Digital PCR: modern solution to parasite diagnostics and population trait genetics.

The use of polymerase chain reaction (PCR)-based diagnostic approaches has steadily increased in the field of parasitology in recent decades. The most recent large-scale technological modification of the PCR formula, also known as third-generation PCR, came in the form of digital PCR (dPCR). Currently, the most common form of dPCR on the market is digital droplet PCR (ddPCR). Unlike quantitative real-time PCR (qPCR), the digital format allows for highly sensitive, absolute quantification of nucleic acid targets and does not require external standards to be included in the developed assays. Dividing each sample into thousands of compartments and using statistical models also eliminates the need for technical replicates. With unprecedented sensitivity and enforcement of binary endpoint reactions, ddPCR not only allows the use of tiny sample volumes (especially important when working with limited amounts of DNA) but also minimises the impact of variations in amplification efficiency and the presence of inhibitors. As ddPCR is characterised by excellent features such as high throughput, sensitivity and robust quantification, it is widely used as a diagnostic tool in clinical microbiology. Due to recent advances, both the theoretical background and the practical, current applications related to the quantification of nucleic acids of eukaryotic parasites need to be updated. In this review, we present the basics of this technology (particularly useful for new users) and consolidate recent advances in the field with a focus on applications to the study of helminths and protozoan parasites.

The presence and relative frequency detection of the levamisole-resistance-associated S168T substitution in hco-acr-8 in Haemonchus contortus.

Parasitic sheep nematodes, among which Haemonchus contortus is often considered to be the most clinically important, exact a significant toll on the animals, not least because of their capacity to evolve drug resistance. Despite decades of research, our understanding of the mechanism of resistance to compounds such as levamisole is fairly limited, which therefore constrains our ability to develop sensitive and efficient molecular diagnostic tools for rapid and accurate resistance detection in field settings. Herein, we investigated the presence and frequency of the newly reported, levamisole-resistance-associated, mutation, yielding a S168T substitution in exon 4 of hco-acr-8, in six different phenotypically described isolates (three susceptible and three resistant), three Swedish field isolates and eight larvae culture samples, the latter two of which originated on farms where levamisole showed complete parasite elimination. For this purpose, we created both an allele-specific and droplet digital PCR approaches and found the mutated allele to be present only in the Kokstad isolate, whereas the other five as well as both the Swedish isolates and larvae cultures displayed only the non-mutated, serine-encoding, allele. While the finding of only the non-mutated allele in the phenotypically susceptible and Swedish isolate and larvae culture samples seemed sensible, we speculate that for the other two phenotypically resistant isolates, different (perhaps secondary) variants are responsible for conferring the resistance to levamisole phenotype, given the polygenic nature of levamisole resistance. All in all, despite the limited number of samples tested here, the mutation causing the S168T substitution in hco-acr-8 represents a plausible levamisole resistance-associated variant in, at least, some isolates of H. contortus.

Signs of multiple anthelmintic resistance in sheep gastrointestinal nematodes in Sweden.

