Transcriptional profiling of the ovine abomasal lymph node reveals a role for timing of the immune response in gastrointestinal nematode resistance.

Gastrointestinal nematodes are a serious cause of morbidity and mortality in grazing ruminants. The major ovine defence mechanism is acquired immunity, with protective immunity developing over time in response to infection. Nematode resistance varies both within and between breeds and is moderately heritable. A detailed understanding of the genes and mechanisms involved in protective immunity, and the factors that regulate this response, is required to aid both future breeding strategies and the development of effective and sustainable nematode control methods. The aim of this study was to compare the abomasal lymph node transcriptome of resistant and susceptible lambs in order to determine biological processes differentially expressed between resistant and susceptible individuals. Scottish Blackface lambs, with divergent phenotypes for resistance, were challenged with 30,000 Teladorsagia circumcincta larvae (L3), and abomasal lymph nodes recovered at 7 and 14days post-infection (dpi). High-throughput sequencing of cDNA from the abomasal lymph node was used to quantitatively sample the transcriptome with an average of 32 million reads per sample. A total of 194 and 144 genes were differentially expressed between resistant and susceptible lambs at 7 and 14 dpi respectively. Differentially expressed networks and biological processes were identified using Ingenuity Pathway Analysis. Genes involved in the inflammatory response, attraction of T lymphocytes and binding of leukocytes were more highly expressed in resistant animals at 7 dpi and in susceptible animals at 14 dpi indicating that resistant animals respond to infection earlier than susceptible animals. Twenty-four Single Nucleotide Polymorphisms (SNP) within 11 differentially expressed genes, were tested for association with gastrointestinal nematode resistance in the Scottish Blackface lambs. Four SNP, in 2 genes (SLC30A2 and ALB), were suggestively associated with faecal egg count. In conclusion, a large number of genes were differentially expressed in the abomasal lymph node of resistant and susceptible lambs responding to gastrointestinal nematode challenge. Resistant Scottish Blackface lambs appear to generate an earlier immune response to T. circumcincta. In susceptible lambs this response appears to be delayed. SNP in 2 differentially expressed genes were suggestively associated with faecal egg count indicating that differentially expressed genes may be considered candidate loci for mediating nematode resistance.

Variations in T Cell Transcription Factor Sequence and Expression Associated with Resistance to the Sheep Nematode Teladorsagia circumcincta.

This study used selected lambs that varied in their resistance to the gastrointestinal parasite Teladorsagia circumcincta. Infection over 12 weeks identified susceptible (high adult worm count, AWC; high fecal egg count, FEC; low body weight, BW; low IgA) and resistant sheep (no/low AWC and FEC, high BW and high IgA). Resistance is mediated largely by a Th2 response and IgA and IgE antibodies, and is a heritable characteristic. The polarization of T cells and the development of appropriate immune responses is controlled by the master regulators, T-bet (TBX21), GATA-3 (GATA3), RORγt (RORC2) and RORα (RORA); and several inflammatory diseases of humans and mice are associated with allelic or transcript variants of these transcription factors. This study tested the hypothesis that resistance of sheep to T. circumcincta is associated with variations in the structure, sequence or expression levels of individual master regulator transcripts. We have identified and sequenced one variant of sheep TBX21, two variants of GATA3 and RORC2 and five variants of RORA from lymph node mRNA. Relative RT-qPCR analysis showed that TBX21, GATA3 and RORC2 were not significantly differentially-expressed between the nine most resistant (AWC, 0; FEC, 0) and the nine most susceptible sheep (AWC, mean 6078; FEC, mean 350). Absolute RT-qPCR on 29 all 45 animals identified RORAv5 as being significantly differentially-expressed (p = 0.038) 30 between resistant, intermediate and susceptible groups; RORAv2 was not differentially- 31 expressed (p = 0.77). Spearman's rank analysis showed that RORAv5 transcript copy number 32 was significantly negatively correlated with parameters of susceptibility, AWC and FEC; and 33 was positively correlated with BW. RORAv2 was not correlated with AWC, FEC or BW but 34 was significantly negatively correlated with IgA antibody levels [corrected]. This study identifies the full length RORA variant (RORAv5) as important in controlling the protective immune response to T. circumcincta infection in sheep.

