Development of two species-specific primer sets to detect the cereal cyst nematodes Heterodera avenae and Heterodera filipjevi

Twelve Heterodera species are of major economic significance in wheat and barley. Of these, H. avenae, H. filipjevi and H. latipons are among the most important ones, and sometimes coexist. The identification of Heterodera species using morphological characteristics is time consuming, requires specialized skill and can be imprecise, especially when they occur mixed in field populations. Molecular techniques can provide a more accurate way for nematode identification. This study reports the results of experiments targeting the mitochondrial cytochrome oxidase subunit 1 (COI) gene to develop species-specific primers that could be used for the identification of H. avenae and H. filipjevi. The COI gene of 9 Heterodera spp. and Punctodera punctata was partially sequenced and the resultant sequences were aligned to find unique sites suitable for the design of primers. The alignment showed variability between H. avenae, H. filipjevi and other Heterodera species. Two sets of species-specific primers were identified for the identification of both species and the conditions for their use in PCR were optimised. The specificity of the designed primers was checked by comparison with one population of P. punctata and populations of 14 other Heterodera species, nine populations of H. avenae and 10 populations of H. filipjevi originating from different countries. To test the sensitivity, the PCR was run with DNA extracted from five second-stage juveniles (J2) of H. avenae or five J2 of H. filipjevi mixed with DNA extracted from varying numbers of J2 of H. latipons. It was possible to detect as few as five J2 of H. avenae or H. filipjevi among 100 J2 of H. latipons. The two primers sets allow the detection of H. avenae and H. filipjevi where they occur in mixed populations with other Heterodera spp.     

New sources of resistance to Meloidogyne incognita race 3 in wild cotton accessions and histological characterization of the defence mechanisms

Accessions of Gossypium spp., some of them never previously tested, were evaluated for resistance to a local isolate of Meloidogyne incognita race 3 in greenhouse experiments. Nematode infection was characterized based on the galling and egg mass indexes and the reproduction factors (RF). Root‐knot nematode reproduction among the newly tested accessions varied from highly susceptible (AS0188, AS0189) to moderately resistant (MT123 no. 3), and some accessions showed highly reduced nematode reproduction (CIR1343, CIR1348, Fai Mui). Histological observations of two resistant accessions (G. barbadense CIR1348 and G. hirsutum TX‐25, respectively) showed that resistance occurs through a two‐stage mechanism in the first accession and through a single‐stage mechanism in the second. Parasitism is blocked early after second‐stage juvenile (J2) penetration or during its initial tissue migration (CIR1348) and the development of later‐stage juveniles into female adults is suppressed at a later stage (TX‐25 and CIR1348). Fluorescence and bright light microscopy showed that root cells surrounding nematodes exhibit a hypersensitivity‐like reaction, with the accumulation of presumably phenolic compounds and the presence of necrotic cells that limit the development of nematodes and the formation of giant cells. Underdeveloped giant cells with degenerated cytoplasmic content were found in small numbers in CIR1348 and in large numbers in TX‐25, along with deformed nematodes. The full characterization of the defence mechanisms of novel sources of resistance to the root‐knot nematode in cotton constitutes a first step towards their use in crop improvement.     

Carbon flow into microbial and fungal biomass as a basis for the belowground food web of agroecosystems

The origin and quantity of plant inputs to soil are primary factors controlling the size and structure of the soil microbial community. The present study aimed to elucidate and quantify the carbon (C) flow from both root and shoot litter residues into soil organic, extractable, microbial and fungal C pools. Using the shift in C stable isotope values associated with replacing C3 by C4 plants we followed root- vs. shoot litter-derived C resources into different soil C pools. We established the following treatments: Corn Maize (CM), Fodder Maize (FM), Wheat + maize Litter (WL) and Wheat (W) as reference. The Corn Maize treatment provided root- as well as shoot litter-derived C (without corn cobs) whereas Fodder Maize (FM) provided only root-derived C (aboveground shoot material was removed). Maize shoot litter was applied on the Wheat + maize Litter (WL) plots to trace the incorporation of C4 litter C into soil microorganisms. Soil samples were taken three times per year (summer, autumn, winter) over two growing seasons. Maize-derived C signal was detectable after three to six months in the following pools: soil organic C (C_org), extractable organic C (EOC), microbial biomass (C_mic) and fungal biomass (ergosterol). In spite of the lower amounts of root- than of shoot litter-derived C inputs, similar amounts were incorporated into each of the C pools in the FM and WL treatments, indicating greater importance of the root- than shoot litter-derived resources for the soil microorganisms as a basis for the belowground food web. In the CM plots twice as much maize-derived C was incorporated into the pools. After two years, maize-derived C in the CM treatment contributed 14.1, 24.7, 46.6 and 76.2% to C_org, EOC, C_mic and ergosterol pools, respectively. Fungi incorporated maize-derived C to a greater extent than did total soil microbial biomass.     

