Serological Evidence and Coexposure of Selected Infections among Livestock Slaughtered at Eastern Cape Abattoirs in South Africa.

Zoonotic infections were investigated in a cross-sectional study on asymptomatic livestock slaughtered in abattoirs in the Eastern Cape. Antibodies against Brucella spp., Coxiella burnetii, Toxoplasma gondii, and the coexposure were investigated in sera using serological tests. A total of 565 animals comprising of 280 cattle, 200 sheep, and 85 pigs were screened using RBT, iELISA, CFT, and AMOS-PCR. The Mast® Toxoreagent test and iELISA were used for the detection of T. gondii and C. burnetii, respectively. The Brucella positivity based on at least two tests was 4.3% (12/280), 1.0% (2/200), and 0.0% (0/85) in cattle, sheep, and pigs, respectively. Toxoplasma gondii seropositivity of 37.90% (106/280), 1.50% (3/200), and 7.10% (6/85) was observed in cattle, sheep, and pigs, respectively. Coxiella burnetii seropositivity of 26.40% (74/280), 15.00% (30/200), and 2.40% (2/85) was observed in cattle, sheep, and pigs, respectively. Coexposure was detected in cattle for positivity against C. burnetii and T. gondii 40.54%, Brucella spp. and T. gondii 1.35%, and Brucella spp. and C. burnetii 4.05%. Coexposure for Brucella spp., C. burnetii, and T. gondii 4.05% was detected in cattle. Coexposure of Brucella spp. and C. burnetii 6.67% was detected in sheep. The AMOS-PCR identified B. abortus in cattle and a mixed infection of B. abortus and B. melitensis in sheep in 64.71% seropositive samples. To our knowledge, the coexposure of Brucella spp., T. gondii, and C. burnetii in cattle has not been reported. Coexposure of Brucella spp. and C. burnetii in cattle and sheep is significant as it results in reproductive losses and constitutes an infectious risk to humans. The detection of antibodies against multiple zoonotic infections in livestock from abattoirs has implications for public health.

Pathogen detection and disease diagnosis in wildlife: challenges and opportunities.

The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology of BTV infection in California and AHSV infection in South Africa. The subsequent evaluation of pan-serotype, real-time, RT-PCR-positive samples through the use of serotype-specific RT-PCR assays allows the rapid identification of virus serotypes, reducing the need for expensive and time-consuming conventional methods, such as virus isolation and serotype-specific virus neutralisation assays. These molecular assays and cELISA platforms provide tools that have enhanced epidemiologic surveillance strategies and improved our understanding of potentially altered Culicoides midge behaviour when infected with BTV. They have also supported the detection of subclinical AHSV infection of vaccinated horses in South Africa. Moreover, in conjunction with whole genome sequence analysis, these tests have clarified that the mechanism behind recent outbreaks of AHS in the AHS-controlled area of South Africa was the result of the reversion to virulence and/or genome reassortment of live attenuated vaccine viruses. This review focuses on the use of contemporary molecular diagnostic assays in the context of recent epidemiologic studies and explores their advantages over historic virus isolation and serologic techniques.

La disponibilité d'essais diagnostiques moléculaires et sérologiques rapides, hautement sensibles et spécifiques tels que l'épreuve immuno-enzymatique de compétition (ELISAc), a accéléré le diagnostic des maladies animales transfrontalières émergentes, dont la fièvre catarrhale ovine (FCO) et la peste équine, et contribué à dresser un tableau épidémiologique plus complet de ces maladies. Grâce à la mise au point d'essais basés sur l'amplification en chaîne par polymérase en temps réel couplée à une transcription inverse (RT­PCR) qui permettent de détecter et d'identifier les nombreux sérotypes du virus de la fièvre catarrhale du mouton et du virus de la peste équine, des études approfondies ont pu être conduites sur l'épidémiologie de l'infection par le virus de la fièvre catarrhale du mouton en Californie et de l'infection par le virus de la peste équine en Afrique du Sud. L'évaluation postérieure des échantillons positifs à une RT­PCR en temps réel de groupe (détectant le virus quel que soit le sérotype) au moyen de RT­PCR spécifiques de chaque sérotype permet d'identifier rapidement le sérotype causal et de limiter le recours à des méthodes classiques onéreuses et chronophages comme l'isolement viral ou les essais de neutralisation virale spécifiques de chaque sérotype. Les outils fournis par ces essais moléculaires et par les plateformes ELISAc ont renforcé les stratégies de surveillance épidémiologique et permis de mieux connaître les altérations potentielles de comportement chez les tiques Culicoides infectées par le virus de la fièvre catarrhale du mouton. Ils ont également contribué à détecter les cas d'infection asymptomatique par le virus de la peste équine chez des chevaux vaccinés en Afrique du Sud. En outre, associés avec l'analyse de séquences du génome entier, ces tests ont révélé que le mécanisme sous-jacent aux récents foyers de peste équine dans la zone de contrôle en Afrique du Sud correspondait à une réversion vers la virulence et/ou à un réassortiment du génome des souches de vaccin à virus vivant atténué. Les auteurs passent en revue l'utilisation des essais de diagnostic moléculaire de nouvelle génération dans le contexte de récentes études épidémiologiques et cherchent à établir leurs avantages par rapport aux techniques classiques d'isolement viral et de recherche sérologique.