Exploring the abomasal lymph node transcriptome for genes associated with resistance to the sheep nematode Teladorsagia circumcincta.

This study exploited Blackface lambs that varied in their resistance to the abomasal nematode parasite, Teladorsagia circumcincta. Infection of these lambs over 3 months identified susceptible (high adult worm count, high faecal egg count and low IgA antibody) and resistant animals that had excluded all parasites. Previous work had shown that susceptibility and resistance is dependent on the differential immune response to the parasite, which occurs within the abomasal (gastric) lymph node (ALN) that drains the site of infection. The Affymetrix ovine gene array was used to interrogate the transcriptome of the ALN to identify genes and physiological pathways associated with resistance. We used a bovine RT-qPCR array of 84 genes to validate the gene array, and also report digital gene expression analysis on the same tissues, reanalysed using the Oar v3.1 sheep genome assembly. These analyses identified Humoral Immune Response, Protein Synthesis, Inflammatory Response and Hematological System Development and Function as the two top-ranked networks associated with resistance. Central genes within these networks were IL4, IL5, IL13RA2 and in particular IL13, which confirmed that differential activation of Th2 polarized responses is critical to the resistance phenotype. Furthermore, in resistant sheep there was up-regulation of genes linked to control and suppression of inflammation. The identity of differentially-expressed chemokines and receptors in the resistant and susceptible sheep also begins to explain the cellular nature of the host response to infection. This work will greatly help in the identification of candidate genes as potential selectable markers of genetic resistance.

Relationship between susceptibility of Blackface sheep to Teladorsagia circumcincta infection and an inflammatory mucosal T cell response.

Teladorsagia circumcincta is the most economically important gastrointestinal (abomasal) nematode parasite of sheep in cool temperate regions, to which sheep show genetically-varying resistance to infection. Lambs, from parents with genetic variation for resistance, were trickle infected with L3 larvae over 12 weeks. 45 lambs were identified with a range of susceptibilities as assessed by: adult worm count at post mortem, faecal egg count (FEC) and IgA antibody levels. This project investigated the correlation of T cell cytokine expression and resistance to infection at the mature stage of response, when the resistant lambs had excluded all parasites.Histopathology showed only minor changes in resistant animals with a low level lymphocyte infiltration; but in susceptible lambs, major pathological changes were associated with extensive infiltration of lymphocytes, eosinophils and neutrophils.Absolute quantitative RT-qPCR assays on the abomasal lymph node (ALN) revealed a significant positive correlation between IL6, IL21 and IL23A transcript levels with adult worm count and FEC. IL23A was also negatively correlated with IgA antibody levels. Significantly positive correlation of TGFB1 levels with adult worm count and FEC were also seen in the abomasal mucosa. These data are consistent with the hypothesis that the inability to control L3 larval colonization, adult worm infection and egg production is due to the activation of the inflammatory Th17 T cell subset.

Infection with the gastrointestinal nematode Ostertagia ostertagi in cattle affects mucus biosynthesis in the abomasum.

The mucus layer in the gastrointestinal (GI) tract is considered to be the first line of defense to the external environment. Alteration in mucus components has been reported to occur during intestinal nematode infection in ruminants, but the role of mucus in response to abomasal parasites remains largely unclear. The aim of the current study was to analyze the effects of an Ostertagia ostertagi infection on the abomasal mucus biosynthesis in cattle. Increased gene expression of MUC1, MUC6 and MUC20 was observed, while MUC5AC did not change during infection. Qualitative changes of mucins, related to sugar composition, were also observed. AB-PAS and HID-AB stainings highlighted a decrease in neutral and an increase in acidic mucins, throughout the infection. Several genes involved in mucin core structure synthesis, branching and oligomerization, such as GCNT3, GCNT4, A4GNT and protein disulphide isomerases were found to be upregulated. Increase in mucin fucosylation was observed using the lectin UEA-I and through the evaluation of fucosyltransferases gene expression levels. Finally, transcription levels of 2 trefoil factors, TFF1 and TFF3, which are co-expressed with mucins in the GI tract, were also found to be significantly upregulated in infected animals. Although the alterations in mucus biosynthesis started early during infection, the biggest effects were found when adult worms were present on the surface of the abomasal mucosa and are likely caused by the alterations in mucosal cell populations, characterized by hyperplasia of mucus secreting cells.