Resistance testing and the effectiveness of difenacoum against Norway rats (Rattus norvegicus) in a tyrosine139cysteine focus of anticoagulant resistance,Westphalia, Germany

BACKGROUND: Anticoagulant resistance in Norway rats at foci in Belgium, Denmark, France, Germany, the Netherlands and the United Kingdom is genetically characterised by the same single nucleotide polymorphism (SNP) and consequent amino acid exchange from tyrosine to cysteine at location 139 of the vkorc1 gene (i.e. tyrosine139cysteine or Y139C). The purpose of this study was to assess the degree of resistance among rats at two infested farm sites in the Y139C focus in Westphalia, Germany, using blood clotting response (BCR) tests, and to determine the practical efficacy of applications of a commercial 50 ppm difenacoum bait (Neokil^?) against them. RESULTS: BCR tests showed that the difenacoum resistance factor (RF) among the Y139C rats was about 2.5. DNA analysis for the Y139C mutation revealed that it was present among rats at the two sites with a prevalence of 75 and 93%. Applications of difenacoum bait at the two sites achieved 86.8 and 59.9% control. The different outcomes did not appear to be due to differences either in the degree and prevalence of resistance or in the quantities of poisoned bait consumed. CONCLUSION: The study showed that, although the RF for difenacoum among rats carrying the Y139C SNP was apparently low, an acceptable level of control of resistant Norway rat infestations was not achieved using difenacoum. Continued use of anticoagulants against rats that are resistant to them will exacerbate resistance problems in terms of both increased severity and prevalence. These conclusions are likely to apply elsewhere in Europe where the Y139C SNP occurs. Copyright © 2012 Society of Chemical Industry     

Spatiotemporal dynamics and risk factors of rotavirus A circulation in backyard pig farms in a Philippine setting

Rotavirus A is one of the leading etiological agents of porcine gastroenteritis, a condition which results to stunted growth among piglets. Moreover, there is increasing evidence for zoonosis of rotavirus A (RVA), which is also the principal cause of diarrhea in children. In the absence of rigorous animal health monitoring in Philippine backyard farms, there is therefore a strong need for RVA surveillance. In this study, 30 randomly selected backyard farms were subjected to surveillance for RVA for 12 months. Results show that RVA detection at a monthly farm-level rate ranged from 0 to 52%, with an overall average of 23%. RVA had higher detection rates in adult pigs compared to young piglets and was most prevalent in non-diarrheic stools, indicating asymptomatic circulation of the virus. Spatiotemporal analysis demonstrated that the viral circulation exhibits a seasonal pattern that peaks and forms geographical clusters during the cooler months of the year, suggesting farm-to-farm transmission. Risk factor analysis identified specific farm conditions that increase the likelihood of RVA circulation: presence of gilts, larger herd size, presence of other animals, and abiotic factors such as low relative humidity and low altitude. The same analysis also revealed three major management practices that can help reduce the pressure of infection in these farms: sanitation and waste disposal, animal grouping, and diet. This new perspective on porcine RVA circulation will benefit the underprivileged backyard farmers and help empower them to protect both animal and public health.

     

Combining anti‐IgM and IgG immunoassays for comprehensive chikungunya virus diagnostic testing

Chikungunya virus (CHIKV) is a mosquito‐borne pathogen that causes CHIKV fever. Definitive diagnosis is crucial for patients experiencing symptoms similar to other arboviral diseases because they can vary in clinical consequences. An increasing number of patients experience long‐term rheumatic effects of CHIKV infection, but these cases may not be optimally detected by molecular assays and anti‐CHIKV IgM ELISAs (M‐ELISAs) used for confirmation and screening, respectively. The subsequent confirmatory serological test, the plaque reduction neutralization test (PRNT), is laborious and time‐consuming. In this study, we evaluated a new diagnostic algorithm in which the M‐ELISA is conducted in parallel with an anti‐CHIKV IgG ELISA (G‐ELISA) and observed that the Euroimmun M‐ELISA combined with the Euroimmun G‐ELISA or the Abcam G‐ELISA exhibited excellent sensitivity and specificity for CHIKV. The combinations demonstrated perfect and near perfect inter‐rater agreement with the PRNT, respectively, suggesting their potential to be used as alternatives to the confirmatory serological PRNT assay for CHIKV.     

The devil is in the details—Host disease and co‐infections are associated with zoonotic pathogen carriage in Norway rats (Rattus norvegicus)

Traditionally, zoonotic pathogen ecology studies in wildlife have focused on the interplay among hosts, their demographic characteristics and their pathogens. But pathogen ecology is also influenced by factors that traverse the hierarchical scale of biological organization, ranging from within‐host factors at the molecular, cellular and organ levels, all the way to the host population within a larger environment. The influence of host disease and co‐infections on zoonotic pathogen carriage in hosts is important because these factors may be key to a more holistic understanding of pathogen ecology in wildlife hosts, which are a major source of emerging infectious diseases in humans. Using wild Norway rats (Rattus norvegicus) as a model species, the purpose of this study was to investigate how host disease and co‐infections impact the carriage of zoonotic pathogens. Following a systematic trap and removal study, we tested the rats for the presence of two potentially zoonotic bacterial pathogens (Bartonella tribocorum and Leptospira interrogans) and assessed them for host disease not attributable to these bacteria (i.e., nematode parasites, and macroscopic and microscopic lesions). We fitted multilevel multivariable logistic regression models with pathogen status as the outcome, lesions and parasites as predictor variables and city block as a random effect. Rats had significantly increased odds of being infected with B. tribocorum if they had a concurrent nematode infection in one or more organ systems. Rats with bite wounds, any macroscopic lesion, cardiomyopathy or tracheitis had significantly increased odds of being infected with L. interrogans. These results suggest that host disease may have an important role in the ecology and epidemiology of rat‐associated zoonotic pathogens. Our multiscale approach to assessing complex intrahost factors in relation to zoonotic pathogen carriage may be applicable to future studies in rats and other wildlife hosts.