IgE,TNFα, IL1 β, IL4 and IFNγ gene polymorphisms in sheep selected for resistance to fleece rot and flystrike

Investigation of restriction fragment length polymorphisms (RFLPs) associated with immunoglobulin E (IgE) and cytokine genes in the sheep genome revealed polymorphisms in the IgE constant heavy chain, interferon γ and interleukin 4 genes. No polymorphisms were found in interleukin 1β or tumour necrosis factor α. PstI and BamHI RFLPs in the IgE gene showed differences in frequency between animals selected for resistance or susceptibility to fleece rot and blowfly strike.     

Transcriptional and Metabolic Changes Associated to the Infection by Fusarium verticillioides in Maize Inbreds with Contrasting Ear Rot Resistance

Fusarium verticillioides causes ear rot and grain mycotoxins in maize (Zea mays L.), which are harmful to human and animal health. Breeding and growing less susceptible plant genotypes is one alternative to reduce these detrimental effects. A better understanding of the resistance mechanisms would facilitate the implementation of strategic molecular agriculture to breeding of resistant germplasm. Our aim was to identify genes and metabolites that may be related to the Fusarium reaction in a resistant (L4637) and a susceptible (L4674) inbred. Gene expression data were obtained from microarray hybridizations in inoculated and non-inoculated kernels from both inbreds. Fungal inoculation did not produce considerable changes in gene expression and metabolites in L4637. Defense-related genes changed in L4674 kernels, responding specifically to the pathogen infection. These results indicate that L4637 resistance may be mainly due to constitutive defense mechanisms preventing fungal infection. These mechanisms seem to be poorly expressed in L4674; and despite the inoculation activate a defense response; this is not enough to prevent the disease progress in this susceptible line. Through this study, a global view of differential genes expressed and metabolites accumulated during resistance and susceptibility to F. verticillioides inoculation has been obtained, giving additional information about the mechanisms and pathways conferring resistance to this important disease in maize.     

Expression of genes in gastrointestinal and lymphatic tissues during parasite infection in sheep genetically resistant or susceptible to Trichostrongylus colubriformis and Haemonchus contortus

Resistance to an acute gastrointestinal nematode (GIN) infection is dependent on the ability of the host to recognise the parasite and mount a protective Th2 response. It is hypothesised that lambs which are genetically susceptible to GIN will differentially up-regulate Th1-type genes and therefore remain susceptible to chronic parasitism compared with genetically resistant lambs which will differentially up-regulate Th2-type genes and clear the parasite infection. Two selection flocks, in which lines of Merino sheep produced lambs genetically resistant or susceptible to GIN, were acutely challenged once or thrice with either Haemonchus contortus or Trichostrongylus colubriformis. Faecal-egg counts (FECs), and plasma and tissue anti-parasite (H. contortus or T. colubriformis) antibody isotype responses showed that resistant animals challenged three times with T. colubriformis established a protective Th2 response (negligible FEC, IgG1 and IgE) whereas susceptible animals required multiple challenges to establish a significant IgG1 response despite FECs remaining high. Trichostrongylus colubriformis elicited a more pronounced host response than H. contortus. RNA extracted from tissues at the site of each parasite infection and associated lymph nodes were interrogated by microarray and quantitative PCR analyses to correlate host gene expression to FECs and antibody responses. The IFN-γ inducible gene cxcl10 was up-regulated in the susceptible line of the Trichostrongylus selection flock sheep after a tertiary challenge with the parasites H. contortus and T. colubriformis. However, a uniform pattern of genes was not up-regulated in resistant animals from both selection flocks during both parasite infections, suggesting that the mode of host resistance to these parasites is different, although some similarities in host susceptibility were apparent.     

萝卜不同抗源对黑腐病抗性的遗传分析

不同抗病基因的挖掘是作物持久抗性遗传改良的基础。本研究利用2份抗黑腐病(Xanthamonas campestris pv.campestris)萝卜(Raphanus sativus L.)材料(KB10Q-22、KB10Q-24)和1份感病材料(KB10Q-33)构建了2个F2群体,采用苗期剪叶+喷雾法接种黑腐病菌Xcc8004进行抗病性鉴定。应用P1、P2、F1、F24个世代的数量性状主基因+多基因混合遗传分析方法,研究了萝卜2个不同抗源抗黑腐病的遗传规律,结果表明2份材料的遗传规律不同。以KB10Q-22为母本的F1植株表现为抗病,其遗传模型为E_0模型,即2对加性-显性-上位性主基因+加性-显性-上位性多基因模型;而以KB10Q-24为母本的F1植株表现为感病,其遗传模型为D_0模型,即1对加性-显性主基因+加性-显性-上位性多基因模型。两群体主基因遗传率分别为87.73%和55.64%,抗性遗传以主基因为主。     

