No description available.

No description available.

Thesis (Ph. D.)--Washington State University, May 2007. / Includes bibliographical references.

The cattle tick, Rhipicephalus microplus, has a debilitating effect on the livestock industry worldwide, owing to its being a vector of the causative agents of bovine babesiosis and anaplasmosis. In South Africa, co-infestation of livestock with R. microplus and R. decoloratus occurs. An alternative to chemical control methods is sought in the form of an anti-tick vaccine. Using microarray technology, this study aimed at identifying genes that are shared between midgut tissues of adult female R. microplus and R. decoloratus ticks. In addition, results from another study were used and a reverse vaccinology pipeline was devised to identify putative novel vaccine candidates. Using a custom oligonucleotide microarray comprising 13 477 R. microplus sequences, 2476 genes were found to be shared between the two abovementioned tick species. In addition, 136 were found to be more abundantly expressed in R. decoloratus and 1084 in R. microplus. Chi-square analysis revealed that genes involved in lipid transport and metabolism are significantly over-represented in R. microplus and R. decoloratus. With vaccine design in mind, considering genes that are expressed in the midgut of both tick species, 6730 genes were identified and of these, 1224 are predicted to contain membrane-spanning helices. One major limitation to anti-tick vaccine discovery in the past has been a lack of candidates to evaluate, combined with limited knowledge of the transcriptome of R. microplus. This study identified a large pool of transcripts that are expressed in the midgut of both R. microplus and R. decoloratus adult females. Of these, those that are expressed in larvae, nymphs and the midgut were identified in another study and an in silico pipeline was used to predict membrane-bound protective antigens using an alignment-free approach, which led to the identification of seven proteins that were predicted to be both glycosylphosphatidylinositol (GPI)-anchored and more likely than Bm86 to be protective antigens. Finally, epitopes were predicted and corresponding synthetic peptides were evaluated using enzyme-linked immunosorbent assay (ELISA), resulting in the identification of three epitopes that are recognized to a greater extent than previously published Bm86 epitopes, when using murine serum raised against membrane proteins from the midgut of R. microplus. These results are significant because novel R. microplus proteins that are also present in R. decoloratus were identified. Trials using recombinant protein are under way and this will ultimately validate the experimental methodology discussed in this dissertation. Finally, regardless of whether the next-generation anti-tick vaccine has been discovered, this study also led to the identification of novel reference genes that can be used for real-time PCR experiments. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Biochemistry / Unrestricted

