Bacillus anthracis in South Africa, 1975-2013: are some lineages vanishing?

The anthrax-causing bacterium Bacillus anthracis comprises the genetic clades A, B, and C. In the northernmost part (Pafuri) of Kruger National Park (KNP), South Africa, both the common A and rare B strains clades occur. The B clade strains were reported to be dominant in Pafuri before 1991, while A clade strains occurred towards the central parts of KNP. The prevalence of B clade strains is currently much lower as only A clade strains have been isolated from 1992 onwards in KNP. In this study 319 B. anthracis strains were characterized with 31-loci multiple-locus variable-number tandem repeat analysis (MLVA-31). B clade strains from soil (n = 9) and a Tragelaphus strepsiceros carcass (n = 1) were further characterised by whole genome sequencing and compared to publicly available genomes. The KNP strains clustered in the B clade before 1991 into two dominant genotypes. South African strains cluster into a dominant genotype A.Br.005/006 consisting of KNP as well as the other anthrax endemic region, Northern Cape Province (NCP), South Africa. A few A.Br.001/002 strains from both endemic areas were also identified. Subclade A.Br.101 belonging to the A.Br.Aust94 lineage was reported in the NCP. The B-clade strains seems to be vanishing, while outbreaks in South Africa are caused mainly by the A.Br.005/006 genotypes as well as a few minor clades such as A.Br.001/002 and A.Br.101 present in NCP. This work confirmed the existence of the rare and vanishing B-clade strains that group in B.Br.001 branch with KrugerB and A0991 KNP strains.

A retrospective sero-epidemiological survey of bovine brucellosis on commercial and communal farming systems in Namibia from 2004 to 2018.

Cattle production is the major livestock production activity and the mainstay of Namibia's economy. Sustained beef exports are contingent on a sound sanitary environment where diseases such as brucellosis are under control. In this retrospective study, 49,718 bovine brucellosis testing results from 2004 to 2018 were analyzed to determine the proportion of sero-positive cattle and herds, and the spatial distribution of positive reactors from commercial and communal areas. In total, 244 positive reactors were identified based on the Rose Bengal Test (RBT) and the Complement Fixation Test (CFT) in series, giving an overall proportion of infected animals of 0.49% (244/49,718; 95% CI, 0.43-0.56%) and an overall proportion of infected herds of 9.26% (78/842; 95% CI, 7.49-11.41%). There was a higher proportion of sero-positive communal herds (33.09%) and cattle (10.27%) than commercial herds (4.67%) and cattle (0.24%; p < 0.05). Annually, the proportion of positive reactors was 0-1.37% in the commercial area and 0-52.38% in the communal areas, with a clear decline in positive reactors in the communal areas. Within the commercial sector, the proportion of positive reactor dairy, beef, and export cattle was 0.19% (51/27,067; 95% CI, 0.14-0.25%), 0.30% (48/16,098; 95% CI, 0.22-0.40%), and 0.33% (16/4811; 95% CI, 0.20-0.54%), respectively. Abortions were found to be the major reason for Brucella testing in the communal areas. About 12.65% (96/759) of abortion-linked sera tested positive in the communal areas, but none were positive in beef or dairy cattle. Widespread vaccination of cattle and robust planned surveillance is recommended to reduce the incidence of the disease, its associated production losses and public health risk.

Whole genome sequencing and identification of Bacillus endophyticus and B. anthracis isolated from anthrax outbreaks in South Africa.