La existencia de ensayos moleculares y serológicos de diagnóstico rápidos y de gran sensibilidad y especificidad, como el ensayo inmunoenzimático de competición (ELISAc), ha acelerado el diagnóstico de enfermedades animales transfronterizas emergentes, como la lengua azul o la peste equina, y facilitado una caracterización más exhaustiva de su epidemiología. La creación de ensayos basados en la reacción en cadena de la polimerasa acoplada a transcripción inversa (RT?PCR) en tiempo real para detectar y caracterizar los numerosos serotipos de los virus de la lengua azul y la peste equina ha ayudado a estudiar a fondo la epidemiología de sendos episodios infecciosos causados por el virus de la lengua azul en California y por el virus de la peste equina en Sudáfrica. El subsiguiente análisis de las muestras positivas a la prueba de RT?PC en tiempo real de cualquier serotipo con empleo de ensayos RT?PCR dirigidos específicamente contra uno u otro serotipo permite identificar rápidamente los serotipos víricos, lo que hace menos necesario el uso de métodos convencionales más caros y largos, como el aislamiento del virus o técnicas de neutralización vírica adaptadas específicamente a un serotipo. Estos dispositivos de ensayo molecular o de ELISAc ponen a nuestra disposición herramientas que potencian las estrategias de vigilancia epidemiológica y ayudan a conocer mejor las eventuales alteraciones del comportamiento de los jejenes Culicoides al ser infectados por el virus de la lengua azul. Estas técnicas han ayudado también a detectar en Sudáfrica casos de infección asintomática por el virus de la peste equina en caballos vacunados. Estas pruebas, además, empleadas en combinación con el análisis de secuencias genómicas completas, han servido para aclarar que el mecanismo subyacente a los recientes brotes de peste equina surgidos en la zona de Sudáfrica donde la enfermedad estaba bajo control fue fruto de la reversión a la virulencia y/o el reordenamiento genómico de virus vacunales atenuados. Los autores, centrándose en el uso de modernos ensayos moleculares de diagnóstico como parte de recientes estudios epidemiológicos, examinan las ventajas que ofrecen en comparación con las tradicionales técnicas serológicas y de aislamiento vírico.

Seroepidemiology of Leptospira infection in slaughtered cattle in Gauteng province, South Africa.

Leptospirosis is an important economical disease of livestock globally, especially in Asia, the Caribbean, and the African continent. Its presence has been reported in a wide range of livestock. However, information on leptospirosis in South Africa is scanty. We conducted a cross-sectional study in 11 randomly selected abattoirs to determine the seroprevalence and risk factors for leptospirosis in slaughtered cattle in Gauteng province, South Africa. During abattoir visits to selected abattoirs, blood samples were collected from 199 cattle and demographic data obtained on the slaughtered animals. The microscopic agglutination test (MAT) was performed on all sera using a 26-serotype panel using cutoff titer ≥ 1:100. Animal- and abattoir-level risk factors were investigated for their association with seropositivity for leptospirosis. The seroprevalence of leptospirosis in the cattle sampled was 27.6% (55/199). The predominant serogroups detected in seropositive cattle were Sejroe (sv. Hardjo) (38.2%) and Mini sv. Szwajizak) (14.5%) but low to Canicola (sv. Canicola) (1.8%) and Pomona (sv. Pomona) (1.8%). The differences were statistically significant (P < 0.05). Of the five variables investigated, only one (abattoirs) had statistically significantly (P < 0.001) differences in the seroprevalence of leptospirosis among abattoirs. The study documented for the first time in South Africa, the occurrence of serogroups Sejroe (Hardjo bovis strain lely 607), Tarassovi, Hebdomadis, and Medanensis in slaughtered cattle. It was concluded that six of the nine serovars (representing seven serogroups) of Leptospira spp. circulating in cattle population in South Africa are not vaccine serogroups. The clinical, diagnostic, and public health importance of the findings cannot be ignored.

A serological survey of anthrax in domestic dogs in Zimbabwe: a potential tool for anthrax surveillance.

