Development of a three-colour digital PCR for early and quantitative detection of benzimidazole resistance-associated single nucleotide polymorphisms in Haemonchus contortus

Haemonchus contortus is the most pathogenic nematode in small ruminants and anthelmintic resistance (AR) hampers its efficient control. Early detection of AR status is required to reduce selection for AR and cannot be achieved using phenotypic tests. For benzimidazoles (BZs), the detection of AR-associated alleles characterised by single nucleotide polymorphisms (SNPs) in the isotype 1 β-tubulin gene allows early AR detection in strongyles. The F200Y, F167Y, E198A and E198L polymorphisms have been described in BZ-resistant populations with a clear variation in frequencies between regions. A novel digital PCR (dPCR) enables the detection of all of the above-described polymorphisms in H. contortus. Assays were validated using synthetic DNA fragments containing these SNPs. Then, larvae obtained and pooled at farm level from 26 Austrian and 10 Italian sheep farms were analysed. For all assays a detection limit of 15 copies/μl of resistance alleles and a high level of accuracy were demonstrated, allowing to detect allele frequencies of 1% in most samples. In Austrian samples, elevated frequencies of F200Y resistance alleles were detected on all farms. Polymorphisms in codon 167 and codon 198 were identified in H. contortus from Austria for the first time. In Italian samples, the frequency of resistance alleles was still comparatively low, but F200Y resistance alleles were traceable. In conclusion we developed for the first time dPCR assays that target all SNPs of relevance associated with BZ-resistance in H. contortus. Future research on AR development could benefit from an early onset of SNP-based surveillance that would include the developed assays for all SNPs of relevance. Improved surveillance in the long term will include other important, though less pathogenic, nematode genera in the analyses

Strongyloides stercoralis infection in dogs in Austria: two case reports.

BACKGROUND Strongyloides stercoralis is endemic in tropical and subtropical regions, but reports of infections in central and northern Europe have been recently increasing. Infections occur mainly in humans and dogs. In dogs, both dog-adapted and zoonotic S. stercoralis genotypes seem to occur. Clinical manifestations mainly include gastrointestinal and respiratory signs. The severity of the disease can vary greatly and depends on the immune status of the host. The infection is potentially fatal in immunosuppressed individuals, either medically induced or due to an underlying disease, in which hyperinfections and disseminated infections with extraintestinal parasite dissemination may occur. METHODS Diagnosis was based on coproscopy, including flotation and the Baermann funnel technique, histology of small intestinal biopsies and molecular analysis of mitochondrial cytochrome oxidase subunit I (COI) and hypervariable regions I and IV (HVR I and HVR IV) of the nuclear 18S rDNA loci. RESULTS Two independent cases of severe canine S. stercoralis infection in Austria are presented. In both cases, S. stercoralis was detected in histological sections of the small intestine and with the Baermann funnel technique. Molecular analysis revealed strains with zoonotic potential. Case 1 was a 1-year-old female French bulldog with a long history of respiratory and gastrointestinal signs, severe emaciation and apathy before S. stercoralis infection was diagnosed. Treatment with moxidectin (2.5 mg/kg body weight [BW], oral route) did not eliminate the infection, but treatment with ivermectin (0.2 mg/kg BW, subcutaneously) was successful. Case 2 consisted of two 2-month-old Pomeranian puppies, one female and one male, from a litter of four, which died soon after presenting dyspnoea and haemorrhagic diarrhoea (female) or torticollis (male); S. stercoralis infection was first diagnosed post-mortem. CONCLUSION More attention should be paid to this nematode because although it appears to be rare in Austria, it is easily overlooked on standard coproscopy unless a Baermann funnel technique is used, and even then, it can be missed. Moxidectin is not always successful in eliminating the infection, and treatment with ivermectin should be considered in cases of infection

Exposition von Bio-Milchvieh gegenüber dem Labmagenwurm Ostertagia ostertagi durch Messung von Antikörpertitern in Tankmilch: vorläufige Ergebnisse in Österreich