BACKGROUND: Bacillus endophyticus is a soil plant-endophytic bacterium, while B. anthracis is the causative agent of anthrax. The virulence factors of B. anthracis are the plasmid encoded tripartite toxins (pXO1) and poly-γ-glutamic acid (PGA) capsule (pXO2). B. endophyticus isolated alongside B. anthracis from animals that died of anthrax in Northern Cape Province (NCP), South Africa, harbored polyglutamate genes. The study compared the characteristics of B. anthracis and B. endophyticus with other Bacillus species with a focus on the presence of the PGA capsule or/and unbound PGA. The morphology and whole genome sequence analysis of B. endophyticus strains and B. anthracis were compared. RESULTS: In conventional microbiology, B. endophyticus showed gram-positive round-shaped rods in single/short chains, which were endospore-forming, non-motile, non-haemolytic with white and dry colonies, and γ-phage resistant. B. anthracis was differentiated from B. endophyticus based on the latter's box-shaped rods in pairs/long chains, white-grey and slimy colonies, encapsulated and γ-phage susceptible. The study identified a PGA polyglutamate synthase operon that consisted of pgsBCA, γ-glutamyltranspeptidase (ggt) and pgsE in B. endophyticus genomes. CONCLUSIONS: PGA regions of B. anthracis contain capBCADE genes located in the pXO2 required for capsulation formation, while B. endophyticus contain the pgsBCAE genes in the chromosome. Whole genome and microbiology analysis identified B. endophyticus, as a non-capsuled endospore-forming bacterium that consists of PGA required for biosynthesis. B. endophyticus strains do not synthesize surface associated PGA, therefore capsule visualization of B. anthracis is a key diagnostic characteristic. The study highlights the significance of using whole genome shotgun sequencing to identify virulence and other important genes that might be present amongst unknown samples from natural outbreaks. None of the B. anthracis related plasmids or virulence genes were found in the B. endophyticus genomes.

Protection of farm goats by combinations of recombinant peptides and formalin inactivated spores from a lethal Bacillus anthracis challenge under field conditions.

BACKGROUND: Bacillus (B.) anthracis, the causal agent of anthrax, is effectively controlled by the Sterne live spore vaccine (34F2) in animals. However, live spore vaccines are not suitable for simultaneous vaccination and antibiotic treatment of animals being at risk of infection in an outbreak situation. Non-living vaccines could close this gap. RESULTS: In this study a combination of recombinant protective antigen and recombinant Bacillus collagen-like antigen (rBclA) with or without formalin inactivated spores (FIS), targeted at raising an immune response against both the toxins and the spore of B. anthracis, was tested for immunogenicity and protectiveness in goats. Two groups of goats received from local farmers of the Kars region of Turkey were immunized thrice in three weeks intervals and challenged together with non-vaccinated controls with virulent B. anthracis, four weeks after last immunization. In spite of low or none measurable toxin neutralizing antibodies and a surprisingly low immune response to the rBclA, 80% of the goats receiving the complete vaccine were protected against a lethal challenge. Moreover, the course of antibody responses indicates that a two-step vaccination schedule could be sufficient for protection. CONCLUSION: The combination of recombinant protein antigens and FIS induces a protective immune response in goats. The non-living nature of this vaccine would allow for a concomitant antibiotic treatment and vaccination procedure. Further studies should clarify how this vaccine candidate performs in a post infection scenario controlled by antibiotics.

Polyphasic Characterization of Brucella spp. in Livestock Slaughtered from Abattoirs in Eastern Cape, South Africa.

In livestock, brucellosis is mainly an asymptomatic disease except when abortion occurs; therefore, two serological tests are used for diagnosis as no single test is suitable. Abattoir samples enable a combination of culture, molecular, and serological tests to detect brucellosis. This study assessed Brucella-specific PCR (ITS-PCR) to detect brucellosis and to conduct a molecular characterization of Brucella spp. isolated from PCR-positive livestock (n = 565) slaughtered at abattoirs and the appropriate sample tissue(s). ITS-PCR detected Brucella DNA in 33.6% of cattle, 14.5% of sheep, and 4.7% of pig tissues. Impure Brucella cultures from PCR-positive tissues were 43.6% (44/94) of cattle, 51.7% (15/29) of sheep, and 50% (2/4) of pigs with predominantly B. abortus identification with AMOS-PCR and low isolation of mixed B. abortus and B. melitensis in all species. In cattle, 33% of isolates were from lymph nodes, while in sheep 38.0% were from the liver and kidney and only from tonsils in pigs (2/4). Brucella infections identified with AMOS-PCR were present in seropositive and mainly seronegative (75.6-100%) livestock with the potential to cause brucellosis during pregnancy or breeding. This study demonstrated the value of the polyphasic approach, especially with chronic infections and the potential risk of these asymptomatic animals.