Insect growth regulator cross-resistance studies in field- and laboratory-selected strains of the Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae)

Abstract Resistance to diflubenzuron in the Australian sheep blowfly, Lucilia cuprina , has rendered this insecticide incapable of preventing flystrike in sheep from a few districts in eastern Australia. Wool producers affected by this situation must find suitable alternatives to protect their flocks. Results of laboratory bioassays against one population demonstrated that, despite extremely high diflubenzuron resistance (Resistance Factor >791), it had only very low (2x) tolerance of cyromazine and dicyclanil. It is unlikely that this level of tolerance would have any practical impact on field control with either insecticide. Consequently, wool producers in districts where diflubenzuron-resistant flies are common can rotate insecticide treatment to either of these compounds to prevent flystrike in their flocks. However, unlike the highly diflubenzuron-resistant field strain, a laboratory strain selected for resistance to diflubenzuron (Resistance Factor = 617) was 10 times more resistant to dicyclanil than a susceptible strain but, like the field strain, was only two times more tolerant of cyromazine. Conversely, a field-derived strain selected in the laboratory for cyromazine resistance was 20 times more resistant to dicyclanil and 362 times more resistant to diflubenzuron than the reference susceptible strain.     

Efficacy of native and recombinant Cry1B protein against experimentally induced and naturally acquired ovine myiasis (fly strike) in sheep

Several hundred strains of Bacillus thuringiensis (Bt), isolated in New Zealand from samples of soil and sheep fleece, were tested for toxicity to larvae of the blowfly Lucilia cuprina Wiedemann. Characterization of the Bt strains revealed that three of the more active strains produced Cry1Ba (an insecticidal protein present in Bt mother cell crystal inclusion) that was toxic to blowflies. These strains were evaluated for the ability to prevent experimentally induced fly strike in a bioassay by using first instars. Results with undiluted spore/crystal preparations were variable, but they generally prevented fly strike on sheep maintained on pasture for 3-6 wk. Spore viability was satisfactory throughout the trials and environmental factors (e.g., precipitation and UV radiation) seemed to have minimal effect on persistence. The loss of fly strike protection in these experiments correlated with the movement of spore/crystal toxicity away from the skin as a result of wool growth. Solubilized protein preparations were not as potent as spore/crystal preparations and fly strike protection lasted only from 1 to 3 wk. Vegetative forms of the Cry1Ba-producing strains of Bt did not establish on the fleece of sheep, did not produce significant sporulation, and no protection against fly strike was achieved. Escherichia coli expressing recombinant Cry1Ba protein was toxic to larvae in vitro but did not effectively protect sheep from fly strike because blowfly larvae were able to establish readily 8 d posttreatment. In a single field experiment involving 80 sheep per group, a spore/crystal preparation from a Bt strain expressing Cry1Ba provided less protection from naturally acquired fly strike than afforded by a commercially available dip.     

Transformation and mineralization of soil organic nitrogen by the humivorous larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

In common bean (Phaseolus vulgaris L.), Fusarium root rot (caused by Fusarium solani f. sp. phaseoli) disease severity is increased by environmental factors that stress the plant. The current study used reciprocal grafting techniques with the resistant cultivar FR266 and the susceptible cultivar Montcalm to determine if the genetic control of resistance is conferred by the rootstock (root genotype) or the scion (shoot genotype) and if root vigor played a role in resistance. The influence of a compacted layer on root and shoot genotype response and root rot resistance was studied. Root rot resistance was found to be controlled by the root genotype, such that on a scale of 1 to 7 (severe disease) the FR266 root had an average score of 2.3 and the Montcalm root had an average score of 4.4. However, when grafted plants were grown in the presence of a compacted layer, the FR266 root and/or shoot genotype in any graft combination with the susceptible Montcalm had reduced root rot (score = 2.4 average) than the Montcalm self graft (score = 4.5). Root mass was shown to be controlled by the root genotype in the absence of compaction such that the FR266 root was 26% larger that the Montcalm root when grafted onto a FR266 shoot or a Montcalm shoot. When a compacted layer was present the root and shoot genotype both contributed to root mass. Average root diameter was controlled by the shoot genotype, as the FR266 shoot grafted to Montcalm or FR266 roots had thicker roots (average diameter 0.455 mm) than the Montcalm shoot (average diameter 0.418 mm). This study shows evidence that root vigor in the presence of Fusarium disease pressure should be evaluated to effectively develop common bean lines resistant to Fusarium root rot across a range of environments.     

