Wilhelm Heinrich Stoltsz

Molecular Characterization of Theileria Species Associated with Mortality in Four Species of African Antelopes

ABSTRACT Pathogen DNA was isolated from roan antelope (Hippotragus equinus), sable antelope (Hippotragus niger), greater kudu (Tragelaphus strepsiceros), and common gray duiker (Sylvicapra grimmia) in South Africa whose deaths were attributed to either theileriosis or cytauxzoonosis. We developed Theileria species-specific probes used in combination with reverse line blot hybridization assays and identified three different species of Theileria in four African antelope species. The close phylogenetic relationship between members of the genera Theileria and Cytauxzoon, similarities in the morphologies of developmental stages, and confusion in the literature regarding theileriosis or cytauxzoonosis are discussed.

Displacement of Boophilus decoloratus by Boophilus microplus in the Soutpansberg region, Limpopo Province, South Africa.

Boophilus ticks (n = 25,987) were collected from cattle at 30 communal dip tanks and five commercial farms in the Soutpansberg region, Limpopo Province, South Africa, between May 1999 and December 2001. Only 6.6% were Boophilus decoloratus, while 93.4% were Boophilus microplus, a tick that had not been reported from this area previously. B. microplus was the most common Boophilus tick on communal dip tank cattle, while B. decoloratus was more commonly found on commercial farms. Where the two species occurred together, B. microplus tended to displace B. decoloratus. The displacement was almost complete at the communal dip tanks, while on the commercial farms the population change was still in progress at the end of the survey. The present study demonstrated that a postulated reproductive interference was insufficient in preventing B. microplus from spreading when the climatic conditions were favourable to this species, as the displacement in most of the areas appeared to be rapid and complete.

Detection of lumpy skin disease virus in saliva of ticks fed on lumpy skin disease virus-infected cattle

Lumpy skin disease is an economically important disease of cattle that is caused by the lumpy skin disease virus (LSDV), which belongs to the genus Capripoxvirus. It is endemic in Africa and outbreaks have also been reported in the Middle-East. Transmission has mostly been associated with blood-feeding insects but recently, the authors have demonstrated mechanical transmission by Rhipicephalus appendiculatus as well as mechanical/intrastadial and transstadial transmission by Amblyomma hebraeum. Saliva is the medium of transmission of pathogens transmitted by biting arthropods and, simultaneously, it potentiates infection in the vertebrate host. This study aimed to detect LSDV in saliva of A. hebraeum and R. appendiculatus adult ticks fed, as nymphs or as adults, on LSDV-infected animals, thereby also demonstrating transstadial or mechanical/intrastadial passage of the virus in these ticks. Saliva samples were tested for LSDV by real-time PCR and virus isolation. Supernatants obtained from virus isolation were further tested by real-time PCR to confirm that the cytopathic effects observed were due to LSDV. Lumpy skin disease virus was detected, for the first time, in saliva samples of both A. hebraeum and R. appendiculatus ticks. At the same time, mechanical/intrastadial and transstadial passage of the virus was demonstrated and confirmed in R. appendiculatus and A. hebraeum.

Evidence of transstadial and mechanical transmission of lumpy skin disease virus by Amblyomma hebraeum ticks.

Lumpy skin disease (LSD) is an economically important disease caused by LSD virus (LSDV), a Capripoxvirus, characterized by fever and circumscribed skin lesions. It is suspected to be transmitted mechanically by biting flies. To assess the vector potential of Amblyomma hebraeum in transmission of LSDV, mechanical/intrastadial and transstadial modes of transmission of the virus by this tick species were investigated. Two cattle were artificially infected as sources (donors) of infection to ticks. Ticks were infected as either nymphs or adults. Male A. hebraeum ticks were partially fed on donor animals and transferred to recipient animals to test for mechanical/intrastadial transmission. Nymphal A. hebraeum were fed to repletion on donor animals. The emergent adult ticks were placed on recipient animals to test for transstadial transmission of the virus. Successful transmission of LSDV infection was determined in recipient animals by monitoring development of clinical signs, testing of blood for the presence of LSDV by real-time PCR, virus isolation and the serum neutralization test. This report provides further evidence of mechanical/intrastadial and, for the first time, transstadial transmission of LSDV by A. hebraeum. These findings implicate A. hebraeum as a possible maintenance host in the epidemiology of the disease.

Theileriosis (Cytauxzoonosis) in Roan antelope ( Hippotragus equinus ): Field exposure to infection and identification of potential vectors