A differential interplay between the expression of Th1/Th2/Treg related cytokine genes in Teladorsagia circumcincta infected DRB1*1101 carrier lambs.

Substantial debate exists on whether the immune response between sheep resistant and susceptible to gastrointestinal nematodes can be differentiated into a Th1 and Th2 phenotype. The present study addresses the hypothesis that variation in resistance to Teladorsagia circumcincta between DRB1*1101 (associated with reduced faecal egg count and worm burden) carriers and non-carriers is due to a differential interplay in the expression of Th1/Th2 and regulatory T (Treg) related cytokine genes. Lambs from each genotype were either slaughtered at day 0 (un-infected control) or infected with 3 × 10(4) Teladorsagia circumcincta L3 and slaughtered at 3, 7, 21, and 35 days later. Lambs carrying the DRB1*1101 allele had a significantly lower worm burden (P < 0.05) compared to the non-carriers. Abomasal mucosal cytokine gene expression was evaluated by quantitative real-time PCR and comparison made for time and genotype effects. The response generated varied through the course of infection and was affected by genotype. DRB1*1101 carriers had an up-regulated expression of the Th1-related cytokine genes (IL-1ß, TNFα, and IFN-γ) at day 3, but this was replaced by an up-regulated expression of Th2-related cytokine genes (IL-10 and IL-13) and Treg-related cytokine genes (IL-2RA-CD25, TGFα, TGFß, Arg2, MIF and FOXP3) by day 7. Conversely, in the non-carriers these changes in gene expression were delayed until days 7 and 21 post infection (pi), respectively. It is concluded that resistance to Teladorsagia circumcincta in animals carrying the DRB1*1101 allele is influenced by an earlier interplay between Th1, Th2 and T regulatory immune response genes.

The dynamic influence of the DRB1*1101 allele on the resistance of sheep to experimental Teladorsagia circumcincta infection.

Suffolk sheep carrying the DRB1*1101 (previously referred to as-DRB1*0203 or G2) allele have been reported to show increased resistance to natural Teladorsagia circumcincta infection compared to non-carriers. The objective of this study was to compare the biochemical and physiological responses of DRB1*1101 carrier and non-carrier twin lambs to an experimental infection with 3 × 10(4) L3 Teladorsagia circumcincta. The variables studied included worm burden, faecal egg count, abomasal mast cells, IgA, IgE, IgG1 plus IgG2 and haematological parameters at 0, 3, 7, 21 and 35 days post infection (dpi), and duodenal smooth muscle contractility at 0 and 35 dpi. DRB1*1101 carrier lambs had significantly lower worm burden, higher mast cell and plasma platelet counts than the DRB1*1101 non-carriers (P < 0.05). Before infection, the non-carrier lambs exhibited significantly higher mucosal levels of all antibody isotypes measured compared to the carriers; these levels remained relatively stable over the course of infection in the non-carriers while there was a slow build up of these antibodies in the carriers up to day 21 post infection (pi). The DRB1*1101 non-carrier lambs had a significantly higher plasma lymphocyte count, and produced greater duodenal contractile force relative to the carrier lambs (P < 0.05). There was no significant difference between genotypes in the level of plasma eosinophils, monocytes, neutrophils or FEC. This evidence suggests that resistance conferred by DRB1*1101 is acquired rather than innate, depends on worm expulsion rather than fecundity and is dependent on mucosal mast cell proliferation, platelet activation, and IgA and IgE antibody responses.

Short communication: Genetic and nongenetic factors influencing Ostertagia ostertagi antibodies in UK Holstein-Friesian cattle.

The purpose of this study was to estimate and discuss the genetic variation, heritability, and effects of nongenetic factors on the ability of Holstein-Friesian cows to produce an immune response by producing IgG antibodies to Ostertagia ostertagi. Total IgG (IgG(1) and IgG(2)) antibody levels were determined using an ELISA and measured using optical density ratio (ODR=OD(sample) - OD(negative control)/OD(positive control) - OD(negative control)) from milk samples collected from 1,276 Holstein-Friesian cattle in 229 commercial dairy farms from 2002 to 2004 during their first (82%) and other (2 to 12) lactations. A sire (n=461) model was fitted to the ODR data using ASREML software, and variance components were estimated. The ability to produce O. ostertagi antibodies as measured by ODR had a heritability of 0.13+/-0.12, and both season of sample and herd had a significant effect on total IgG levels. To conclude, this study has ascertained that genetic variation is present in the ability of dairy cows to mount an immune response to the parasite O. ostertagi. Inasmuch as evidence exists that IgG is linked to protective immunity against the parasite via a reduction in its reproductive ability, this trait may be of potential interest to genetic selection programs as an aid to reduce the effect of O. ostertagi in dairy herds.