Gastrointestinal nematodes in small ruminants are clinically and economically important parasites that often are controlled with anthelmintics. In this study, we compiled information on the anthelmintic efficacy collected on sheep farms according to routines established by Farm & Animal Health in Sweden. The efficacies of benzimidazoles (i.e. albendazole or fenbendazole, n = 30), ivermectin (n = 47), levamisole (n = 2) or moxidectin (n = 2) were examined between 2015 and 2021 in 81 treatment groups on 49 non-randomly selected farms in south-central Sweden. Drug efficacies were estimated with the faecal egg count reduction test. In addition, efficacy data were in most cases supplemented with data on the abundance of the three most common nematode genera in sheep by performing droplet digital (dd) PCR on coprocultures. Efficacies of <95% for benzimidazoles or ivermectin were identified in 37% and 77% of the tested groups, respectively. In addition, on 27 (55%) of the 49 farms where both benzimidazoles and ivermectin were tested, multiple resistance was found on 8 (30%). In contrast, on each of the two farms tested for levamisole and moxidectin both drugs proved to be 100% effective. However, because post-sampling was performed earlier than recommended in several susceptible groups (benzimidazoles = 15, and ivermectin = 10 groups), this could have underestimated the severity of the situation. Mainly larvae from the genus Haemonchus were detected in post-treatment coprocultures, in all groups with declared resistance, suggesting that this parasite was primarily associated with anthelmintic resistance. Unexpectedly, the DNA of larvae, which survived treatment, was also detected on farms declared as susceptible. Taken together, this indicates that the situation regarding the anthelmintic efficacy has deteriorated compared with the latest nationwide study on Swedish sheep farms conducted more than a decade ago. Unlike the previous study, the farm selection here was not strictly randomized but rather opportunistic i.e., only farms with a recognized parasite problem were included. Thus, there is a need for a truly randomized study to get an update on the extent of the situation of anthelmintic resistance at a national level, as well as to identify risk factors involved in the resistance selection. Research is also required to establish the optimal intervals for sampling post-treatment.

Parasitic strongyle nemabiome communities in wild ruminants in Sweden.

BACKGROUND: Wildlife hosts may serve as reservoirs for strongyles, which can be transmitted to domestic livestock. Therefore, studies evaluating nemabiome compositions in wildlife ruminants are of great use in assessing the possibility of transmission of important nematode pathogens to domestic sheep in Sweden. METHODS: First, fecal samples were collected from roe deer (n = 125), fallow deer (n = 106), red deer (n = 18) and mouflon (n = 13) in south central Sweden during the hunting season in 2019. Second, after fecal examination samples were cultured and the larvae were harvested, followed by DNA extractions. Third, all samples were barcoded and processed for sequence analysis on the PacBio platform. Finally, bioinformatic sequence analysis was conducted with DADA2, while species diversity and richness, as well as interactions between the different hosts, were calculated and analyzed in R. RESULTS: Nematode ITS2 sequences were found in 225 of 262 (86%) samples. In total, 31 taxa were identified, among which 26 (86%) to the species level. These were found in different combinations, among which 24 (77%) occurred in roe deer, 19 (61%) in fallow deer, 20 (65%) in red deer and 10 (32%) in mouflon. Five of the species found are known to be associated with livestock (Chabertia ovina, Haemonchus contortus, Oesophagostomum venulosum, Teladorsagia circumcincta and Trichostrongylus axei). However, in the present study the relative abundance and prevalence of most of these species were low. The most striking exception was T. axei, which was relatively abundant in all wildlife hosts. Mostly a wide range of wildlife specific nematodes such as Ostertagia leptospicularis and Spiculopteragia spp. were identified including the invasive nematode Spiculopteragia houdemeri, which was found for the first time in red deer, fallow deer, and mouflon in Sweden. The difference in the number of shared species between mouflon and all cervids (n = 6) was less than among all three cervids (n = 8). CONCLUSION: In this study, we investigated the community structure of parasitic intestinal nematodes in four wildlife hosts, and we found that the majority of the parasite species identified were wildlife specific. We also found a new, potentially invasive species not reported before. After comparing the nemabiome of the wildlife hosts in this study with a previous study in sheep from the same geographical region, we conclude that the horizontal transmission potential appears to be relatively low. Still, cross-infections of nematodes between game and sheep cannot be completely ignored.

Estimation of the impact of three different bioinformatic pipelines on sheep nemabiome analysis.

