Variation In Faecal Egg Count Research Articles

Association between variation in faecal egg count for a natural mixed field-challenge of nematode parasites and TLR4 variation

Research has shown that Toll-like receptor 4 (TLR4) is important in immune responses to some helminth parasites. In sheep, variation in the PAMP region of TLR4 may result in structurally and thus functionally different TLR4 molecules, and this may consequently lead to variation in the TLR4 response to parasite infections. This study involved three separate, but related sheep breeds (Merino, Polwarth and Corriedale sheep) and a total of 885 lambs from five New Zealand farms that underwent a mixed field-challenge from gastro-intestinal parasites. Faecal samples were collected at approximately 4 and 9 months of age and faecal egg counts (FECs) for Nematodirus spp. and Strongyle species determined, along with the total number of eggs per gram (EPG). Analysis of the five farms collectively revealed an association (P=0.023) between the presence of TLR4 variant *02 (mean 24 EPG) and the absence of the variant (mean 32 EPG) at 9 months of age. Conversely the presence of *03 had a significantly (P=0.047) higher mean Nematodirus spp. FEC (mean 42 EPG) compared to the absence (mean 28 EPG) at 9 months of age. More associations were revealed when the data were split according to the dominant faecal parasite species. With a predominantly Trichostrongylus spp. FEC group of lambs at 9 months of age, the presence of TLR4 variant *02 was found to have significantly (P=0.003) lower Nematodirus spp. FEC (mean 4 EPG), and also significantly (P=0.033) lower total FEC (mean 312 EPG) when compared to sheep without the variant (mean 15 EPG and 449 EPG, respectively). The presence of TLR4 variant *03 and *04 were associated or tended to be associated (P=0.010 and P=0.088, respectively) with higher Nematodirus spp. FEC (mean 25 EPG and 22 EPG, respectively) when compared to lambs without the variant (mean 10 EPG and 11 EPG, respectively). These results suggest that TLR4 variation may be affecting the immune response to gastro-intestinal parasites in sheep, although principally to Nematodirus spp. infections and not Strongyle species infections.

A joint analysis to identify loci underlying variation in nematode resistance in three European sheep populations

Gastrointestinal nematode infections are one of the main health/economic issues in sheep industries, worldwide. Indicator traits for resistance such as faecal egg count (FEC) are commonly used in genomic studies; however, published results are inconsistent among breeds. Meta (or joint)-analysis is a tool for aggregating information from multiple independent studies. The aim of this study was to identify loci underlying variation in FEC, as an indicator of nematode resistance, in a joint analysis using data from three populations (Scottish Blackface, Sarda × Lacaune and Martinik Black-Belly × Romane), genotyped with the ovine 50k SNP chip. The trait analysed was the average animal effect for Strongyles and Nematodirus FEC data. Analyses were performed with regional heritability mapping (RHM), fitting polygenic effects with either the whole genomic relationship matrix or matrices excluding the chromosome being interrogated. Across-population genomic covariances were set to zero. After quality control, 4123 animals and 38 991 SNPs were available for the analysis. RHM identified genome-wide significant regions on OAR4, 12, 14, 19 and 20, with the latter being the most significant. The OAR20 region is close to the major histocompatibility complex, which has often been proposed as a functional candidate for nematode resistance. This region was significant only in the Sarda × Lacaune population. Several other regions, on OAR1, 3, 4, 5, 7, 12, 19, 20 and 24, were significant at the suggestive level.

Comparative kinetics of serological and coproantigen ELISA and faecal egg count in cattle experimentally infected with Fasciola hepatica and following treatment with triclabendazole

