Serum proteomes of Santa Gertrudis cattle before and after infestation with Rhipicephalus australis ticks.

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.

Percutaneous tibial nerve stimulation for the treatment of interstitial cystitis/bladder pain syndrome: a pilot study.

OBJECTIVES: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic pain condition that requires multimodal management. The American Urologic Association includes sacral neuromodulation in the treatment algorithm for refractory IC/BPS. We sought to determine the rate of overall symptom improvement of IC/BPS symptoms, using validated measures, after treatment with percutaneous tibial nerve stimulation (PTNS), a form of peripheral neuromodulation. METHODS: This was a single-arm, dual-center, pilot study examining a standard PTNS protocol in subjects with IC/BPS. Our primary outcome was subject response of "moderately" or "markedly improved" on the Global Response Assessment (GRA) scale after 12-weekly PTNS sessions. Assuming a 60% response rate, 24 subjects were needed to detect a response rate between 40 and 80% with 95% confidence. Secondary objectives included change in urinary frequency on a 24-h bladder diary, bladder pain as measured by VAS and responses to validated questionnaires for pelvic pain and IC/BPS. RESULTS: Of 21 subjects enrolled, 16 initiated and 10 completed the PTNS treatment course. The GRA response rate was 40% at week 6 and 30% at week 12. Seventy percent of the cohort had some degree of improvement. There were no adverse events. CONCLUSIONS: While only a minority of subjects with IC/BPS were responders to PTNS per GRA criteria, 70% of the cohort had some degree of improvement. Due to low recruitment and loss to follow-up, we did not achieve our predetermined significance. However, our promising findings add to the limited literature on this subject.

Vaginal Hysterectomy in a Large Health Maintenance Organization: Retrospective Application of a Clinical Algorithm.

STUDY OBJECTIVE: The primary objective was to describe practice patterns of benign hysterectomy within a large community health maintenance organization (HMO) and evaluate clinical and surgeon characteristics associated with the performance of vaginal hysterectomy (VH). Secondary objectives were to retrospectively apply a VH algorithm to determine how our practice patterns conform, and compare rates of perioperative complications among patients who did and did not meet the algorithm. Patient and surgeon characteristics, and perioperative complications, were compared between patients who underwent VH and did or did not meet the VH algorithm. DESIGN: Retrospective cohort study. SETTING: Large community HMO. PATIENTS: Women undergoing benign hysterectomy. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Route of hysterectomy, patient and surgeon characteristics, perioperative complications. One hundred and thirty-one of 984 (13.3%) benign hysterectomies from January 1, 2013 to June 30, 2015 were vaginal. Patients who were vaginally parous, Hispanic, had normal preoperative uterine size and documentation of uterine descent were more likely to have VH (all p <.05). High-volume surgeons performed 18.8% of their hysterectomies vaginally, as compared to low-volume surgeons who performed 11.4% of their hysterectomies vaginally (p <.01). VH were more likely to be performed by surgeons with longer practice durations than non-vaginal hysterectomies (16.3 vs 12.2 years, p <.01). Seventy-five percent of patients who met the VH algorithm underwent non-vaginal hysterectomy and they had longer operative durations and higher rates of postoperative complications compared to patients who underwent VH. Conversely, patients who underwent VH despite not meeting the VH algorithm did not have significantly different rates of perioperative complications or blood loss than patients who met the VH algorithm. CONCLUSION: Seventy-five percent of patients deemed appropriate for VH by our algorithm underwent non-vaginal hysterectomy and had more postoperative complications and longer operative durations. Our data suggest that surgeon characteristics, including surgical volume and duration of practice, may explain some of this performance gap. These findings contribute additional insight into current practice patterns and describe clinical factors that should be included in VH algorithms.

Impact of surgical training on the performance of proposed quality measures for hysterectomy for pelvic organ prolapse.