A genome-based investigation of the Priestia species isolated from anthrax endemic regions in Kruger National Park.

Priestia is a genus that was renamed from the genus Bacillus based on the conserved signature indels (CSIs) in protein sequences that separate Priestia species from Bacillus, with the latter only including species closely related to B. subtilis and B. cereus. Diagnosis of anthrax, a zoonotic disease, is implicated by tripartite anthrax virulence genes (lef, pagA, and cya) and poly-γ-D-glutamic acid capsular genes cap-ABCDE of Bacillus anthracis. Due to the amplification of anthrax virulence genes in Priestia isolates, the search for homologous anthrax virulence genes within the Priestia genomes (n = 9) isolated from animal blood smears was embarked upon through whole genome sequencing. In silico taxonomic identification of the isolates was conducted using genome taxonomy database (GTDB), average nucleotide identity (ANI), and multi-locus sequence typing (MLST), which identified the genomes as P. aryabhattai (n = 5), P. endophytica (n = 2) and P. megaterium (n = 2). A pan-genome analysis was further conducted on the Priestia genomes, including the screening of virulence, antibiotic resistance genes and mobile genetic elements on the sequenced genomes. The oligoribonuclease NrnB protein sequences showed that Priestia spp. possess a unique CSI that is absent in other Bacillus species. Furthermore, the CSI in P. endophytica is unique from other Priestia spp. Pan-genomic analysis indicates that P. endophytica clusters separately from P. aryabhattai and P. megaterium. In silico BLASTn genome analysis using the SYBR primers, Taqman probes and primers that target the chromosomal marker (Ba-1), protective antigen (pagA), and lethal factor (lef) on B. anthracis, showed partial binding to Priestia regions encoding for hypothetical proteins, pyridoxine biosynthesis, hydrolase, and inhibitory proteins. The antibiotic resistance genes (ARG) profile of Priestia spp. showed that the genomes contained no more than two ARGs. This included genes conferring resistance to rifamycin and fosfomycin on P. endophytica, as well as clindamycin on P. aryabhattai and P. megaterium. Priestia genomes lacked B. anthracis plasmids and consisted of plasmid replicon types with unknown functions. Furthermore, the amplification of Priestia strains may result in false positives when qPCR is used to detect the virulence genes of B. anthracis in soil, blood smears, and/or environmental samples.

Clinical Coxiella burnetii infection in sable and roan antelope in South Africa.

Various zoonotic microorganisms cause reproductive problems such as abortions and stillbirths, leading to economic losses on farms, particularly within livestock. In South Africa, bovine brucellosis is endemic in cattle, and from 2013-2018, outbreaks of Brucella melitensis occurred in sable. Coxiella burnetii, the agent responsible for the zoonotic disease known as Q-fever and/or coxiellosis, also causes reproductive problems and infects multiple domestic animal species worldwide, including humans. However, little is known of this disease in wildlife. With the expansion of the wildlife industry in South Africa, diseases like brucellosis and coxiellosis can significantly impact herd breeding success because of challenges in identifying, managing and treating diseases in wildlife populations. This study investigated samples obtained from aborted sable and roan antelope, initially suspected to be brucellosis, from game farms in South Africa using serology tests and ruminant VetMAX™ polymerase chain reaction (PCR) abortion kit. The presence of C. burnetii was confirmed with PCR in a sable abortion case, while samples from both sable and roan were seropositive for C. burnetii indirect enzyme-linked immunosorbent assay (iELISA). This study represents the initial report of C. burnetii infection in sable and roan antelope in South Africa. Epidemiological investigations are crucial to assess the risk of C. burnetii in sable and roan populations, as well as wildlife and livestock in general, across South Africa. This is important in intensive farming practices, particularly as Q-fever, being a zoonotic disease, poses a particular threat to the health of veterinarians and farm workers as well as domestic animals.Contribution: A report of clinical C. burnetii infection in the wildlife industry contributes towards the limited knowledge of this zoonotic disease in South Africa.

