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Previous studies have applied genomics and transcriptomics to identify immune and genetic markers as key indicator traits for cattle tick susceptibility/resistance; however, results differed between breeds, and there is lack of information on the use of host proteomics. Serum samples from Santa Gertrudis cattle (naïve and phenotyped over 105 days as tick‐resistant [TR] or tick‐susceptible [TS]) were used to conduct differential abundance analyses of protein profiles. Serum proteins were digested into peptides followed by identification and quantification using sequential window acquisition of all instances of theoretical fragment ion mass spectrometry. Before tick infestation, abundance of 28 proteins differed significantly (adjusted P < 10−5) between TR and TS. These differences were also observed following tick infestation (TR vs TS) with a further eight differentially abundant proteins in TR cattle, suggesting possible roles in adaptive responses. The intragroup comparisons (TS‐0 vs TS and TR‐0 vs TR) showed that tick infestation elicited quite similar responses in both groups of cattle, but with relatively stronger responses in TR cattle. Many of the significantly differentially abundant proteins in TR Santa Gertrudis cattle (before and after tick infestation) were associated with immune responses including complement factors, chemotaxis for immune cells and acute‐phase responses.

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 host's haemostasis mechanisms.

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 24 h 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 24 h 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.

Resistance to cattle tick infestation in single-host ticks is primarily manifested against the larval stage and results in the immature tick failing to attach successfully and obtain a meal. This study was conducted to identify immune responses that characterize the tick-resistant phenotype in cattle. Thirty-five tick-naive Santa Gertrudis heifers were used in this study, thirty of which were artificially infested for thirteen weeks with tick larvae while five animals remained at a tick-free quarantine property to serve as a control group. Following thirteen weeks of tick infestation, the animals in this trial exhibited highly divergent tick-resistant phenotypes. Blood samples collected throughout the trial were used to measure peripheral immune parameters: haematology, the percentage of cellular subsets comprising the peripheral blood mononuclear cell (PBMC) population, tick-specific IgG1 and IgG2 antibody titres, IgG1 avidity for tick antigens and the ability of PBMC to recognize and proliferate in response to stimulation with tick antigens in vitro. The tick-susceptible cattle developed significantly higher tick-specific IgG1 antibody titres compared to the tick-resistant animals. These results suggest that the heightened antibody response either does not play a role in resistance or might contribute to increased susceptibility to infestation.

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.

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.

This study investigated the local immune response at larval attachment sites in Santa Gertrudis cattle with low and high levels of tick resistance. Skin samples with tick larvae attached were collected from Santa Gertrudis cattle at the end of a period of 25 weekly infestations, when the animals manifested highly divergent tick-resistant phenotypes. There was a tendency for more CD3+ , CD4+ , CD8+ , CD25+ , γδ T cells and neutrophils to concentrate at larval tick attachment site in susceptible cattle than in resistant cattle but the differences were significant only for γδ T cells and CD4+ cells. Most of the cattle developed intra-epidermal vesicles at the larval attachment site but the predominant cell within or around the vesicles was the neutrophil in susceptible animals and eosinophil in the resistant animals. The monoclonal antibodies (mAbs) specific for CD45 and CD45 RO antigens reacted with skin leucocytes from a higher number of susceptible cattle than resistant cattle. Our data suggest that some of the cellular responses mounted at larval attachment site are not involved in tick protection. The mAbs specific for CD45 and CD45 RO directly, or a test for CD45 genotype might be developed as markers of tick susceptibility or resistance.

The bovine genome consists of about 22 000 protein-coding genes, coded by about three billion base pairs (2.87 Gbp), organised into 30 chromosomes (29 autosomes plus X, Y), giving a diploid number (2n) of 60 chromosomes. Domestication, formation of breeds, and selection of animals within breed for milk production or other traits, have caused tight genetic bottlenecks and relatively low effective population sizes in most modern breeds. The accuracy of parentage tests is important to stud producers and commercial cattle producers who wish to use multiple-sire mating systems in their herds and allocate calves to their correct sires. Parentage and paternity tests are performed using genetic (DNA) markers. Many inherited diseases caused by single autosomal genes can now be detected by PCR targeting the causal mutation responsible for the disorder, usually a SNP or insertion/deletion mutation.

