Prediction of Potato Tuber Damage by Root-Knot Nematodes using Quantitative DNA Assay of Soil.

Root-knot nematodes (Meloidogyne fallax and M. hapla) cause significant reductions in potato yield by reducing tuber quality. Concentrations of M. fallax and M. hapla DNA in soil were determined by quantitative polymerase chain reaction following sampling at planting and harvest within 78 fields across 3 years in Australia. Meloidogyne spp. were also detected using a tomato bioassay. M. fallax was more prevalent than M. hapla and DNA concentrations of M. fallax in soil were significantly higher in samples collected at harvest compared with those at planting. In contrast, M. hapla DNA in soil did not significantly change from planting to harvest. Using receiver operating characteristic curve analysis, M. fallax DNA in soil at planting and harvest was a highly accurate predictor of tuber damage at harvest and galling on tomato. Prediction accuracy for tuber damage was highest for M. fallax DNA compared with M. hapla or M. fallax + M. hapla. Both Meloidogyne spp. were detected in the peel of asymptomatic certified seed. For M. fallax, the addition of seedborne inoculum did not improve tuber damage predictions. This suggested that soilborne M. fallax populations contributed most substantially to tuber damage. These findings highlight the utility of this approach for predicting risk of crop damage from nematodes. The use of this technique as a practical management tool is discussed.

Influence of dietary docosahexaenoic acid supplementation on the overall rumen microbiota of dairy cows and linkages with production parameters.

The rumen microbiota contributes to greenhouse gas emissions and has an impact on feed efficiency and ruminant product fatty acid composition. Dietary fat supplements have shown promise in reducing enteric methane production and in altering the fatty acid profiles of ruminant-derived products, yet in vivo studies on how these impact the rumen microbiota are limited. In this study, we investigated the rumen bacterial, archaeal, fungal, and ciliate protozoan communities of dairy cows fed diets supplemented with 4 levels of docosahexaenoic acid (DHA) (0, 25, 50, and 75 g·cow(-1)·day(-1)) and established linkages between microbial communities and production parameters. Supplementation with DHA significantly (P < 0.05) altered rumen bacterial and archaeal, including methanogenic archaeal, communities but had no significant (P > 0.05) effects on rumen fungal or ciliate protozoan communities. Rumen bacterial communities of cows receiving no DHA were correlated with increased saturated fatty acids (C18:0 and C11:0) in their milk. Furthermore, rumen bacterial communities of cows receiving a diet supplemented with 50 g DHA·cow(-1)·day(-1) were correlated with increases in monounsaturated fatty acids (C20:1n-9) and polyunsaturated fatty acids (C22:5n-3; C22:6n-3; C18:2 cis-9, trans-11; C22:3n-6; and C18:2n-6 trans) in their milk. The significant diet-associated changes in rumen archaeal communities observed did not result in altered enteric methane outputs in these cows.

Investigating Dirofilaria immitis isolates infecting domestic canines and their susceptibility/resistance patterns to macrocyclic lactones in the northern region of the Mississippi Delta area (southeast Missouri).

Previous reports of macrocyclic lactone (ML) resistance in Dirofilaria immitis, the parasitic nematode which causes heartworm disease, have mainly been from the southern Mississippi Delta region. Southeast Missouri (SEMO), forming the northern boundary of this region, has not previously been well studied. The area is an ideal propagation region for heartworm infection and possibly for the spread of ML resistance. To assess whether D. immitis isolates infecting domestic canines in SEMO exhibit evidence of resistance to MLs, domestic canines, presented to veterinary facilities testing positive for heartworms through antigen and microfilariae (MF) examination, were utilized in the study. Using a descriptive epidemiological cross-sectional study, from March 2021 through February 2022, blood sample collection from 96 canines living in SEMO testing positive for heartworms were analyzed. MiSeq technology was utilized to sequence specific genetic markers associated with susceptibility/resistance for MLs in D. immitis isolates. Genomic data revealed most D. immitis isolates had genotypic profiles consistent with resistance to MLs. Of the 96 samples tested, 91 (94.8%) had a resistant genotype, 4 (4.2%) had a mixed genotype, and 1 sample (1%) genotyped as susceptible. While detailed and reliable medical histories were not available for most canines, detailed medical history from 2 canines indicated evidence of phenotypic resistance that was consistent with their genotypes. However, in vivo preventive tests are needed to confirm a high frequency of phenotypic ML resistance in D. immitis from this region. Increasing resistance patterns to MLs indicate the approach to heartworm prevention/treatment protocol should be reconsidered. New measures may be required to stop heartworm disease.