Wir untersuchten die Exposition von Bio-Milchvieh gegenüber O. ostertagi in Österreich durch Messung von parasitenspezifischer Antikörper in Tankmilch. Es wurde eine hohe Parasiten-Exposition bestätigt, was ein Monitoring und die Einführung nachhaltiger, lokal relevanten Kontrolle rechtfertigt

Best-bet integrated strategies for containing drug-resistant trypanosomes in cattle

Background African animal trypanosomosis is a major constraint to the rearing of productive livestock in the sub-humid Sudan-Sahel zone of West Africa where cotton is grown. Trypanosomosis is mainly controlled using trypanocidal drugs, but the effective use of drugs is threatened by the development of widespread resistance. This study tested integrated best-bet strategies for containment and/ or reversal of trypanocide resistance in villages in south-east Mali where resistance has been reported. Methods Four sentinel villages each from an intervention area (along the road from Mali to Burkina Faso) and a control area (along the road from Mali to Côte d’Ivoire) were selected for the study. Tsetse control was based on deltamethrin-treated stationary attractive devices and targeted cattle spraying between March 2008 and November 2009. Trypanosome-positive cattle were selectively treated with 3.5 mg/kg diminazene aceturate. Strategic helminth control using 10 mg/kg albendazole was also undertaken. During the intervention, tsetse densities along drainage lines, trypanosome infections and faecal egg counts in risk cattle (3 to 12 months of age) were monitored. Results Catch reductions of 66.5 % in Glossina palpalis gambiensis and 90 % in G. tachinoides were observed in the intervention area. Trypanosome prevalence was significantly (p < 0.05) lower in the intervention area (2.3 %; 1.3-3.6 %) compared to the control area (17.3 %; 14.8-20.1 %). Albendazole treatment resulted in a faecal egg count reduction of 55.6 % and reduced trypanosome infection risk (2.9 times lower than in the placebo group) although not significantly (p > 0.05). Further studies are required before confirming the existence of albendazole resistant strongyles in the study area. Conclusion Integration of best-bet strategies in areas of multiple drug- resistance is expected to reduce trypanosome infection risk thus contributing to containment of trypanocidal drug resistance. Integrated best-bet strategies could therefore be considered a viable trypanosomosis control option especially in areas where multiple drug-resistance has been reported

Presence of Equine and Bovine Coronaviruses, Endoparasites, and Bacteria in Fecal Samples of Horses with Colic

Acute abdominal pain (colic) is one of the major equine health threats worldwide and often necessitates intensive veterinary medical care and surgical intervention. Equine coronavirus (ECoV) infections can cause colic in horses but are rarely considered as a differential diagnosis. To determine the frequency of otherwise undetected ECoV infections in horses with acute colic, fresh fecal samples of 105 horses with acute colic and 36 healthy control horses were screened for viruses belonging to the Betacoronavirus 1 species by RT-PCR as well as for gastrointestinal helminths and bacteria commonly associated with colic. Horses with colic excreted significantly fewer strongyle eggs than horses without colic. The prevalence of anaerobic, spore-forming, gram-positive bacteria (Clostridium perfringens and Clostridioides difficile) was significantly higher in the feces of horses with colic. Six horses with colic (5.7%) and one horse from the control group (2.8%) tested positive for Betacoronaviruses. Coronavirus-positive samples were sequenced to classify the virus by molecular phylogeny (N gene). Interestingly, in three out of six coronavirus-positive horses with colic, sequences closely related to bovine coronaviruses (BCoV) were found. The pathogenic potential of BCoV in horses remains unclear and warrants further investigation

Two New Loci for Body-Weight Regulation Identified in a Joint Analysis of Genome-Wide Association Studies for Early-Onset Extreme Obesity in French and German Study Groups