Anthrax is an important disease caused by the bacterium Bacillus anthracis affecting both domestic and wild animals at the wildlife/livestock interface, defined here as a physical space in which wild and domestic species overlap in range and potentially interact. In endemic regions, sporadic anthrax outbreaks occur, causing significant deaths of both wildlife and livestock and sporadically, humans. However, it may also occur as isolated outbreaks with a few animals affected. Such isolated anthrax outbreaks maybe missed. High seroprevalence among carnivores suggests either regular non-fatal exposure to the pathogen circulating in a given environment, or contact with missed cases through consumption of anthrax carcases. To investigate the relevance of this potential indicator, a cross-sectional study was conducted to determine anthrax seroprevalence in domestic dogs (Canis lupus familiaris) from selected interface and non-interface areas of Zimbabwe with known history of anthrax outbreaks. Based on past anthrax outbreaks in the respective areas, the sites were further classified as high or low risk areas for anthrax outbreaks. Sera were collected from domestic dogs (n = 186) and tested for antibodies against B. anthracis protective antigens (PA) using an ELISA test. The overall seroprevalence was 51.6% (96/186; 95% CI 44.2-59.0). Sites from the non-interface areas recorded a significantly (P < 0.001) higher (72.1%) anthrax seroprevalence compared with those from the wildlife -livestock interface (41.5%). The results demonstrated a strong association (χ2 = 14.3; OR = 3.2, 1.6 < OR < 6.2, P < 0.001) between anthrax seropositivity and interface type. Low-risk sites (42.5%) had a significantly (P = 0.044) lower seroprevalence compared with high-risk sites (58.5%) but still demonstrated high seroprevalence for areas where anthrax was last reported more than 20 years back. Dogs from Tsholotsho South were more than 90-times (OR = 96.5, 13.5 < OR < 690.8) more likely to be seropositive compared with those from Hwange. The study demonstrated the potential to use domestic dogs as indicators of anthrax in the study areas to survey anthrax circulation in supposed low-risk areas and calls for a redefinition of both low and high risk areas for anthrax in Zimbabwe based on an improved surveillance.

The reporting of a Bacillus anthracis B-clade strain in South Africa after more than 20 years.

OBJECTIVES: Anthrax is a disease with an age old history in Africa caused by the Gram-positive endospore forming soil bacterium Bacillus anthracis. Epizootics of wild ungulates occur annually in the enzootic region of Pafuri, Kruger National Park (KNP) in the Limpopo Province of South Africa. Rigorous routine surveillance and diagnostics in KNP, has not revealed these rare isolates since the 1990s, despite unabated annual outbreaks. In 2011 a cheetah was diagnosed as anthrax positive from a private game reserve in Limpopo Province and reported to State Veterinary Services for further investigation. Isolation, molecular diagnostics, whole genome sequencing and comparative genomics were carried out for B. anthracis KC2011. RESULTS: Bacteriological and molecular diagnostics confirmed the isolate as B. anthracis. Subsequent typing and whole genome single nucleotide polymorphisms analysis indicated it clustered alongside B. anthracis SA A0091 in the B.Br.010 SNP branch. Unlike B. anthracis KrugerB strain, KC2011 strain has unique SNPs and represents a new branch in the B-clade. The isolation and genotypic characterisation of KC2011 demonstrates a gap in the reporting of anthrax outbreaks in the greater Limpopo province area. The identification of vulnerable and susceptible cheetah mortalities due to this strain has implications for conservation measures and disease control.

Use of the mice passive protection test to evaluate the humoral response in goats vaccinated with Sterne 34F2 live spore vaccine.

The Sterne live spore vaccine (34F2) is the most widely used veterinary vaccine against anthrax in animals. Antibody responses to several antigens of Bacillus anthracis have been described with a large focus on those against protective antigen (PA). The focus of this study was to evaluate the protective humoral immune response induced by the live spore anthrax vaccine in goats. Boer goats vaccinated twice (week 0 and week 12) with the Sterne live spore vaccine and naive goats were used to monitor the anti-PA and toxin neutralizing antibodies at week 4 and week 17 (after the second vaccine dose) post vaccination. A/J mice were passively immunized with different dilutions of sera from immune and naive goats and then challenged with spores of B. anthracis strain 34F2 to determine the protective capacity of the goat sera. The goat anti-PA ELISA titres indicated significant sero-conversion at week 17 after the second doses of vaccine (p = 0.009). Mice receiving undiluted sera from goats given two doses of vaccine (twice immunized) showed the highest protection (86%) with only 20% of mice receiving 1:1000 diluted sera surviving lethal challenge. The in vitro toxin neutralization assay (TNA) titres correlated to protection of passively immunized A/J mice against lethal infection with the vaccine strain Sterne 34F2 spores using immune goat sera up to a 1:10 dilution (rs ≥ 0.522, p = 0.046). This study suggests that the passive mouse protection model could be potentially used to evaluate the protective immune response in livestock animals vaccinated with the current live vaccine and new vaccines.

