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During the United States Department of Agriculture (USDA) National Animal Health Monitoring System's (NAHMS) 2007-2008 beef study, 567 producers from 24 US States were offered the opportunity to collect fecal samples from weaned beef calves and have them evaluated for the presence of parasite eggs (Phase 1). Participating producers were provided with instructions and materials for sample collection. Up to 20 fresh fecal samples were collected from each of the 99 participating operations. Fresh fecal samples were submitted to one of 3 randomly assigned laboratories for evaluation. Upon arrival at the laboratories, all samples were processed for the enumeration of strongyle, Nematodirus, and Trichuris eggs using the modified Wisconsin technique. The presence or absence of coccidian oocysts and tapeworm eggs was also noted. In submissions where the strongyle eggs per gram exceeded 30, aliquots from 2 to 6 animals were pooled for DNA extraction. Extracted DNA was subjected to genus level polymerase chain reaction (PCR) identification for the presence of Ostertagia, Cooperia, Haemonchus, Oesophagostomum, and Trichostrongylus. In this study, 85.6% of the samples had strongyle type, Nematodirus, and Trichuris eggs. Among the samples evaluated, 91% had Cooperia, 79% Ostertagia, 53% Haemonchus, 38% Oesophagostomum, 18% Nematodirus, 7% Trichuris, and 3% Trichostrongylus. The prevalence of coccidia and tapeworm eggs was 59.9% and 13.7%, respectively.

During the United States Department of Agriculture (USDA) National Animal Health Monitoring System's (NAHMS) 2007-2008 beef study, producers from 24 states were offered the opportunity to evaluate their animals for internal parasites and for overall responses to treatment with anthelmintics. A lapse of 45 d was required between initial sampling and any previous treatments. Choice of anthelmintic (oral benzimidazoles, and both injectable and pour-on endectocides) was at the discretion of the producer so as not to alter the local control programs. Fresh fecal samples were collected from 20 animals, or from the entire group if less than 20, then randomly assigned to 1 of 3 participating laboratories for examination. Analyses consisted of double centrifugation flotation followed by enumeration of strongyle, Nematodirus, and Trichuris eggs (the presence of coccidian oocysts and tapeworm eggs was also noted). Where strongyle eggs per gram (epg) exceeded 30, aliquots from 2 to 6 animals were pooled for egg isolation and polymerase chain reaction (PCR) analysis for the presence of Ostertagia, Cooperia, Haemonchus, Oesophagostomum, and Trichostrongylus. Results from 72 producers (19 States) indicated that fecal egg count reductions were < 90% in 1/3 of the operations. All operations exhibiting less than a 90% reduction had used pour-on macrocyclic lactones as the anthelmintic treatment. While some of these less than expected reductions could have been the result of improper drug application, PCR analyses of the parasite populations surviving treatment, coupled with follow-up studies at a limited number of sites, indicated that less than expected reductions were most likely due to anthelmintic resistance in Cooperia spp. and possibly Haemonchus spp.

Genetic markers associated with parasite indicator traits are ideal targets for study of marker assisted selection aimed at controlling infections that reduce herd use of anthelminthics. For this study, we collected gastrointestinal (GI) nematode fecal egg count (FEC) data from post-weaning animals of an Angus resource population challenged to a 26 week natural exposure on pasture. In all, data from 487 animals was collected over a 16 year period between 1992 and 2007, most of which were selected for a specific DRB1 allele to reduce the influence of potential allelic variant effects of the MHC locus. A genome-wide association study (GWAS) based on BovineSNP50 genotypes revealed six genomic regions located on bovine Chromosomes 3, 5, 8, 15 and 27; which were significantly associated (-log10 p=4.3) with Box-Cox transformed mean FEC (BC-MFEC). DAVID analysis of the genes within the significant genomic regions suggested a correlation between our results and annotation for genes involved in inflammatory response to infection. Furthermore, ROH and selection signature analyses provided strong evidence that the genomic regions associated BC-MFEC have not been affected by local autozygosity or recent experimental selection. These findings provide useful information for parasite resistance prediction for young grazing cattle and suggest new candidate gene targets for development of disease-modifying therapies or future studies of host response to GI parasite infection.

Fetal and maternal immune responses 3, 6 and 9 weeks post infection (wpi) were investigated in cows experimentally infected with Neospora caninum on day 110 of gestation. Descriptive analysis showed that the fetuses had lower percentages of spleen T cell subpopulations (CD3+, CD4+ and CD8+) at 6 wpi compared to 3 wpi and/or 9 wpi, with the lowest percentages observed in a dead fetus found upon euthanasia at that time. Increased expression of most cytokines over levels recorded at 3 and 9 wpi were found in fetuses that were alive at 6 wpi. Up-regulated Th1, Th2 and Treg expression was also observed at 6 wpi in the spleen and in the lymph nodes draining the placenta of the cows. At the placental level, while most cytokines were down-regulated from 6 wpi, up-regulation of IL-4 expression was observed at 6 wpi in the caruncle. Our results suggest that the immune response at 6 wpi was crucial for fetal survival in this model of bovine neosporosis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The first documented case of macrocyclic lactone resistance in gastrointestinal (GI) nematodes of cattle was seen in the US approximately 10 years ago. Since that time the increase incidence of anthelmintic resistance has continued at an alarming rate. Currently parasites of the genera Cooperia and/or Haemonchus resistant to generic or brand-name macrocyclic lactones have be demonstrated in more than half of all operations examined. Both of these parasite genera are capable of causing economic losses by decreasing food intake and subsequently animal productivity. Currently, there are no easy and quick means to detect anthelmintic resistant GI nematodes. Definitive identification requires killing of cattle. The most commonly used field detection method is the fecal egg count reduction test (FECRT). This method can be adapted for use as a screening agent for Veterinarians and producers to identify less than desired clearance of the parasites after anthelmintic treatment. Further studies can then define the reasons for persistence of the egg counts. The appearance of anthelmintic resistance is largely due to the development of very effective nematode control programs that have significantly improved the productivity of the US cattle industry, but at the same time has placed a high level of selective pressure on the parasite genome. The challenges ahead include the development of programs that control the anthelmintic resistant nematodes but at the same time result in more sustainable parasite control. The goal is to maintain high levels of productivity but to exert less selective pressures on the parasites. One of the most effective means to slow the development of drug resistance is through the simultaneous use of multiple classes of anthelmintics, each of which has a different mode of action. Reduction of the selective pressure on the parasites can be attained through a more targeted approach to drug treatments where the producer's needs are met by selective treatment of different classes of animals and not by blanket treatment at a location. The implementation of such programs will vary by the sector of the industry and the individual site. In general, the feedlot will be the easiest sector for developing of programs, while stocker/backgrounder operations will provide the most challenging problems. A major question that must be addressed is whether it is important that parasite control programs to be sustainable over time, or if the pharmaceutical industry develop new control agents at a rate sufficient to render sustainability unimportant., (Copyright © 2014. Published by Elsevier B.V.)