BACKGROUND: Recent healthcare reform has led to increased emphasis on standardized provision of quality care. Use of government- and organization-approved quality measures is 1 way to document quality care. Quality measures, to improve care and aid in reimbursement, are being proposed and vetted in many areas of medicine. OBJECTIVES: We aimed to assess performance of proposed quality measures that pertain to hysterectomy for pelvic organ prolapse stratified by surgical training. The 4 quality measures that we assessed were (1) the documentation of offering conservative treatment of pelvic organ prolapse, (2) the quantitative assessment of pelvic organ prolapse (Pelvic Organ Prolapse-Quantification or Baden-Walker), (3) the performance of an apical support procedure, and (4) the performance of cystoscopy at time of hysterectomy. STUDY DESIGN: Patients who underwent hysterectomy for pelvic organ prolapse from January 1 to December 31, 2008, within a large healthcare maintenance organization were identified by diagnostic and procedural codes within the electronic medical record. Medical records were reviewed extensively for demographic and clinical data that included the performance of the 4 proposed quality measures and the training background of the primary surgeon (gynecologic generalist, fellowship-trained surgeon in Female Pelvic Medicine and Reconstructive Surgery, and "grandfathered" Female Pelvic Medicine and Reconstructive Surgery). Data were analyzed with the use of descriptive statistics. Inferential statistics with chi-squared tests were used to compare performance rates of quality measures that were stratified by surgical training. Probability values <.05 were considered statistically significant. RESULTS: Six hundred thirty patients who underwent hysterectomy for pelvic organ prolapse in 2008 had complete records available for analysis. Fellowship-trained surgeons performed 302 hysterectomies for pelvic organ prolapse; grandfathered Female Pelvic Medicine and Reconstructive Surgery surgeons performed 98 hysterectomies, and gynecologic generalist surgeons performed 230 hysterectomies. Fellowship-trained surgeons had the highest performance rates for individual quality measures (91.4-98.7%) and cumulative performance of all measures (80.8% of cases). Grandfathered Female Pelvic Medicine and Reconstructive Surgery surgeons performed significantly fewer measures (80.6-95.9% performance rate for individual measures; 65.3% cumulatively for all measures) than fellowship-trained surgeons and more than gynecologic generalists (64.3-70% for individual measures; 29.1% cumulatively for all measures). There was an association between surgeon training background and number of hysterectomies performed for pelvic organ prolapse, with specialist surgeons performing more hysterectomies. When quality measure performance was stratified by surgeon volume, similar significant associations were found, with high-volume surgeons performing more quality measures than low-volume surgeons. CONCLUSION: Within a large healthcare maintenance organization, fellowship-trained Female Pelvic Medicine and Reconstructive Surgery surgeons were more likely to perform proposed quality measures in women who underwent hysterectomy for pelvic organ prolapse compared with those surgeons without such training. Grandfathered Female Pelvic Medicine and Reconstructive Surgery surgeons performed measures more frequently than gynecologic generalists but less than fellowship-trained surgeons. Further study is indicated to correlate the proposed quality measures with clinical outcomes.

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

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 ß-adrenergic octopamine receptor gene (RmßAOR). 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 RmßAOR, 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 RmßAOR mutation while increasing the prevalence of amitraz-resistance. We conclude that polymorphisms in the RmßAOR gene are likely to confer resistance to amitraz.

Efficacy of toltrazuril 5 % suspension against Eimeria bovis and Eimeria zuernii in calves and observations on the associated immunopathology.

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.

Allelic Variation in Protein Tyrosine Phosphatase Receptor Type-C in Cattle Influences Erythrocyte, Leukocyte and Humoral Responses to Infestation With the Cattle Tick Rhipicephalus australis.