Local inflammation as a possible mechanism of resistance to gastrointestinal nematodes in Angus heifers.

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.

Genomic tools to improve parasite resistance.

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.

Role of the bovine immune system and genome in resistance to gastrointestinal nematodes.

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.

How lambs control infection with Ostertagia circumcincta.

Two of the most important questions for immunologists studying nematode infections are what effect does the host response have on the parasite and which components of the host response are responsible for these effects. The number of nematodes and the mean length of adult female Ostertagia circumcincta was measured in over 500 6-7 month old lambs of the Scottish Blackface breed. Quantitative genetic analyses indicated that there was substantial genetic variation among lambs in faecal egg counts and in worm length but the analyses provided no evidence for genetic variation in worm numbers. Separate analyses have shown a strong relationship between decreased worm length and decreased worm fecundity. Therefore, genetic resistance in lambs appears to be due to control of worm growth and not to control of worm numbers. The only immune response that is consistently associated with reduced worm length is the IgA response to fourth-stage larvae. The association is remarkably strong. After allowing for the influence of worm number on worm length (density-dependence) essentially all of the variation among deliberately infected lambs in worm length can be accounted for, in a statistical sense, by variation in the strength and specificity of the local IgA response. Therefore, the IgA mediated suppression of worm growth and fecundity appears to be the major mechanism of resistance to O. circumcincta in lambs.

Quantification of cytokine gene expression in lamina propria lymphocytes of cattle following infection with Ostertagia ostertagi.

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.

The genetic basis of resistance to Ostertagia circumcincta in lambs.

The relationship between Ostertagia (Teladorsagia) circumcincta and sheep is one of the best understood host-parasite relationships in any species. The key components of resistance have been quantified, the extent of genetic control has been established for lambs, and methods now exist to breed lambs which will be both more resistant to worms and more productive than unselected lambs. A major gene for resistance has been identified within or around the major histocompatibility complex, and this gene appears to be the strongest yet identified for resistance to any parasite species. The most important mechanisms of resistance are local IgA responses which regulate worm fecundity and immediate hypersensitivity responses which regulate worm burdens. IgA responses develop before effective immediate hypersensitivity responses. Good simulation models now exist to predict, for example, outbreaks of disease and the response of sheep to selection. The challenge now is to use our improved understanding of the population biology to develop even better simulation models and to produce expert systems based on these models which can be used by veterinarians and others to determine optimal procedures for individual farms to control disease and reduce sub-clinical economic losses.

An ovine major histocompatibility complex DRB1 allele is associated with low faecal egg counts following natural, predominantly Ostertagia circumcincta infection.

Infection with Ostertagia circumcincta is a major constraint on sheep production in temperate areas of the world. A potential control strategy is the use of genetically resistant sheep. Therefore we examined the association between MHC-DRB1 alleles and faecal egg counts following natural, predominately O. circumcincta infection in a flock of Scottish Blackface sheep. Nineteen DRB1 alleles were identified by a combination of variation in the length of simple repetitive sequences within the intron between exons 2 and 3 and hybridisation of selected oligonucleotides to polymorphisms within exon 2. Faecal samples were taken from 200 lambs from one to six months of age at intervals of 4 weeks. Genetic effects were strongest at 6 months of age. Least-squares analysis indicated that substitution of the most common allele (I) by allele G2 would result in a 58-fold reduction in faecal egg counts in 6-month-old lambs and a 22-fold reduction in 5-month-old lambs. These results suggest that the major histocompatibility complex plays an important role in the development of resistance to O. circumcincta.

Indirect gene diagnoses for complex (multifactorial) diseases--a review.