Four hand-reared, naïve roan antelope, 4 months of age, were exposed to naturally infected pasture on a game farm in Mpumalanga Province, South Africa, where roan are known to die from theileriosis. Various clinical parameters were recorded during this period. The predominant ticks parasitising these animals at the time (January to February), were Rhipicephalus appendiculatus and Rhipicephalus evertsi evertsi adults. After a period of 5 weeks the animals developed signs of clinical theileriosis and were treated with buparvaquone to prevent mortality. Primary hyperplasia of the local draining lymph nodes (Lnn. anorectales) near the feeding site of adult R. evertsi evertsi indicated possible transmission of Theileria sp. (sable) by this tick species. After recovery from theileriosis, these animals were confirmed carriers of Theileria sp. (sable) by PCR (polymerase chain reaction) and DNA probe analysis. Laboratory-bred larvae and nymphs of R. evertsi evertsi and R. appendiculatus respectively, were fed on the ears of these roan antelope. Salivary glands from moulted and prefed adult ticks of each species were dissected and stained for Theileria spp., and the PCR and DNA probe applied to a representative batch of dissected glands. R. appendiculatus adults collected from grass in infected camps were also dissected after prefeeding them on rabbits. Salivary glands of both tick species showed infected acini on staining and were also positive for Theileria sp. (sable) only, on multiprotozoal PCR-screening analysis. There was no statistical significant difference between the infection rate and the intensity of infection between the two tick species. R. appendiculatus ticks collected from grass were also PCR-positive for Theileria sp. (sable).

Genetic diversity, acaricide resistance status and evolutionary potential of a Rhipicephalus microplus population from a disease-controlled cattle farming area in South Africa

The Southern cattle tick, Rhipicephalus microplus is a hematophagous ectoparasite of great veterinary and economic importance. Along with its adaptability, reproductive success and vectoring capacity, R. microplus has been reported to develop resistance to the major chemical classes of acaricides currently in use. In South Africa, the Mnisi community in the Mpumalanga region offers a unique opportunity to study the adaptive potential of R. microplus. The aims of this study therefore included characterising acaricide resistance and determining the level and pattern of genetic diversity for R. microplus in this region from one primary population consisting of 12 communal dip-stations. The level of acaricide resistance was evaluated using single nucleotide polymorphisms (SNPs) in genes that contribute to acaricide insensitivity. Additionally, the ribosomal internal transcribed spacer 2 (ITS2) gene fragments of collected individuals were sequenced and a haplotype network was constructed. A high prevalence of alleles attributed to resistance against formamidines (amitraz) in the octopamine/tyramine (OCT/Tyr) receptor (frequency of 0.55) and pyrethroids in the carboxylesterase (frequency of 0.81) genes were observed. Overall, the sampled tick population was homozygous resistant to pyrethroid-based acaricides in the voltage-gated sodium channel (VGS) gene. A total of 11 haplotypes were identified in the Mnisi R. microplus population from ITS2 analysis with no clear population structure. From these allele frequencies it appears that formamidine resistance in the Mnisi community is on the rise, as the R. microplus populations is acquiring or generating these resistance alleles. Apart from rearing multi-resistant ticks to commonly used acaricides in this community these ticks may pose future problems to its surrounding areas.

Demonstration of lumpy skin disease virus infection in Amblyomma hebraeum and Rhipicephalus appendiculatus ticks using immunohistochemistry.

Lumpy skin disease (LSD) is caused by lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus. Transmission of the virus has been associated with haematophagous insects such as Stomoxys calcitrans as well as Aedes and Culex species of mosquitoes. Recent studies have reported the transmission of the virus by Amblyomma hebraeum, Rhipicephalus appendiculatus, and Rhipicephalus decoloratus ticks and the presence of LSDV in saliva of A. hebraeum and R. appendiculatus ticks. The aim of this study was to determine which tick organs become infected by LSDV following intrastadial infection and transstadial persistence of the virus in A. hebraeum and R. appendiculatus ticks. Nymphal and adult ticks were orally infected by feeding them on LSDV-infected cattle. Partially fed adult ticks were processed for testing while nymphs were fed to repletion and allowed to moult to adults before being processed for testing. The infection in tick organs was determined by testing for the presence of the viral antigen using monoclonal antibodies with immunohistochemical staining. The viral antigen was detected in salivary glands, haemocytes, synganglia, ovaries, testes, fat bodies, and midgut. Since the virus was shown to be able to cross the midgut wall and infect various tick organs, this may indicate potential for biological development and transmission of LSDV in ticks. This study strengthens the previously reported evidence of the occurrence of LSDV in tick saliva.

Transovarial passage and transmission of LSDV by Amblyomma hebraeum, Rhipicephalus appendiculatus and Rhipicephalus decoloratus

Lumpy skin disease (LSD), an acute, sub-acute or inapparent disease of cattle, is caused by lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus in the family Poxviridae. LSD is characterised by high fever, formation of circumscribed skin lesions and ulcerative lesions on the mucous membranes of the mouth, respiratory and digestive tracts. It is an economically important disease due to the permanent damage to hides, the reduction in productivity and trade restrictions imposed on affected areas. Transmission has been associated with blood-feeding insects such as stable flies (Stomoxysis calcitrans) and mosquitoes (Aedes aegypti). Mechanical (intrastadial) and transstadial transmission by Amblyomma hebraeum and Rhipicephalus appendiculatus as well as transovarial transmission by R. decoloratus have been reported. In this study transovarial passage of LSDV to larvae and subsequent transmission to recipient animals were demonstrated. The finding of transovarial passage of LSDV in female ticks shows the potential for A. hebraeum, R. appendiculatus and R. decoloratus to be reservoir hosts for LSDV.