Predicting Take-All Severity in Second-Year Wheat Using Soil DNA Concentrations of Gaeumannomyces graminis var. tritici Determined with qPCR.

The lack of accurate detection of Gaeumannomyces graminis var. tritici inoculum in soil has hampered efforts to predict the risk of severe take-all for wheat growers. The current study used a molecular method to quantify soil G. graminis var. tritici concentrations in commercial wheat fields in New Zealand and to compare them with the proportion of crops surpassing the thresholds for visible and moderate to severe take-all over three growing seasons. The study evaluated a soil G. graminis var. tritici DNA-based take-all prediction system developed in Australia, with four take-all risk categories. These categories were found to be useful for predicting disease severity in second wheat but did not clearly separate risk between fields in medium- and high-risk categories. A sigmoidal relationship was identified between inoculum concentration and the proportion of fields exceeding the two disease thresholds. A logistic response curve was used to further examine this relationship and evaluate the boundaries between take-all risk categories. G. graminis var. tritici boundaries between medium- and high-risk categories were clustered near or within the upper plateau of the relationship. Alternative G. graminis var. tritici boundaries for a three-category system were identified that provided better separation of take-all risk between categories. This information could improve prediction of the risk of severe take-all.

Influence of antimicrobial feed additives on broiler commensal posthatch gut microbiota development and performance.

The effects of avilamycin, zinc bacitracin, and flavophospholipol on broiler gut microbial community colonization and bird performance in the first 17 days posthatch were investigated. Significant differences in gut microbiota associated with gut section, dietary treatment, and age were identified by terminal restriction fragment length polymorphism (T-RFLP), although no performance-related differences between dietary treatments were detected. Similar age-related shifts in the gut microbiota were identified regardless of diet but varied between the ilea and ceca. Interbird variabilities in ileal bacterial communities were reduced (3 to 7 days posthatch) in chicks fed with feed containing antimicrobial agents. Avilamycin and flavophospholipol had the most consistent effect on gut microbial communities. Operational taxonomic units (OTU) linked to changes in gut microbiota in birds on antimicrobial-supplemented diets were characterized and identified. Some OTUs could be identified to the species level; however, the majority could be only tentatively classified to the genus, family, order, or domain level. OTUs 140 to 146 (Lachnospiraceae), OTU 186/188 (Lactobacillus johnsonii), OTU 220 (Lachnospiraceae), OTUs 284 to 288 (unclassified bacterial spp. or Ruminococcaceae), OTU 296/298 (unclassified bacterium or Clostridiales), and OTU 480/482 (Oxalobacteraceae) were less prevalent in the guts of chicks fed antimicrobial-supplemented diets. OTU 178/180 (Lactobacillus crispatus), OTU 152 (Lactobacillus reuteri or unclassified Clostridiales), OTU 198/200 (Subdoligranulum spp.), and OTU 490/492 (unclassified bacterium or Enterobacteriaceae) were less prevalent in the gut of chicks raised on the antimicrobial-free diet. The identification of key bacterial species influenced by antimicrobial-supplemented feed immediately posthatch may assist in the formulation of diets that facilitate beneficial gut microbial colonization and, hence, the development of alternatives to current antimicrobial agents in feed for sustainable poultry production.

Identification and characterization of potential performance-related gut microbiotas in broiler chickens across various feeding trials.