Meta-analyses of population-based genome-wide association studies (GWAS) in adults have recently led to the detection of new genetic loci for obesity. Here we aimed to discover additional obesity loci in extremely obese children and adolescents. We also investigated if these results generalize by estimating the effects of these obesity loci in adults and in population-based samples including both children and adults. We jointly analysed two GWAS of 2,258 individuals and followed-up the best, according to lowest p-values, 44 single nucleotide polymorphisms (SNP) from 21 genomic regions in 3,141 individuals. After this DISCOVERY step, we explored if the findings derived from the extremely obese children and adolescents (10 SNPs from 5 genomic regions) generalized to (i) the population level and (ii) to adults by genotyping another 31,182 individuals (GENERALIZATION step). Apart from previously identified FTO, MC4R, and TMEM18, we detected two new loci for obesity: one in SDCCAG8 (serologically defined colon cancer antigen 8 gene; p = 1.85610 x 10(-8) in the DISCOVERY step) and one between TNKS (tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase gene) and MSRA (methionine sulfoxide reductase A gene; p = 4.84 x 10(-7)), the latter finding being limited to children and adolescents as demonstrated in the GENERALIZATION step. The odds ratios for early-onset obesity were estimated at similar to 1.10 per risk allele for both loci. Interestingly, the TNKS/MSRA locus has recently been found to be associated with adult waist circumference. In summary, we have completed a meta-analysis of two GWAS which both focus on extremely obese children and adolescents and replicated our findings in a large followed-up data set. We observed that genetic variants in or near FTO, MC4R, TMEM18, SDCCAG8, and TNKS/MSRA were robustly associated with early-onset obesity. We conclude that the currently known major common variants related to obesity overlap to a substantial degree between children and adults

Understanding the uptake of diagnostics for sustainable gastrointestinal nematode control by European dairy cattle farmers: a multi-country cross-sectional study

To mitigate emerging anthelmintic resistance (AR) in cattle, sustainable gastrointestinal nematode control strategies should be adopted. A multi-centre study was set up to understand the factors affecting European dairy cattle farmers’ adoption of diagnostics and to gauge for differences between regions. The data were collected through a multi-lingual survey by participating countries of the European Co-operation in Science and Technology (COST) action COMbatting Anthelmintic Resistance in ruminants (COMBAR). Four countries provided sufficient data to be included in the data analysis: Norway, Italy, Germany and Austria. Three models were estimated and validated through structural equation modelling. Norway, along with Germany and Austria (pooled dataset) showed similar trends that align with previous studies. AR risk perception had no influence on the adoption intention of diagnostics, a positive influence was found for attitude towards diagnostics and subjective norms (i.e., perceived opinion of others), and a negative influence of attitudes towards anthelminthics. Additionally, routine (i.e., perception of the current treatment) had an indirect effect on adoption intention through attitudes. Italy’s data deviated from these findings, presenting a positive effect of the perceived severity of AR, and perceived behavioural control (i.e., perceived ability to perform a specific behaviour) on adoption intention of diagnostics. Finally, Norway’s data set allowed for inclusion of a measurement of current behaviour in the model, identifying a direct positive effect of the perceived actual behaviour of other farmers on their own behaviour

The Association of a SNP Upstream of INSIG2 with Body Mass Index is Reproduced in Several but Not All Cohorts

A SNP upstream of the INSIG2 gene, rs7566605, was recently found to be associated with obesity as measured by body mass index (BMI) by Herbert and colleagues. The association between increased BMI and homozygosity for the minor allele was first observed in data from a genome-wide association scan of 86,604 SNPs in 923 related individuals from the Framingham Heart Study offspring cohort. The association was reproduced in four additional cohorts, but was not seen in a fifth cohort. To further assess the general reproducibility of this association, we genotyped rs7566605 in nine large cohorts from eight populations across multiple ethnicities (total n = 16,969). We tested this variant for association with BMI in each sample under a recessive model using family-based, population-based, and case-control designs. We observed a significant (p < 0.05) association in five cohorts but saw no association in three other cohorts. There was variability in the strength of association evidence across examination cycles in longitudinal data from unrelated individuals in the Framingham Heart Study Offspring cohort. A combined analysis revealed significant independent validation of this association in both unrelated (p = 0.046) and family-based (p = 0.004) samples. The estimated risk conferred by this allele is small, and could easily be masked by small sample size, population stratification, or other confounders. These validation studies suggest that the original association is less likely to be spurious, but the failure to observe an association in every data set suggests that the effect of SNP rs7566605 on BMI may be heterogeneous across population samples

Meta-Analysis of the INSIG2 Association with Obesity Including 74,345 Individuals: Does Heterogeneity of Estimates Relate to Study Design?