The protein tyrosine phosphatase receptor type-C (PTPRC) gene encodes the common leukocyte antigen (CD45) receptor. CD45 affects cell adhesion, migration, cytokine signalling, cell development, and activation state. Four families of the gene have been identified in cattle: a taurine group (Family 1), two indicine groups (Families 2 and 4) and an African "taurindicine" group (Family 3). Host resistance in cattle to infestation with ticks is moderately heritable and primarily manifests as prevention of attachment and feeding by larvae. This study was conducted to describe the effects of PTPRC genotype on immune-response phenotypes in cattle that display a variable immune responsiveness to ticks. Thirty tick-naïve Santa-Gertrudis cattle (a stabilized composite of 5/8 taurine and 3/8 indicine) were artificially infested with ticks weekly for 13 weeks and ranked according to their tick counts. Blood samples were taken from control and tick-challenged cattle immediately before, then at 21 d after infestation and each subsequent week for 9 weeks. Assays included erythrocyte profiles, white blood cell counts, the percentage of cellular subsets comprising the peripheral blood mononuclear cell (PBMC) population, and the ability of PBMC to recognize and proliferate in response to stimulation with tick antigens in vitro. The cattle were PTPRC genotyped using a RFLP assay that differentiated Family 1 and 3 together (220 bp), from Family 2 (462 bp), and from Family 4 (486 bp). The PTPRC allele frequencies were Family 1/3 = 0.34; Family 2 = 0.47; Family 4 = 0.19. There was no significant association between PTPRC genotype and tick count. Each copy of the Family 1/3 allele significantly decreased total leucocyte count (WCC) and CD8+ cells. Increasing dosage of Family 2 alleles significantly increased red blood cell count (RCC), haematocrit (PCV), and haemoglobin (Hb) concentration in blood. Increasing dosage of the Family 4 allele was associated with increased WCC, reduced RCC, reduced PCV and reduced Hb. Homozygote Family 1/3 animals had consistently lower IgG1 in response to tick Ag than homozygote Family 2 animals. The PTPRC genotype influences the bovine immune response to ticks but was not associated with the observed variation in resistance to tick infestation in this study.

Vaginal Estrogen for the Prevention of Recurrent Urinary Tract Infection in Postmenopausal Women: A Randomized Clinical Trial.

OBJECTIVES: We aimed to compare the efficacy of 2 commonly used contemporary vaginal estrogen administrations versus placebo for the prevention of urinary tract infection (UTI) in postmenopausal women with a clinical diagnosis of recurrent UTI (rUTI). METHODS: This was an investigator-initiated, multicenter, single-blind, randomized, placebo-controlled trial of vaginal estrogen (delivered via ring or cream) compared with placebo. Postmenopausal women with documented rUTI were randomized to receive either vaginal estrogen (via ring or cream) or placebo cream in a 1:1:1 fashion. The primary outcome was occurrence of UTI at 6 months. After 6 months, open-label use of ring or active cream was offered to all participants for an additional 6 months. Because of slower than expected recruitment, sample size calculations and block randomization schema were revised to combine estrogen groups (ring or cream) for statistical comparisons to placebo cream in a 1:1 fashion. RESULTS: Thirty-five women were randomized with 9 dropouts (1 ring, 2 cream, and 6 placebo) prior to the 6 months. Intention-to-treat analysis (assuming dropouts as failures) revealed fewer women treated with vaginal estrogen had a UTI within 6 months versus placebo (11/18 vs 16/17, respectively; P = 0.041). Per-protocol analysis revealed fewer subjects treated with vaginal estrogen had a UTI at 6 months (8/15 vs 10/11, respectively; P = 0.036). CONCLUSIONS: Commonly prescribed forms of vaginal estrogen with contemporary dosing schedules prevent UTIs in postmenopausal women with an active diagnosis of rUTI.

In Search of Mobile Applications for Patients With Pelvic Floor Disorders.