DNA markers associated with quantitative trait loci (QTL) affecting host tolerance to gastrointestinal (GI) parasite infection are ideal targets for marker-assisted selection. However, few studies in cattle have attempted to identify this type of QTL due to the difficulty of generating accurate phenotypic data from a resource population with adequate statistical power for detection. For this effort, we amassed fecal egg count (FEC) measures from annual natural field challenges with GI nematodes that spanned 12 different contemporary groups of Angus calves (1992-2000) derived from a closed breeding population. FEC and blood pepsinogen measures were taken weekly over a 26-week period post-weaning, and the FEC data were Box-Cox transformed to normalize the distribution of phenotypes. These 305 test animals and more than 100 founding animals from the extended pedigree were genotyped across 190 microsatellites markers. The genome-wide analyses identified a suggestive genome-wide QTL on bovine chromosome (Chr) 8 (P < 0.002) and nominal QTL on Chr 4, 12 and 17 (P < 0.05). These findings were unique for cattle, and some corresponded to previously identified QTL locations for parasite-related traits in sheep to provide genome locations for further fine mapping of parasite resistance/susceptibility in Angus cattle., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Thirty-three colostrum-deprived Holstein bull calves (initial BW of 131 ± 4 kg) were used to determine the effect of timing of anthelmintic administration relative to vaccination on antibody titer response to vaccine component antigens. When calves were at least 3 mo of age, they were sorted randomly into individual pens and assigned to 1 of 3 treatment groups, treatments consisted of 1) dewormed 2 wk before vaccination (DPV), 2) dewormed at the time of vaccination (DV), or 3) control, vaccinated but not dewormed (CONT). All calves were inoculated with infective larvae of brown stomach worms (Ostertagia ostertagi) and intestinal worms (Cooperia spp.) on d 1, 7, 10, 14, and 18 for a total dose of 235,710 infective larvae per calf. Calves (DPV and DV) were dewormed on d 21 or 35 with a 10% fenbendazole suspension at 5 mg/kg of BW. On d 35, all calves were vaccinated with a modified-live virus respiratory vaccine containing IBRV (infectious bovine rhinotracheitis virus), BVDV-1 (bovine viral diarrhea virus genotype 1), BVDV-2 (BVDV genotype 2), PI-3 (parainfluenza-3), and BRSV (bovine respiratory syncytial virus). During the 103-d experiment, weekly fecal egg counts, blood, and rectal temperatures were collected and health status was recorded daily. Blood samples were obtained weekly to determine serum neutralizing (SN) antibody titers to IBRV, BVDV-1, BVDV-2, and PI-3 and cytokine levels for IL-4, IL-6, TNF-α (tumor necrosis factor-α), and IFN-γ (interferon-gamma). There was a tendency (P < 0.09) for CONT calves to have greater IL-4 concentrations. By design, control calves had greater (P < 0.01) fecal egg counts during the experiment. All calves developed antibody titers to IBRV, BVDV-1, BVDV-2, and PI-3 by d 15 postvaccination. On d 88, all calves were challenged with IBRV and blood samples were obtained on d 88, 89, 90, 93, 95, 98, 99, and 103. All calves had increased rectal temperatures during the final 7 d of the IBRV challenge. However, the CONT group had greater (P < 0.01) rectal temperatures on each sampling day except d 90 compared with the DPV and DV treatments. Therefore, deworming before or at vaccination reduced parasite burden and decreased rectal temperature increase after an IBRV challenge. Deworming strategy had no effect on antibody response to vaccination or IBRV challenge.

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. We previously reported an initial analysis of copy number variations (CNVs) in Angus cattle selected for resistance or susceptibility to gastrointestinal nematodes. In this study, we performed a large-scale analysis of CNVs using SNP genotyping data from 472 animals of the same population. We detected 811 candidate CNV regions, which represent 141.8 Mb (~4.7%) of the genome. To investigate the functional impacts of CNVs, we created 2 groups of 100 individual animals with extremely low or high estimated breeding values of eggs per gram of feces and referred to these groups as parasite resistant (PR) or parasite susceptible (PS), respectively. We identified 297 (~51 Mb) and 282 (~48 Mb) CNV regions from PR and PS groups, respectively. Approximately 60% of the CNV regions were specific to the PS group or PR group of animals. Selected PR- or PS-specific CNVs were further experimentally validated by quantitative PCR. A total of 297 PR CNV regions overlapped with 437 Ensembl genes enriched in immunity and defense, like WC1 gene which uniquely expresses on gamma/delta T cells in cattle. Network analyses indicated that the PR-specific genes were predominantly involved in gastrointestinal disease, immunological disease, inflammatory response, cell-to-cell signaling and interaction, lymphoid tissue development, and cell death. By contrast, the 282 PS CNV regions contained 473 Ensembl genes which are overrepresented in environmental interactions. Network analyses indicated that the PS-specific genes were particularly enriched for inflammatory response, immune cell trafficking, metabolic disease, cell cycle, and cellular organization and movement.

Cooperia spp. have become the most prevalent parasites in United States cow/calf operations as observed in the USDA NAHMS (National Animal Health Monitoring System) Beef Cow/Calf survey in 2008. This is at least in part due to the widespread use of macrocyclic lactones that have recently been shown to have a reduced activity against these parasites. The effects of Cooperia spp. on cattle productivity are largely unknown. This study was conducted to assess their effect upon cattle housed under conditions found in American feedlots. Two hundred yearling calves (average weight 460 lb/209 kg) were acquired from northwestern Arkansas and northeastern Oklahoma and were vaccinated and dewormed upon arrival at the feedlot. Animals were comingled and preconditioned for approximately one month, and were fed a standard growing ration throughout the study. Calves were randomly divided into two groups (n=80, infected and control) and each group was further divided into two replicate pens (n=40). Calves from the two infected pens were orally inoculated with a gavage of 1 × 10(5) and 0.825 × 10(5) infective larvae of a recent isolate of Cooperia punctata on day 0 and 14, respectively, with the two control pens receiving a similar volume of tap water. Data collected included biweekly fecal egg counts, daily individual feed consumption and weight gain over the 60-day test period. The presence of C. punctata (>99% of recovered worms) was confirmed by necropsy and recovery from the small intestine on days 35 and 60 post infection (PI) in a subset of animals. Egg counts were positive by day 14 PI and remained at numbers similar to values seen in field studies. The control group gained weight 7.5% more rapidly (p=0.02) than infected animals (3.24 lb/1.47 kg per day vs. 3.0 lb/1.36 kg per day, respectively). The Cooperia-infected calves also consumed 1.5 lb (0.68 kg) less dry feed per day than the control animals (p=0.02). These data suggest that C. punctata has a deleterious effect on both appetite and nutrient uptake or utilization. At necropsy (days 35 and 60), the draining mesenteric lymph nodes of infected animals were increased in size and the small intestinal mucosa was thickened and covered with a thick layer of mucus in the infected animals. The most prominent histological changes in the Cooperia- infected animals included a moderate increase in the number of intraepithelial lymphocytes and globule leukocytes, as well as aggregates of eosinophils within the lower lamina propria. The only significant difference was an increase in the goblet cell density at day 60. Anthelmintic sensitivity/resistance of the Cooperia isolate used was determined by treatment of one pen of infected calves with a macrocyclic lactone and the other pen with a benzimidazole at the completion of the study. The macrocyclic lactone treatment (n=40) did not remove the parasites (FECRT=8.8%), while treatment with a benzimidazole was very effective (FECRT=98.1%). This study demonstrated that C. punctata has a significant effect on cattle productivity, both reduced weight gain and decreased feed intake compared to controls., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Accurate genetic evaluation of livestock is based on appropriate modeling of phenotypic measurements. In ruminants, fecal egg count (FEC) is commonly used to measure resistance to nematodes. FEC values are not normally distributed and logarithmic transformations have been used in an effort to achieve normality before analysis. However, the transformed data are often still not normally distributed, especially when data are extremely skewed. A series of repeated FEC measurements may provide information about the population dynamics of a group or individual. A total of 6375 FEC measures were obtained for 410 animals between 1992 and 2003 from the Beltsville Agricultural Research Center Angus herd. Original data were transformed using an extension of the Box-Cox transformation to approach normality and to estimate (co)variance components. We also proposed using random regression models (RRM) for genetic and non-genetic studies of FEC. Phenotypes were analyzed using RRM and restricted maximum likelihood. Within the different orders of Legendre polynomials used, those with more parameters (order 4) adjusted FEC data best. Results indicated that the transformation of FEC data utilizing the Box-Cox transformation family was effective in reducing the skewness and kurtosis, and dramatically increased estimates of heritability, and measurements of FEC obtained in the period between 12 and 26 weeks in a 26-week experimental challenge period are genetically correlated.

Neospora caninum is a major cause of abortion in cattle, but the reasons why only some animals abort remain unclear. The immunological control of the parasite in the placenta or by the foetus could be the key to determining the mechanism of abortion and/or transplacental transmission to the foetus. In this study, cytokine gene expression, analysed by real-time RT-PCR, at the maternal (caruncle) and foetal placenta (cotyledon) of heifers infected at 110 days of gestation by intravenous inoculation of N. caninum tachyzoites was compared with the responses in uninfected heifers. Animals were euthanized 3 weeks after infection. Upregulated Th1, Th2 and T-regulatory (Treg) cytokine gene expression was observed in both the maternal and the foetal placenta in the infected group. In the caruncle of infected animals, the main changes included upregulation of IFN-γ, IL-12p40, IL-6 and IL-10. In the cotyledon, the main changes included upregulation of IFN-γ and downregulation of TGF-β, being the later the only cytokine downregulated in the infected group. The observed cytokine expression pattern was associated with alive but transplacentally infected foetuses, suggesting that such cytokine pattern is beneficial to foetal survival, but could have a role in the transplacental transmission of the parasite., (© 2011 Blackwell Publishing Ltd.)