Three methods of diagnosing Fasciola hepatica (F. hepatica) infection (a coproantigen ELISA, Bio-X Diagnostics, Belgium, Faecal Egg Count (FEC), and a serum IgG ELISA,Bio-X Diagnostics, Belgium) were evaluated in artificially infected cattle, with and without drug treatment. Specifically, the potential value of the coproantigen ELISA in the quantitation of F. hepatica infection was sought. Twelve steers were each infected with 100, 200 or 500 metacercariae (n=4 cattle/group). On day 84, post infection (PI), 2 animals from each group were treated orally with triclabendazole (TCBZ). Faecal and blood samples were collected weekly after infection from all animals, as well as over 5 consecutive days (days 105–109 PI) for the six animals remaining infected to determine the repeatability of these assays. Cattle were killed 126 days PI and the coproantigen, FEC and IgG levels were compared with the number of fluke recovered. Animals first tested positive for infection with the serum ELISA, with 11/12 animals positive on day 28, and IgG responses increased to day 42 PI. The coproantigen ELISA was first positive on day 42 (3/12 animals), with all animals positive by day 56 PI. The first F. hepatica egg was detected on day 49 from an animal infected with 500 metacercariae; however only on one occasion (day 84) did all animals return positive FEC. Within one week of treatment with TCBZ, all six treated animals had returned to negative status by coproantigen ELISA and FEC whereas IgG levels persisted. Weekly variation in both coproantigen level and FEC was evident throughout the trial. Results from the consecutive daily collections varied greatly between days for both methods, with 2–6-fold differences in coproantigen levels and 2–4-fold variation in FEC. Strong correlations were observed between fluke burdens (day 126) and day 125 coproantigen levels (R2=0.8718) and FEC (R2=0.8368). The coproantigen ELISA was more sensitive than FEC (FEC displayed false negatives) and detected infection earlier. This ELISA showed good correlation to fluke burdens in these cattle and has promise as a test for detecting low fluke burdens.

The contribution of simple random sampling to observed variations in faecal egg counts

It has been over 100 years since the classical paper published by Gosset in 1907, under the pseudonym “Student”, demonstrated that yeast cells suspended in a fluid and measured by a haemocytometer conformed to a Poisson process. Similarly parasite eggs in a faecal suspension also conform to a Poisson process. Despite this there are common misconceptions how to analyse or interpret observations from the McMaster or similar quantitative parasitic diagnostic techniques, widely used for evaluating parasite eggs in faeces. The McMaster technique can easily be shown from a theoretical perspective to give variable results that inevitably arise from the random distribution of parasite eggs in a well mixed faecal sample. The Poisson processes that lead to this variability are described and illustrative examples of the potentially large confidence intervals that can arise from observed faecal eggs counts that are calculated from the observations on a McMaster slide. Attempts to modify the McMaster technique, or indeed other quantitative techniques, to ensure uniform egg counts are doomed to failure and belie ignorance of Poisson processes. A simple method to immediately identify excess variation/poor sampling from replicate counts is provided.

The bias, accuracy and precision of faecal egg count reduction test results in cattle using McMaster, Cornell-Wisconsin and FLOTAC egg counting methods

The faecal egg count reduction test (FECRT) is the recommended method to monitor anthelmintic drug efficacy in cattle. There is a large variation in faecal egg count (FEC) methods applied to determine FECRT. However, it remains unclear whether FEC methods with an equal analytic sensitivity, but with different methodologies, result in equal FECRT results. We therefore, compared the bias, accuracy and precision of FECRT results for Cornell-Wisconsin (analytic sensitivity=1egg per gram faeces (EPG)), FLOTAC (analytic sensitivity=1EPG) and McMaster method (analytic sensitivity=10EPG) across four levels of egg excretion (1–49EPG; 50–149EPG; 150–299EPG; 300–600EPG). Finally, we assessed the sensitivity of the FEC methods to detect a truly reduced efficacy. To this end, two different criteria were used to define reduced efficacy based on FECR, including those described in the WAAVP guidelines (FECRT <95% and lower limit of 95%CI <90%) (Coles et al., 1992) and those proposed by El-Abdellati et al. (2010) (upper limit of 95%CI <95%). There was no significant difference in bias and accuracy of FECRT results across the three methods. FLOTAC provided the most precise FECRT results. Cornell-Wisconsin and McMaster gave similar imprecise results. FECRT were significantly underestimated when baseline FEC were low and drugs were more efficacious. For all FEC methods, precision and accuracy of the FECRT improved as egg excretion increased, this effect was greatest for McMaster and least for Cornell-Wisconsin. The sensitivity of the three methods to detect a truly reduced efficacy was high (>90%). Yet, the sensitivity of McMaster and Cornell-Wisconsin may drop when drugs only show sub-optimal efficacy. Overall, the study indicates that the precision of FECRT is affected by the methodology of FEC, and that the level of egg excretion should be considered in the final interpretation of the FECRT. However, more comprehensive studies are required to provide more insights into the complex interplay of factors inherent to study design (sample size and FEC method) and host-parasite interactions (level of egg excretion and aggregation across the host population).