Three broiler feeding trials were investigated in order to identify gut bacteria consistently linked with improvements in bird performance as measured by feed efficiency. Trials were done in various geographic locations and varied in diet composition, broiler breed, and bird age. Gut microbial communities were investigated using microbial profiling. Eight common performance-linked operational taxonomic units (OTUs) were identified within both the ilea (180, 492, and 564-566) and ceca (140-142, 218-220, 284-286, 312, and 482) across trials. OTU 564-566 was associated with lower performance, while OTUs 140-142, 482, and 492 were associated with improved performance. Targeted cloning and sequencing of these eight OTUs revealed that they represented 26 bacterial species or phylotypes which clustered phylogenetically into seven groups related to Lactobacillus spp., Ruminococcaceae, Clostridiales, Gammaproteobacteria, Bacteroidales, Clostridiales/Lachnospiraceae, and unclassified bacteria/clostridia. Where bacteria were identifiable to the phylum level, they belonged predominantly to the Firmicutes, with Bacteroidetes and Proteobacteria also identified. Some of the potential performance-related phylotypes showed high sequence identity with classified bacteria (Lactobacillus salivarius, Lactobacillus aviarius, Lactobacillus crispatus, Faecalibacterium prausnitzii, Escherichia coli, Gallibacterium anatis, Clostridium lactatifermentans, Ruminococcus torques, Bacteroides vulgatus, and Alistipes finegoldii). The 16S rRNA gene sequence information generated will allow quantitative assays to be developed which will enable elucidations of which of these phylotypes are truly performance related. This information could be used to monitor strategies to improve feed efficiency and feed formulation for optimal gut health.

Application of methods for identifying broiler chicken gut bacterial species linked with increased energy metabolism.

A high-throughput microbial profiling tool based on terminal restriction fragment length polymorphism was developed to monitor the poultry gut microbiota in response to dietary manipulations. Gut microbial communities from the duodena, jejuna, ilea, and ceca of 48 birds fed either a barley control diet or barley diet supplemented with exogenous enzymes for degrading nonstarch polysaccharide were characterized by using multivariate statistical methods. Analysis of samples showed that gut microbial communities varied significantly among gut sections, except between the duodenum and jejunum. Significant diet-associated differences in gut microbial communities were detected within the ileum and cecum only. The dissimilarity in bacterial community composition between diets was 73 and 66% within the ileum and cecum, respectively. Operational taxonomic units, representing bacterial species or taxonomically related groups, contributing to diet-associated differences were identified. Several bacterial species contributed to differences between diet-related gut microbial community composition, with no individual bacterial species contributing more than 1 to 5% of the total. Using canonical analysis of principal coordinates biplots, we correlated differences in gut microbial community composition within the ileum and cecum to improved performance, as measured by apparent metabolizable energy. This is the first report that directly links differences in the composition of the gut microbial community with improved performance, which implies that the presence of specific beneficial and/or absence of specific detrimental bacterial species may contribute to the improved performance in these birds.

Developing and testing a diagnostic probe for grape phylloxera applicable to soil samples.

Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera Phylloxeridae) is a damaging pest of grapevines (Vitis spp.) around the world, and the management of this pest requires early detection of infestations. Here, we describe the development and validation of a sensitive DNA test for grape phylloxera that can be applied to soil. Species-specific primers were developed for grape phylloxera in the internal transcribed space region 2, and their specificity was confirmed after thorough screening by using a wide range of vineyard organisms and aphid genera. Preliminary testing of the detection limits of the grape phylloxera-specific primers was conducted using field-sourced soil types spiked with a known number of grape phylloxera. The assay was converted to a real-time polymerase chain reaction format (TaqMan MGB). This assay, in combination with DNA extraction from soil, can detect phylloxera crawlers added to soil. The assay was evaluated in the field at a recently detected grape phylloxera infestation site from the Yarra Valley in Victoria, Australia. The DNA assay proved to be substantially more sensitive than a standard ground survey for detecting grape phylloxera presence on vine roots in the infested vineyard. Moreover, unlike the ground survey, the assay provided quantitative information on grape phylloxera infestations, because grape phylloxera DNA concentrations in samples from vines closely matched the numbers of grape phylloxera crawlers collected with emergence traps placed at the base of vines. Unlike other detection techniques, the method can be applied at any time of the year, and it can be potentially modified to provide specific information on the virulence levels of the particular grape phylloxera genotypes responsible for any new infestations.

Clinical validation of molecular markers of macrocyclic lactone resistance in Dirofilaria immitis.