Cooperia oncophora is an economically important gastrointestinal nematode in ruminants. Acquired resistance to Cooperia oncophora infection in cattle develops rapidly as a result of prior infections. Naïve cattle, when given a primary infection of high-dose infective L3 larvae, develop a strong immunity to subsequent reinfection. Compared to primary infection, reinfection resulted in a marked reduction in worm establishment. In order to understand molecular mechanisms underlying the development of acquired resistance, we characterized the transcriptomic responses of the bovine small intestine to a primary infection and reinfection. A total of 23 pathways were significantly impacted during infection. The vitamin D receptor activation was strongly induced only during reinfection, suggesting that this pathway may play an important role in the development of acquired resistance via its potential roles in immune regulation and intestinal mucosal integrity maintenance. The expression of inducible nitric oxide synthase (NOS2) was strongly induced during reinfection but not during primary infection. As a result, several canonical pathways associated with NOS2 were impacted. The genes involved in eicosanoid synthesis, including prostaglandin synthase 2 (PTGS2 or COX2), remained largely unchanged during infection. The rapid development of acquired resistance may help explain the lack of relative pathogenicity by Cooperia oncophora infection in cattle. Our findings facilitate the understanding of molecular mechanisms underlying the development of acquired resistance, which could have an important implication in vaccine design.

Apyrases (ATP-diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyse extracellular ATP and ADP to orthophosphate and AMP. One class of newly-described, Ca2+-dependent, salivary apyrases known to counteract blood-clotting, has been identified in haematophagous arthropods. Herein, we have identified a gene (Oos-apy-1) encoding a protein that structurally conforms to the Ca2+-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library. The expressed protein (rOos-APY-1) was biochemically functional in the presence of Ca2+ only, with greatest activity on ATP, ADP, UTP and UDP. Host antibodies to the fusion protein appeared as early as 14 days post-infection (p.i.) and increased through 30 days p.i. Immunohistochemical and Western blot analyses demonstrated that the native Oos-APY-1 protein is present in the glandular bulb of the oesophagus and is confined to the L4. A putative signal sequence at the N-terminus and near 100% identity with a Teladorsagia circumcincta L4 secreted protein is consistent with the native protein being secreted at the cellular level. Predicated upon substrate specificity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides released by locally-damaged tissues in an effort to modulate immune intervention and inflammation.

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. We report an initial analysis of copy number variations (CNVs) in cattle selected for resistance or susceptibility to intestinal nematodes. We performed three array comparative genomic hybridization (CGH) experiments to compare Angus cattle with extreme phenotypes for fecal egg count and serum pepsinogen level. We identified 20 CNVs in total, of which 12 were within known chromosomes harboring or adjacent to gains or losses. About 85% of the CNV identified (17/20) overlapped with cattle CNV regions that were reported recently. Selected CNVs were further validated by independent methods using quantitative PCR (qPCR) and FISH. Pathway analyses indicated that annotated cattle genes within these variable regions are particularly enriched for immune function affecting receptor activities, signal transduction, and transcription. Analysis of transcription factor binding sites (TFBS) within the promoter regions of differentially expressed genes suggested that common transcription factors are probably involved in parasite resistance. These results provide valuable hypotheses for the future study of cattle CNVs underlying economically important health and production traits.

Infections in cattle by the abomasal nematode Ostertagia ostertagi result in impaired gastrointestinal function. Six partially immune animals were developed using multiple drug-attenuated infections, and these animals displayed reduced worm burdens and a slightly elevated abomasal pH upon reinfection. In this study, we characterized the abomasal microbiota in response to reinfection using metagenomic tools. Compared to uninfected controls, infection did not induce a significant change in the microbial community composition in immune animals. 16S rRNA gene-based phylogenetic analysis identified 15 phyla in the bovine abomasal microbiota with Bacteroidetes (60.5%), Firmicutes (27.1%), Proteobacteria (7.2%), Spirochates (2.9%), and Fibrobacteres (1.5%) being the most predominant. The number of prokaryotic genera and operational taxonomic units (OTU) identified in the abomasal microbial community was 70.8±19.8 (mean ± SD) and 90.3±2.9, respectively. However, the core microbiome comprised of 32 genera and 72 OTU. Infection seemingly had a minimal impact on the abomasal microbial diversity at a genus level in immune animals. Proteins predicted from whole genome shotgun (WGS) DNA sequences were assigned to 5,408 Pfam and 3,381 COG families, demonstrating dazzling arrays of functional diversity in bovine abomasal microbial communities. However, none of COG functional classes were significantly impacted by infection. Our results demonstrate that immune animals may develop abilities to maintain proper stability of their abomasal microbial ecosystem. A minimal disruption in the bovine abomasal microbiota by reinfection may contribute equally to the restoration of gastric function in immune animals.

The abomasal nematode Ostertagia ostertagi is a major causal agent contributing to production inefficiencies in the cattle industry in temperate regions of the world. Protective immunity to infections develops very slowly and resistance to reinfection manifests only after prolonged exposure. Mechanisms underlying the development of protective immunity remain largely unexplored. Immune animals, which have significantly reduced worm burdens, were developed after multiple drug-attenuated experimental infections and were compared to a primary infected group and their respective uninfected controls. In this study, transcriptomic analysis identified three signaling pathways significantly impacted during both primary and repeat infections, the complement system, leukocyte extravasation and acute phase responses. Increased mRNA levels of complement components C3, factor B (CFB) and factor I (CFI) in the abomasal mucosa of the infected cattle were confirmed using quantitative PCR while Western blot analysis established the presence of elevated levels of activated C3 proteins in the mucosa. One of the initiators of local complement activation could be related to secretory IgA and IgM because infections significantly up-regulated expression of J chain (IGJ), as well as polymeric Ig receptor (PIGR) and an IgM-specific receptor (FAIM3), suggesting sustained increases in both synthesis and transepithelial transport of IgA and IgM during the infection. The elevated levels of pro-inflammatory cytokines, such as IL-4 and IL-1β, during infection may be involved in gene regulation of complement components. Our results suggest enhanced tissue repair and mucin secretion in immune animals may also contribute to protective immunity. These results are the first evidence that local complement activation may be involved in the development of long-term protective immunity and provide a novel mechanistic insight into resistance against O. ostertagi in cattle., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Neospora caninum is a major cause of abortion in cattle, but the reasons why some animals abort and not others remain unclear. Most of the N. caninum experimental primary infections in cattle late in gestation, after 120 days of pregnancy, result in birth of full-term congenitally infected fetuses. In the present study, the distribution of parasites and pathogenesis of infection in both dams and fetuses after inoculation with 10(7) culture derived tachyzoites of N. caninum NC-Illinois cattle strain at 110 days of gestation were analyzed at 3 weeks, 6 weeks and 9 weeks after infection (WAI) in eight Angus heifers. One dam from the group euthanized at 6 WAI had a dead fetus at necropsy. Extensive lesions were observed in the placenta and tachyzoites were detected in both the placenta and the fetus. The fetus was seropositive and had high IFN-gamma g production in fetal fluids. Another fetus, still alive when euthanized at 3 WAI, had severe lesions and high IFN-gamma production and a similar fate could have been expected if the experimental period would have been longer. Lesions in the placenta of the remaining six dams that had live fetuses at necropsy were mild. In those dams, the fetal and maternal placentas had not separated and contained focal areas of placentitis at the materno-fetal junction. Transplacental infection took place on all fetuses based on detection of parasitic DNA in fetal tissues. The present study shows that experimental N. caninum infection of naïve dams after 110 days of pregnancy can lead to fetal death. The results suggest that the severity of placental lesions and the strong IFN-gamma response in some fetuses, possibly as part of the immune response trying to control the high parasitemia, might, in fact, be the cause of their death., ((c) 2010 Elsevier B.V. All rights reserved.)

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here, we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, three Bos indicus, and three composite breeds for beef, dairy, or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 megabases or approximately 1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions, such as immunity, lactation, reproduction, and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in their CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research.