BACKGROUND: Next-generation sequencing (NGS) has provided an alternative strategy to study the composition of nematode communities with increased resolution and sensitivity. However, the handling and processing of gigabytes worth of amplicon sequence data produced by an NGS platform is still a major hurdle, limiting the use and adoption of faster and more convenient analysis software. METHODS: In total 32 paired, fecal samples from Swedish sheep flocks were cultured and the larvae subsequently harvested subjected to internal transcribed spacer 2 (ITS2) amplicon sequencing using the PacBio platform. Samples were analyzed with three different bioinformatic pipelines, i.e. the DADA2, Mothur and SCATA pipelines, to determine species composition and richness. RESULTS: For the the major species tested in this study (Haemonchus contortus, Teladorsagia circumcinta and Trichostrongylus colubriformis) neither relative abundances nor species diversity differed significantly between the three pipelines, effectively showing that all three analysis pipelines, although different in their approaches, yield nearly identical outcomes. In addition, the samples analyzed here had especially high frequencies of H. contortus (90-95% across the three pipelines) both before and after sample treatment, followed by T. circumcinta (3.5-4%). This shows that H. contortus is the parasite of primary importance in contemporary Swedish sheep farms struggling with anthelmintic resistance. Finally, although on average a significant reduction in egg counts was achieved post-treatment, no significant shifts in major species relative frequencies occurred, indicating highly rigid community structures at sheep farms where anthelmintic resistance has been reported. CONCLUSIONS: The findings presented here further contribute to the development and application of NGS technology to study nemabiome compositions in sheep, in addition to expanding our understanding about the most recent changes in parasite species abundances from Swedish sheep farms struggling with anthelmintic resistance.

Assessment of three DNA extraction kits for the absolute quantification of strongyle nematode eggs in faecal samples.

BACKGROUND: Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes of small ruminants. The current diagnostic approach for the detection of this species relies on coproscopic methods, which both have low sensitivity and are time consuming. Methods employing detection through DNA amplification, such as droplet digital polymerase chain reaction (ddPCR), offer an advantageous approach to the diagnosis of H. contortus. However, DNA extraction protocols need to be constantly updated for the optimal retrieval of diagnostically usable template. Here, we describe the evaluation of three genomic DNA extraction kits for the detection and quantification of H. contortus ITS2 amplicon DNA from faecal samples, using droplet digital PCR. RESULTS: DNA samples, extracted from faecal material with the Nucleospin DNA Stool kit, produced the highest amounts of ITS2 amplicon copies and had the lowest coefficient of variation across different dilutions and sample types (fresh or frozen) out of the tested kits (Nucleospin DNA Stool, E.Z.N.A.® Stool DNA Kit and QIAamp Fast DNA Stool Mini Kit). Furthermore, the protocol of this kit has the fewest number of steps and the price of DNA extraction per sample is reasonable (2.77 €). CONCLUSIONS: The Nucleospin DNA Stool kit is an attractive option for the detection and quantification of H. contortus DNA in faecal samples of small ruminants in a diagnostic setting.

Genome-wide analysis of the response to ivermectin treatment by a Swedish field population of Haemonchus contortus.

Haemonchus contortus is a pathogenic gastrointestinal nematode of small ruminants and, in part due to its capacity to develop resistance to drugs, contributes to significant losses in the animal production sector worldwide. Despite decades of research, comparatively little is known about the specific mechanism(s) driving resistance to drugs such as ivermectin in this species. Here we describe a genome-wide approach to detect evidence of selection by ivermectin treatment in a field population of H. contortus from Sweden, using parasites sampled from the same animals before and seven days after ivermectin exposure followed by whole-genome sequencing. Despite an 89% reduction in parasites recovered after treatment measured by the fecal egg count reduction test, the surviving population was highly genetically similar to the population before treatment, suggesting that resistance has likely evolved over time and that resistance alleles are present on diverse haplotypes. Pairwise gene and SNP frequency comparisons indicated the highest degree of differentiation was found at the terminal end of chromosome 4, whereas the most striking difference in nucleotide diversity was observed in a region on chromosome 5 previously reported to harbor a major quantitative trait locus involved in ivermectin resistance. These data provide novel insight into the genome-wide effect of ivermectin selection in a field population as well as confirm the importance of the previously established quantitative trait locus in the development of resistance to ivermectin.

