E.K. Piper

Immunological Profiles of Bos taurus and Bos indicus Cattle Infested with the Cattle Tick, Rhipicephalus (Boophilus) microplus

ABSTRACT The cattle tick, Rhipicephalus (Boophilus) microplus, is a major threat to the improvement of cattle production in tropical and subtropical countries worldwide. Bos indicus cattle are naturally more resistant to infestation with the cattle tick than are Bos taurus breeds, although considerable variation in resistance occurs within and between breeds. It is not known which genes contribute to the resistant phenotype, nor have immune parameters involved in resistance to R. microplus been fully described for the bovine host. This study was undertaken to determine whether selected cellular and antibody parameters of the peripheral circulation differed between tick-resistant Bos indicus and tick-susceptible Bos taurus cattle following a period of tick infestations. This study demonstrated significant differences between the two breeds with respect to the percentage of cellular subsets comprising the peripheral blood mononuclear cell population, cytokine expression by peripheral blood leukocytes, and levels of tick-specific immunoglobulin G1 (IgG1) antibodies measured in the peripheral circulation. In addition to these parameters, the Affymetrix bovine genome microarray was used to analyze gene expression by peripheral blood leukocytes of these animals. The results demonstrate that the Bos indicus cattle developed a stabilized T-cell-mediated response to tick infestation evidenced by their cellular profile and leukocyte cytokine spectrum. The Bos taurus cattle demonstrated cellular and gene expression profiles consistent with a sustained innate, inflammatory response to infestation, although high tick-specific IgG1 titers suggest that these animals have also developed a T-cell response to infestation.

Tick-susceptible Bos taurus cattle display an increased cellular response at the site of larval Rhipicephalus (Boophilus) microplus attachment, compared with tick-resistant Bos indicus cattle

Cattle demonstrate divergent and heritable phenotypes of resistance and susceptibility to infestation with the cattle tick Rhipicephalus (Boophilus) microplus. Bos indicus cattle are generally more resistant to tick infestation than Bos taurus breeds although large variations in resistance can occur within subspecies and within breed. Increased tick resistance has been previously associated with an intense hypersensitivity response in B. taurus breeds; however, the mechanism by which highly resistant B. indicus cattle acquire and sustain high levels of tick resistance remains to be elucidated. Using the commercially available Affymetrix microarray gene expression platform, together with histological examination of the larval attachment site, this study aimed to describe those processes responsible for high levels of tick resistance in Brahman (B. indicus) cattle that differ from those in low-resistance Holstein-Friesian (B. taurus) cattle. We found that genes involved in inflammatory processes and immune responsiveness to infestation by ticks, although up-regulated in tick-infested Holstein-Friesian cattle, were not up-regulated in Brahman cattle. In contrast, genes encoding constituents of the extracellular matrix were up-regulated in Brahmans. Furthermore, the susceptible Holstein-Friesian animals displayed a much greater cellular inflammatory response at the site of larval R. microplus attachment compared with the tick-resistant Brahman cattle.

Gene expression in the skin of Bos taurus and Bos indicus cattle infested with the cattle tick, Rhipicephalus (Boophilus) microplus

The cattle tick Rhipicephalus microplus (formerly Boophilus microplus) is responsible for severe production losses to the cattle industry worldwide. It has long been known that different breeds of cattle can resist tick infestation to varying degrees; however, the mechanisms by which resistant cattle prevent heavy infestation are largely unknown. The aim of this study was to determine whether gene expression varied significantly between skin sampling sites (neck, chest and tail region), and whether changes in gene expression could be detected in samples taken at tick attachment sites (tick attached to skin sample) compared with samples taken from non-attachment sites (no tick attachment). We present here the results of an experiment examining the expression of a panel of forty-four genes in skin sections taken from Bos indicus (Brahman) cattle of known high resistance, and Bos taurus (Holstein-Friesian) cattle of known low resistance to the cattle tick. The forty-four genes chosen for this study included genes known to be involved in several immune processes, some structural genes, and some genes previously suggested to be of importance in tick resistance by other researchers. The expression of fifteen gene transcripts increased significantly in Holstein-Friesian skin samples at tick attachment sites. The higher expression of many genes involved in innate inflammatory processes in the Holstein-Friesian animals at tick attachment sites suggests this breed is exhibiting a non-directed pathological response to infestation. Of the forty-four genes analysed, no transcripts were detected in higher abundance at tick attachment sites in the Brahman cattle compared with similar samples from the Holstein-Friesian group, nor difference between attachment site and non-attachment site samples within the Brahman group. The results presented here suggest that the means by which these two cattle breeds respond to tick infestation differ and warrant further investigation.

