Breeding for resistance to gastrointestinal nematodes - the potential in low-input/output small ruminant production systems

AbstractThe control of gastrointestinal nematodes (GIN) is mainly based on the use of drugs, grazing management, use of copper oxide wire particles and bioactive forages. Resistance to anthelmintic drugs in small ruminants is documented worldwide. Host genetic resistance to parasites, has been increasingly used as a complementary control strategy, along with the conventional intervention methods mentioned above. Genetic diversity in resistance to GIN has been well studied in experimental and commercial flocks in temperate climates and more developed economies. However, there are very few report outputs from the more extensive low-input/output smallholder systems in developing and emerging countries. Furthermore, results on quantitative trait loci (QTL) associated with nematode resistance from various studies have not always been consistent, mainly due to the different nematodes studied, different host breeds, ages, climates, natural infections versus artificial challenges, infection level at sampling periods, among others. The increasing use of genetic markers (Single Nucleotide Polymorphisms, SNPs) in GWAS or the use of whole genome sequence data and a plethora of analytic methods offer the potential to identify loci or regions associated nematode resistance. Genomic selection as a genome-wide level method overcomes the need to identify candidate genes. Benefits in genomic selection are now being realised in dairy cattle and sheep under commercial settings in the more advanced countries. However, despite the commercial benefits of using these tools, there are practical problems associated with incorporating the use of marker-assisted selection or genomic selection in low-input/output smallholder farming systems breeding schemes. Unlike anthelmintic resistance, there is no empirical evidence suggesting that nematodes will evolve rapidly in response to resistant hosts. The strategy of nematode control has evolved to a more practical manipulation of host-parasite equilibrium in grazing systems by implementation of various strategies, in which improvement of genetic resistance of small ruminant should be included. Therefore, selection for resistant hosts can be considered as one of the sustainable control strategy, although it will be most effective when used to complement other control strategies such as grazing management and improving efficiency of anthelmintics currently

Evaluation of Diagnostic Potential of Echinococcus granulosus Recombinant EgAgB8/1, EgAgB8/2 and EPC1 Antigens for Cystic Echinococcosis in Goats

Echinococcus granulosus recombinant proteins including two antigen B sub-units EgAgB8/1 and EgAgB8/2 and Echinococcus protoscolex calcium binding protein (EPC1) were expressed in prokaryotic expression vectors. The diagnostic potential of these three recombinant proteins was evaluated in the detection of cystic echinococcosis in goats in IgG-ELISA. The EgAgB8/1 and EgAgB8/2 recombinant proteins reacted fairly with the hydatid infected goats with sensitivity of 66.7% and 80.0% and specificity of 71.3% and 73.3%, respectively while EPC1 recombinant protein showed lower sensitivity (60%) but comparable specificity (72.3%). Cross-reactivity of these three antigens with goat gastro-intestinal strongyle nematodes and Taenia hydatigena under field conditions was studied. Results showed that EgAgB8/1, EgAgB8/2 and EPC1 antigens cross-reacted with most of the parasites in the goat host

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Not AvailableMultidrug-resistant bacteria have now emerged as a global threat due to the emergence of new or re-emergence of old pathogens. This alarming situation demands either to find new/modified effective antibiotics or alternatives to reduce the overall load of infectious diseases both in the veterinary and public domain. Antibiotic adjuvants are non-antibiotic compounds that potentiate the existing antibiotic activity. The Himalayan region being a big basket and hot spot of biodiversity, inhabiting a diverse flora of medicinal plants/herbs can be evaluated for antibiotic adjuvants. These adjuvants work either by blocking the main bacterial resistance mechanisms or enhancing the antimicrobial action of the drug. The adjuvants assist in polypharmacy on bacteria by de- energizing the efflux pump channel of bacteria to escape antibiotic action and by allowing antibiotics to have greater internal access. Thus, the plant-based antibiotic adjuvants have the potency to re-empower the existing antibiotics, restoring their activities against target pathogen that too with minimal side effects/toxicity being natural in origin.Not Availabl

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RNA interference in Fasciola gigantica: Establishing and optimization of experimental RNAi in the newly excysted juveniles of the fluke.

Fasciolosis caused by Fasciola gigantica is a neglected tropical disease but a constraint on the growth and productivity of cattle, buffaloes and sheep in the tropical countries of Asia and Africa. Resistance to commonly used anthelmintics in Fasciola has increased the need to search for alternative therapeutic targets. RNA interference is the current tool of choice in the search for such targets in Fasciola. The susceptibility of juvenile Fasciola hepatica to double stranded (ds) RNA induced RNAi has been established but in F. gigantica a single preliminary report on RNAi induced mRNA transcript knockdown is available. Here we optimized conditions for RNAi in the liver fluke F.gigantica targeting six genes including superoxide dismutase (SOD), σ class of glutathione-s-transferase (GST), cathepsin (Cat) L1-D, Cat B1, Cat B2 and Cat B3 that showed robust transcriptional silencing of the targets following exposure of the newly excysted juveniles (NEJs) to long (170-223 nt) dsRNA. Knockdown was shown to be concentration dependent with significant mRNA transcript suppression occurring at 5 ng / μl that showed further suppression with the increase in the dsRNA concentration. The dsRNA induced persistent silencing of the mRNA transcript of SOD and σGST up to 15 days of observation. Delivery of the long dsRNA and siRNA to the newly excysted juveniles by soaking method was found to be efficient by tracking the uptake and diffusion of Cy3 labelled siRNA and long dsRNA in the flukes. Off-target effects of dsRNA trigger on some of the non-target genes were detected in the present investigation on RNAi in F. gigantica. The dsRNA induced superoxide dismutase protein suppression while impact of RNAi on other target proteins was not studied. There is no in vitro culture system for prolonged survival of the F. gigantica and in the present study in vitro maintenance of the NEJs is reported for a period of 3 weeks. The present study is the first attempt on optimization of RNAi protocols in F. gigantica where long dsRNA allowed for an efficient and persistent gene silencing, opening prospects for functional validation of putative vaccine and therapeutic targets in this neglected parasite