We previously documented the appearance of cattle nematode parasites resistant to avermectins, milbemycin, and a benzimidazole at the end of a grazing season in a backgrounding operation in the upper Midwestern US. To further characterize the pattern of drug resistance, we extended the study to (1) monitor the animals over the course of the grazing season; (2) increase the number of animals slaughtered at the end of the season to minimize the effect of potential outlying observations; (3) increase the time interval between treatment and slaughter to ensure sufficient time for drug action; (4) utilize repeated fecal sampling in the fecal egg reduction test to minimize procedural variation; (5) increase the number of drugs tested. The results of the present study were in agreement with those of the previous study and demonstrated that during the course of the grazing season the pastures harbored significant numbers of parasites that were refractory to avermectins, milbemycin, and a benzimidazole at the label recommended doses. As seen previously, Haemonchus contortus resistant to all these anthelmintics were present over the course of the study period. In contrast, Cooperia sp., mainly punctata, and Haemonchus placei were resistant only to the macrocyclic lactones. There was no apparent resistance against the older anthelmintic levamisole, which had not been used for >20 years in the operation. However, animals treated with this drug continued to harbor small but measurable numbers of Ostertagia ostertagi.

Resistance to modern anthelmintics by ruminant nematode parasites is an increasing problem throughout the world. To date the problem has largely been reported in parasites of small ruminants, but there are increasing reports of such resistance in nematodes recovered from cattle. Until now there have been no published reports of drug resistant parasites from cattle in North America. In 2002 a producer in the upper Midwest who backgrounds young cattle acquired from the southeastern US experienced lower than expected weight gain as well as apparent parasitic gastroenteritis in his cattle during the fall. Fecal sample results supported the suspicion that decreased productivity and diarrhea were the result of GI nematode parasitism. The operation used intensive grazing management and practiced strategically timed deworming for >17 year. In 2003, all animals were dewormed the first week of May with Ivomec Plus, then with Dectomax Injectable on 4 June and 17 July. On 31 July, 10 randomly taken fecal samples showed EPG values from 0 to 55. To assess whether the apparent decreased drug efficacy was the result of drug resistance in the nematode population, on 18 August approximately 150 heads, previously strategic timed dewormed, of 9-11 month old cattle from one pasture were selected for study. The calves were randomly assigned to 1 of 6 treatment groups: untreated (U), ivermectin injectable (I), moxidectin pour-on (M), doramectin injectable (D), eprinomectin pour-on (E), albendazole oral (A). Cattle were weighed prior to treatment and the drug was dosed according to label directions. Seven days later, 3 calves from each group were slaughtered for worm recovery. Fecal samples taken from the remaining animals at 14 days after treatment showed that the reduction of mean fecal EPG value for each group was: U-46%, I-52%, M-72%, D-61%, E-8%, and A-68%. Worm recovery from the slaughter calves showed that all groups harbored significant numbers of Haemonchus placei and H. contortus. In addition, all avermectin-treated groups contained significant numbers of Cooperia punctata, and smaller numbers of C. oncophora and C. spatulata. These results imply that the pastures studied contain substantial numbers of H. contortus resistant to both avermectins and benzimidazoles, and H. placei and Cooperia sp. resistant to all the commonly used avermectin anthelmintics. This is the first report of anthelmintic resistance in American cattle parasites.

Mucin hypersecretion is considered to be one of the most common components of the immune response to gastrointestinal nematode infection. However, investigations have not been conducted in the Cattle-Cooperia oncophora system to verify the findings largely derived from murine models. In this study, we examined the expression of seven mucins and seven enzymes in the mucin biosynthesis pathway involved in O-linked glycosylation in the bovine small intestine including goblet cells enriched using laser capture microdissection during a primary C. oncophora infection. At the mRNA level, MUC2 expression was significantly higher in both lamina propria and goblet cells at 28 days post-infection compared to the naive control. MUC5B expression at the mRNA level was also higher in lamina propria at 28dpi. Expression of MUC1, MUC4, MUC5AC, and MUC6 was extremely low or not detectable in goblet cells, columnar epithelial cells, and lamina propria from both naive control and infected animals. Among the seven enzymes involved in post-translational O-linked glycosylation of mucins, GCNT3, which may represent one of the key rate-limiting steps in mucin biosynthesis, was up-regulated in goblet cells, columnar epithelial cells, lamina propria, and gross small intestine tissue during the course of infection. Western blot analysis revealed that MUC2 glycoprotein was strongly induced by infection in both gross small intestine tissue and its mucosal layer. In contrast, the higher MUC5B protein expression was observed only in the mucosal layer. Immunohistochemistry provided further evidence of the mucin glycoprotein production and localization. Our results provided insight into regulation of mucin biosynthesis in various cell types in the bovine small intestine during gastrointestinal nematode infection and will facilitate our understanding of mucins and their role in immune response against parasitic nematodes.

In 1990, the Human Genome Sequencing Project was established. This laid the ground work for an explosion of sequence data that has since followed. As a result of this effort, the first complete genome of an animal, Caenorhabditis elegans was published in 1998. The sequence of Drosophila melanogaster was made available in March, 2000 and in the following year, working drafts of the human genome were generated with the completed sequence (92%) being released in 2003. Recent advancements and next-generation technologies have made sequencing common place and have infiltrated every aspect of biological research, including parasitology. To date, sequencing of 32 apicomplexa and 24 nematode genomes are either in progress or near completion, and over 600k nematode EST and 200k apicomplexa EST submissions fill the databases. However, the winds have shifted and efforts are now refocusing on how best to store, mine and apply these data to problem solving. Herein we tend not to summarize existing X-omics datasets or present new technological advances that promise future benefits. Rather, the information to follow condenses up-to-date-applications of existing technologies to problem solving as it relates to parasite research. Advancements in non-parasite systems are also presented with the proviso that applications to parasite research are in the making.

Cooperia oncophora is an important parasitic nematode of cattle with a wide distribution in temperate areas. Twenty Holstein nematode-naïve bull calves were experimentally infected with approximately 100,000 infective L3s and infection was allowed to progress for 7, 14, 28, 42 days, respectively. This experiment was conducted to identify putative recognition and inflammatory pathways in the host-parasite relationship. Gene expression profiles of the small intestine were compared using a high-density bovine 60 mer oligo microarray. A total of 310 genes were differentially expressed during the course of infection. The pathways and regulatory networks significantly impacted by the infection were analysed. A total of 22 canonical pathways and nine regulatory networks were significantly affected during infection. During the early phase of the infection (7 days p.i.), parasites suppressed the acute phase response and the complement system of the host. At 14 days p.i., three out of the six pathways impacted were related with retinoid X receptor (RXR) functions. At 28 days p.i., the effects on RXR were less evident. The host response shifted to lipid metabolism and signalling, especially eicosanoid production and signalling, suggesting that eicosanoid-mediated inflammation might be a major host defence mechanism. By 42 days p.i., the pathways impacted involved glycosphingolipid biosynthesis and transforming growth factor beta (TGFbeta signalling. The expression of cadherin-like 26 (CDH26) was strongly up-regulated starting at 14 days p.i. and peaked at 28 days p.i. The extent of its expression is positively correlated with the infiltration of eosinophils (R=0.82) and coincides with the number of adult parasites in the tissue. CDH26 demonstrated an expression profile similar to two other cell adhesion molecules involved in recognition of carbohydrates on foreign organisms, collectin and galectin, suggesting that it may serve as a pattern recognition molecule for C. oncophora. These results provide a potential molecular roadmap for future studies aimed at defining host immune responses and understanding protective immunity against gastrointestinal nematodes.

In the present study, we use microarray technology to investigate the expression patterns of 381 genes with known association to host immune responses. Hybridization targets were derived from previously characterized bovine cDNAs. A total of 576 reporters (473 sequence-validated cDNAs and 77 controls) were spotted onto glass slides in two sets of four replicates. Two color, comparative hybridizations across both mesenteric lymph node (MLN) and small intestine mucosa (SIM) RNA samples were done between animals with previously demonstrated phenotypic differences based on natural exposure to gastrointestinal (GI) nematodes over a 6-month exposure period. A total of 138 significant hybridization differences were detected by mixed model analysis of variance. A subset of these significant differences was validated by quantitative, real-time RT-PCR to assay transcript levels for 18 genes. These results confirmed that in the SIM, susceptible animals showed significantly higher levels in the genes encoding IGHG1, CD3E, ACTB, IRF1, CCL5 and C3, while in the MLN of resistant animals, higher levels of expression were confirmed for PTPRC, CD1D and ITGA4. Combined, the results indicate that immune responses against GI nematode infections involve multiple response pathways. Higher levels of expression for IgE receptor, integrins, complement, monocyte/macrophage and tissue factors are related to resistance. In contrast, higher levels of expression for immunoglobulin chains and TCRs are related to susceptibility. Identification of these genes provides a framework to better understand the genetic variation underlying parasite resistance.