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The broad objective of this study was to identify single nucleotide polymorphisms (SNP) markers associated with tick resistance in South African Nguni cattle and it was addressed by three specific objectives. The first objective was to assess tick load and prevalence in Nguni cattle in different agro-climatic regions of South Africa using tick count data collected monthly from 586 Nguni cattle reared under natural grazing conditions, over two years. Tick counts were assessed under natural challenge at ARC Roodeplaat and Loskop farms (warm climate), and Mukhuthali Nguni Community and University of Fort Hare farms (cool climate). The second objective was to estimate genetic parameters for tick counts in Nguni cattle. The third objective was to identify SNPs associated with tick resistance in Nguni cattle. Counts for each tick species were conducted on each animal in the herd once a month on different body locations, including the head, ears, neck, back, legs, belly, perineum and tail. Distribution of counts was determined using the PROC FREQ (SAS, 2002 - 2010). The tick counts were then analysed with the PROC GLM procedure using the two fixed effect models. Genetic parameters for log-transformed counts were estimated from univariate animal and sire models and bivariate sire models using the ASREML program. Animals were genotyped using Illumina BovineSNP50K assay. After Quality Control (call rate >90%, minor allele frequency > 0.02), 40 436 SNPs were retained for analysis. Association analysis for tick resistance was carried out using two approaches: genome-wide association (GWA) analysis using the GenABEL package and a Regional Heritability Mapping (RHM) analysis. Six tick species were identified: Amblyomma hebraeum (42%), Rhipicephalus evertsi evertsi (22%), Rhipicephalus (Boophilus) spp. (16%), Rhipicephalus appendiculatus (11%), Hyalomma marginatum (5%) and Rhipicephalus simus (4%). Tick infestation was significantly affected by location, season, year, month of counting and age of the animal. Loskop farm, as the warmest location, had the highest tick counts and also showed the largest variation in tick loads. Higher tick counts were also observed in the hot-dry (September to November) and hot-wet (December to February) seasons compared to the other seasons. Amblyomma hebraeum was the dominant tick species across all four locations. Heritability estimates for tick count varied according to season and trait (body part or tick species) and ranged from 0.01±0.01 to 0.26±0.01. Genetic correlations ranged from -0.79±0.33 to 1.00±0.00 among counts for different body parts and 0.00±0.00 to 0.99±0.00 among tick species. Phenotypic correlations ranged from 0.06±0.01 to 0.72±0.01 among body parts and 0.01±0.02 to 0.44±0.01 for tick species. Whole body count was highly correlated to the perineum and the belly. These two traits appear to be the most suitable surrogates for whole body count. Several genomic regions of interest were identified for different traits by both the GWA and RHM approaches. Three genome-wide significant regions on chromosomes 7, 10 and 19 were identified for total tick count on the head, total A. hebraeum ticks and for total number of A. hebraeum in the perineum region. Suggestive significant regions were identified on chromosomes 1, 3, 6, 7, 8, 10, 11, 12, 14, 15, 17, 19 and 26 for several of the tick traits analysed. The GWA approach identified more genomic regions than did the RHM approach. These findings provide information that would be useful in developing strategies for genetic improvement of tick resistance through selection. The chromosome regions identified as harbouring quantitative trait loci (QTL) underlying variation in tick burden form the basis for further analyses to identify specific candidate genes related to cattle tick resistance and provide the potential for marker-assisted selection in Nguni. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om enkel nukleotied polimorfismes (ENPs) merkers te identifiseer wat verwant is aan bosluisweerstand in Suid-Afrikaanse Nguni beeste; dit is aangespreek deur drie doelwitte. Die eerste doelwit was om bosluislading en -voorkoms van bosluise in Nguni beeste in verskillende landbou-klimaatstreke van Suid-Afrika te bepaal deur die gebruik van bosluistelling data wat maandeliks van 586 Nguni beeste, grootgemaak op natuurlike weiding, oor 'n tydperk van twee jaar versamel was. Die tweede doelwit van die studie, was om die genetiese parameters te bepaal vir die bosluistellings in die Nguni beesras. Om hierdie doelwit aan te spreek, is vier verskillende datastelle onderskei in die bosluistelling data wat oor die twee jaar periode versamel was. Genetiese parameters is derhalwe beraam vir die telling van bosluise om sodoende die beste seisoen te identifiseer vir die insameling van bosluistelling data om ten einde strategieë te ontwikkel vir die genetiese seleksie vir vehoogde weerstand teen bosluise. Die derde doelwit was om ENP streke te identifiseer wat verband hou met bosluisweerstand in Nguni beeste. Verskillende bosluisspesies was getel op elke dier in die kudde een keer per maand op verskillende plekke op die liggaam, insluitend die kop, ore, nek, rug, bene, maag, perineum en stert. Bosluistelling data is ontleed met behulp van die SAS program om bosluislading variasie te bepaal. Genetiese parameter skattings vir log getransformeerde bosluistellings data was bereken vanaf twee-veranderlike vaar modelle en een-veranderlike dier- en vaar modelle met behulp van die ASREML program. Om ‘n genomiese wye assosiasie studie (GWAS) uit te voer, is DNS geïsoleer en genotipering gedoen met behulp van die Illumina BovineSNP50K toets. Na kwaliteit kontrole (oproep frekwensie>90%, klein alleelfrekwensie>0.02) is 40.436 ENPs behou vir ontleding. Assosiasie analise vir bosluisweerstand is uitgevoer met behulp twee benaderings, d.i. 'n genoom-wye assosiasie (GWA) analise met behulp van die GenABEL pakket en 'n plaaslike oorerflikheid karterings (POK) analise. Ses bosluisspesies is geïdentifiseer, d.i. Amblyomma hebraeum (42%), Rhipicephalus evertsi evertsi (22%), Rhipicephalus (Boophilus) spp. (16%), Rhipicephalus appendiculatus (11%), Hyalomma marginatum (5%) en Rhipicephalus simus (4%). Bosluis besmetting was beduidend beïnvloed deur die plek, seisoen, jaar, maand tel en ouderdom van die dier. Loskop plaas het die warmste weer ervaar en het die hoogste bosluis tellings en ook die grootste variasie in bosluislading gehad. Hoër bosluistellings is ook waargeneem in die warm droë (September tot November) en warm nat (Desember-Februarie) seisoene in vergelyking met die ander seisoene. Amblyomma hebraeum is geïdentifiseer as die mees dominante bosluisspesies oor al vier lokaliteite. Die voorkeur aanhegtingsarea vir die bosluise was onder die stert, perineum en maag areas op die liggaam. Die oorerflikheid beraming vir bosluistelling, soos beïnvloed deur die seisoen en eienskap (d.i. deel van die liggaam of bosluisspesies), het gewissel van 0.01±0.01 tot 0.26±0.01. Genetiese korrelasies het gewissel van -0.79±0.33 tot 1.04±0.01 vir bosluistellings op verskillende liggaamsdele en tussen 0.00±0.00 en 0.99±0.19 vir bosluisspesies. Fenotipiese korrelasies was laag tot matig en het gewissel van 0.06±0.01 tot 0.72±0.01 vir liggaamsdele en 0.01±0.02 to 0.44±0.01 vir bosluisspesies. Die datastel D wat September-Januarie bosluistellings bevat het die hoogste genetiese variasie aangedui. Heel liggaam bosluistellings was hoogs gekorreleerd met bosluistellings rondom die perineum en maag. Hierdie twee lokaliteite blyk die mees geskikte plaasvervanger vir die heel liggaam bosluistelling te wees. Verskeie genoom gebiede van belang is geïdentifiseer vir die verskillende eienskappe van beide die GWA en RHM benaderings. Drie genoom-wye beduidende streke (op chromosome 7, 10 en 19) is geïdentifiseer vir die totale bosluistelling op die kop, totale A. hebraeum bosluise en vir die totale aantal A. hebraeum in die perineum streek. Aanbevelende beduidende streke is geïdentifiseer op chromosome 1, 3, 6, 7, 8, 10, 11, 12, 14, 15, 17, 19 en 26 vir 'n paar van die bosluis eienskappe wat ontleed was. Die GWA benadering identifiseer meer genoom gebiede as die POK benadering. Hierdie bevindinge bied nuttige inligting vir die ontwikkeling van strategieë vir die genetiese verbetering van bosluisweerstand deur seleksie. Die chromosome streke hier geïdentifiseer is skuiling kwantitatiewe eienskap loki (KEL) vir die onderliggende variasie in bosluislading en vorm die basis vir verdere ontledings vir spesifieke kandidaat gene te identifiseer wat verband hou met die vee bosluisweerstand en bied die potensiaal vir merkerbemiddelde seleksie in Nguni.