Subclinical nematode parasitism affects activity and rumination patterns in first-season grazing cattle.

Sickness behaviour has been suggested as an applicable indicator for monitoring disease. Deviating feeding behaviour and activity can provide information about animals' health and welfare status. Recent advances in sensor technology enable monitoring of such behaviours and could potentially be utilized as an indicator of gastrointestinal nematode (GIN) infections. This study investigated activity and rumination patterns in first-season grazing steers exposed to subclinical infection levels of the GIN Ostertagia ostertagi and Cooperia oncophora. At turnout, animals were allocated to one of four experimental groups and were faced with "high" (H1, n = 15; H2, n = 17) or "low" (L1, n = 17; L2, n = 11) levels of parasite exposure by grazing in similar enclosures contaminated with overwintering third stage (L3) GIN larvae. Animals in H1 and H2 (HP) received a 1:1 mix of approximately 10,000 O. ostertagi and C. oncophora L3 at turnout; whereas the animals in L1 and L2 (LP) were treated monthly with ivermectin. Activity and rumination patterns were monitored by fitting animals with leg- (IceQube) and neck-mounted (Heatime) sensors. BW was recorded every fortnight, whereas faecal and blood samples were examined every four weeks for nematode faecal egg count and serum pepsinogen concentrations (SPCs). There was an interaction effect of exposure level and period (P < 0.0001) on average lying daily time across the entire grazing time. A higher mean daily lying time (P = 0.0037) was found in HP compared with LP during the first 40 days on pasture. There was also interaction effects of treatment and day since turnout on rumination time (P < 0.0001) and rumination change (P = 0.0008). Also mean daily steps (P < 0.0001) and mean daily motion index (P < 0.0001) were markedly higher in HP during days 62-69, coinciding with peaking SPC in HP. Strongyle eggs were observed both in HP and LP from 31 days after turnout. Eggs per gram (EPG) differed between parasite exposure levels (P < 0.0001), with mean EPG remaining low in LP throughout the experiment. Similarly, an increase in SPC was observed (P < 0.0001), but only in HP where it peaked at day 56. In contrast, no difference in BW gain (BWG) (P = 0.78) between HP and LP was observed. In conclusion, this study shows that behavioural measurements monitored with sensors were affected even at low infection levels not affecting BWG. These combined results demonstrate the potential of automated behavioural recordings as a tool for detection of subclinical parasitism.

Using droplet digital PCR for the detection of hco-acr-8b levamisole resistance marker in H. contortus.

The nematode Haemonchus contortus is one of the most prevalent and pathogenic parasites in small ruminants. Although usually controlled using anthelmintics, the development of drug resistance by the parasite has become a major issue in livestock production. While the molecular detection of benzimidazole resistance in H. contortus is well developed, the molecular tools and protocols are far less advanced for the detection of levamisole resistance. The hco-acr-8 gene encodes a critical acetylcholine susceptible subunit that confers levamisole-sensitivity to the receptor. Here, we report the development of a droplet digital PCR assay as a molecular tool to detect a 63 bp deletion in the hco-acr-8 that has been previously associated with levamisole resistance. Sanger sequencing of single adult H. contortus yielded 56 high-quality consensus sequences surrounding the region containing the deletion. Based on the sequencing data, new primers and probes were designed and validated with a novel droplet digital PCR assay for the quantification of the deletion containing "resistant" allele in genomic DNA samples. Single adult worms from six phenotypically described isolates (n = 60) and from two Swedish sheep farms (n = 30) where levamisole was effective were tested. Even though a significant difference in genotype frequencies between the resistant and susceptible reference isolates was found (p = 0.01), the homozygous "resistant" genotype was observed to be abundantly present in both the susceptible isolates as well as in some Swedish H. contortus samples. Furthermore, field larval culture samples, collected pre- (n = 7) and post- (n = 6) levamisole treatment on seven Swedish sheep farms where levamisole was fully efficacious according to Fecal Egg Count Reduction Test results, were tested to evaluate the frequency of the "resistant" allele in each. Frequencies of the deletion ranged from 35 to 80% in the pre-treatment samples, whereas no amplifiable H. contortus genomic DNA was detected in the post-treatment samples. Together, these data reveal relatively high frequencies of the 63 bp deletion in the hco-acr-8 both on individual H. contortus and field larval culture scales, and cast doubt on the utility of the deletion in the hco-acr-8 as a molecular marker for levamisole resistance detection on sheep farms.