As a complement to the Bovine HapMap Consortium project, we initiated a systematic study of the copy numbervariation (CNV) within the same cattle population using array comparative genomic hybridization (array CGH). Oligonucleotide CGH arrays were designed and fabricated to cover all chromosomes with an average interval of 6 kb using the latest bovine genome assembly. In the initial screening, three Holstein bulls were selected to represent major paternal lineages of the Holstein breed with some maternal linkages between these lines. Dual-label hybridizations were performed using either Hereford L1 Dominette 01449 or L1 Domino 99375 as reference. The CNVs were represented by gains and losses of normalized fluorescence intensities relative to the reference. The data presented here, for the first time, demonstrated that significant amounts of germline and fewer somatic CNVs exist in cattle, that many CNVs are common both across diverse cattle breeds and among individuals within a breed, and that array CGH is an effective tool to systematically detect bovine CNV. Selected CNVs have been confirmed by independent methods using real-time (RT) PCR. The strategy used in this study, based on genome higher-orderarchitecture variation, is a powerful approach to generating resources for the identification of novel genomic variation and candidate genes for economically important traits.

Understanding mechanisms of resistance to gastrointestinal nematodes is important in developing effective and sustainable control programs. A resource population of Angus cattle consisting of approximately 600 animals with complete pedigree records has been developed. The majority of these animals were completely characterized for their resistance to natural challenge by gastrointestinal nematodes. As the first step towards understanding the molecular basis of disease resistance, we investigated expression profiles of 17 cytokines, cytokine receptors, and chemokines using real-time RT-PCR in animals demonstrating resistance or susceptibility to pasture challenge. The animals exposed to natural infection for approximately 6 months were treated to remove existing parasites and then experimentally challenged with both Ostertagia ostertagi and Cooperia oncophora. The mRNA expression profiles of these genes in abomasal and mesenteric lymph nodes (ALN, MLN), fundic and pyloric abomasa (FA, PA), and small intestine (SI) were compared between resistant and susceptible animals. Resistant heifers exhibited elevated expression of inflammatory cytokines such as TNFalpha, IL-1beta, and MIP-1alpha in fundic and pyloric abomasa 7 days post infection. Expression levels of IL-10, polymeric immunoglobullin receptor gene (PIGR), and WSX-1 were also 2.7-19.9-folds higher in resistant than susceptible heifers in these tissues. No difference in expression of CXCL6, CXCL10, IFN-gamma, IL-2, IL-4, IL-6, IL-8, IL-12 p40, IL-13, IL-15 and IL-18 was observed between the two groups. The expression of MIP-1alpha, IL-6, and IL-10 was also elevated in small intestines in resistant animals. In contrast, little difference in expression of these genes was detected between resistant and susceptible groups in the draining lymph nodes. These data indicate that resistant animals can better maintain inflammatory responses at the site of infection, suggesting a possible novel mechanism of resistance.

MicroRNAs are small approximately 22 nucleotide-long noncoding RNAs capable of controlling gene expression by inhibiting translation. Alignment of human microRNA stem-loop sequences (mir) against a recent draft sequence assembly of the bovine genome resulted in identification of 334 predicted bovine mir. We sequenced five tissue-specific cDNA libraries derived from the small RNA fractions of bovine embryo, thymus, small intestine, and lymph node to validate these predictions and identify new mir. This strategy combined with comparative sequence analysis identified 129 sequences that corresponded to mature microRNAs (miR). A total of 107 sequences aligned to known human mir, and 100 of these matched expressed miR. The other seven sequences represented novel miR expressed from the complementary strand of previously characterized human mir. The 22 sequences without matches displayed characteristic mir secondary structures when folded in silico, and 10 of these retained sequence conservation with other vertebrate species. Expression analysis based on sequence identity counts revealed that some miR were preferentially expressed in certain tissues, while bta-miR-26a and bta-miR-103 were prevalent in all tissues examined. These results support the premise that species differences in regulation of gene expression by miR occur primarily at the level of expression and processing.

Control strategies for nematode parasites rely on knowledge of the relationships between the parasites and their hosts. Specifically, these programs are based on identifying crucial points of interaction in the environment provided by the host, including genetics and the immune response, and critical periods in the physical environment in which the eggs and larval stages must develop. When these targets are identified and the interactions understood, cost-effective sustainable programs can be developed using currently available antiparasitic compounds. Resistance to the major classes of anthelmintic compounds requires consideration of new approaches, such as immunity or genetics of the host. Additionally, the efficacy of these compounds can be expanded with combined or concomitant use. Increased study of the use of novel approaches, including fungi, elements such as copper, and plant products, has also occurred. This article explores each of these areas to allow readers to appreciate how various approaches may be developed and incorporated into an effective parasite control program.

This study was undertaken to determine if administration of recombinant bovine interleukin-12 (rBoIL-12) could stimulate a cellular immune response that protected calves from an oral challenge inoculation with Cryptosporidium parvum oocysts. In a first experiment, rBoIL-12 intraperitoneally administered as a single dose 1 day before challenge inoculation, did not alter the course of infection. The percentage of immune competent cells and levels of cytokine gene expression in the ileo-cecal mucosa and in the draining lymph nodes of treated calves were similar to those of untreated control calves. However, when rBoIL-12 was subcutaneously administered daily from 2 days before infection to 2 days after infection, a consistent increase of T lymphocytes and an higher expression of interferon-gamma (IFN-gamma) was detected. Again, treatment did not alter the course of infection. Similar results were obtained when rBoIL-12 was administered daily for 4 days beginning 2 days after oral inoculation. These data indicate that although rBoIL-12 stimulated a strong immune response in the gut of neonatal calves, the response was not able to provide protection from challenge inoculation with C. parvum oocysts.

The following three papers are a very small window onto the types of research being pursued by members of the American Association of Veterinary Parasitologists. They are related by the fact that newer areas in the biology of parasites and their hosts are discussed. The first paper by Dr. Tom Klei, gives a brief view of the interactions between host and parasite of the fascinating organism Wolbachia, a parasite of parasites. The second paper by Dr. Gloria Solano-Aguilar addresses the use of probiotics to alter the host–parasite interface and influence host resistance. The final paper by Dr. Lou Gasbarre outlines an example of integration of the genomics revolution into Veterinary Parasitology. While the subjects are diverse, they demonstrate the vitality of the AAVP.

Neosporosis is an important cause of pregnancy loss in cattle worldwide. Protective immunity against Neospora caninum infection may include both cell-mediated (CMI) and humoral immune responses. This study was to establish short-term antigen-specific T cell lines composed of primarily CD4(+)T cells from peripheral blood lymphocytes (PBL) of infected cows, which may be used to identify immunodominant antigens for the development of N. caninum vaccines. Crude N. caninum tachyzoite antigen was prepared from in vitro derived N. caninum tachyzoites. Multiple T cell lines were established and maintained for 11 weeks by weekly re-stimulation with N. caninum antigen and antigen-presenting cells. All cell lines responded highly to antigen between weeks 3 and 11. Phenotypically, these cells were composed primarily of CD4(+)T cells between weeks 2-8, with a gradual expansion of gamma/delta(+)T cells thereafter. The results indicate that N. caninum-specific T cell lines can be established and maintained without exogenous T cell growth factors and may be used to identify N. caninum antigens. This research will enhance our understanding of bovine CMI to neosporosis and may facilitate development of a proven neosporosis vaccine.