Prophylaxis with macrocyclic lactone (ML) endectocides is the primary strategy for heartworm control. Recent evidence has confirmed that ML-resistant Dirofilaria immitis isolates have evolved. Comparison of genomes of ML-resistant isolates show they are genetically distinct from wild-type populations. Previously, we identified single nucleotide polymorphisms (SNPs) that are correlated with phenotypic ML resistance. Since reliable in vitro assays are not available to detect ML resistance in L3 or microfilarial stages, the failure to reduce microfilaraemia in infected dogs treated with an ML has been proposed as a surrogate clinical assay for this purpose. The goal of our study was to validate the genotype-phenotype correlation between SNPs associated with ML resistance and failure to reduce microfilaraemia following ML treatment and to identify a minimal number of SNPs that could be used to confirm ML resistance. In this study, 29 participating veterinary clinics received a total of 148 kits containing supplies for blood collection, dosing and prepaid shipping. Patients recruited after a diagnosis of heartworm infection were treated with a single standard dose of Advantage Multi® and a blood sample taken pre- and approximately 2-4 weeks post-treatment. Each sample was processed by performing a modified Knott's Test followed by isolation of microfilariae, genomic DNA extraction and MiSeq sequencing of regions encompassing 10 SNP sites highly correlated with ML resistance. We observed significant correlation of SNP loci frequencies with the ML microfilaricidal response phenotype. Although all predictive SNP combination models performed well, a 2-SNP model was superior to other models tested. The predictive ability of these markers for ML-resistant heartworms should be further evaluated in clinical and epidemiological contexts.

Genetic profiles of ten Dirofilaria immitis isolates susceptible or resistant to macrocyclic lactone heartworm preventives.

BACKGROUND: For dogs and cats, chemoprophylaxis with macrocyclic lactone (ML) preventives for heartworm disease is widely used in the United States and other countries. Since 2005, cases of loss of efficacy (LOE) of heartworm preventives have been reported in the U.S. More recently, ML-resistant D. immitis isolates were confirmed. Previous work identified 42 genetic markers that could predict ML response in individual samples. For field surveillance, it would be more appropriate to work on microfilarial pools from individual dogs with a smaller subset of genetic markers. METHODS: MiSeq technology was used to identify allele frequencies with the 42 genetic markers previously reported. Microfilaria from ten well-characterized new isolates called ZoeKY, ZoeMI, ZoeGCFL, ZoeAL, ZoeMP3, ZoeMO, ZoeAMAL, ZoeLA, ZoeJYD-34, and Metairie were extracted from fresh blood from dogs. DNA were extracted and sequenced with MiSeq technology. Allele frequencies were calculated and compared with the previously reported susceptible, LOE, and resistant D. immitis populations. RESULTS: The allele frequencies identified in the current resistant and susceptible isolates were in accordance with the allele frequencies previously reported in related phenotypes. The ZoeMO population, a subset of the ZoeJYD-34 population, showed a genetic profile that was consistent with some reversion towards susceptibility compared with the parental ZoeJYD-34 population. The Random Forest algorithm was used to create a predictive model using different SNPs. The model with a combination of three SNPs (NODE_42411_RC, NODE_21554_RC, and NODE_45689) appears to be suitable for future monitoring. CONCLUSIONS: MiSeq technology provided a suitable methodology to work with the microfilarial samples. The list of SNPs that showed good predictability for ML resistance was narrowed. Additional phenotypically well characterized D. immitis isolates are required to finalize the best set of SNPs to be used for large scale ML resistance screening.

ABC-B transporter genes in Dirofilaria immitis.

Dirofilaria immitis is a filarial nematode causing infection and heartworm disease in dogs and other canids, cats, and occasionally in humans. Prevention with macrocyclic lactones (ML) is recommended during the mosquito transmission season. Recently, ML resistance has been reported. ABC-B transporter genes are thought to be involved in the mechanism of ML resistance in other nematodes. This study aimed to identify all the ABC-B transporter genes in D. immitis using as a reference the nDi.2.2 D. immitis whole genome, which is not completely annotated. Using bioinformatic tools and PCR amplification on pooled D. immitis genomic DNA and on pooled cDNA, nine ABC transporter genes including one pseudogene were characterized. Bioinformatic and phylogenetic analyses allowed identification of three P-glycoproteins (Pgps) (Dim-pgp-3 Dim-pgp-10, Dim-pgp-11), of two ABC-B half transporter genes (one ortholog of Cel-haf-4 and Cel-haf-9; and one ortholog of Cel-haf-1 and Cel-haf-3), of one ABC half transporter gene (ortholog of Cel-haf-5) that contained an ABC-C motif, and of one additional half transporter that would require functional study for characterization. The number of ABC-B transporter genes identified was lower than in Caenorhabditis elegans and Haemonchus contortus. Further studies are needed to understand their possible role in ML resistance in D. immitis. These ABC transporters constitute a base for ML resistance investigation in D. immitis and advance our understanding of the molecular biology of this parasite.