O carrapato do boi Rhipicephalus microplus é um ectoparasita de bovinos de grande importância sanitária e econômica, que produz grandes prejuízos na bovinocultura do Brasil. O uso de ivermectina como meio de controle químico, ao longo dos anos, levou à seleção de linhagens de R. microplus resistentes a ivermectina. A consequência desse processo é a perda da eficácia do acaricida. Este trabalho teve por objetivo determinar as bases moleculares da resistência a ivermectina. Para o estudo dos mecanismos envolvidos na resistência a ivermectina de R. microplus foram utilizadas duas cepas de referência: a cepa Mozo, suscetível a ivermectina, e a cepa Juarez, resistente, e duas abordagens experimentais complementares: ensaios enzimáticos com sinergistas e estudos de expressão gênica por meio de sequenciamento massivo em paralelo do RNA ou RNA-Seq. Os resultados deste trabalho permitem afirmar que o mecanismo de desintoxicação metabólica de maior importância é mediado por transportadores ABC, seguido de esterases, glutation-S-transferases e citocromo-oxidases. As diferenças de expressão gênica observadas entre as cepas, e dentro das cepas a diferentes concentrações de ivermectina, confirmam os resultados e permitem concluir que R. microplus apresenta uma resposta poligênica à ivermectina / The cattle tick Rhipicephalus microplus is one of the most important ectoparasites with great sanitary and economic importance for cattle rearing in Brazil. Ivermectin is a drug used in the chemical control of R. microplus. The utilization of ivermectin in the last 30 years has led to the selction of resistant populations of R. microplus, and thus, the loss of efficacy in the cattle tick control. To study the molecular basis of ivermectin resistance in R. microplus, two strains of ticks were used: the susceptible strain Mozo, and the resistant strain Juarez. Two complementary approaches were carried out: enzimatic assays using sinergists and gene expression studies using massive parallel sequencing of RNA or RNA-Seq. The results of this work indicate the involvement of various mechanisms of metabolic resistance. Detoxification mechanisms mediated by ABC transporters are probably the most important. Esterases, gluthathion-S-transferases and citochrome-oxidases play a less important role in detoxification. Differences in gene expression were observed between strains and among samples of the same strain exposed to different concentrations of ivermectin, confirming the result with the assays. Hence, the response of R. microplus to ivermectin is, probably, complex and poligenic

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

Thesis (MSc (Veterinary Science)--University of Pretoria, 2007. / Includes bibliographical references.

The cattle tick, Rhipicephalus (Boophilus) microplus is a tick of veterinary and health importance globally, transmitting Babesia bovis and B. bigemina. Tick control is important and needed to prevent livestock diseases caused by tick-transmitted pathogens. Traditionally, tick control methods have resulted in development of acaricide-resistant ticks, environmental pollution and meat and milk contamination. Therefore, there is a need for alternative method and vaccines directed against tick feeding. The aim of this study was to identify proteins involved in tick feeding, tickhost-pathogen interactions and tick reproduction. Consequently, these will help in identification of antigens with the ultimate goal of developing anti-tick vaccines. R. (B.) microplus female ticks were collected at five different feeding stages. RNA was isolated from the salivary gland extracts (SGEs). The cDNA libraries were synthesized and sequenced with the Illumina MiSeq technology. Transcriptome data was analyzed with CLC Genomics Workbench, Trinity and Minia. The SGEs were also used to isolate the fractions: membrane, soluble and pellet protein for proteomic analysis. The proteomics data was analysed with Mascot, X!Tandem and Scaffold. Both the transcriptome and proteome analysis revealed the presence of major secretory protein families such as Kunitz, lipocalins, serpins, cement proteins and metalloproteases, while the majority of transcripts coded for housekeeping genes. / National Research Foundation (South Africa) / Life and Consumer Science / M. Sc. (Life Science)