Lowered immune responses during bovine ostertagiosis have been reported in both in vivo and in vitro assay systems. In the present study we have employed three different life cycle stages of the nematode Ostertagia ostertagi to determine if products of this economically important parasite inhibit in vitro proliferation of Con A-stimulated cells from uninfected animals. We have demonstrated an inhibitory effect upon the growth of Con A-stimulated lymphocytes after addition of fourth stage larval (L4) soluble extract (L4SE) to the cultures. In contrast, extracts from the third stage larvae (L3) had little or no inhibitory activity. The suppressive products were also shown to be secreted by the late L4. The suppressive activity is reversible if the L4 products are removed from culture. There is no immediate effect on proliferating cells and the L4SE must be in culture for 24-48 h before suppression is observable. The L4SE caused slight but not statistically significant decreases in the percentage of T cells and increases in B cell percentages in cultures when compared with cultures stimulated with Con A alone. No changes were seen in percentage of cells positive for markers for CD4, CD8, gammadelta T cells, or monocytes/macrophages as a consequence of the addition of L4SE. In contrast, there was a strong and significant reduction in the expression of the IL-2 receptors in cells cultured in the presence of the worm extract. There was no evidence of either necrosis or apoptosis resulting from the presence of L4 products in culture. The expression of messenger RNA for interleukin-2, -4, -13, tumor necrosis factor-alpha (TNF-alpha), and gamma-interferon (gamma-IFN) was decreased when L4SE was included in cultures of Con A-stimulated cells compared to cultures stimulated with Con A only. In contrast, messenger RNA expression of transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) was increased in cells growing in the presence of L4 products. The potential role of these cytokines during ostertagiosis is discussed.

Neospora caninum is a major cause of abortion in cattle. An essential role for Th1 cytokines, such as IFN-gamma and IL-12 in protective immunity against N. caninum in murine models has been indicated. However, little is known about immunity to Neospora in pregnant cattle where a considerable level of immunomodulation may exist. In this study, the immune response of heifers infected early in the second trimester of pregnancy by intravenous inoculation of N. caninum tachyzoites was compared with immune responses in uninfected pregnant heifers. Animals were killed 3 weeks after infection. No abortion was observed in any infected dam, however, transplacental infection was shown to have already taken place. Infection with N. caninum during pregnancy induced significant immune responses in both dams and their foetuses. Infected dams showed significant changes in lymphocyte subpopulations compared with uninfected pregnant animals and these changes were compartmentalized. Increased levels of T lymphocytes were observed in the infected foetuses. Cytokine gene expression analysed by real time RT-PCR showed increased expression of both Th1 and Th2 cytokines in N. caninum infected animals. This cytokine expression could have a role in the transplacental transmission of the parasite and/or mediate tissue damage.

In this study we demonstrated that combined inoculation of interleukin-12 (IL-12) and IL-18 reduced the number of bacteria in the spleens of mice infected with Brucella abortus 2308 and that the effect of the treatment was mediated by an increased capability of spleen cells to produce gamma interferon at the early phase of infection.

The natural genetic variability of the ruminant immune system provides a feasible means to control gastrointestinal (GI) parasite infection without anthelmintics. However, the paradigm of traditional selection has not been effectively applied to the moderately heritable traits of parasite resistance (h approximately equal to 0.3) due to the difficulty and expense of gathering accurate phenotypes in a commercial production setting. These characteristics make host traits related to GI nematode infection ideal candidates for genomics-based research. To initiate explanation of important allelic differences, economic trait loci (ETL) are being identified and mapped using a resource population of Angus cattle segregating for GI nematode resistance and susceptibility to the two most common nematode parasites of US cattle, Ostertagia ostertagi and Cooperia oncophora. The population is composed of five generations of half-sib progeny with complete phenotypic records produced from controlled infections. To detect the genomic locations of the three distinct phenotypic traits being expressed (innately immune, acquired immune, and immunologically non-responsive), genotypes have been generated for DNA markers (N=199) spaced at regular intervals (approximately 20cm intervals) throughout the entire genome (3000cm). Although initial ETL detection may be limited by half-sib family size, the unique structure of this population provides additional statistical power for refining map position of potential ETL. After allele frequency and contribution to phenotype are determined in this population, marker tests associated with ETL most beneficial for controlling parasite infection can be accurately used for selection. Comparative map and functional genomic information from humans and other species of biomedical importance will be utilized in further investigations to elucidate the genes underlying ETL.

A questionnaire covering management practices and producer perception of the effects of gastrointestinal nematode infections was sent to dairy and beef producers in the northeastern US. The mailing list was derived from membership in grazing groups and attendance at grazing events. A final total of 474 responses were suitable for analyses. These responses covered 14 states, but for the purpose of analysis were broken into five groups: New England (NE), Vermont (VT), New York (NY), Pennsylvania (PA), and south and west (S and W) of Pennsylvania. Two-thirds of the responses were from dairy producers. The average number of animals for the farms was 50 cows, 27 heifers, and 20 calves. The average acreage used for grazing was 70 acres, and about two-thirds of the responses used rotational grazing for at least the cows. About one-half of the rotational grazers had been practicing rotational grazing for more than 5 years. Most rotational programs for cows involved a daily rotation, but the rotational interval for other age groups was longer. There was a difference of about 2 months (5.25-7.27) in the length of the grazing season as one moved from New England to south and west of Pennsylvania. Parasite control practices varied greatly by location and animal class. Most producers used anthelmintics one to two times per year, but 10-30% of responses said they did not deworm their cattle. The most common time to deworm was in the spring, and the second most common time was the fall. Between 10 and 20% of respondents reported deworming as a response to decreased productivity or body condition. The use of anthelmintics increased as the location moved from New England to south and west of Pennsylvania. Producer perception of parasite effects was closely related to their anthelmintic use, and also increased as the location moved to the south, and is most likely the result of the increased length of the grazing season. Of producers who ascribed estimated a cost of the parasite, the majority estimated this cost to be between US$ 5 and 20 per animal per year.

This study indicated that mice immunized with Brucella abortus RB51 bacteria and subsequently challenged with B. abortus 2308 were protected from reinfection. After vaccination, both Th1 and Th2 cytokine patterns were observed. Of those, the early production of gamma interferon seems to have the prominent role in inducing an immunologically based protection.