Quantifying the sources of variability in equine faecal egg counts: Implications for improving the utility of the method

The faecal egg count (FEC) is the most widely used means of quantifying the nematode burden of horses, and is frequently used in clinical practice to inform treatment and prevention. The statistical process underlying the FEC is complex, comprising a Poisson counting error process for each sample, compounded with an underlying continuous distribution of means between samples. Being able to quantify the sources of variability contributing to this distribution of means is a necessary step towards providing estimates of statistical power for future FEC and FECRT studies, and may help to improve the usefulness of the FEC technique by identifying and minimising unwanted sources of variability. Obtaining such estimates require a hierarchical statistical model coupled with repeated FEC observations from a single animal over a short period of time. Here, we use this approach to provide the first comparative estimate of multiple sources of within-horse FEC variability. The results demonstrate that a substantial proportion of the observed variation in FEC between horses occurs as a result of variation in FEC within an animal, with the major sources being aggregation of eggs within faeces and variation in egg concentration between faecal piles. The McMaster procedure itself is associated with a comparatively small coefficient of variation, and is therefore highly repeatable when a sufficiently large number of eggs are observed to reduce the error associated with the counting process. We conclude that the variation between samples taken from the same animal is substantial, but can be reduced through the use of larger homogenised faecal samples. Estimates are provided for the coefficient of variation (cv) associated with each within animal source of variability in observed FEC, allowing the usefulness of individual FEC to be quantified, and providing a basis for future FEC and FECRT studies.

Association between variation in faecal egg count for a mixed field-challenge of nematode parasites and ovine MHC- DQA2 polymorphism

The selection of sheep that are resistant to gastrointestinal parasites and have lower faecal egg counts (FECs) has been the subject of extensive research. This has led to the speculation that the Major Histocompatibility Complex (MHC) genes could be used as markers to reduce FEC. In this study, associations between variation in ovine MHC- DQA2 and various measures of FEC recorded at two times (approximately 4 and 9 months of age) were investigated in a large group of New Zealand lambs ( n = 4676), derived from 185 different sire-lines, of a variety of breeds and raised on 25 separate farms. Pair-sample t-tests revealed that FEC for Nematodirus spp., Strongyle spp. and total FEC differed significantly between the two assessments. A total of twenty one DQA2 alleles or DQA2– DQA2-like haplotypes were identified, with allele/haplotype presence and frequency varying significantly between farms. For example, allele *0103 was observed on all farms, ranging in frequency from 0.2 to 60.9%, while haplotype *0101–*1601 was only present on one farm, in two lambs. A number of associations between the presence/absence of these alleles and egg counts were observed, but nearly all the allelic/haplotypic associations were age and parasite specific, suggesting that immune response is both age and challenge (parasite species mix) dependent. The exception was allele *1201 which was associated with increased total FECs at both 4 and 9 months of age; with it either being, or tending toward being, significantly associated with both increased Strongyle spp. and Nematodirus spp. counts as well. However, the observed increases in egg counts were small and ranged between 5 and 32 eggs per gram. In conclusion, we believe that the MHC plays an important role in parasite resistance, but that the MHC–nematode interaction is complex and thus the development of a single gene-marker based on the “MHC effect” is unlikely.

Divergent ghrelin expression patterns in sheep genetically resistant or susceptible to gastrointestinal nematodes

Gastrointestinal nematodes are a major problem for pastoral ruminant production systems. This problem could be reduced by the application of breeding strategies that select for nematode resistant sheep, but no suitable molecular markers are available. Research selection flocks containing lines that are resistant (R) or susceptible (S) to gastrointestinal nematodes provide an excellent resource for discovering selectable markers, and for studying the underlying mechanisms of an effective anti-nematode response. In this study we have used a combination of quantitative real time PCR assays and ELISA to determine if nematode challenge impacts on the expression of the satiety-regulating hormone ghrelin. The expression responses were then compared between the selection flock R and S lines. The results show that the basal levels of ghrelin in plasma were greater than 2-fold higher in nematode naïve S line sheep. Three days after a primary nematode challenge divergent ghrelin expression patterns were observed between the selection lines, with levels increasing in R sheep while decreasing in S sheep. After a secondary challenge this trend was repeated, but following a third challenge ghrelin expression levels rose in both R and S sheep, by which time the S animals had acquired an effective immune response to the nematodes, as measured by a significant reduction in faecal egg output. Importantly, this phenomenon was observed in gene expression studies in gut tissues and also in ELISA measurements of ghrelin peptide levels in plasma. A regression analysis showed that ghrelin transcript expression in the gut accounted for >40% of the variation in faecal egg count measured following Haemonchus or Trichostrongylus infection. We therefore hypothesise that the direction of ghrelin expression (up or down) immediately following nematode exposure may play an important role in regulating the differing anti-nematode responses that occur in the R and S lines. Such differences identify ghrelin as a previously unrecognized factor influencing the acquisition of immunity to nematodes.