Stents and stone disease.

The use of stents in renal stone disease is relatively new. The main advantage is reduced pressure in the renal collecting system during times of infected obstructed collecting systems, surgery or obstructing stones. As much pain relieve theses stents offer when indicated, equally much morbidity is caused when stent materials interface with the human urothelium in terms of symptoms, perforations and the "forgotten stent". This review aim to summarize some of the most important considerations when stents are used in stone disease.

Genetic identification of two sweet-potato-infecting begomoviruses in South Africa.

The complete genome sequences of two monopartite begomovirus isolates (genus Begomovirus, family Geminiviridae) that occurred either alone or in mixed infection in sweet potato (Ipomoea batatas) plants collected in Waterpoort, South Africa, are presented. One of the isolates corresponds to sweet potato mosaic-associated virus (SPMaV; SPMaV-[ZA:WP:2011]), with which it shared 98.5 % nucleotide identity, whereas the second isolate corresponds to a new variant of sweet potato leaf curl Sao Paulo virus (SPLCSPV; SPLCSPV-[ZA:WP:2011]), with which it shared 91.4 % nucleotide identity. The phylogenetic and recombination relationships of these isolates to other monopartite Ipomoea-infecting begomoviruses were also investigated. SPLCSPV-[ZA:WP:2011] was found to be a natural recombinant of swepoviruses consisting of two distinct parental genomic sequences from SPLCSPV and sweet potato leaf curl Georgia virus (SPLCGV).

Characterization of the pCS20 region of different Ehrlichia ruminantium isolates.

Heartwater is a serious tick-borne disease of ruminants caused by the rickettsial organism Ehrlichia (Cowdria) ruminantium. A diagnostic test, targeting the pCS20 genomic region and using PCR amplification and probe hybridization, detects E. ruminantium infection in ticks and animals. However, only the pCS20 sequence of the Crystal Springs E. ruminantium isolate is available and the existence of sequence variation amongst different E. ruminantium isolates has not been determined. Primers were designed from the published pCS20 sequence to obtain sequences of the pCS20 region of various E. ruminantium isolates. These primers were unable to amplify the pCS20 region from genomic Welgevonden DNA and genome walking was used to characterize the pCS20 region. This technique showed that the published pCS20 sequence is from a chimeric clone. Sequences of the pCS20 region of 14 different E. ruminantium isolates were determined after amplification with newly designed primers. Sequencing data indicated that West African E. ruminantium isolates are highly conserved, whereas more variation occurs amongst the southern African isolates. These results facilitated the design of a short pCS20 probe and a large PCR target that improved the sensitivity of the E. ruminantium detection assay.

Phylogenetic relationships among Ehrlichia ruminantium isolates.

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.

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

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.

Ehrlichia ruminantium major antigenic protein gene (map1) variants are not geographically constrained and show no evidence of having evolved under positive selection pressure.

In a search for tools to distinguish antigenic variants of Ehrlichia ruminantium, we sequenced the major antigenic protein genes (map1 genes) of 21 different isolates and found that the sequence polymorphisms were too great to permit the design of probes which could be used as markers for immunogenicity. Phylogenetic comparison of the 21 deduced MAP1 sequences plus another 9 sequences which had been previously published did not reveal any geographic clustering among the isolates. Maximum likelihood analysis of codon and amino acid changes over the phylogeny provided no statistical evidence that the gene is under positive selection pressure, suggesting that it may not be important for the evasion of host immune responses.

Health surveillance of employees on a lead mine, 1979-1989.

Health surveillance of employees at a lead mine in the northern Cape, employing about 1,400 people, is specifically aimed at early detection of excessive lead absorption, which is the main chemical hazard. Over a period of 9 years the blood lead level distribution showed very few values (2.5%) that exceeded 60 micrograms/100 ml. The predictive validity (calculated according to the method of Alessio) of zinc protoporphyrin (ZPP) levels, at a cut-off level of 4 micrograms/g haemoglobin, to screen exposed workers in order to determine whether their blood lead level would exceed 50 micrograms/100 ml proved to be high (198). In 1988 a significant correlation between ZPP and blood lead levels was found in 195 employees at a low level of absorption manifested by an incidence of only 4% exceeding the cut-off level of 4 micrograms ZPP/g haemoglobin and only 2% exceeding a blood lead level of 50 micrograms/100 ml in that year. Monitoring by ZPP provides a high degree of safety for workers and is a relatively inexpensive, well-accepted and effective method.