Gastrointestinal nematode infections of cattle remain a constraint on the efficient raising of cattle on pasture throughout the world. Most of the common genera of parasites found in cattle stimulate an effective level of protective immunity in most animals within the herd after the animals have been on pasture for several months. In contrast, cattle remain susceptible to infection by Ostertagia for many months, and immunity that actually reduces the development of newly acquired larvae is usually not evident until the animals are more than 2 years old. This prolonged susceptibility to reinfection is a major reason that this parasite remains the most economically important GI nematode in temperate regions of the world. Although, animals remain susceptible to reinfection for a prolonged period of time, there are a number of manifestations of the immune response that result in an enhanced level of herd immunity. These include a delay in the development time of the parasites, an increase in the number of larvae that undergo an inhibition in development, morphological changes in the worms, stunting of newly acquired worms, and most importantly a reduction in the number of eggs produced by the female worms. The overall result of these manifestations of immunity is a reduction in parasite transmission within the cattle herd. The immune mechanisms responsible for these different types of functional immunity remain to be defined. In general, GI nematode infections in mammals elicit very strong Th2-like responses characterized by high levels of Interleukin 4 (IL4), high levels of IgG1 and IgE antibodies, and large numbers of mast cells. In cattle, the most extensively studied GI nematode, in regards to host immune responses, is Ostertagia ostertagi. In Ostertagia infections, antigens are presented to the host in the draining lymph nodes very soon after infection, and within the first 3-4 days of infection these cells have left the nodes, entered the peripheral circulation, and have homed to tissues immediately surrounding the parasite where they become established. The immune response seen in the abomasum is in many ways are similar to that seen other mammalian hosts, with high levels of expression of IL4 in the draining lymph nodes and in lymphocytes isolated from the mucosa. But unlike a number of other systems, lymphocyte populations taken from Ostertagia infected cattle seem to be up-regulated for a number of other cytokines, most notably Interferon (IFN, implying that in Ostertagia infections, the immune response elicit is not simply a stereotypic Th2 response. In addition, effector cell populations in the tissues surrounding the parasites, are not typical, inferring the Ostertagia has evolved means to suppress or evade protective immune mechanisms. Studies have also demonstrated that the number of nematode eggs/gram (EPG) in feces of pastured cattle is strongly influenced by host genetics and that the heritability of this trait is approximately 0.30. In addition, EPG values are not "normally" distributed and a small percentage of a herd is responsible for the majority of parasite transmission. This suggests that genetic management of a small percentage of the herd can considerably reduce overall parasite transmission. A selective breeding program has been initiated to identify the host genes controlling resistance/susceptibility to the parasites. The best indicator of the number of Cooperia infecting a host is the EPG value, while Ostertagia is best measured by serum pepsinogen levels, weight gain, and measures of anemia. Other phenotypic measures are either not significantly associated with parasite numbers or are very weakly correlated. In addition, calves can be separated into three types: (1) Type I which never demonstrates high EPG values, (2) Type II which shows rises in EPG values through the first 2 months on pasture which then fall and remain at levels associated with Type I calves, and (3) Type III calves which maintain high EPG levels. The approximate percentage of these calves is 25:50:25 respectively. Because these cattle are segregating for traits involved in resistance and susceptibility to GI nematodes, this resource population is being used to effectively detect the genomic locations of these Economic Trait Loci (ETL). For relational analysis between phenotype and genome location, over 80,000 genotypes have been generated by PCR amplification, and marker genotypes have been scored to produce inheritance data. The marker allele inheritance data is currently being statistically analyzed to detect patterns of co-segregation between allele haplotype and EPG phenotypes. Statistical power of this genome-wide scan has been strengthened by including genotypic data from the historic pedigree. In our herd, paternal half-sib families range from 5-13 progeny/sire, and extensive marker genotypes are available from ancestors of the population most of which are paternally descended from a single founding sire. Once ETL have been identified the next will be to refine ETL map resolution in attempt to discover the genes underlying disease phenotypes. Accurate identification of genes controlling resistance will offer the producer several alternatives for disease control. For a non-organic producer, the small percentage of susceptible animals can be targeted for drug administration. This approach would reduce both the cost of anthelmintics used and the odds for selection of drug resistant mutants, because the selective agent (drug) would not be applied over the entire parasite population. A second treatment option would be based on correcting a heritable immunologic condition. In this case, susceptible animals could be the targets for immunotherapy involving vaccines of immunomodulation. A final option would be genetic selection to remove susceptible animals from the herd. Producers with a high degree of risk for parasite-induced production losses, such as organic producers of producers in geographic areas with environmental conditions favorable to high rates of transmission would benefit the most from this strategy. In contrast, producers at low risk could take a more conservative approach and select against susceptibility when other factors were equal.

A multiplex polymerase chain reaction (PCR) test was developed for identifying gastrointestinal (GI) nematodes that commonly infect cattle. This assay was developed using adult-derived genomic DNA and shown capable of discriminating parasite eggs from the feces of experimentally-infected animals at both the species and genus levels. Sequence data from internal (ITS) and external (ETS) transcribed spacers of the ribosomal DNA (rDNA) repeats as well as the 3'-end of the small subunit rDNA and 5'-end of the large subunit rDNA were used to generate five primer sets which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern characterized by a single DNA fragment for Ostertagia ostertagi (257bp), Haemonchus placei (176bp), Oesophagostomum radiatum (329bp), Trichostrongylus colubriformis (243bp) and Cooperia oncophora (151bp). In a similar manner, the constructed primer sets amplified DNA from Ostertagia lyrata, Haemonchus contortus, Trichostrongylus axei, Cooperia surnabada and Cooperia punctata. With respect to H. contortus, a closely migrating doublet was generated suggesting size heterogeneity in the ETS which is consistent with multiple rDNA repeat units within this species. PCR analyses using mixtures of monospecifically-purified nematode eggs indicated a sensitivity of less than 0.5 egg-DNA equivalent per species. Although, not designed as a quantitative technique, relative PCR signal intensities corresponded to relative egg burdens within the DNA samples from mixed species of eggs.

The acceptance of the fetal allograft by pregnant women and mice seems to be associated with a shift from a Th 1 dominated to a Th 2 dominated immune response to certain infectious agents. The goal of this study was to examine cytokine expression in peripheral blood mononuclear cells (PBMCs) from cattle immune to bovine viral diarrhea virus (BVDV) to determine whether pregnancy also has an influence on the type of immune response in this species. Forty-six heifers and cows between 14 months and 13 years of age were included in this study. Twenty-four were seropositive and 22 seronegative for BVDV. Eleven of the seropositive animals and 11 of the seronegative animals were in the eighth month of gestation, the remaining animals were virgin heifers. PBMC from these animals were analyzed for Interferon (IFN)-gamma and Interleukin (IL)-4 mRNA expression by real-time RT-PCR after stimulation with a non-cytopathic strain of BVDV. Additionally, an ELISA was performed to measure IFN-gamma in the supernatants of stimulated cell cultures. In BVDV seropositive animals, IFN-gamma mRNA levels were significantly higher than in BVDV seronegative animals and there was a significant positive correlation between the changes in IFN-gamma and IL-4 mRNA expression. There was, however, no significant difference in IFN-gamma and IL-4 mRNA levels between pregnant and non-pregnant animals. These results are inconsistent with BVDV inducing a Th1 or Th2 biased immune response. Furthermore, a shift in the cytokine pattern during bovine pregnancy was not evident.

In the present study, cell-surface markers and cytokine gene expression of lymphocytes from the local lymph nodes were studied 9 days after primary infection with Ostertagia ostertagi in previously naive calves or in calves previously immunized with multiple, chemically attenuated infections. Changes in lymphocyte populations were assessed by flow cytometry utilizing monoclonal antibodies specific for bovine cell-surface markers. Changes observed in the percentages of lymphoid populations after challenge were similar in animals immunized by either three or five drug-attenuated infections. In both immunized groups, the CD4+/CD8+ ratio was significantly higher than in naive animals after challenge infections. In addition, both immunized groups showed significantly lower levels of Ig-bearing cells upon experimental challenge when compared to animals with a primary experimental infection. No differences were observed in the number of gammadelta or interleukin 2 receptor (IL-2R) positive cells. The levels of mRNA for IL-4, IL-10, IL-15, IFN-gamma and TGF-beta1 were examined by competitive RT-PCR. After challenge, the levels of these cytokines were lower in animals immunized by five drug-attenuated infections, and in the case of IL-4 and TGF-beta1, these differences were statistically significant. These results indicate that animals exhibiting protection from reinfection with O. ostertagi do not show a shift to higher percentages of Ig+ cells characteristic of a primary infection. In addition, protected animals appear to show a decreased IL4 and TGF-beta1 response upon challenge when compared to non-immune animals.

A region within the first internal transcribed spacer (ITS-1) of the ribosomal DNA repeat of Ostertagia ostertagi has been identified that is 408 base pairs (bp) in length and is comprised of a 2 x 204 bp repeat. Universal polymerase chain reaction (PCR) primers which span this region, as well as a portion of the 5.8S rDNA, generate a 1011 bp fragment using genomic DNA from O. ostertagi. However, these same primers generate only a 600 bp (approximate) fragment using DNA from Haemonchus contortus, Cooperia oncophora and Oesophagostomum radiatum, as well as other species within the genus Ostertagia. When DNA samples derived from adult parasites of the different genera were mixed and simultaneously amplified, the O. ostertagi component could be identified within the mixed DNA populations. Furthermore, a correlation was observed between relative fluorescence intensities of the 1011 bp and the 600 bp PCR fragments and the percentage of O. ostertagi DNA within a mixture of parasite DNAs. A similar high correlation was obtained between the percentage of O. ostertagi DNA and percent O. ostertagi eggs in feces containing eggs of other nematode genera. This resulted in the generation of a protocol that can determine the percentage of O. ostertagi eggs within a mixed population of gastrointestinal nematode eggs. Results indicate a detection equivalent to 0.05 eggs.

In the present study, localized changes in cytokine transcription profiles were examined in neonatal calves following a primary infection with Cryptosporidium parvum, using competitive reverse transcriptase polymerase chain reaction (RT-PCR). Total RNA was prepared from ileocecal lymph nodes (LN), lamina propria lymphocytes (LPL), and intraepithelial lymphocytes (IEL) isolated from neonatal calves 7 days after C. parvum infection. Competitive RT-PCR performed on cDNA samples containing internal cytokine gene competitor molecules showed increases in the levels of interferon-gamma and interleukin-12 (IL-12) (P40) mRNA in both LPL and IEL populations but not in the draining LN. In addition, the levels of mRNA of the newly identified growth factor IL-15 decreased in the IEL of the infected animals. No consistent differences were seen in any of the cell populations when the samples were analyzed for IL-10 and levels of mRNA for IL-2 and IL-4 were low and highly variable in both infected and control groups in all 3 lymphocyte populations.