Interaction of macrocyclic lactones with a Dirofilaria immitis P-glycoprotein.

Dirofilaria immitis, a filarial nematode, causes dirofilariasis or heartworm disease in dogs, cats and wild canids. Effective prevention of the disease is mainly by the use of the macrocyclic lactone class of drugs as heartworm preventives, and no other class of drugs is effective for preventing infection. Macrocyclic lactones have been used for prevention of heartworm infection for more than 26years. However, prevention has been compromised by the development of resistance in recent years. The mechanism of macrocyclic lactone resistance in D. immitis has yet to be established. In other parasitic nematodes, P-glycoproteins (PGPs) have been implicated in macrocyclic lactone resistance. The presence of two polymorphic loci on D. immitis P-glycoprotein-11 (Dim-pgp-11) correlated with loss of efficacy of macrocyclic lactone anthelmintics, suggesting that PGPs may be involved in macrocyclic lactone resistance in D. immitis. We have identified the full length of Dim-Pgp-11 cDNA, expressed it in mammalian cells, and studied the functional activity of the expressed protein. We have characterised its interaction with the four macrocyclic lactone preventives, ivermectin, selamectin, moxidectin and milbemycin oxime, using the transport of different fluorescent substrates. The inhibitory effect of these macrocyclic lactones on the transport of two fluorophore probes, Rhodamine 123 and Hoechst 33342, by Dim-PGP-11 has been studied. The avermectins, ivermectin and selamectin, markedly inhibited Rhodamine 123 transport in a concentration-dependent and saturable manner, whereas the milbemycins, moxidectin and milbemycin oxime, were found to have different inhibition profiles with Rhodamine 123 transport. However, both avermectins and milbemycin preventives inhibited the transport of Hoechst 33342 by Dim-PGP-11 in a concentration-dependent and apparently saturable manner, although differences existed in terms of efficiency and potency of inhibition between the two sub-classes of macrocyclic lactones. We postulate that Dim-PGP-11 may have two to three drug binding sites, as with mammalian Pgp, including the 'R' site for Rhodamine 123 and the 'H' site for Hoechst 33342. The avermectins appear to bind the 'R' binding site unlike the milbemycins, whereas both sub-classes of macrocyclic lactones might interact with the 'H' site of D. immitis PGP-11.

Polymorphism in ion channel genes of Dirofilaria immitis: Relevant knowledge for future anthelmintic drug design.

Dirofilaria immitis, a filarial parasite, causes cardiopulmonary dirofilariasis in dogs, cats and wild canids. The macrocyclic lactone (ML) class of drugs has been used to prevent heartworm infection. There is confirmed ML resistance in D. immitis and thus there is an urgent need to find new anthelmintics that could prevent and/or control the disease. Targeting ion channels of D. immitis for drug design has obvious advantages. These channels, present in the nematode nervous system, control movement, feeding, mating and respond to environmental cues which are necessary for survival of the parasite. Any new drug that targets these ion channels is likely to have a motility phenotype and should act to clear the worms from the host. Many of the successful anthelmintics in the past have targeted these ion channels and receptors. Knowledge about genetic variability of the ion channel and receptor genes should be useful information for drug design as receptor polymorphism may affect responses to a drug. Such information may also be useful for anticipation of possible resistance development. A total of 224 ion channel genes/subunits have been identified in the genome of D. immitis. Whole genome sequencing data of parasites from eight different geographical locations, four from ML-susceptible populations and the other four from ML-loss of efficacy (LOE) populations, were used for polymorphism analysis. We identified 1762 single nucleotide polymorphic (SNP) sites (1508 intronic and 126 exonic) in these 224 ion channel genes/subunits with an overall polymorphic rate of 0.18%. Of the SNPs found in the exon regions, 129 of them caused a non-synonymous type of polymorphism. Fourteen of the exonic SNPs caused a change in predicted secondary structure. A few of the SNPs identified may have an effect on gene expression, function of the protein and resistance selection processes.

Isothermal Diagnostic Assays for Monitoring Single Nucleotide Polymorphisms in Necator americanus Associated with Benzimidazole Drug Resistance.

