The genetic relationship between R. microplus and R. decoloratus ticks in South Africa and their population structure.

Rhipicephalus microplus and R. decoloratus are one-host ticks that preferentially feed on cattle. They are capable of transmitting various tick-borne pathogens which may be detrimental to the agricultural and livestock industry in South Africa. Previous studies have shown that R. microplus forms five lineages in the R. microplus complex, segregating into different geographical areas based on mitochondrial markers. This study examined the phylogenetic relationship within and between R. microplus and R. decoloratus using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase subunit I (COI) genes. The results showed that the nuclear ITS2 marker is informative for interspecific variation but lacks the resolution for intraspecific variation. Analysis of the mitochondrial COI gene revealed that R. microplus ticks from South Africa grouped into a clade comprised of ticks from Asia and South America. The population structure of these two tick species was also investigated using novel microsatellite markers. Population structure analyses revealed that both the R. microplus and R. decoloratus populations presented with two genetic clusters. Rhipicephalus microplus ticks from the Kwa-Zulu Natal (KZN) province belonged to cluster 1, and those from the Eastern Cape (EC) province predominantly grouped into cluster 2. No observable population structure was noted for R. decoloratus. The overlap of genetic clusters in both species could be attributed to inbreeding between the regions by unrestricted movement of cattle across provinces. Such movement promotes tick mobility, gene flow and the homogenisation of tick populations.

Differentially expressed genes in response to amitraz treatment suggests a proposed model of resistance to amitraz in R. decoloratus ticks.

The widespread geographical distribution of Rhipicephalus decoloratus in southern Africa and its ability to transmit the pathogens causing redwater, gallsickness and spirochaetosis in cattle makes this hematophagous ectoparasite of economic importance. In South Africa, the most commonly used chemical acaricides to control tick populations are pyrethroids and amitraz. The current amitraz resistance mechanism described in R. microplus, from South Africa and Australia, involves mutations in the octopamine receptor, but it is unlikely that this will be the only contributing factor to mediate resistance. Therefore, in this study we aimed to gain insight into the more complex mechanism(s) underlying amitraz resistance in R. decoloratus using RNA-sequencing. Differentially expressed genes (DEGs) were identified when comparing amitraz susceptible and resistant ticks in the presence of amitraz while fed on bovine hosts. The most significant DEGs were further analysed using several annotation tools. The predicted annotations from these genes, as well as KEGG pathways potentially point towards a relationship between the α-adrenergic-like octopamine receptor and ionotropic glutamate receptors in establishing amitraz resistance. All genes with KEGG pathway annotations were further validated using RT-qPCR across all life stages of the tick. In susceptible ticks, the proposed model is that in the presence of amitraz, there is inhibition of Ca2+ entry into cells and subsequent membrane hyperpolarization which prevents the release of neurotransmitters. In resistant ticks, we hypothesize that this is overcome by ionotropic glutamate receptors (NMDA and AMPA) to enhance synaptic transmission and plasticity in the presence of neurosteroids. Activation of NMDA receptors initiates long term potentiation (LTP) which may allow the ticks to respond more rapidly and with less stimulus when exposed to amitraz in future. Overactivation of the NMDA receptor and excitotoxicity is attenuated by the estrone, NAD+ and ATP hydrolysing enzymes. This proposed pathway paves the way to future studies on understanding amitraz resistance and should be validated using in vivo activity assays (through the use of inhibitors or antagonists) in combination with metabolome analyses.

Metazoan Parasite Vaccines: Present Status and Future Prospects.

Eukaryotic parasites and pathogens continue to cause some of the most detrimental and difficult to treat diseases (or disease states) in both humans and animals, while also continuously expanding into non-endemic countries. Combined with the ever growing number of reports on drug-resistance and the lack of effective treatment programs for many metazoan diseases, the impact that these organisms will have on quality of life remain a global challenge. Vaccination as an effective prophylactic treatment has been demonstrated for well over 200 years for bacterial and viral diseases. From the earliest variolation procedures to the cutting edge technologies employed today, many protective preparations have been successfully developed for use in both medical and veterinary applications. In spite of the successes of these applications in the discovery of subunit vaccines against prokaryotic pathogens, not many targets have been successfully developed into vaccines directed against metazoan parasites. With the current increase in -omics technologies and metadata for eukaryotic parasites, target discovery for vaccine development can be expedited. However, a good understanding of the host/vector/pathogen interface is needed to understand the underlying biological, biochemical and immunological components that will confer a protective response in the host animal. Therefore, systems biology is rapidly coming of age in the pursuit of effective parasite vaccines. Despite the difficulties, a number of approaches have been developed and applied to parasitic helminths and arthropods. This review will focus on key aspects of vaccine development that require attention in the battle against these metazoan parasites, as well as successes in the field of vaccine development for helminthiases and ectoparasites. Lastly, we propose future direction of applying successes in pursuit of next generation vaccines.

Population structure and genetic diversity of Rhipicephalus microplus in Zimbabwe.

