the enhanced transcription of GhLecRK-2 upon treatment with the cell wall extract suggests a role in perception and the activation of defence responses.

Quantitative anti-PA IgG ELISA; assessment and comparability with the anthrax toxin neutralization assay in goats

BackgroundPresently, few data exist on the level and duration of anti-protective antigen (PA) IgG in vaccinated livestock. Various adaptation of enzyme-linked immunosorbent assays (ELISAs) have been developed in studies to assess immune response following vaccination, albeit mostly in laboratory rodent models. The quantitative anti-anthrax IgG ELISA in this study describes a method of enumerating the concentration of anti-PA specific IgG present in sera of immunized goats, with the aid of an affinity-purified caprine polyclonal anti-anthrax PA-83 IgG standard. This was compared with the anthrax toxin neutralization assay (TNA) which measures a functional subset of toxin neutralizing anti-PA IgG.ResultsThe measured concentrations obtained in the standard curve correlated with the known concentration at each dilution. Percentage recovery of the standard concentrations ranged from 89 to 98% (lower and upper asymptote respectively). Mean correlation coefficient (r2) of the standard curve was 0.998. Evaluation of the intra-assay coefficient of variation showed ranges of 0.23-16.90% and 0.40-12.46% for days 28 and 140 sera samples respectively, following vaccination. The mean inter-assay coefficient of variation for triplicate samples repeated on 5 different days was 18.53 and 12.17% for days 28 and 140 sera samples respectively. Spearman’s rank correlation of log-transformed IgG concentrations and TNA titres showed strong positive correlation (rs = 0.942; p = 0.01).ConclusionThis study provides evidence that an indirect ELISA can be used for the quantification of anti-anthrax PA IgG in goats with the added advantage of using single dilutions to save time and resources. The use of such related immunoassays can serve as potential adjuncts to potency tests for Sterne and other vaccine types under development in ruminant species. This is the first report on the correlation of polyclonal anti-anthrax PA83 antibody with the TNA in goats.

Detection of Brucella abortus in Chiredzi district in Zimbabwe

Brucellosis is an endemic disease in Zimbabwe caused by the genus Brucella. Brucella seroprevalence was recently reported to be high in the wildlife-livestock interface in the Chiredzi district and the neighbouring Gonarezhou National Park (GNP) in Zimbabwe, and higher amongst communal cattle with an abortion history and access to grazing in GNP than amongst communal cattle with no abortion history or access to grazing in GNP. The aim of this study was to investigate Brucella species in brucellosis seropositive cattle in the Chiredzi district with access to GNP using isolation and identification. Isolation of Brucella species from whole blood (n = 18) and milk samples (n = 10) from seropositive animals with an abortion history was based on the rose Bengal test (RBT) and enzyme-linked immunoassays (enzyme- linked immunosorbent assay [ELISA]; indirect ELISA and complement ELISA), using microbiology and polymerase chain reaction (PCR) methods. Brucella abortus was cultured and identified from blood and milk collected from seropositive cows in both communal areas. The Brucella-specific 16-23S intergenic spacer (ITS) PCR and multiplex AMOS-PCR assays verified the identification of the cultures. Our results confirmed that B. abortus is present in cattle on communal farms in the Chiredzi district in Zimbabwe and might cause cattle abortions. The need for implementing control measures and raising public awareness on zoonotic transmission of brucellosis are recommended.

Variability of pCS20 gene sequences among different Ehrlichia ruminantium isolates.

Ehrlichia ( Cowdria ) ruminantium is the causative agent of heartwater, a tick-borne disease affecting wild and domestic ruminants in sub-Saharan Africa and on some islands in the Caribbean. It is transmitted by ticks of the genus Amblyomma . The detection of E. ruminantium in ticks and ruminants is essential for epidemiological studies and for implementing control measures. Various techniques have been used for this purpose, including electron or fluorescent microscopy and various serological tests. The disadvantage of serological testing is that false negative and false positive results are known to occur. The most reliable diagnostic test for E. ruminantium is based on polymerase chain reaction (PCR) amplification of the pCS20 region, followed by probing. 1,2 The test is highly sensitive and appears to be E. ruminantium -specific. 3 No amplification or probe hybridization occurs with the closely related E. canis or E. chaffeensis nor with Anaplasma species. The test can be applied to DNA extracted from blood and other tissues and also to tick DNA. Nothing is known, however, about the level of pCS20 polymorphism, which may exist between different E. ruminantium isolates. The aim of the study was to obtain sequence information from a range of isolates to confirm the specificity of the test. The published pCS20 sequence is 1,306 base pairs (bp) 4 long and contains two open reading frames, one of which is homologous to the ribonuclease III gene and the other of which is homologous to the cytochrome c oxidase assembly protein gene of various bacteria. Primers were designed from the published sequence, for amplification of the pCS20 region of the Welgevonden E. ruminantium isolate, which is the type specimen. 5 Using these primers, amplification of the positive control sample, the plasmid containing the pCS20 region of the E. ruminantium (Crystal Springs) isolate, gave a single band of the expected size. Multiple bands, none of which was of the expected size, were produced when attempts were made to amplify Welgevonden genomic DNA (see F IGURE 1). To investigate the reason for this anomaly, genome walking was carried out on digested genomic Welgevonden E. ruminantium DNA according to the protocol described in the Genome Walker Kits user manual (Clontech Laboratories, USA, 1999). A Welgevonden-specific amplicon yielded a pCS20 sequence similar to that of the Crystal Springs sequence as far as nucleotide 1161. Beyond this point, the