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Not AvailableApart from the tick-borne pathogens affecting human and animal health, ticks also harbor various non-pathogenic endosymbionts with dynamic ecological interactions. These endosymbionts are unexplored from the Indian ticks; hence this pilot study was conducted. Seventy-nine ticks were collected from Nainital district of Uttarakhand state of north India and were identified as Rhipicephalus microplus morphologically and by molecular analysis. PCR and sequence analysis were carried out to detect the presence of Rickettsia-like, Coxiella-like and Francisella-like endosymbionts in these ticks. Based on the partial 16S rRNA gene sequence, Coxiella-like endosymbiont (CLE) was detected in the adult and other life-cycle stages of ticks with 96.6–97.7% nucleotide sequence identity with the published CLE sequences from GenBank. The phylogenetic analysis revealed that the CLE from R. microplus were clustered with the CLE from other Rhipicephalus species. All these CLE formed distinct clades from the pathogenic Coxiella burnetii. None of the tick samples was found positive for Rickettsia-like and Francisella-like endosymbionts in the present study. We also demonstrated the vertical transmission of CLE from surface sterilized and laboratory reared fully engorged adult females to the eggs and the larvae. However, large scale studies are to be conducted to detect various endosymbionts and endosymbiont-tick associations in the Indian tick species and to explore these associations for tick and tick-borne disease control.Not Availabl

RNA interference in <i>Fasciola gigantica</i>: Establishing and optimization of experimental RNAi in the newly excysted juveniles of the fluke

<div><p>Fasciolosis caused by <i>Fasciola gigantica</i> is a neglected tropical disease but a constraint on the growth and productivity of cattle, buffaloes and sheep in the tropical countries of Asia and Africa. Resistance to commonly used anthelmintics in <i>Fasciola</i> has increased the need to search for alternative therapeutic targets. RNA interference is the current tool of choice in the search for such targets in <i>Fasciola</i>. The susceptibility of juvenile <i>Fasciola hepatica</i> to double stranded (ds) RNA induced RNAi has been established but in <i>F</i>. <i>gigantica</i> a single preliminary report on RNAi induced mRNA transcript knockdown is available. Here we optimized conditions for RNAi in the liver fluke <i>F</i>.<i>gigantica</i> targeting six genes including superoxide dismutase (SOD), σ class of glutathione-s-transferase (GST), cathepsin (Cat) L1-D, Cat B1, Cat B2 and Cat B3 that showed robust transcriptional silencing of the targets following exposure of the newly excysted juveniles (NEJs) to long (170–223 nt) dsRNA. Knockdown was shown to be concentration dependent with significant mRNA transcript suppression occurring at 5 ng / μl that showed further suppression with the increase in the dsRNA concentration. The dsRNA induced persistent silencing of the mRNA transcript of SOD and σGST up to 15 days of observation. Delivery of the long dsRNA and siRNA to the newly excysted juveniles by soaking method was found to be efficient by tracking the uptake and diffusion of Cy³ labelled siRNA and long dsRNA in the flukes. Off-target effects of dsRNA trigger on some of the non-target genes were detected in the present investigation on RNAi in <i>F</i>. <i>gigantica</i>. The dsRNA induced superoxide dismutase protein suppression while impact of RNAi on other target proteins was not studied. There is no <i>in vitro</i> culture system for prolonged survival of the <i>F</i>. <i>gigantica</i> and in the present study <i>in vitro</i> maintenance of the NEJs is reported for a period of 3 weeks. The present study is the first attempt on optimization of RNAi protocols in <i>F</i>. <i>gigantica</i> where long dsRNA allowed for an efficient and persistent gene silencing, opening prospects for functional validation of putative vaccine and therapeutic targets in this neglected parasite.</p></div

Survival of <i>F</i>. <i>gigantica</i> NEJs in RPMI-1640, DMEM and DME/F-12 culture media supplemented with 10% foetal bovine serum, 2% glucose and 25 mM HEPES.

<p>NEJs showed higher % survival (≥ 80%) in RPMI-1640 which was lower in DMEM and DME/F-12 medium (≥75% and ≥72%), respectively at 3 weeks of culture. All flukes died by 28–30 days of culture.</p

Primers designed for qRT-PCR estimation of the mRNA transcript knockdown of <i>F</i>. <i>gigantica</i> target genes post-RNAi.

<p>Primers designed for qRT-PCR estimation of the mRNA transcript knockdown of <i>F</i>. <i>gigantica</i> target genes post-RNAi.</p

Sense and anti-sense primers tailored with T7 promoter sequence at 5' end (in bold) for <i>in vitro</i> transcription of dsRNA trigger against six target genes of <i>F</i>. <i>gigantica</i> and for irrelevant control dsRNA against <i>Plasmodium falciparum</i> KAHRP gene.

<p>Sense and anti-sense primers tailored with T7 promoter sequence at 5' end (in bold) for <i>in vitro</i> transcription of dsRNA trigger against six target genes of <i>F</i>. <i>gigantica</i> and for irrelevant control dsRNA against <i>Plasmodium falciparum</i> KAHRP gene.</p