Recently there was an expansion in the geographic range of Rhipicephalus microplus in Zimbabwe. In order to understand gene flow patterns and population structure in this highly invasive and adaptable cattle tick, a population genetics study was carried out. Eighty-seven R. microplus tick samples drawn from 5 distinct populations were genotyped using eight polymorphic microsatellite loci. Genetic diversity (He) was high (0.755-0.802) in all the populations, suggesting high levels of gene flow with 97% of genetic variation found within populations and 3% amongst populations. No isolation by distance was observed with low but significant genetic differentiation amongst the populations (0-0.076). Most of the sampled individuals had admixed genetic backgrounds, except for those from Matabeleland North whose genetic makeup appeared different from the rest. Rhipicephalus microplus was recently recorded in this area and the environmental conditions do not support survival of the tick there. These results confirm recent range expansion of the tick and the lowest genetic diversity recorded in the Matabeleland North population is suggestive of a founder effect, which may lead to genetic drift. Generally, the very low levels of genetic differentiation amongst the populations could be a result of the frequent movement of livestock from one area to another, which will have implications for disease control. This study offers further opportunities to study evolutionary adaptation of R. microplus in Zimbabwe and southern Africa.

Genotyping acaricide resistance profiles of Rhipicephalus microplus tick populations from communal land areas of Zimbabwe.

Acaricide resistance is one of the greatest threats towards the successful control of vector ticks worldwide. Communal farmers of Zimbabwe use amitraz as the most common acaricide with occasional usage of pyrethroids and organophosphates. As a strategy towards developing an effective acaricide resistance management system in Zimbabwe, screening was done by genotyping Rhipicephalus microplus tick populations using molecular markers associated with resistance to these chemicals. The frequency of the mutant allele for the octopamine/tyramine receptor marker associated with amitraz resistance was high (0.55) and a large proportion 78.5% (288/367) of heterozygote genotypes were observed indicating balancing selection. Of the communal dipping tanks where R. microplus occurred 37.8% (39/103) showed complete resistance genotypes for amitraz. The carboxylesterase marker that has been associated with resistance to organophosphate and pyrethroids indicated no selection pressure in these chemical groups with a low frequency (0.052) of the mutant allele and 89.6% (329/367) sampled ticks showing homozygous susceptibility genotypes. Heterozygous genotypes were present at 27.2% (28/103) of the dipping tanks. The L641 mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance was not detected in Zimbabwean samples. This would suggest a different mechanism of resistance to pyrethroids in these tick populations. Sequence analysis of the octopamine/tyramine receptor gene revealed the presence of other mutations in this region, it will be important to investigate their association with amitraz resistance. These results present the first molecular genotyping of resistance profiles of R. microplus tick populations from Zimbabwe.

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.

An integrative approach to understanding pyrethroid resistance in Rhipicephalus microplus and R. decoloratus ticks.

Rhipicephalus microplus and Rhipicephalus decoloratus species occur in regions with savannah and temperate climates, typically in grassland and wooded areas used as cattle pasture. Both species are associated with the transmission of Anaplasma and Babesia spp., impacting livestock health and quality of livestock-associated products. In Africa, tick control is predominantly mediated with the use of acaricides, such as synthetic pyrethroids. After several years on the market, reports of resistance to synthetic pyrethroids escalated but limited field data and validation studies have been conducted to determine the extent of acaricide resistance in Africa. Without this data, knowledge-based tick control will remain problematic and selection pressure will remain high increasing the rate of resistance acquisition. To date, several pyrethroid resistance associated single nucleotide polymorphisms (SNPs) have been reported for arthropods within the voltage-gated sodium channel. Three SNPs have been identified within this channel in pyrethroid resistant R. microplus ticks, but none has been reported for R. decoloratus. This study is the first to report the presence of a shared SNP within the voltage-gated sodium channel in both R. microplus and R. decoloratus, which is directly linked to pyrethroid resistance in R. microplus. As the mode of action by which these SNPs mediate pyrethroid resistance remains unknown, this study aims to set hypotheses by means of predictive structural modelling. This not only paves the way forward to elucidating the underlying biological mechanisms involved in pyrethroid resistance, but also improvement of existing acaricides and ultimately sustainable tick control management.

SNP Analysis Infers that Recombination Is Involved in the Evolution of Amitraz Resistance in Rhipicephalus microplus.

Rhipicephalus microplus, better known as the Asiatic cattle tick, is a largely invasive ectoparasite of great economic importance due to the negative effect it has on agricultural livestock on a global scale, particularly cattle. Tick-borne diseases (babesiosis and anaplasmosis) transmitted by R. microplus are alarming as they decrease the quality of livestock health and production. In sub-Saharan Africa, cattle represent a major source of meat and milk, but this region of the world is severely affected by the Rhipicephalus microplus tick. The principal method for tick control is the use of chemical acaricides, notably amitraz, which was implemented in the 1990's after resistance to other acaricides surfaced. However, the efficiency of chemical control is hindered by an increase in the frequency of mutant resistance alleles to amitraz in tick populations. Presently, the only way to assess amitraz resistance is by means of larval packet tests, but this technique is time-consuming and not particularly cost effective. The main aims of this study were three-fold. First, we attempted to correlate two known SNPs in the octopamine/tyramine (OCT/Tyr) receptor with amitraz resistance in South African field samples of R. microplus. Second, we calculated gametic disequilibrium for these SNPs to determine whether they are randomly associated. Lastly, we conducted a study to assess the evolutionary effects of recombination within the OCT/Tyr receptor. Our results confirmed that the two SNPs are associated with amitraz resistance in the South African tick strain, and that they are in gametic disequilibrium. Additionally, recombination was detected in the OCT/Tyr receptor generating two recombinant haplotypes. These results are of concern to farmers in sub-Saharan Africa, and the emergence of amitraz resistance should be closely monitored in future. Therefore, we present a quick and affordable RFLP based diagnostic technique to assess amitraz resistance in field samples of R. microplus.