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3(+), CD4(+), CD8(+) and gammadelta T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for gammadelta T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.

Mutation in the RmβAOR gene is associated with amitraz resistance in the cattle tick Rhipicephalus microplus

Significance Amitraz is a widely used acaricide for the control of the cattle tick Rhipicephalus microplus, an important parasite of cattle in the tropics and subtropics. Here we describe in detail the evolution of amitraz resistance in replicated populations of ticks in the field, using divergent selection pressures with amitraz. We also demonstrate a close association between resistance to amitraz and a specific allele of the β-adrenergic octopamine receptor gene, which we propose confers resistance to amitraz. We aimed to describe the evolution of resistance to amitraz in Rhipicephalus microplus in the field and to test the association between amitraz resistance and the frequency of a mutation in the β-adrenergic octopamine receptor gene (RmβAOR). We established six populations of Rhipicephalus microplus ticks in similar paddocks by the admixture of ticks from strains known to be susceptible and resistant to amitraz and synthetic pyrethroids. Each population was managed using one of three acaricide treatment regimes: always amitraz, always spinosad, or rotation between amitraz and spinosad. We used microsatellites to elucidate population structure over time, an SNP in the para-sodium channel gene previously demonstrated to confer resistance to synthetic pyrethroids to quantify changes in resistance to synthetic pyrethroids over time, and a nonsynonymous SNP in the RmβAOR, a gene that we proposed to confer resistance to amitraz, to determine whether selection with amitraz increased the frequency of this mutation. The study showed panmixia of the two strains and that selection of ticks with amitraz increased the frequency of the RmβAOR mutation while increasing the prevalence of amitraz-resistance. We conclude that polymorphisms in the RmβAOR gene are likely to confer resistance to amitraz.

Host resistance in cattle to infestation with the cattle tick Rhipicephalus microplus

Resistance to Rhipicephalus microplus infestation in cattle has many effector mechanisms, each of which is likely to be modulated by complex, interacting factors. Some of the mechanisms of host resistance and their modulating factors have been identified and quantified, although much remains to be explained. The variation in resistance to tick infestation is most marked between Bos taurus and Bos indicus cattle, taurine cattle given the same exposure carrying between five and 10 times as many ticks as indicine cattle. Tick resistance is mostly manifest against attaching larvae, which attempt to feed often and without success, death occurring mostly within 24 h of finding a host. There is evidence of innate and adaptive immune response to tick infestation, and it appears that the relative importance of each differs between indicine and taurine cattle. There is conflicting information regarding the role of humoral immunity in tick resistance, and recent studies indicate that strong IgG responses to tick antigens are not protective. A strong T‐cell‐mediated response directed against larval stages, as mounted by indicine cattle, seems to be protective. Variation in the extracellular matrix of skin (epidermal growth factors, collagens and other matrix components such as lumican) also contributes to variation in host resistance.

Rhipicephalus microplus lipocalins (LRMs): genomic identification and analysis of the bovine immune response using in silico predicted B and T cell epitopes.

The attachment to host skin by Rhipicephalus microplus larvae induces a series of physiological events at the attachment site. The host-parasite interaction might induce a rejection of the larvae, as is frequently observed in Bos taurus indicus cattle, and under certain conditions in Bos taurus taurus cattle. Ticks deactivate the host rejection response by secreting specific proteins and lipids that play an essential role in manipulation of the host immune response. The available genomic information on the R. microplus tick was mined using bioinformatics approaches to identify R. microplus lipocalins (LRMs). This in silico examination revealed a total of 12 different putative R. microplus LRMs (LRM1-LRM12). The identity of the LRM family showed high sequence variability: from 6% between LRM7 and LRM8 to 55.9% between LRM2 and LRM6. However, the three-dimensional structure of the lipocalin family was conserved in the LRMs. The B and T cell epitopes in these lipocalins were then predicted, and six of the LRMs (5, 6, 9, 10, 11 and 12) were used to examine the host immune interactions with sera and peripheral blood mononuclear cells (PBMCs) collected from tick-susceptible and tick-resistant cattle challenged with R. microplus. On days 28-60 after tick infestation, the anti-LRM titres were higher in the resistant group compared with the susceptible cattle. After 60 day, the anti-LRM titres (except LRM9 and LRM11) decreased to zero in the sera of both the tick-resistant and tick-susceptible cattle. Using cell proliferation assays, the PBMCs challenged with some of the predicted T cell epitopes (LRM1_T1, T2; LRM_T1, T2 and LRM12_T) exhibited a significantly higher number of IFN-γ-secreting cells (Th1) in tick-susceptible Holstein-Friesians compared with tick-resistant Brahman cattle. In contrast, expression of the Th2 cytokine (IL-4) was lower in Holstein-Friesians cattle compared with Brahman cattle. Moreover, this study found that LRM6, LRM9 and LRM11 play important roles in the mechanism by which R. microplus interferes with the hosts haemostasis mechanisms.