THE OLFACTORY BEHAVIOUR OF LUCILIA SPECIES (DIPTERA) UNDER NATURAL CONDITIONS

Using an apparatus of the 'two-choice' type under field conditions, it has been demonstrated that a wool-factor forms an important part of the mechanism whereby female Lucilia sericata may be attracted to sheep. The 'wool-factor' could not wholly be replaced by ammonium- or sulphydryl-type attractants.
L. sericata was strongly attracted by the combination of ammonium carbonate/indole with sheep wool but less strongly attracted when ethyl mercaptan/hydrogen sulphide was used with sheep wool. L. caesar and L. illustris , on the other hand, were not markedly responsive to the ammonium-type attractants but were strongly attracted by the sulphydryl mixture used either alone or in combination with sheep wool.
Temperature is an important element in the oviposition stimulus. The amount of oviposition by L. sericata was increased when substances known to induce oviposition were exposed at 30–40† C.
Sheep wool kept moist and incubated at approx. 38† C. under field conditions for 3 weeks did not attract L. sericata and oviposition did not occur on such wool. This suggests that bacteria normally present on the wool fibre as distinct from the skin, do not play an important part in rendering sheep susceptible to blowfly attack.     

The effect of host nutrition on itch mite, Psorergates ovis, populations and fleece derangement in sheep

Abstract. A group of thirty-two Merino sheep infested with itch mites (Psorergates ovis) and fed a maintenance diet which imposed moderate nutritional stress had a significantly higher mite population, significantly more skin scurf, and significantly more fleece damage or derangement (P < 0.05) than a second group of thirty-two infested sheep fed a diet designed for unrestricted body weight gain and wool growth. Histologically there were no significant differences between the groups in the numbers of mast cells, neutrophils or eosinophils observed in skin sections, but sheep that had high mite counts (>10 per 200 cm² of skin area) in both groups, had more dermal mast cells than sheep with fewer mites irrespective of the plane of nutrition. Skin thickness and greasy fleece weight in die group maintained on the low plane of nutrition were significantly less (P < 0.05) than in die well-nourished group, reflecting the difference in protein and energy content of the two diets. Within the nutritionally stressed group, the sheep with low mite counts had a significantly lower (P < 0.05) greasy fleece weight and a shorter mean staple length than the sheep with high mite counts. There was no significant difference in greasy fleece weight between sheep with low or high mite counts in the group fed on the high plane of nutrition.     

Novel Pathways Revealed in Bursa of Fabricius Transcriptome in Response to Extraintestinal Pathogenic Escherichia coli (ExPEC) Infection

Extraintestinal pathogenic Escherichia coli (ExPEC) has major negative impacts on human and animal health. Recent research suggests food-borne links between human and animal ExPEC diseases with particular concern for poultry contaminated with avian pathogenic E. coli (APEC), the avian ExPEC. APEC is also a very important animal pathogen, causing colibacillosis, one of the world’s most widespread bacterial diseases of poultry. Previous studies showed marked atrophy and lymphocytes depletion in the bursa during APEC infection. Thus, a more comprehensive understanding of the avian bursa response to APEC infection will facilitate genetic selection for disease resistance. Four-week-old commercial male broiler chickens were infected with APEC O1 or given saline as a control. Bursas were collected at 1 and 5 days post-infection (dpi). Based on lesion scores of liver, pericardium and air sacs, infected birds were classified as having mild or severe pathology, representing resistant and susceptible phenotypes, respectively. Twenty-two individual bursa RNA libraries were sequenced, each yielding an average of 27 million single-end, 100-bp reads. There were 2469 novel genes in the total of 16,603 detected. Large numbers of significantly differentially expressed (DE) genes were detected when comparing susceptible and resistant birds at 5 dpi, susceptible and non-infected birds at 5 dpi, and susceptible birds at 5 dpi and 1 dpi. The DE genes were associated with signal transduction, the immune response, cell growth and cell death pathways. These data provide considerable insight into potential mechanisms of resistance to ExPEC infection, thus paving the way to develop strategies for ExPEC prevention and treatment, as well as enhancing innate resistance by genetic selection in animals.     

Using Drosophila melanogaster to validate metabolism-based insecticide resistance from insect pests

Identifying molecular mechanisms of insecticide resistance is important for preserving insecticide efficacy, developing new insecticides and implementing insect control. The metabolic detoxification of insecticides is a widespread resistance mechanism. Enzymes with the potential to detoxify insecticides are commonly encoded by members of the large cytochrome P450, glutathione S-transferase and carboxylesterase gene families, all rapidly evolving in insects. Here, we demonstrate that the model insect Drosophila melanogaster is useful for functionally validating the role of metabolic enzymes in conferring metabolism-based insecticide resistance. Alleles of three well-characterized genes from different pest insects were expressed in transgenic D. melanogaster : a carboxylesterase gene (αE7) from the Australian sheep blowfly Lucilia cuprina, a glutathione S-transferase gene (GstE2) from the mosquito Anopheles gambiae and a cytochrome P450 gene (Cyp6cm1) from the whitefly Bemisia tabaci. For all genes, expression in D. melanogaster resulted in insecticide resistance phenotypes mirroring those observed in resistant populations of the pest species. Using D. melanogaster to assess the potential for novel metabolic resistance mechanisms to evolve in pest species is discussed.     