No evidence for a universal association between variation in faecal egg count for a mixed field-challenge of gastrointestinal parasites and the presence of the Ovar-DQA1null haplotype in sheep

We tested the association of the Ovar-DQA1 null haplotype with faecal egg count (FEC) in 4676 male lambs sourced from 185 different sire-lines and four breeds. For each lamb a faecal sample was collected at weaning (approx. 4 months of age) and mid-autumn (approx. 9 months of age) and each was typed for the presence of Ovar-DQA1 null. General Linear Mixed Models (GLMMs) were used to analyse the relationship between FEC and Ovar-DQA1 null, both across and within breeds. Two models were used, the first testing the effect of the presence/absence of Ovar-DQA1 null and the second testing the effect of the number of copies of Ovar-DQA1 null present in an animal's genotype (i.e. dominant/recessive or additive effects). Across the breeds, no significant effect of the presence of Ovar-DQA1 null on FEC for Nematodirus sp., Strongyle genera, or total FEC was detected at either 4 or 9 months of age using either of the models. Within breeds, significant associations were detected at 4 months in South African Meat Merinos, with the presence of Ovar-DQA1 null in an animal's genotype being associated with a lower strongyle (absent: n = 23, geometric mean = 135 epg; present: n = 10, geometric mean = 67 epg; P = 0.025) and total FEC (absent: n = 23, geometric mean = 220 epg; present: n = 10, geometric mean = 121 epg; P = 0.017). These associations were not detected at 9 months of age.

Association between variation in faecal egg count for a mixed field-challenge of nematode parasites and IGHA gene polymorphism

Research has shown that variation in ovine immunoglobulin A (IgA) levels are associated with reduced faecal egg counts (FECs) in sheep hosting gastro-intestinal (GI) parasites. Variation in the constant region of the ovine IgA heavy alpha chain gene ( IGHA) may result in structurally and functionally different IgA molecules and may consequently lead to variation in the IgA response to parasitisation. This study involved three sheep breeds (Merino, Polwarth and Corriedale) and a total of 2098 lambs from eight New Zealand farms that underwent a mixed field-challenge of nematode parasites. Faecal samples were taken at approximately 4 and 9 months of age and FECs for Nematodirus and Strongyle species determined along with total eggs per gram (EPG). Analysis of all eight farms collectively revealed no significant differences in FECs associated with the presence or absence of a particular IGHA allele. However, when the data was split into predominant challenge type groups, associations were detected. In 4-month-old lambs predominantly challenged by Nematodirus sp., the presence of the IGHA allele *01 was associated ( P < 0.05) with higher Strongyle FECs. In 9-month-old lambs predominantly challenged by Trichostrogylus sp., the presence of IGHA allele *02 was associated ( P < 0.006) with a higher mean total EPG at 9 months of age. These results suggest that IGHA gene variation will not be an effective gene-marker for reducing overall FEC but may be useful in defined or specific species challenges.

Major Histocompatibility ComplexDRB1 gene: its role in nematode resistance in Suffolk and Texel sheep breeds

A potential control strategy for nematode infection in sheep is the implementation of a breeding programme to select for genes associated with resistance. The Texel breed is more resistant to gastrointestinal nematode infection than the Suffolk breed, based on faecal egg count, and this difference should enable the identification of some of the genes responsible for resistance. The objective of this study was to determine if variation at the ovine MHC-DRB1 locus was associated with variation in faecal egg count in Suffolk and Texel sheep. Ovar-DRB1 alleles and faecal egg count were determined for Texel (n = 105) and Suffolk (n = 71) lambs. Eight Ovar-DRB1 alleles, including 1 previously unknown allele, were identified in the Texel breed by sequence-base-typing. Seven Ovar-DRB1 alleles were identified in the Suffolk breed. Two Ovar-DRB1 alleles were common to both breeds, but were among the least frequent in the Suffolk population. In the Suffolk breed 1 Ovar-DRB1 allele was associated with a decrease in faecal egg count and 2 alleles with an increase in faecal egg count. This locus accounted for 14% of the natural variation in faecal egg count in Suffolks. There was no evidence for an association between Ovar-DRB1 alleles and faecal egg count in the Texel breed and the Ovar-DRB1 locus accounted for only 3% of the phenotypic variation in faecal egg count. These results suggest that the Ovar-DRB1 gene plays an important role in resistance to nematode infection in the Suffolk breed. The difference in faecal egg counts between these breeds may be attributable in part to the different allele profile at the Ovar-DRB1 locus.