Comparison of metabolic, hematological, and peripheral blood leukocyte cytokine profiles of dairy cows and heifers during the periparturient period.

The periparturient period presents major physiological challenges for the dairy cow. It is a period that is affected by metabolic stressors, major changes in endocrine status, and altered immune function, which together result in an increased risk of disease. Immunological, hematological, and metabolic profiles from the periparturient period of heifers (primipara) were compared with those of cows (pluripara) to test the hypothesis that at the time of calving they have qualitatively different peripheral blood profiles. Blood samples were collected from 22 Holstein-Friesian animals on 3 occasions: approximately 2 wk before calving, within 24h after calving, and approximately 2 wk after calving. Quantitative PCR was used to measure the expression of a selected set of cytokines and receptors by peripheral blood leukocytes. Additional analyses included hemoglobin concentration, red cell, platelet and white cell counts (total and differentiated), and clinical diagnostic biochemical profiles. Total leukocyte counts, neutrophils, and lymphocytes were higher in heifers than cows before calving and within 24h after calving. Alkaline phosphatase was consistently higher in heifers than cows and several significant differences were observed between the 2 groups with regards to cytokine and cytokine-receptor mRNA expression. The results warrant further investigation from the perspective of identifying risk factors for metabolic and parturient disease in dairy cattle.

Generation of Full-Length cDNAs for Eight Putative GPCnR from the Cattle Tick, R. microplus Using a Targeted Degenerate PCR and Sequencing Strategy

We describe here a rapid and efficient method for the targeted isolation of specific members of gene families without the need for cloning. Using this strategy we isolated full length cDNAs for eight putative G-protein coupled neurotransmitter receptors (GPCnR) from the cattle tick Rhipicephalus (Boophilus) microplus. Gene specific degenerate primers were designed using aligned amino acid sequences of similar receptor types from several insect and arachnid species. These primers were used to amplify and sequence a section of the target gene. Rapid amplification of cDNA ends (RACE) PCR was used to generate full length cDNA sequences. Phylogenetic analysis placed 7 of these sequences into Class A G-protein coupled receptors (GPCR) (Rm_α2AOR, Rm_β2AOR, Rm_Dop1R, Rm_Dop2R, Rm_INDR, Rm_5-HT7R and Rm_mAchR), and one into Class C GPCR (Rm_GABABR). Of the 7 Class A sequences, only Rm_mAchR is not a member of the biogenic amine receptor family. The isolation of these putative receptor sequences provides an opportunity to gain an understanding of acaricide resistance mechanisms such as amitraz resistance and might suggest possibilities for the development of new acaricides.

Identification of Genes Involved with Tick Infestation in Bos taurus and Bos indicus

Tick resistant cattle could provide a potentially sustainable and environmentally sound method of controlling cattle ticks. Advances in genomics and the availability of the bovine genome sequence open up opportunities to identify useful and selectable genes controlling cattle tick resistance. Using quantitative real-time PCR and theAffymetrix bovine array platform, differences in gene expression of skin biopsies from tick resistant Bos indicus (Brahman) and tick susceptible Bos taurus (Holstein-Friesian) cattle following tick challenge were examined. We identified 138 significant differentially-expressed genes, including several immunologicallhost defence genes, extracellularmatrix proteins, and transcription factors as well as genes involved in lipid metabolism. Three key pathways, represented by genes differentially expressed in resistant Brahmans, were identified; the development of the cell-mediated immune response, structural integrity of the dermis and intracellular Ca2+ levels. Ca2+, which is implicated in host responses to microbial stimuli, may be required for the enhancement or fine-tuning of transcriptional activation of Ca2+ -dependant host defence signalling pathways.