BACKGROUND: Soil-transmitted helminths (STHs) are the most prevalent intestinal helminths of humans, and a major cause of morbidity in tropical and subtropical countries. The benzimidazole (BZ) drugs albendazole (ABZ) and mebendazole (MBZ) are used for treatment of human STH infections and this use is increasing dramatically with massive drug donations. Frequent and prolonged use of these drugs could lead to the emergence of anthelmintic resistance as has occurred in nematodes of livestock. Previous molecular assays for putative resistance mutations have been based mainly on PCR amplification and sequencing. However, these techniques are complicated and time consuming and not suitable for resource-constrained situations. A simple, rapid and sensitive genotyping method is required to monitor for possible developing resistance to BZ drugs. METHODS: To address this problem, single nucleotide polymorphism (SNP) detection assays were developed based on the Smart amplification method (SmartAmp2) to target codons 167, 198, and 200 in the ß-tubulin isotype 1 gene for the hookworm Necator americanus. FINDINGS: Diagnostic assays were developed and applied to analyze hookworm samples by both SmartAmp2 and conventional sequencing methods and the results showed high concordance. Additionally, fecal samples spiked with N. americanus larvae were assessed and the results showed that the Aac polymerase used has high tolerance to inhibitors in fecal samples. CONCLUSION: The N. americanus SmartAmp2 SNP detection assay is a new genotyping tool that is rapid, sensitive, highly specific and efficient with the potential to be used as a field tool for monitoring SNPs associated with BZ resistance. However, further validation on large numbers of field samples is required.

Macrocyclic lactone resistance in Dirofilaria immitis: Failure of heartworm preventives and investigation of genetic markers for resistance.

Macrocyclic lactone (ML) endectocides are used as chemoprophylaxis for heartworm infection (Dirofilaria immitis) in dogs and cats. Claims of loss of efficacy (LOE) of ML heartworm preventives have become common in some locations in the USA. We directly tested whether resistance to MLs exists in LOE isolates of D. immitis and identified genetic markers that are correlated with, and therefore can predict ML resistance. ML controlled studies showed that LOE strains of D. immitis established infections in dogs despite chemoprophylaxis with oral ivermectin or injectable moxidectin. A whole genome approach was used to search for loci associated with the resistance phenotype. Many loci showed highly significant differences between pools of susceptible and LOE D. immitis. Based on 186 potential marker loci, Sequenom(®) SNP frequency analyses were conducted on 663 individual parasites (adult worms and microfilariae) which were phenotypically characterized as susceptible (SUS), confirmed ML treatment survivors/resistant (RES), or suspected resistant/loss of efficacy (LOE) parasites. There was a subset of SNP loci which appears to be promising markers for predicting ML resistance, including SNPs in some genes that have been associated with ML resistance in other parasites. These data provide unequivocal proof of ML resistance in D. immitis and identify genetic markers that could be used to monitor for ML resistance in heartworms.

Pulmonary glial heterotopia in a monoamniotic twin.

Brain heterotopia in the lungs is rare, but when it occurs in an otherwise healthy newborn, it presents a difficult diagnostic problem and uncertain pathophysiology. We report on a 2-week-old premature infant who presented with respiratory distress and widespread cystic lung changes identified by chest imaging studies. Autopsy demonstrated that the cyst walls were composed of well-differentiated neuroglial tissue, which was confirmed by immunohistochemistry. The cysts were partially lined by bronchial epithelium and contained keratinous debris. For the first time, we demonstrate that the debris stain for human chorionic gonadotropin, compatible with aspirated amnion. There were no other congenital abnormalities. Her monoamniotic twin was anencephalic and died at birth. Although the etiology of glial heterotopia in the lungs is unknown, the majority of cases are associated with anencephalic newborns. Some authors postulated that this heterotopia may therefore be a consequence of fetal aspiration of brain tissue. Other possibilities include glial predominant teratomas, hamartomatous malformations, and abnormal neural crest migration. Our review of the 21 cases reported over the past century suggests that in utero aspiration of glial cells, or abnormal neural crest migration, are the most likely explanations for this rare and fatal disease.

Fungal community structure in disease suppressive soils assessed by 28S LSU gene sequencing.

Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. Soil microbe and phytopathogen interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soil-borne disease. Fungi were analyzed in co-located soils 'suppressive' or 'non-suppressive' for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. DNA was extracted from a minimum of 125 g of soil per replicate to reduce the micro-scale community variability, and from soil samples taken at sowing and from the rhizosphere at 7 weeks to cover the peak Rhizoctonia infection period. A total of ∼ 994,000 reads were classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Statistical analyses and community ordinations revealed significant differences in fungal community composition between suppressive and non-suppressive soil and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites such as Xylaria spp. Non-suppressive soils were dominated by Alternaria, Gibberella and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and identified candidate taxa that may influence pathogen-plant interactions in stable disease suppression.

Efficiency of a genetic test to detect benzimidazole resistant Haemonchus contortus nematodes in sheep farms in Quebec, Canada.

Haemonchus contortus is a hemophilic nematode which infects sheep and causes anemia and death to lambs. Benzimidazole drugs are used to remove these parasites, but the phenomenon of resistance has arisen worldwide. A sensitive test to detect resistance before treatment would be a useful tool to enable farmers to anticipate the efficiency of the drug before drenching the flock. In this study, we compared a test for benzimidazole resistance based on detection of genetic markers in H. contortus before treatment with the common method of fecal egg count reduction test (FECRT). We recruited 11 farms from different regions of Quebec for this study. Fecal samples from animals were collected per rectum before and after treatment in control and treated groups (10 animals per group). The 10 sheep were treated with fenbendazole at the recommended dose rate. Among the 11 farms participating in the study, we found H. contortus in 8 of them and it was the most predominant nematode species detected by egg count. Using the genetic test, we found benzimidazole resistance in each of these 8 farms. In 5 of these 8 farms there were sufficient sheep with an egg count for H. contortus above 150 eggs per gram to allow the FECRT test to be conducted. Benzimidazole resistance was observed in each of these 5 farms by the FECRT. When we compared the results from the genetic test for samples off pasture and from individual sheep, with the results from the FECRT, we concluded that the genetic test can be applied to samples collected off pasture to estimate benzimidazole resistance levels before treatment for H. contortus infections.

The role of several ABC transporter genes in ivermectin resistance in Caenorhabditis elegans.

The functions of nine ATP-binding cassette (ABC) transporter genes, mrp-1, mrp-4, mrp-6, pgp-2, pgp-3, pgp-4, pgp-5, haf-2 and haf-9, in an ivermectin (IVM) resistant strain of Caenorhabditis elegans were screened by comparing transcription levels between the resistant (IVR10) and wild-type (Bristol N2) strains, and by measuring the effects of RNA interference (RNAi) on the IVM resistant strain, on motility, pharyngeal pumping, egg production and death in the presence or varying concentrations of IVM (0-20 ng/ml). mRNA levels of mrp-1, 2, 4, 5, 6, 7, pgp-1, 2, 4, 12, 14, haf-1, 2 and 3 were significantly increased in IVR10 compared with the N2 strain. At 15 or 20 ng/ml IVM, down regulation of mrp-1, pgp-4, haf-2 and haf-9 significantly increased the effect of IVM to reduce egg production. At low to moderate IVM concentrations, down regulation of mrp-1 and haf-2 reduced the motility of C. elegans. However, at high IVM concentrations motility was increased by down regulation of transcription of pgp-3, pgp-4 and haf-9. Down regulation of expression of mrp-1, pgp-2 and pgp-5 resulted in reduced pharyngeal pumping in the presence of varying concentrations of IVM, while down regulation of mrp-6 and haf-2 increased pharyngeal pumping of the resistant strain irrespective of the IVM concentration used. Although the IVR10 strain was markedly resistant to IVM, compared with the unselected N2 strain, IVM led to the death of the C. elegans in a concentration dependent manner. However, differences in the IVM induced death rate, following RNAi, were not significantly different from the IVR10 strain without RNAi. The study shows that different ABC transporter genes may play a role in modulating the effects of IVM on pharyngeal pumping, motility and egg production, with down regulation of mrp-1 and haf-2 perhaps having the greatest effects. However, down regulation of expression of no individual ABC transporter gene profoundly affected the effect of IVM on mortality in the IVR10 strain. This suggests that some of these ABC transporter genes and their products may play a role in modulating the effects of IVM, but are not, individually, the critical gene responsible for IVM resistance. This study provides a model that may help to understand drug resistance in parasitic nematodes.