Changes in cell surface markers and cytokine transcription were analyzed in lamina propria lymphocytes from control animals (noninfected calves) and calves after a single high but nonprotective primary infection with Ostertagia ostertagi. Flow cytometry of cells recovered from the lamina propria showed an increase in the percentages of IgM+, WC1+, and IL-2R+-bearing cells 10 days after infection; however, 2 mo after infection, cell staining was comparable to preinfection levels. Transcription levels of interleukins IL-2, IL-4, IL-10, and interferon (IFN)-gamma mRNA were measured using a competitive reverse transcription-polymerase chain reaction. Results indicated elevated levels of IL-4 and IFN-gamma in the infected animals at 10 days and at 60 days after infection. Transcription of IL-10 also increased; however, this change was not observed until 60 days postinfection.

Gastrointestinal (GI) nematodes of ruminants evoke a wide variety of immune responses in their hosts. In terms of specific immune responses directed against parasite antigens, the resulting immune responses may vary from those that give strong protection from reinfection after a relatively light exposure (e.g. Oesophagostomum radiatum) to responses that are very weak and delayed in their onset (e.g. Ostertagia ostertagi). The nature of these protective immune responses has been covered in another section of the workshop and the purpose of this section will be to explore the nature of changes that occur in the immune system of infected animals and to discuss the effect of GI nematode infections upon the overall immunoresponsiveness of the host. The discussion will focus primarily on Ostertagia ostertagi because this parasite has received the most attention in published studies. The interaction of Ostertagia and the host immune system presents what appears to be an interesting contradiction. Protective immunity directed against the parasite is slow to arise and when compared to some of the other GI nematodes, is relatively weak. Although responses that reduce egg output in the feces or increase the number of larvae undergoing inhibition may occur after a relatively brief exposure (3-4 months), immune responses which reduce the number of parasites that can establish in the host are not evident until the animal's second year. Additionally, even older animals that have spent several seasons on infected pastures will have low numbers of Ostertagia in their abomasa, indicating that sterilizing immune responses against the parasite are uncommon. In spite of this apparent lack of specific protective immune responses, infections with Ostertagia induce profound changes in the host immune system. These changes include a tremendous expansion of both the number of lymphocytes in the local lymph nodes and the number of lymphoid cells in the mucosa of the abomasum. This expansion in cell numbers involves a shift away from a predominant classic T cell population (CD2 and CD3 positive), to a population where T cell percentages are decreased and B cells (immunoglobulin-bearing) and gamma-delta cells are increased. At the same time the expression of messenger RNAs for T cell cytokines (IL2, IL4, IL10 and gamma-interferon) is changed to that of increased expression of IL4 and IL10 and decreased expression of IL2 and perhaps of gamma-interferon. The reasons for these changes remain to be elucidated, but it is evident that the lack of protective immune responses is not the result of a poor exposure of the host to parasite products, or to the stomach being an immunoprivileged site. In fact, a superficial look at the responses elicited indicates that Ostertagia induces responses (the so-called TH2 mediated responses) that are widely considered to be the type of responses necessary for protection against GI nematodes. There are many factors that could lead to this apparent lack of immunity in the face of a strong stimulation of immune responses including: (1) the elicitation of suboptimal responses; (2) the failure of the abomasum to function as an efficient effector organ; (3) active evasion of the functional immune response by the parasite; and (4) that these classic responses are not protective in this particular ruminant-parasite system and that novel protective mechanisms may be required. The strong stimulation of the host gut immune system by Ostertagia and perhaps by other GI nematode infections, raises questions about the potential effects of such infections on the overall well-being of the host. A number of authors have indicated that Ostertagia infections may diminish the host's ability to mount subsequent immune responses to antigenic challenges such as vaccination against other infectious organisms. In addition, recent studies have indicated that infections with GI nematodes may result in increased circulatory levels of stress-related hormo

Performance of Angus cows and calves was compared between two methods of bahiagrass (Paspalum notatum Flügge) grazing management, rotational stocking (ROT) and continuous intensive stocking (CIS), where stocking rate was varied by adjusting available pasture area as forage growth rate changed during the growing season. Effects of sire type (low [LO] vs high [HI] EPD for nematode egg shedding rate [EPG]) also were studied. Data were analyzed for two complete cycles of calf production from breeding through weaning. There was no effect of pasture grazing management method on cow BW, cow body condition score, adjusted 205-d calf weaning weight, and preweaning calf ADG. Five genera of nematodes (Ostertagia, Haemonchus, Trichostrongylus, Cooperia, and Oesophagostomum) were recovered from calves removed and killed at times throughout the grazing season. Mean nematode numbers recovered increased (P < .05) for all species as the grazing season progressed from spring to fall, consistent with results on EPG (P < .001). Effect of sire type on EPG was not significant; however, sire type did affect mean EPG (P < .05) from a subset of calves placed in drylot after weaning and sampled for three consecutive days. Sire type affected IgG1 titer to H. placei (LO = .50 +/- .012, HI = .45 +/- .011) and IgA titer to O. radiatum (LO = .28 +/- .006, HI = .26 +/- .005), and there was a sire type x pasture grazing method interaction on IgG1 titer to H. placei (LO-ROT = .49 +/- .016, HI-ROT = .49 +/- .017, LO-CIS = .50 +/- .017, HI-CIS = .41 +/- .014). Increased anti-parasite antibody titers in progeny of sires with EPD for low nematode egg shedding rates may reflect increased host resistance to these parasites.

Interleukin-15 (IL-15) is a recently identified growth and differentiation factor with an important potential role in the initial immune responses to infection. To enable the study of the role of this cytokine in the protective immune-mechanisms generated against parasitic diseases of swine, cDNA was generated from a macrophage enriched adherent cell population from peripheral blood mononuclear cells (PBMC). This cDNA was used for the enzymatic amplification of the porcine IL-15 sequence using human IL-15-derived primers. The open-reading frame of the porcine IL-15 cDNA is 486 base pairs (bp) in length and encodes a 162-amino-acid (aa) protein. Comparisons of the predicted swine protein sequence with those predicted from human, bovine and mouse IL-15 sequences indicate similarities of 82.1, 84.6, and 71.6%, respectively.

Changes that occur in the local draining lymph nodes including, changes in cell surface markers and cytokine gene expression were studied over the first 4 weeks of a primary, Ostertagia ostertagi infection of the abomasum. Cells recovered from the abomasal lymph nodes (ABLN) after infection showed a decrease in the percentage of CD3+ cells, and an increase in the percentage of IgM+ cells and cells bearing the TcR1 marker. These changes were coincident with an increase in the proportion of activated cells (II-2R). Analysis of mitogen-stimulated ABLN cells by RNase protection assay (RPA) showed a dramatic reduction in IL-2 and IFN-gamma transcription after infection. In addition, analysis of unstimulated ABLN cells by competitive RT-PCR showed a similar decrease in demonstrable levels of IL-2 mRNA, but IL-10, IL-4 and IFN-gamma mRNA levels were elevated.

The bovine interleukin-15 (IL-15) sequence was cloned from abomasal lymph node mRNA by enzymatic amplification of cDNA using human primers proximal to and including the translation start and stop sites. The open reading frame is 486 base pairs in length, and the proposed protein sequence shows 78.4% and 73.5% similarity with that predicted for the human and mouse sequences, respectively. Expressed and purified recombinant bovine IL-15 in the absence of the 48-amino acid leader sequence stimulated the proliferation of bovine lymphoblast cells at least 12-fold over background at maximum concentration levels. Competitive reverse transcriptase-polymerase chain reaction analysis showed constitutive levels of IL-15 mRNA within a broad range of tissues and cell types. Lipopolysaccharide addition to adherent lymph node populations caused moderate increases in IL-15 transcription, whereas the addition of phorbol 12-myristate 13-acetate and calcium ionophore failed to induce gene expression for this cytokine. Transcription of IL-15 was also downregulated in the presence of low concentrations of human recombinant interleukin-2.