Brucellosis Seropositivity Using Three Serological Tests and Associated Risk Factors in Abattoir Workers in Gauteng Province, South Africa.

Abattoir workers are liable to zoonotic infections from animals and animal products, primarily to diseases with asymptomatic and chronic clinical manifestations in animals, such as brucellosis. No published reports exist on the seroprevalence of brucellosis in abattoir workers in South Africa. Therefore, this cross-sectional study was conducted to estimate the occurrence and risk factors for Brucella exposure in abattoir workers in Gauteng Province. A total of 103 abattoir workers and managers from 6 abattoirs, where brucellosis-positive slaughtered cattle and sheep were previously detected, were interviewed and tested with serological assays using the Rose Bengal test (RBT), BrucellaCapt, and IgG-ELISA. A pre-tested questionnaire was administered to consenting respondents to obtain information on risk factors for brucellosis. Of the 103 respondents tested, the distribution of female and male workers was 16 (15.5%) and 87 (84.5%), respectively. The seroprevalence for exposure to brucellosis was 21/103 (20.4%, 95%CI: 13.1-29.5) using a combination of RBT, BrucellaCapt, or IgG-ELISA. For test-specific results, seroprevalences by RBT, BrucellaCapt, and IgG-ELISA were 13/103 (12.6%, 95%CI: 6.9-20.6), 9/103 (8.74%, 95%CI: 4.1-15.9), and 18/103 (17.5%, 95%CI: 10.7-26.2), respectively. Low-throughput abattoirs were identified as associated risks, as 29.3% of workers were seropositive compared with 12.7% of workers in high-throughput abattoirs, which highlights that direct contact at abattoirs poses higher risk to workers than indirect and direct contact outside abattoirs. This study confirms the occurrence of Brucella spp. antibodies among abattoir workers in South Africa, possibly due to occupational exposure to Brucella spp., and highlights the occupational hazard to workers. Furthermore, findings underscore that abattoir facilities can serve as points for active and passive surveillance for indicators of diseases of public health importance. We recommend periodic implementation of brucellosis testing of abattoir workers country-wide to establish baseline data for informing appropriate preventive practices and reducing the potential burden of infection rates among these high-risk workers.

Whole Genome Sequence Analysis of Brucella spp. from Human, Livestock, and Wildlife in South Africa.

Brucellosis is an economically important zoonotic disease affecting humans, livestock, and wildlife health globally and especially in Africa. Brucella abortus and B. melitensis have been isolated from human, livestock (cattle and goat), and wildlife (sable) in South Africa (SA) but with little knowledge of the population genomic structure of this pathogen in SA. As whole genome sequencing can assist to differentiate and trace the origin of outbreaks of Brucella spp. strains, the whole genomes of retrospective isolates (n = 19) from previous studies were sequenced. Sequences were analysed using average nucleotide identity (ANI), pangenomics, and whole genome single nucleotide polymorphism (wgSNP) to trace the geographical origin of cases of brucellosis circulating in human, cattle, goats, and sable from different provinces in SA. Pangenomics analysis of B. melitensis (n = 69) and B. abortus (n = 56) was conducted with 19 strains that included B. abortus from cattle (n = 3) and B. melitensis from a human (n = 1), cattle (n = 1), goat (n = 1), Rev1 vaccine strain (n = 1), and sable (n = 12). Pangenomics analysis of B. melitensis genomes, highlighted shared genes, that include 10 hypothetical proteins and genes that encodes for acetyl-coenzyme A synthetase (acs), and acylamidase (aam) amongst the sable genomes. The wgSNP analysis confirmed the B. melitensis isolated from human was more closely related to the goat from the Western Cape Province from the same outbreak than the B. melitensis cattle sample from different cases in the Gauteng Province. The B. melitensis sable strains could be distinguished from the African lineage, constituting their own African sub-clade. The sequenced B. abortus strains clustered in the C2 lineage that is closely related to the isolates from Mozambique and Zimbabwe. This study identified genetically diverse Brucella spp. among various hosts in SA. This study expands the limited known knowledge regarding the presence of B. melitensis in livestock and humans in SA, further building a foundation for future research on the distribution of the Brucella spp. worldwide and its evolutionary background.