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.

The International Society for Animal Genetics (ISAG) proposed a panel of single nucleotide polymorphisms (SNPs) for parentage testing in cattle (a core panel of 100 SNPs and an additional list of 100 SNPs). However, markers specific to East Asian taurine cattle breeds were not included, and no information is available as to whether the ISAG panel performs adequately for these breeds. We tested ISAG's core (100 SNP) and full (200 SNP) panels on two East Asian taurine breeds: the Korean Hanwoo and the Japanese Wagyu, the latter from the Australian herd. Even though the power of exclusion was high at 0.99 for both ISAG panels, the core panel performed poorly with 3.01% false-positive assignments in the Hanwoo population and 3.57% in the Wagyu. The full ISAG panel identified all sire-offspring relations correctly in both populations with 0.02% of relations wrongly excluded in the Hanwoo population. Based on these results, we created and tested two population-specific marker panels: one for the Wagyu population, which showed no false-positive assignments with either 100 or 200 SNPs, and a second panel for the Hanwoo, which still had some false-positive assignments with 100 SNPs but no false positives using 200 SNPs. In conclusion, for parentage assignment in East Asian cattle breeds, only the full ISAG panel is adequate for parentage testing. If fewer markers should be used, it is advisable to use population-specific markers rather than the ISAG panel.

Feed efficiency and growth are the most important traits in pig production, and very few genetic markers have been reported to be associated with feed efficiency. The suppressor of cytokine signalling-2 (encoded by SOCS2) is the main negative regulator of somatic growth, and the knockout of SOCS2 and naturally mutant mice have high-growth phenotypes. Porcine SOCS2 was selected as a primary positional candidate for feed efficiency, because it is located on chromosome 5q, in the vicinity of a Quantitative Trait Locus (QTL) region for food conversion ratio in pigs. Here, we report five single nucleotide polymorphisms identified by sequencing of the promoter region and exon 1. One PCR-RFLP assay was designed for genotyping the polymorphism c.1667A > G (GenBank Accession No AY312266). Association analyses were performed in an Australian mapping resource pedigree population (PRDC-US43) for food conversion ratio, backfat, IGF1 level and growth traits and showed significant effects on average daily gain on test (ADG2) (P < 0.01) and marginal association with food conversion ratio (FCR) (P < 0.08). © 2010 The Authors, Animal Genetics

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.

The immuno-staining patterns of skin leukocytes were investigated in three breeds of cattle: Holstein-Friesian, Brahman and Santa Gertrudis of similar age before and after tick infestation. The antibodies specific for CD45 and CD45RO reacted with cells in the skin of all Holstein-Friesian cattle but did not react with cells in the skin of any Brahman cattle. The same antibodies reacted with cells from the skin of four (CD45) and seven (CD45RO) of twelve Santa Gertrudis cattle. The antibodies specific for T cells and γδ subset of T cells recognized cells from all three breeds of cattle. The antibody specific for MHC class II molecules labelled cells of mostly irregular shape, presumably dermal dendritic cells and/or macrophages and Langerhans cells. The antibody specific for granulocytes (mAb CH138) reacted with cells only in sections cut from skin with lesions. The antibody specific for CD25(+) cells labelled regularly shaped cells that showed a wide range of intensities of staining.

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.

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-HT(7)R and Rm_mAchR), and one into Class C GPCR (Rm_GABA(B)R). 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.