OBJECTIVES: The aims of the study were to identify patient-centered applications (apps) in female pelvic medicine and reconstructive surgery and to evaluate them using a modified APPLICATIONS scoring system. METHODS: A comprehensive list of female pelvic medicine and reconstructive surgery search terms was used to identify apps in the iTunes store. Apps that were eligible for scoring were in English, pertinent to the search term, patient centered, and accurate. All five authors independently evaluated eligible apps using a modified APPLICATIONS scoring system and subsequently reconciled differences by discussion. RESULTS: One hundred and fifty-seven terms were generated and searched in the iTunes Store to reveal 4127 apps. Twenty-three eligible apps were scored. Approximately half (47%, 11/23) of the apps were free. Twelve (52%) apps cited scientific literature or expert source. Only 8 (35%) of the apps were also available in the Google Play Store. "Squeezy," "LeakFreeMe," and "Stop UTI" received the highest score of 14. CONCLUSIONS: Few identified apps were accurate and subsequently scored. Our results demonstrate that when searching for apps, patients are likely to obtain information that is not relevant or inaccurate, even if the appropriate search term is used. By carefully excluding inaccurate apps, providers should feel confident that the 23 scored apps can be shared with patients.

Efficacy of Repeat Midurethral Sling for Persistent or Recurrent Stress Urinary Incontinence: A Fellows Pelvic Research Network Study.

OBJECTIVE: The objective of this study is to compare quality of life and success rates of repeat midurethral slings (RMUS) using retropubic (RP) and transobturator (TO) routes. MATERIALS AND METHODS: Multicenter retrospective cohort with prospective follow-up of patients undergoing RMUS from 2003 to 2016. Prospective Urinary Distress Inventory (UDI-6) and Patient Global Impression of Improvement (PGI-I) were collected by phone. Primary outcome was success of repeat sling by approach (RP vs TO), defined as responses of no to UDI-6 number 3 and very much better or much better on PGI-I. RESULTS: A total of 122 patients prospectively completed UDI-6. Average ± SD time to failure after initial sling was 51.6 ± 56.1 months; mean follow-up after repeat sling was 30.7 months. Route of initial sling was RP 30.3%, TO 49.2%, and minisling 16.4%. Of the patients, 55.8% met our success definition following RMUS. About 71.3% were very much better or much better on PGI-I, and 30.3% reported stress urinary incontinence (SUI) on UDI-6. Of the RMUS, 73.8% were RP versus 26.2% TO.There was no difference in success between repeat RP and TO routes (53.3% versus 63.3%, P = 0.34), nor for individual components: PGI-I response of very much better or much better (68.9% vs 78.1%), UDI-6 total score (25.9 vs 22.7, P = 0.29), or SUI on UDI-6 number 3 (32.2% vs 25.0%, P = 0.45), although the predetermined sample size was not met. No predictors of success or failure of RMUS were identified. CONCLUSIONS: Majority of patients are very much better or much better after RMUS, although 30% still report bothersome SUI. No difference in success was observed between RP and TO RMUS.

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.

Paraurethral Leiomyoma in a 20 Year-old Woman: A Case Report.

We present the case of a 20 year-old woman with a vulvar mass, found to be a paraurethral leiomyoma. She subsequently underwent supermedial-approach paraurethral mass excision, distal urethral reconstruction and cystourethroscopy. Paraurethral leiomyoma make up approximately five percent of urethral tumors. This case depicts the presentation and treatment of a paraurethral leiomyoma in one of the youngest women reported in the literature.

A Large Retrospective Series of Pregnancy and Delivery After Midurethral Sling for Stress Urinary Incontinence.

OBJECTIVES: The aim of this study was to review the complete medical course, including safety, efficacy, and medical decision making, of patients who delivered a pregnancy after the placement of midurethral sling (MUS) within a large health maintenance organization. METHODS: We conducted a retrospective case series of patients within the Northern and Southern California Permanente Medical Group who were identified based on procedural codes for placement of MUS for stress urinary incontinence. Electronic medical records with MUS code were referenced against records of live births within the health care system. Demographic and clinical data were abstracted through extensive, standardized physician review of the electronic medical records of all identified patients. RESULTS: Twenty-six patients were identified and included in analysis. Sixteen patients had a retropubic MUS, and 10 had a transobturator MUS. Twenty-one of 26 patients reported subjective resolution of urinary incontinence after MUS. Fourteen of the 25 first deliveries after MUS were cesarean section (CS). Of these, 5 CS were elective primary for the indication of previous MUS, 5 were elective repeat CS, and the remaining 4 were for fetal indications. Eleven patients had spontaneous vaginal deliveries. There were no MUS-related pregnancy complications. One patient developed recurrent stress urinary incontinence postpartum, for which she underwent repeat MUS within a year resulting in cure of incontinence. CONCLUSIONS: This case series corroborates previous series demonstrating the safety and durability of MUS after subsequent pregnancy.