Molecular characterisation of two homoeologous elicitor-responsive lipin genes in cotton.

The identification and molecular characterisation of two lipin-like gene copies (GhLIPN) in cotton, Gossypium hirsutum, an allotetraploid derived from two progenitor diploid Gossypium species, is described. Sequence analyses of the GhLIPN copies, designated GhLIPN-1 and -2, revealed that they contain 11 exons, separated by ten introns. They each have a 2,643 bp open reading frame that encodes 880 aa proteins, and share a 97.7 and 95.5 % sequence similarity at the translated nucleotide and amino acid level, respectively. The GhLIPN genes have a distinct domain architecture consisting of an archetypical N-terminal lipin domain, followed by a haloacid dehalogenase (HAD) domain towards the C-terminus. A Southern blot did not distinguish between the two gene copies, which suggests that they may be homoeologs rather than paralogs. GhLIPN-2 is more similar to a homoeologous sequence from G. raimondii, representing the ancestral D-genome, compared to GhLIPN-1 that matches G. herbaceum and that represents the A-genome. Our data indicate that GhLIPN-1 and GhLIPN-2 are homoeologs that derive from the A- and the D-diploid genomes, respectively. The promoter sequences of GhLIPN-1 and -2 differ by 56 %, as a result of multiple indels. In silico analysis of the promoter regions revealed that both genes contain numerous putative defence-related and elicitor-responsive cis-elements that support a role for GhLIPN in defence responses. Relative quantification real-time PCR confirmed the up-regulation in response to a cell-wall-derived V. dahliae elicitor, which supported the association of GhLIPN with defence signalling. The results add a new dimension to the proposed roles of lipins in plants by suggesting that lipins may have a role in defence signalling.

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

BACKGROUND: Presently, 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. RESULTS: The 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). CONCLUSION: This 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.

Assessment of knowledge, attitudes and practices relating to brucellosis among cattle farmers, meat handlers and medical professionals in Namibia.

BACKGROUND: Brucellosis is a re-emerging zoonosis of significant socio-economic, animal and public health importance. It is principally a foodborne or occupation-associated infection of humans, whose effective control depends on maximum cooperation of high-risk populations. OBJECTIVES: The study assessed knowledge, attitudes and practices relating to brucellosis among cattle farmers (communal and commercial), meat handlers (abattoir and butchery workers) and medical professionals (nurses and doctors) in Namibia. METHODS: Between June 2019 and September 2020, self-administered questionnaires and questionnaire interviews were carried out in cattle farmers (n = 264), meat handlers (n = 143) and medical professionals (n = 124) in Namibia. RESULTS: Overall, 43.50% (231/531) of respondents were aware of brucellosis, with the highest awareness among medical professionals (73.39%, 91/124) and the least in meat handlers (13.99%, 20/143). Awareness of brucellosis was associated with tertiary education (p < 0.001) and the medical profession (p < 0.001). However, most medical professionals (98.39%, 122/124) did not consider brucellosis as a differential diagnosis in cases of persistent febrile illness. A proportion of communal (85.60%) and commercial (71.00%) farmers; abattoir workers (44.40%); butchers (53.50%); nurses (55.60%); and medical doctors (28.00%) consumed raw milk. CONCLUSIONS: The study identified the purchase of animals of unknown health status; assisting cow delivery; handling of aborted fetuses with no protective wear; consumption of raw milk, homemade cheese, cattle testes and undercooked livers, as risk factors for Brucella infection in cattle and humans. Thus, intensified risk communication, including public health education, is recommended, in particular, among meat handlers and communal farmers, to promote awareness and discourage risky practices.

Development of conjugated secondary antibodies for wildlife disease surveillance.