SOCS (suppressor of cytokine signalling) proteins, also known as SSI (stat-induced stat-inhibitor) proteins, are negative regulators of cytokine-induced signal transduction. Cytokines regulate cellular behaviour by interacting with receptors on the plasma membrane of target cells and activating intracellular signal transduction cascades such as the JAK-STAT pathway (Krebs and Hilton, 2000.) Metcalf et al. (2000) generated mice unable to express Socs2, to examine the function of the gene in vivo. Socs2–/– mice grew significantly larger than their wildtype littermates (Metcalf et al., 2000). Characteristics of deregulated growth hormone and insulin-like growth factor-1 (IGF-1) signalling were observed in the Socs2–/– mice, indicating that SOCS2 may have an essential negative regulatory role in the growth hormone/IGF-1 pathway (Metcalf et al., 2000). Greenhalgh et al. (2003) provided compelling evidence that the gigantism displayed in the Socs2–/– phenotype is a result of negative regulation of growth hormone (GH) signalling by creating Socs2–/– mice that were also deficient in endogenous growth hormone production due to a point mutation in the GH-releasing hormone receptor. These Socs2–/–Ghrhrlit/lit mice displayed almost double the response to exogenous growth hormone administration as Socs2+/+Ghrhrlit/lit mice. Genome sequences show that SOCS2 maps to human chromosome 12q (GenBank accession number NM003877) and mouse chromosome 10 (GenBank accession number NM007706). SOCS2 has not previously been mapped in the pig, and this is the first step in further investigation into the gene’s action in a large production animal. Materials and methods

16 calves were each infected with suspensions containing a mixture of approximately 230,000 Eimeria bovis and 70,000 E. zuernii oocysts, which resulted in detection of oocysts in faeces of 12 of 16 calves by day +14 after infection. On day +14 after infection calves were either treated (n = 8) with toltrazuril at 15 mg/kg body weight or with a placebo. Observations were made on the clinical condition, faecal score and liveweight of calves daily from one day post infection (pi) until 24 days pi when all calves were euthanised and examined post mortem. Samples were collected from ileum and colon for histological, immunohistochemical and gene expression studies. The study demonstrated an efficacy of toltrazuril for the treatment of E. bovis and E. zuernii infections in calves reaching 99 % (based on arithmetic mean oocyst counts in faeces) within three days of treatment and remaining at or above this level for six days. Toltrazuril did not have a significant effect on the pattern and extent of immune cellular infiltration in the mucosa of ileum and colon, but the expression of the genes coding IL-2, IL-10 and TNF-α in the ileum and TNF-α in the colon were elevated in calves treated with toltrazuril. Higher levels of oocyst shedding were significantly associated with lower expression of genes coding for IL-2, IL-10 and higher IP-10. It is concluded that toltrazuril is effective for the treatment of coccidiosis due to E. bovis and E. zuernii in calves and enables the development of a normal or enhanced immune response to infection.

Growth factors such as bone morphogenetic proteins 6, 7, 15, and two isoforms of transforming growth factor-beta (BMP6, BMP7, BMP15, TGFB1, and TGFB2), and insulin-like growth factor system act as local regulators of ovarian follicular development. To elucidate if these factors as well as others candidate genes, such as estrogen recep- tor 1 (ESR1), growth differentiation factor 9 (GDF9), follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), bone morphogenetic protein receptor, type 2 (BMPR2), type 1 insulin-like growth factor receptor (IGFR1), and key steroidogenic enzymes cytochrome P450 aromatase and 3-β-hydroxysteroid dehydrogenase (CYP19A1 and HSD3B1) could modulate or influence diestrus on the onset of puberty in Brahman heifers, their ovarian mRNA expression was measured before and after puberty (luteal phase). Six postpubertal (POST) heifers were euthanized on the luteal phase of their second cycle, confirmed by corpus luteum observation, and six prepubertal (PRE) heifers were euthanized in the same day. Quantitative real-time PCR analysis showed that the expression of FSHR, BMP7, CYP19A1, IGF1, and IGFR1 mRNA was greater in PRE heifers, when contrasted to POST heifers. The expression of LHR and HSD3B1 was lower in PRE heifers. Differential expression of ovarian genes could be associated with changes in follicular dynamics and different cell populations that have emerged as consequence of puberty and the luteal phase. The emerging hypothesis is that BMP7 and IGF1 are co-expressed and may modulate the expression of FSHR, LHR and IGFR1, and CYP19A1. BMP7 could influence the downregulation of LHR and upregulation of FSHR and CYP19A1, which mediates the follicular dynamics in heifer ovaries. Upregulation of IGF1 expression prepuberty, compared to postpuberty diestrus, correlates with increased levels FSHR and CYP19A1. Thus, BMP7 and IGF1 may play synergic roles and were predicted to interact, from the expression data (P = 0.07, r = 0.84). The role of these co-expressed genes in puberty and heifers luteal phase merits further research.