Utilization of droplet digital PCR to survey resistance associated polymorphisms in the ß tubulin gene of Haemonchus contortus in sheep flocks in Sweden.

Infections with gastrointestinal nematodes (GINs) in small ruminants are becoming increasingly harder to treat due to the development of anthelmintic resistance. Across Swedish sheep farms, Haemonchus contortus is one of the more persistent and pathogenic species encountered. Benzimidazole drugs, such as albendazole, are still widely used to control the GIN burden in small ruminants. However, the decline in efficacy of this drug has been observed across the country. In this study, we aimed to continue to investigate the presence of single nucleotide polymorphisms in the ß tubulin gene associated with benzimidazole drug resistance in H. contortus. This was carried out for sheep flocks from 67 farms around Sweden by screening for the two most commonly encountered SNPs at codons 167 and 200 in the isotype 1 ß tubulin gene utilizing the droplet digital PCR technology. We first established a good agreement (Lin's concordance correlation coefficient = 0.987) between the previously widely used pyrosequencing assay for the detection of the SNP at codon 200 (otherwise known as mutation F200Y) and our assay, as well as developed and validated primer-probe pairs for the detection of the mutation at codon 167 (mutation F167Y) in the ß tubulin gene of H. contortus. We then screened 174 pooled larval culture samples, collected either pre- or post-treatment, for the frequencies of the mutations F167Y and F200Y. Not only did we find the latter to be present at much higher frequencies, but the overall levels of this resistance conferring mutation have stayed stable throughout the years 2014-2019 at an average value of 88.5 ±â€¯20.3% in the pre-treatment samples across the tested farms (p = 0.61, Kruskal-Wallis test). Furthermore, after establishing a mixed model and fitting our data, we found a significant (p < 0.01) difference in the average frequency of the mutation F200Y between paired, pre- and post-treatment with albendazole, samples. Although the frequency difference in samples treated with albendazole was relatively minor (88.5% in pre- and 95.6% in post-treatment), no significant (p = 0.15) change in F200Y mutation frequency was observed between the samples from the flocks treated with ivermectin (90.8% and 92.6 %, respectively).

Assessment of the F200Y mutation frequency in the ß tubulin gene of Haemonchus contortus following the exposure to a discriminating concentration of thiabendazole in the egg hatch test.

The ruminant livestock production sector is under threat due to the infections with gastrointestinal nematode parasites and the subsequent development of anthelmintic resistance. One of most common and pathogenic species in small ruminants is Haemonchus contortus. The ability to control the infections with this and other gastrointestinal nematodes relies heavily on the use of anthelmintic drugs. Although resistance to all major classes of anthelmintics has been shown in H. contortus, the precise mechanism of resistance acquisition is only known for benzimidazoles. F200Y (TAC) is a common point mutation in the isotype 1 ß tubulin gene which is associated with an effective increase in the resistance towards benzimidazole drugs. Here, we show the utility of using this mutation as a marker in a droplet digital PCR assay to track how two H. contortus laboratory strains, characterized by different resistance levels, change with respect to this mutation, when subjected to increasing concentrations of thiabendazole. Additionally, we wanted to investigate whether exposure to a discriminating dose of thiabendazole in the egg hatch test resulted in the death of all H. contortus eggs with a susceptible genotype. We found the MHco5 strain to maintain an overall higher frequency of the F200Y mutation (80-100%) over all drug concentrations, whilst a steady, gradual increase from around 30%-60% was observed in the case of the MHco4 strain. This is further supported by the dose-response curves, displaying a much higher tolerance of the MHco5 strain (LD50 = 0.38 µg/ml) in comparison to the MHco4 strain (LD50 = 0.07 µg/ml) to the effects of thiabendazole. All things considered, we show that the F200Y mutation is still a viable and reliable marker for the detection and surveillance of benzimidazole drug resistance in H. contortus in Europe.