Disease monitoring in free-ranging wildlife is a challenge and often relies on passive surveillance. Alternatively, proactive surveillance that relies on the detection of specific antibodies could give more reliable and timely insight into disease presence and prevalence in a population, especially if the evidence of disease occurs below detection thresholds for passive surveillance. Primary binding assays, like the indirect ELISA for antibody detection in wildlife, are hampered by a lack of species-specific conjugates. In this study, we developed anti-kudu (Tragelaphus strepsiceros) and anti-impala (Aepyceros melampus) immunoglobulin-specific conjugates in chickens and compared them to the binding of commercially available protein-G and protein-AG conjugates, using an ELISA-based avidity index. The conjugates were evaluated for cross-reaction with sera from other wild herbivores to assess future use in ELISAs. The developed conjugates had a high avidity of >70% against kudu and impala sera. The commercial conjugates (protein-G and protein-AG) had significantly low relative avidity (<20%) against these species. Eighteen other wildlife species demonstrated cross-reactivity with a mean relative avidity of >50% with the impala and kudu conjugates and <40% with the commercial conjugates. These results demonstrate that species-specific conjugates are important tools for the development and validation of immunoassays in wildlife and for the surveillance of zoonotic agents along the livestock-wildlife-human interface.

Seroprevalence, and molecular detection of Brucella abortus in cattle tissues from an abattoir in Namibia.

Brucellosis is a worldwide zoonosis that is endemic in Namibia. This study estimated seroprevalence of brucellosis, and determined the presence of Brucella infection in slaughtered cattle using the genus-specific 16-23S rRNA interspacer PCR (ITS-PCR), and the species-specific AMOS-PCR. Between December 2018 and May 2019, sera (n = 304), pooled lymph nodes (n = 304), and individual spleen (n = 304) were collected from slaughtered cattle from 52 farms. Sera were tested for anti-Brucella antibodies using the Rose Bengal test (RBT), and the complement fixation test (CFT). Seroprevalence was 2.3% (7/304) (RBT) and 1.6% (5/304) (CFT). Prevalence of positive herds was 9.6% (5/52). Lymph node (n = 200) and spleen (n = 200) samples from seronegative cattle tested negative for Brucella spp. DNA on ITS-PCR, but Brucella spp. DNA was detected in lymph nodes (85.7%, 6/7) and spleen (85.7%, 6/7) from RBT positive cattle. ITS-PCR confirmed isolates from lymph node (51.4%, 4/7) and spleen (85.7%, 6/7) as Brucella spp.; while AMOS-PCR and Brucella abortus species specific (BaSS) PCR confirmed the isolates as Brucella abortus, and field strains, respectively. Provision of adequate protective gear, and the promotion of brucellosis awareness among abattoir workers is recommended to prevent zoonotic infection.

Variation in herbivore space use: comparing two savanna ecosystems with different anthrax outbreak patterns in southern Africa.

BACKGROUND: The distribution of resources can affect animal range sizes, which in turn may alter infectious disease dynamics in heterogenous environments. The risk of pathogen exposure or the spatial extent of outbreaks may vary with host range size. This study examined the range sizes of herbivorous anthrax host species in two ecosystems and relationships between spatial movement behavior and patterns of disease outbreaks for a multi-host environmentally transmitted pathogen. METHODS: We examined range sizes for seven host species and the spatial extent of anthrax outbreaks in Etosha National Park, Namibia and Kruger National Park, South Africa, where the main host species and outbreak sizes differ. We evaluated host range sizes using the local convex hull method at different temporal scales, within-individual temporal range overlap, and relationships between ranging behavior and species contributions to anthrax cases in each park. We estimated the spatial extent of annual anthrax mortalities and evaluated whether the extent was correlated with case numbers of a given host species. RESULTS: Range size differences among species were not linearly related to anthrax case numbers. In Kruger the main host species had small range sizes and high range overlap, which may heighten exposure when outbreaks occur within their ranges. However, different patterns were observed in Etosha, where the main host species had large range sizes and relatively little overlap. The spatial extent of anthrax mortalities was similar between parks but less variable in Etosha than Kruger. In Kruger outbreaks varied from small local clusters to large areas and the spatial extent correlated with case numbers and species affected. Secondary host species contributed relatively few cases to outbreaks; however, for these species with large range sizes, case numbers positively correlated with outbreak extent. CONCLUSIONS: Our results provide new information on the spatiotemporal structuring of ranging movements of anthrax host species in two ecosystems. The results linking anthrax dynamics to host space use are correlative, yet suggest that, though partial and proximate, host range size and overlap may be contributing factors in outbreak characteristics for environmentally transmitted pathogens.