Intron 1 of the interferon γ gene: Its role in nematode resistance in Suffolk and Texel sheep breeds

Genetic variation at intron 1 of the interferon γ gene has recently been associated with variation in faecal egg count in Romney, Merino and Soay breeds of sheep. The Texel breed is more resistant to gastrointestinal nematode infection than the Suffolk breed, based on faecal egg count. Hence, the objective of this experiment was to characterise the polymorphisms of intron 1 of the interferon γgene in Suffolk and Texel sheep, and to determine if the characterised haplotype variants were associated with faecal egg count variation in these breeds. Intron 1 haplotypes and faecal egg measurements were determined for Suffolks ( n = 113) and Texels ( n = 135). Four haplotypes were identified in Suffolks (A, B, C and D), two haplotypes as previously described, and two previously unknown haplotypes. However, there was no association between interferon γ intron 1 haplotype and faecal egg count in Suffolks. In contrast, two previously identified haplotypes (A and B) were present in the Texel breed, and the B haplotype was associated with resistance to nematode infection ( P = 0.02). These results suggest that intron 1 of the interferon γ gene has an important role in resistance to nematode infection in the Texel breed.

Mechanisms underlying resistance to nematode infection

Lambs show considerable genetic variation in faecal egg count following natural, predominantly Ostertagia circumcincta infection. This genetic variation is acquired and not innate. Worm length is positively associated with worm fecundity. The genetic variation in faecal egg count is a consequence of genetic variation in worm length and hence worm fecundity, and not of genetic variation in worm burdens. In contrast to lambs, mature sheep may be able to regulate both fecundity and worm numbers. In lambs, three factors account for the majority of the variation in worm length: the strength of the local IgA response against fourth-stage larvae, the specificity of this response against four molecules in particular, and the density-dependent influence of worm number.

Resistance to nematode parasites in Merino sheep: sources of genetic variation

Merino sheep representing a range of bloodlines in resource flocks located across Australia were tested for resistance to gastro-intestinal nematodes. These flocks included the JB Pye Flock (Camden, NSW), Katanning Base Flock (Katanning, WA), Turretfield Merino Resource Flock (Rosedale, SA), CSIRO Finewool Flock (Armidale, NSW), and the Trangie D Flock (Trangie, NSW). Faecal egg count (FEC) was used to measure relative resistance of sheep to nematode parasites after either natural or artificial infection with Haemonchus contortus and Trichostrongylus colubriformis. Differences in FEC 0' 33 between strains and between and within bloodlines were examined and the heritability of this trait was estimated. A low proportion of the total variation in parasite resistance could be attributed to strain and bloodline effects (1 and 3.5%, respectively) after either natural or artificial infection. The major source of genetic variation was found within bloodlines (22.2% of total variation), with individual sires showing a wide range in parasite resistance. Paternal half-sib heritability estimates for FEC 0' 33 were significant (P &lt; 0.05) in 9 of the 11 analyses and ranged from 0.07 to 0.42, with a weighted average of 0.22. The influence of the environmental effects of sex, age of dam, birth-rearing rank, and day of birth were also investigated, and were found to be only occasionally significant, accounting for a small proportion (0.3-2.2%) of variation. Management group effects both prior to and at the time of measurement were often significant, and accounted for 2.2-19.4% of variation in FEC. Correction of FEC for effects other than management group would seem to add little to precision of selection. These results have demonstrated that significant genetic variation for nematode parasite resistance exists within a wide range of Merino bloodlines, and within-flock selection of resistant sires appears to be an effective method of improving this trait in Merino sheep.

Variations in Faecal Egg-counts in Paramphistome Infections as Determined by a New Technique

Although paramphistomes have been known to occur in cattle and sheep in the British Isles for-many years little precise information on the incidence, distribution and degree of infection is available. The diagnosis, except in exceptional cases, can be made only post mortem, or by the detection of eggs in the faeces. Before undertaking an investigation into this problem it was necessary to devise a simple standard technique for the collection of eggs from cattle faeces, which would be practicable not only in the laboratory but also in the field. This done, it became desirable to know firstly, within what limits egg counts were reliable criteria on which to base estimates of the number of parasites present; secondly, what was the smallest number of parasites which could be detected by faecal examination; thirdly, how much variation occurred in egg-counts from week to week in the same animals; and fourthly, if there were any variation in counts made on faeces collected at different hours of the day.