Candidate Gene Expression in Bos indicus Ovarian Tissues: Prepubertal and Postpubertal Heifers in Diestrus.

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 receptor 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-ß-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.

Pregnancy after transvaginal sling for stress urinary incontinence: a case series.

OBJECTIVES: The aim of this study is to assess the incidence, medical course, and outcomes of women who carried a pregnancy to delivery after the placement of a transvaginal mesh sling for treatment of stress urinary incontinence (SUI) in a closed health care system. METHODS: We conducted a retrospective case series of 15 women identified within the Kaiser Permanente Northern California region based on coding for placement of a sling for SUI. Records were cross-matched against the records of live births. The electronic medical records of subjects were extensively reviewed. RESULTS: Fifteen patients were identified and included in the analysis. Twelve patients had retropubic midurethral slings, 2 patients had transobturator slings, and one had a single-incision sling procedure. Ten of 15 were delivered via cesarean delivery. Five of the 15 patients had concomitant prolapse surgery at the time of sling placement; all of these patients had cesarean deliveries. There were no reports of voiding dysfunction or sling-related complications during pregnancy. Eleven of 15 patients were dry after the sling procedure, and 9 of the 15 were dry after delivery. No patients have had additional procedures or interventions for SUI after pregnancy. CONCLUSIONS: Our case series may suggest that vaginal delivery is not contraindicated and sling-related complications during pregnancy are not prevalent.

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 host's haemostasis mechanisms.

Imputation of microsatellite alleles from dense SNP genotypes for parentage verification across multiple Bos taurus and Bos indicus breeds.

To assist cattle producers transition from microsatellite (MS) to single nucleotide polymorphism (SNP) genotyping for parental verification we previously devised an effective and inexpensive method to impute MS alleles from SNP haplotypes. While the reported method was verified with only a limited data set (N = 479) from Brown Swiss, Guernsey, Holstein, and Jersey cattle, some of the MS-SNP haplotype associations were concordant across these phylogenetically diverse breeds. This implied that some haplotypes predate modern breed formation and remain in strong linkage disequilibrium. To expand the utility of MS allele imputation across breeds, MS and SNP data from more than 8000 animals representing 39 breeds (Bos taurus and B. indicus) were used to predict 9410 SNP haplotypes, incorporating an average of 73 SNPs per haplotype, for which alleles from 12 MS markers could be accurately be imputed. Approximately 25% of the MS-SNP haplotypes were present in multiple breeds (N = 2 to 36 breeds). These shared haplotypes allowed for MS imputation in breeds that were not represented in the reference population with only a small increase in Mendelian inheritance inconsistancies. Our reported reference haplotypes can be used for any cattle breed and the reported methods can be applied to any species to aid the transition from MS to SNP genetic markers. While ~91% of the animals with imputed alleles for 12 MS markers had ≤1 Mendelian inheritance conflicts with their parents' reported MS genotypes, this figure was 96% for our reference animals, indicating potential errors in the reported MS genotypes. The workflow we suggest autocorrects for genotyping errors and rare haplotypes, by MS genotyping animals whose imputed MS alleles fail parentage verification, and then incorporating those animals into the reference dataset.

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_ß2AOR, 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.

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.

Immunological profiles of Bos taurus and Bos indicus cattle infested with the cattle tick, Rhipicephalus (Boophilus) microplus.

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.