Identification of potential functional variants underlying ovine resistance to gastrointestinal nematode infection by using RNA-Seq

In dairy sheep flocks from Mediterranean countries, replacement and adult ewes are the animals most affected by gastrointestinal nematode (GIN) infections. In this study, we have exploited the information derived from an RNA-Seq experiment with the aim of identifying potential causal mutations related to GIN resistance in sheep. Considering the RNA-Seq samples from 12 ewes previously classified as six resistant and six susceptible animals to experimental infection by Teladorsagia circumcincta, we performed a variant calling analysis pipeline using two different types of software, gatk version 3.7 and Samtools version 1.4. The variants commonly identified by the two packages (high-quality variants) within two types of target regions – (i) QTL regions previously reported in sheep for parasite resistance based on SNP-chip or sequencing technology studies and (ii) functional candidate genes selected from gene expression studies related to GIN resistance in sheep – were further characterised to identify mutations with a potential functional impact. Among the genes harbouring these potential functional variants (930 and 553 respectively for the two types of regions), we identified 111 immune-related genes in the QTL regions and 132 immune-related genes from the initially selected candidate genes. For these immune-related genes harbouring potential functional variants, the enrichment analyses performed highlighted significant GO terms related to apoptosis, adhesion and inflammatory response, in relation to the QTL related variants, and significant disease-related terms such as inflammation, adhesion and necrosis, in relation to the initial candidate gene list. Overall, the study provides a valuable list of potential causal mutations that could be considered as candidate causal mutations in relation to GIN resistance in sheep. Future studies should assess the role of these suggested mutations with the aim of identifying genetic markers that could be directly implemented in sheep breeding programmes considering not only production traits, but also functional traits such as resistance to GIN infections.     

Gastrointestinal nematode challenge induces some conserved gene expression changes in the gut mucosa of genetically resistant sheep

Sheep have a varying ability to resist infection with gastrointestinal nematodes. This ability is due in part to genetic differences that exist between individuals. In order to define these differences we have used real-time PCR to quantify gene expression responses in the gut mucosal surface of genetically resistant and susceptible sheep, following a nematode challenge. Expression profiles were determined in response to two different nematode species, Haemonchus contortus and Trichostrongylus colubriformis, and in divergent sheep originating from two different genetic backgrounds. Results show that the response generated differs between resistant and susceptible animals and is further impacted by the origin of the sheep and nematode species used for challenge. However, some conserved features of a response mounted by a resistant or a susceptible animal were identified. Genes found to be more abundantly expressed in resistant animals include markers of an early inflammatory response, several Toll-like receptors (TLR2, 4, 9) and free radical producing genes (DUOX1 and NOS2A). Conversely, genes differentiating susceptible animals indicate a prolonged response and development of a chronic inflammatory state, characterised by elevated expression of members of the NF-kappabeta signalling pathway (IKBKB and NFKBIA) together with delayed expression of regulatory markers such as IL2RA (CD25), IL10 and TGFbeta2. While multiple nematode response pathways were identified, the identification of conserved aspects of the response which associate with resistance provides evidence that alternative nematode control strategies, such as breeding for resistant animals, may be feasible.     

Role of Pseudomonas fluorescens and INA against Black Rot of Cabbage

Cabbage (Brassica oleracea var. capitata) is an important vegetable crop among crucifers. It is affected by a bacterial disease known as black rot. Black rot is caused by Xanthomonas campestris pv. campestris a disease of worldwide importance. The present study highlights the effect of biotic inducer—Pseudomonas fluorescens—and an abiotic inducer—2,6‐dichloro‐isonicotinic acid—in combating black rot, followed by their effect on the seed treatment and disease incidence, role of antioxidant enzymes followed by validation of the defence‐related genes by quantitative real‐time PCR. The resistant (Pusa mukta) and the highly susceptible (NBH boss) cabbage cultivars were analysed for defence‐related enzymes such as peroxidase and superoxide dismutase. An increase in total peroxidase and superoxide dismutase activity was observed upon inoculation with X. campestris pv. campestris. The activity was greater in resistant cultivar when compared to susceptible ones. Both enzyme activity assays and qPCR analyses for the expression of the defence genes in susceptible and resistant cultivars demonstrated that the peroxidase gene was up‐regulated in resistant cultivar compared to susceptible cultivar. The present study proved that P. fluorescens‐induced resistance against X. campestris pv. campestris in cabbage seedlings is more efficient as compared to the use of INA—abiotic inducer.