The persistence of time: the lifespan of Bacillus anthracis spores in environmental reservoirs.

Anthrax is a lethal bacterial zoonosis primarily affecting herbivorous wildlife and livestock. Upon host death Bacillus anthracis vegetative cells form spores capable of surviving for years in soil. Anthrax transmission requires host exposure to large spore doses. Thus, conditions that facilitate higher spore concentrations or promote spore survival will increase the probability that a pathogen reservoir infects future hosts. We investigated abiotic and pathogen genomic variation in relation to spore concentrations in surface soils (0-1 cm depth) at 40 plains zebra (Equus quagga) anthrax carcass sites in Namibia. Specifically, how initial spore concentrations and spore survival were affected by seasonality associated with the timing of host mortality, local soil characteristics, and pathogen genomic variation. Zebras dying of anthrax in wet seasons-the peak season for anthrax in Etosha National Park-had soil spore concentrations 1.36 orders of magnitude higher than those that died in dry seasons. No other variables considered affected spore concentrations, and spore survival rates did not differ among sites. Surface soils at these pathogen reservoirs remained culture positive for a range of 3.8-10.4 years after host death. Future research could evaluate if seasonal patterns in spore concentrations are driven by differences in sporulation success or levels of terminal bacteremia.

Distribution and Prevalence of Anaplasmataceae, Rickettsiaceae and Coxiellaceae in African Ticks: A Systematic Review and Meta-Analysis.

In Africa, ticks continue to be a major hindrance to the improvement of the livestock industry due to tick-borne pathogens that include Anaplasma, Ehrlichia, Rickettsia and Coxiella species. A systemic review and meta-analysis were conducted here and highlighted the distribution and prevalence of these tick-borne pathogens in African ticks. Relevant publications were searched in five electronic databases and selected using inclusion/exclusion criteria, resulting in 138 and 78 papers included in the qualitative and quantitative analysis, respectively. Most of the studies focused on Rickettsia africae (38 studies), followed by Ehrlichia ruminantium (27 studies), Coxiella burnetii (20 studies) and Anaplasma marginale (17 studies). A meta-analysis of proportions was performed using the random-effects model. The highest prevalence was obtained for Rickettsia spp. (18.39%; 95% CI: 14.23-22.85%), R. africae (13.47%; 95% CI: 2.76-28.69%), R. conorii (11.28%; 95% CI: 1.77-25.89%), A. marginale (12.75%; 95% CI: 4.06-24.35%), E. ruminantium (6.37%; 95% CI: 3.97-9.16%) and E. canis (4.3%; 95% CI: 0.04-12.66%). The prevalence of C. burnetii was low (0%; 95% CI: 0-0.25%), with higher prevalence for Coxiella spp. (27.02%; 95% CI: 10.83-46.03%) and Coxiella-like endosymbionts (70.47%; 95% CI: 27-99.82%). The effect of the tick genera, tick species, country and other variables were identified and highlighted the epidemiology of Rhipicephalus ticks in the heartwater; affinity of each Rickettsia species for different tick genera; dominant distribution of A. marginale, R. africae and Coxiella-like endosymbionts in ticks and a low distribution of C. burnetii in African hard ticks.

Molecular Characterization of Leptospira Species Detected in the Kidneys of Slaughtered Livestock in Abattoirs in Gauteng Province, South Africa.