Molecular detection of two major gastrointestinal parasite genera in cattle using a novel droplet digital PCR approach.

Cooperia sp. and Ostertagia sp. are two cosmopolitan parasitic nematodes often found in mixed gastrointestinal infections in cattle across temperate regions. In light of the recent increase in the emergence of anthelmintic resistance in these and other nematodes derived from cattle around the globe, and their negative impact on animal health and productivity, novel molecular assays need to be put forth in order to facilitate the monitoring of parasite burden in infected herds, using pasture and/or fecal samples. Here, we describe a novel droplet digital PCR platform-based concept for precise identification and quantification of the two most abundant and important parasite genera in grazing western European cattle. By exploiting a single nucleotide difference in the two parasites' ITS2 sequence regions, we have developed two specific hydrolysis probes labeled with FAM™ or HEX™ fluorophores, which can not only distinguish between the DNA sequences of the two, but also quantify them in mixed DNA samples. A third, newly developed universal probe was also tested along the genus-specific probes to provide a robust and accurate reference. It was evident that the universal probe displayed congruent results to those obtained by the genus-specific probes when used with DNA from both parasites in a single sample. All in all, the results of our assay suggest that this novel protocol could be used to distinguish and quantify cattle parasites belonging to the two most important genera (i.e., Cooperia and Ostertagia) in a single mixed DNA sample.

Exploring benzimidazole resistance in Haemonchus contortus by next generation sequencing and droplet digital PCR.

Anthelmintic resistance in gastrointestinal nematode (GIN) parasites of grazing ruminants is on the rise in countries across the world. Haemonchus contortus is one of most frequently encountered drug-resistant GINs in small ruminants. This blood-sucking abomasal nematode contributes to massive treatment costs and poses a serious threat to farm animal health. To prevent the establishment of resistant strains of this parasite, up-to-date molecular techniques need to be proposed which would allow for quick, cheap and accurate identification of individuals infected with resistant worms. The effort has been made in the previous decade, with the development of the pyrosequencing method to detect resistance-predicting alleles. Here we propose a novel droplet digital PCR (ddPCR) assay for rapid and precise identification of H. contortus strains as being resistant or susceptible to benzimidazole drugs based on the presence or absence of the most common resistance-conferring mutation F200Y (TAC) in the ß tubulin isotype 1 gene. The newly developed ddPCR assay was first optimized and validated utilizing DNA templates from single-worm samples, which were previously sequenced using the next generation PacBio RSII Sequencing (NGS) platform. Subsequent NGS results for faecal larval cultures were then used as a reference to compare the obtained values for fractional abundances of the resistance-determining mutant allele between ddPCR and NGS techniques in each sample. Both methods managed to produce highly similar results and ddPCR proved to be a reliable tool which, when utilized at full capacity, can be used to create a powerful mutation detection and quantification assay.

Characterization of a lytic cyanophage that infects the bloom-forming cyanobacterium Aphanizomenon flos-aquae.

Vb-AphaS-CL131 is a novel cyanosiphovirus that infects harmful Aphanizomenon flos-aquae. This cyanophage has an isometric head, 97 nm in diameter and a long, flexible non-contractile tail, 361 nm long. With a genome size of ~120 kb, it is the second largest cyanosiphovirus isolated to date. The latent period was estimated to be ~36 h and a single infected cell produces, on average, 218 infectious cyanophages. Cyanophage infection significantly suppresses host biomass production and alters population phenotype.