The analysis of multifactorial diseases requires the efficient investigation of large numbers of (gene) loci and patient (family) samples. Since simple repetitive DNA markers are dispersed all over the chromosomes, molecular techniques employing these tools render most conventional screening procedures obsolete. Examples of tumors, autoimmune diseases and infections are presented to validate concepts of indirect gene diagnoses via simple, tandemly arranged, repetitive DNA sequences. The salient advantages of microsatellite technologies vs. those of multilocus DNA fingerprinting are weighed.

Genetic resistance to parasitic disease: particularly of resistance in ruminants to gastrointestinal nematodes.

There is substantial variation among individuals in susceptibility to a wide variety of parasitic diseases and part of this variation in susceptibility is due to genetic factors. The challenge now is to determine the best methods of using the variation to improve our understanding of parasitic infection and to reduce the ravages of parasitic disease. Scientific and commercial applications will depend upon the type of genetic variation. Variation among breeds can be easily exploited by a policy of breed substitution. Variation within a breed can be exploited by selective breeding to improve resistance to infection or to disease, but more work is needed to develop selection indices which are acceptable to livestock breeders. Identifying genes which contribute to the variation in resistance provides a better understanding of the mechanisms of resistance but more work is needed to determine if such genes, alone or in combination, account for a sufficient proportion of the variation in resistance to allow marker assisted selection. A comparison of responses in susceptible and resistant stock provides a powerful tool to distinguish among protective, irrelevant and pathological responses. These themes have been illustrated by three studies of gastrointestinal nematode infections in ruminants.

Influence of host genetics upon antibody responses against gastrointestinal nematode infections in cattle.

Previous studies have shown that the number of gastrointestinal nematode eggs released per gram of feces (EPG) of calves is strongly influenced by host genetics. The purpose of this study was to determine if host genetics also influenced immune recognition of parasite antigens in these same calves. Serum samples were taken at monthly intervals from calves during their first grazing season, from approximately 4 months after the onset of calving and were continued until weaning. Serum samples were analyzed for antibodies against Ostertagia ostertagi, Haemonchus placei, Cooperia oncophora, and Oesophagostomum radiatum. Significant rises in antibodies of the IgG1 class were seen against Ostertagia ostertagi, H. placei, and C. oncophora. In addition, rises in anti-Ostertagia antibodies of the IgG2 and IgM isotypes were also noted. During periods of elevated antibody responses, the sire of the individual calves was found to influence significantly the level of circulating antibody. The heritability of serum anti-parasite antibody levels was demonstrated to be between 70 and 80%, depending upon the time and antibody isotype. The antibody levels did not appear to be correlated with parasite EPG values. These results indicate that the ability of calves to recognize parasite antigens is strongly influenced by genetic factors, and that the genetic factors which control antibody responses may differ from those controlling EPG values.

Genetic control of immunity to gastrointestinal nematodes of cattle.

Previous studies have indicated that host genetics significantly affects the number of gastrointestinal nematode eggs per gram (epg) in the feces of calves during their first grazing season. An entire calf crop of approximately 190 animals was monitored monthly until weaning to verify these earlier results, and to begin to discern the basis for this phenomenon. A significant genetic effect on fecal epg values was not observed until calves had been on pasture for 2-3 months, and was demonstrable until late in the grazing season when the effect was lost. The loss of a genetic effect coincided with the appearance of significant numbers of the more highly fecund nematode species Haemonchus placei and Oesophagostomum radiatum, and with an apparent increase in Ostertagia ostertagi transmission, indicating that the observed genetic control of epg values may be species specific, dose dependent or both. Calves were selected from the population, and grouped according to their epg phenotype over the grazing season as either high or low epg calves. Postmortem examination of some of these calves indicated that worm burdens in the low epg calves were 60% of those of the high epg calves. Experimental challenge inoculation of the remaining calves indicated that: (1) challenge with Cooperia oncophora resulted in low epg calves harboring worm numbers that were 65% of those of high epg calves; (2) challenge with O. ostertagi resulted in similar numbers of worms in both groups, but the fecundity of worms in the low epg groups was significantly lower (P less than 0.05) than in the high epg group. Analysis of serum anti-Ostertagia antibody levels in the grazing calf population showed rises in serum IgG1, IgG2, IgM and IgA antibody levels during the grazing season. Peak serum IgG2 and IgG1 anti-Ostertagia antibody levels were found to be significantly affected by host genetic factors while IgA and IgM levels were not under such control.