Leptospira was investigated in kidneys (n = 305) from slaughtered livestock in the Gauteng Province abattoirs, South Africa, using a culture medium to isolate Leptospira, followed by the LipL32 qPCR to detect Leptospira DNA. The SecY gene region was amplified, sequenced, and analyzed for LipL32 qPCR-positive samples or Leptospira isolates. The overall frequency of isolation of Leptospira spp. was 3.9% (12/305), comprising 4.8% (9/186), 4.1% (3/74), and 0% (0/45) from cattle, pigs, and sheep, respectively (p > 0.05). However, with LipL32 qPCR, the overall frequency of Leptospira DNA was 27.5%, consisting of 26.9%, 20.3%, and 42.2% for cattle, pigs, and sheep, respectively (p = 0.03). Based on 22 SecY sequences, the phylogenetic tree identified the L. interrogans cluster with serovar Icterohaemorrhagiae and the L. borgpetersenii cluster with serovar Hardjo bovis strain Lely 607. This study is the first molecular characterization of Leptospira spp. from livestock in South Africa. The reference laboratory uses an eight-serovar microscopic agglutination test panel for leptospirosis diagnosis, of which L. borgpetersenii serovar Hardjo bovis is not part. Our data show that pathogenic L. interrogans and L. borgpetersenii are circulating in the livestock population. Diagnostic use of molecular methods will eliminate or reduce the under-reporting of leptospirosis in livestock, particularly sheep, in South Africa.

Molecular characterization of an elicitor-responsive Armadillo repeat gene (GhARM) from cotton (Gossypium hirsutum).

Only a few Armadillo (ARM) repeat proteins have been characterized in plants where they appear to have diverse functions, including the regulation of defence responses. In this study, the identification, cloning and characterization of a gene, encoding an ARM repeat protein (GhARM), is described. GhARM exists as multiple copies in cotton, with an 1713 bp ORF encoding 570 amino acids. The predicted protein contains three consecutive ARM repeats within an Armadillo-type fold, with no other distinguishing domains. Sequence alignments and phylogenetic analysis revealed that GhARM has a high homology with other ARM proteins in plants. The predicted three dimensional model of GhARM displayed a characteristic right-handed superhelical twist. In silico analysis of the promoter sequence revealed that it contains several defence- and hormone-responsive cis-regulatory elements. Expression of GhARM was significantly down-regulated in response to treatment with a V. dahliae elicitor suggesting that GhARM may function as a negative-regulator of cotton defence signalling against V. dahliae. To date, GhARM is the only ARM repeat gene that has been completely sequenced and characterized in cotton.

Seroprevalence of brucellosis and molecular characterization of Brucella spp. from slaughtered cattle in Rwanda.

Bovine brucellosis is endemic in Rwanda, although, there is a paucity of documented evidence about the disease in slaughtered cattle. A cross-sectional study was conducted in slaughtered cattle (n = 300) to determine the seroprevalence of anti-Brucella antibodies using the Rose Bengal Test (RBT), and indirect enzyme-linked immunosorbent assay (i-ELISA). Corresponding tissues were cultured onto a modified Centro de Investigación y Tecnología Agroalimentaria (CITA) selective medium and analysed for Brucella spp. using the 16S-23S ribosomal interspacer region (ITS), AMOS, and Bruce-ladder PCR assays. The seroprevalence was 20.7% (62/300) with RBT, 2.9% (8/300) with i-ELISA, and 2.9% (8/300) using both tests in series. Brucella-specific 16S-23S ribosomal DNA interspace region (ITS) PCR detected Brucella DNA in 5.6% (17/300; Brucella culture prevalence). AMOS-PCR assay identified mixed B. abortus and B. melitensis (n = 3), B. abortus (n = 3) and B. melitensis (n = 5) while Bruce-ladder PCR also identified B. abortus (n = 5) and B. melitensis (n = 6). The gold standard culture method combined with PCR confirmation identified 5.6% Brucella cultures and this culture prevalence is higher than the more sensitive seroprevalence of 2.9%. This emphasizes the need to validate the serological tests in Rwanda. The mixed infection caused by B. abortus and B. melitensis in slaughtered cattle indicates cross-infection and poses a risk of exposure potential to abattoir workers. It is essential to urgently strengthen a coordinated national bovine brucellosis vaccination and initiate a test-and-slaughter program that is not presently applicable in Rwanda.