Ecological suitability modeling for anthrax in the Kruger National Park, South Africa

The spores of the soil-borne bacterium, Bacillus anthracis, which causes anthrax are highly resistant to adverse environmental conditions. Under ideal conditions, anthrax spores can survive for many years in the soil. Anthrax is known to be endemic in the northern part of Kruger National Park (KNP) in South Africa (SA), with occasional epidemics spreading southward. The aim of this study was to identify and map areas that are ecologically suitable for the harboring of B. anthracis spores within the KNP. Anthrax surveillance data and selected environmental variables were used as inputs to the maximum entropy (Maxent) species distribution modeling method. Anthrax positive carcasses from 1988–2011 in KNP (n = 597) and a total of 40 environmental variables were used to predict and evaluate their relative contribution to suitability for anthrax occurrence in KNP. The environmental variables that contributed the most to the occurrence of anthrax were soil type, normalized difference vegetation index (NDVI) and precipitation. Apart from the endemic Pafuri region, several other areas within KNP were classified as ecologically suitable. The outputs of this study could guide future surveillance efforts to focus on predicted suitable areas for anthrax, since the KNP currently uses passive surveillance to detect anthrax outbreaks.

Major outer membrane proteins of Ehrlichia ruminantium encoded by a multigene family.

Abstract: Immune responses of infected animals and humans have been reported to be directed against variable outer membrane proteins of Ehrlichia species that are encoded by polymorphic multigene families. In Ehrlichia (= Cowdria) ruminantium, two immunodominant proteins have been identified, namely major antigenic protein 1 (MAP1) and open reading frame 2 (ORF2). The aim of the present study was to identify additional map1‐like genes in the E. ruminantium genome. A 12 kb clone that hybridized with the map1 probe was amplified using long template PCR. The PCR product was partially digested, cloned, and sequenced. Four map1‐like genes are located in tandem, namely map1−1 (orf2) and map1−2 upstream of map1 as well as map1+1 downstream of map1. A large ORF (2.4 kb) at the 3′ end is homologous to secA genes of other organisms. The sequence data in this study support other findings that outer membrane proteins are located in tandem and are encoded by a polymorphic multigene family.

Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model

The Sterne 34F2 live spore vaccine (SLSV) developed in 1937 is the most widely used veterinary vaccine against anthrax. However, literature on the immunogenicity of this vaccine in a target ruminant host is scarce. In this study, we evaluated the humoral response to the Bacillus anthracis protective antigen (rPA), a recombinant bacillus collagen-like protein of anthracis (rBclA), formaldehyde inactivated spores (FIS) prepared from strain 34F2 and a vegetative antigen formulation prepared from a capsule and toxin deficient strain (CDC 1014) in Boer goats. The toxin neutralizing ability of induced antibodies was evaluated using an in vitro toxin neutralization assay. The protection afforded by the vaccine was also assessed in vaccinates. Anti-rPA, anti-FIS and lethal toxin neutralizing titres were superior after booster vaccinations, compared to single vaccinations. Qualitative analysis of humoral responses to rPA, rBclA and FIS antigens revealed a preponderance of anti-FIS IgG titres following either single or double vaccinations with the SLSV. Antibodies against FIS and rPA both increased by 350 and 300-fold following revaccinations respectively. There was no response to rBclA following vaccinations with the SLSV. Toxin neutralizing titres increased by 80-fold after single vaccination and 700-fold following a double vaccination. Lethal challenge studies in naïve goats indicated a minimum infective dose of 36 B. anthracis spores. Single and double vaccination with the SLSV protected 4/5 and 3/3 of goats challenged with>800 spores respectively. An early booster vaccination following the first immunization is suggested in order to achieve a robust immunity. Results from this study indicate that this crucial second vaccination can be administered as early as 3 months after the initial vaccination.

Draft Genome Sequences of Two South African Bacillus anthracis Strains

ABSTRACT Bacillus anthracis is a Gram-positive bacterium that causes anthrax, mainly in herbivores through exotoxins and capsule produced on plasmids, pXO1 and pXO2. This paper compares the whole-genome sequences of two B. anthracis strains from an endemic region and a sporadic outbreak in South Africa. Sequencing was done using next-generation sequencing technologies.