The INSIG2 rs7566605 polymorphism was identified for obesity (BMI≥30 kg/m2) in one of the first genome-wide association studies, but replications were inconsistent. We collected statistics from 34 studies (n = 74,345), including general population (GP) studies, population-based studies with subjects selected for conditions related to a better health status (‘healthy population’, HP), and obesity studies (OB). We tested five hypotheses to explore potential sources of heterogeneity. The meta-analysis of 27 studies on Caucasian adults (n = 66,213) combining the different study designs did not support overall association of the CC-genotype with obesity, yielding an odds ratio (OR) of 1.05 (p-value = 0.27). The I2 measure of 41% (p-value = 0.015) indicated between-study heterogeneity. Restricting to GP studies resulted in a declined I2 measure of 11% (p-value = 0.33) and an OR of 1.10 (p-value = 0.015). Regarding the five hypotheses, our data showed (a) some difference between GP and HP studies (p-value = 0.012) and (b) an association in extreme comparisons (BMI≥32.5, 35.0, 37.5, 40.0 kg/m2 versus BMI<25 kg/m2) yielding ORs of 1.16, 1.18, 1.22, or 1.27 (p-values 0.001 to 0.003), which was also underscored by significantly increased CC-genotype frequencies across BMI categories (10.4% to 12.5%, p-value for trend = 0.0002). We did not find evidence for differential ORs (c) among studies with higher than average obesity prevalence compared to lower, (d) among studies with BMI assessment after the year 2000 compared to those before, or (e) among studies from older populations compared to younger. Analysis of non-Caucasian adults (n = 4889) or children (n = 3243) yielded ORs of 1.01 (p-value = 0.94) or 1.15 (p-value = 0.22), respectively. There was no evidence for overall association of the rs7566605 polymorphism with obesity. Our data suggested an association with extreme degrees of obesity, and consequently heterogeneous effects from different study designs may mask an underlying association when unaccounted for. The importance of study design might be under-recognized in gene discovery and association replication so far

Meta-Analysis of the INSIG2 Association with Obesity Including 74,345 Individuals: Does Heterogeneity of Estimates Relate to Study Design?

The INSIG2 rs7566605 polymorphism was identified for obesity (BMI≥30 kg/m2) in one of the first genome-wide association studies, but replications were inconsistent. We collected statistics from 34 studies (n = 74,345), including general population (GP) studies, population-based studies with subjects selected for conditions related to a better health status (‘healthy population’, HP), and obesity studies (OB). We tested five hypotheses to explore potential sources of heterogeneity. The meta-analysis of 27 studies on Caucasian adults (n = 66,213) combining the different study designs did not support overall association of the CC-genotype with obesity, yielding an odds ratio (OR) of 1.05 (p-value = 0.27). The I2 measure of 41% (p-value = 0.015) indicated between-study heterogeneity. Restricting to GP studies resulted in a declined I2 measure of 11% (p-value = 0.33) and an OR of 1.10 (p-value = 0.015). Regarding the five hypotheses, our data showed (a) some difference between GP and HP studies (p-value = 0.012) and (b) an association in extreme comparisons (BMI≥32.5, 35.0, 37.5, 40.0 kg/m2 versus BMI<25 kg/m2) yielding ORs of 1.16, 1.18, 1.22, or 1.27 (p-values 0.001 to 0.003), which was also underscored by significantly increased CC-genotype frequencies across BMI categories (10.4% to 12.5%, p-value for trend = 0.0002). We did not find evidence for differential ORs (c) among studies with higher than average obesity prevalence compared to lower, (d) among studies with BMI assessment after the year 2000 compared to those before, or (e) among studies from older populations compared to younger. Analysis of non-Caucasian adults (n = 4889) or children (n = 3243) yielded ORs of 1.01 (p-value = 0.94) or 1.15 (p-value = 0.22), respectively. There was no evidence for overall association of the rs7566605 polymorphism with obesity. Our data suggested an association with extreme degrees of obesity, and consequently heterogeneous effects from different study designs may mask an underlying association when unaccounted for. The importance of study design might be under-recognized in gene discovery and association replication so far