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This article highlights anthelmintic drug resistance starting from its origin to its detection procedures at parasitic population genetic level. Main goal of this article is to explore the population genetics of parasites involved in development of anthelmintic resistance. It is analyzed in this research that how we can use this information in identification of molecular markers which can be further utilized in development of accurate diagnostic tests. In veterinary clinics these molecular markers can be used for routine diagnostic tests not only for diagnosing the parasitic infection but also in surveillance of parasitic spread. This research would help in studying progress of parasitic drug resistance in relation to migration of resistant alleles from one generation to next generation. Candidate gene responsible of carrying and transferring mutation to next generation is analyzed for determining root cause of anthelmintic resistance. This information can be used in recognition of genetic loci that involved in anthelmintic resistance but it can only be possible when full genome sequence develop. Unluckily a little information is available about nematodes. Finally techniques involved in detection of candidate gene are discussed.
Key words: levamizole resistance, Haemonchus contortus, genetic linkage, trichostrongylid nematodes, genetic marker, ivermectin, Benzonedazole.
Table of contents
Parasitic Nematodes (roundworms) are extremely important in both human and animal diseases. The gastro-intestinal tract (GIT) harboring parasites especially nematodes are prevalent worldwide in livestock as well as in agricultural sector and responsible for major economical losses. It has been estimated that GIT nematodes infestation cost the North American cattle industry more than $2 billion per year (Pedro et al., 2003). Control is heavily dependent upon the routine prophylactic use of broad spectrum anthelmintics include benzimidazoles, imidazothiazoles, tetrahydropyrimidines and macrocyclic lactones. In many parts of the world, parasites are resistant to these three classes of anthelmintic drugs along with major clinical problems in sheep, goats and cattle (Subash 1990). Although last two decades, anthalmentic efficacy against nematodes is reduce due to rise of parasitic resistance. The anthalmentic resistance has been seen in almost all countries of the world (Riou et al. 2003).in domestic livestock the most frequently anthelmintic has been experienced in Trichostrongylidea superfamily than others. Blood sucking parasites belong to genus Haemonchus known as important parasites; normally reside in abomasum of ruminants with greater socio-economical losses (Urquhart et al., 1996). The following factors of anthlementic resistance are normally associated with parasites such as maturity stage of parasite, sexual phase of parasite, immune response of host which can be age related and history of infected animal and distribution of worms (Cornell et al., 2004).
The trichostrongylid nematodes like Haemonchus contortus, parasite of sheep and goats residing in abomasum show highest anthelmintic resistance (Mcghee et al., 1981). The focus on genetic variation studying is become necessary now days with unnecessary mutations in genetics of already existing gene population. The high variations have been studied by different labs till today in H.contortus population with different genetic differentiation between isolates of different regions and even some time in same country. However, there is still little information on genetic sub-structuring at a fine scale or on the significance of these differences in terms of phenotype or the potential for cryptic speciation.
For studying genetic variations, effects, impacts and spread of anthelmintic resistances nematodes are used against the treatment of anthlemintics drugs (Coles, 1999; Kaplan, 2004). This methodology was developed and improved as time elapsed. But now days this procedure is extensively used for better studying in parasites of small ruminant livestock. Now a day resistance has been developed against all known broad spectrum anthelmintic of small ruminants with heavy burden on agricultural sector (Bartley et al. 2001, 2006; Pomroy, 2006).
A basic idea of this review article is to explore about specific genetic changes which are accountable and correlate with anthelmintic resistance.
These may then provide as genetic markers of resistance handling. Such markers can be used in early diagnosis and observation of anthelmintic resistance. These markers can help us in recognizing resistance before genetic changes in the parasite which will prevent failure of anthelmintic treatment. This review article provides us detail about number of characteristics of parasite genetics which are directly involved in the classification of genetic markers of anthelmintic resistance and finally a comprehensive discussion on applications of these markers as molecular diagnosis or tests in the field.
Furthermore, parasite genetics and population biology are very important features for understanding the genetic variations in candidate gene studies. However many ideas discussed in this review have impact for the future application of genome wide approaches in field of diagnostic procedures which ultimately reduce anthalmentic resistance.
ORIGIN OF RESISTANCE
Main issue is the transfer of resistance from one generation to next generation after treatment with anthelmintic drug. Different stages of developmental larve of parasite shows different intensity of resistance. In UK farmers treated their grazing animals with massive ivermectin and benzonedazole drugs resulting into development of resistance (Coles., 1999).
Treatment using anthelmintic drugs gives direct benefit to parasites either in their survival or in carrying mutation. It directly involves in reducing survival of parasites carrying these mutations and transfers them into their offspring's resulting into increase frequently of anthelmintic resistance if selection is upheld. This genetic transfer ultimately resulting into production of large proportion of parasites which are resistant to therapeutic doses of anthelmintic drugs resulting into treatment failures
Development of resistance against anthelmintic drug requires accessibility to resistance alleles which have direct source of the selection. Early drug resistance rate determination requires the amount of frequency in development of these alleles. As a result of these anthelmintic resistance mutations at genetic level occur and their early existence that can originate in parasitic nematode population can be studied by different ways as discussed bellow.
Trichostrongylid nematodes are incompatible and inconsistent just like other invertebrate species. The probability of presence of a resistance mutant allele in Trichostrongylid nematodes is very common. Anthelmintic treatment is parallel, positively with this inconsistency and so we may suppose their presence to be common. It is practically observed that resistant alleles are actually pre-exist in trichostrongylid nematodes. And when use of anthelmintic increases it resulted into increase of resistant allels. It can be observed experimentally by the treatment of susceptible population with anthelmintic drug e.g. when experimentally produced infection of H. contortus was treated with anthelmintic drug i.e. ivermectin for continues three generations (by selecting previously treated organism), strong resistance against drug was observed (Coles, 2005).
Evidence in favour of pre existing alleles is benzimidazole (BZ) resistance, but in spite of all the proof needs careful study and analysis. Samples of H. contortus from different geographical areas were taken where benzimidazole was used against parasite for continuous three generations and development of resistance was observed in those areas against the drug (Roos et al. 1990).
The selection for susceptible population can be done by southern blot technique along with isotype 1 beta-tubulin probe, which finally yield number of hybridization fragments. Hybridization fragments gradually decreased from five to one fragment as resistance increase as a result of treatment of susceptible population. RELP fragment present at position 200 on beta-tubulin protein loss the property of benzimidazole binding which ultimately resulted into resistance against drug (Kwa, 1994). Same results are reported for Trichostrongylus colubriformis (Grant and Mascord, 1996).
Spontaneous and recurrent mutations
Spontaneous mutation has also been reported but it is believed that it may not be a key mechanism for anthelmintic resistance. on the other hand, theoretical basis and some experimental evidences suggested that these may in fact occur. Trichostrongylid nematodes population shows high spontaneous mutations rate, give us an opportunity for research and study anthelmintic resistance. Two mutations per genome per generation has been observed in nematode C. elegans and the fruit fly D. Melanogaster (Denver et al. 2004; Haag-Liautard et al. 2007).
Studies conducted on trichostrongylid nematodes larvae in which sample size of 5 x 107 larvae having haploid genome size of 1 x 108 bp showing results that each larvae has mutation in every nucleotide. Sheep infected with very high worm infection of H. contortus shows resistance allele in eggs of H. contortus which increases with use of drug and producing resistance in infected agent against drug used. This evidence is no doubt difficult in its quantification but it gives us evidence to mutation. It is very hard to attain clear cut facts for spontaneous mutation for anthelmintic resistance alleles in the populations of parasite. But these above studies give sport to the hypothesis of spontaneous mutation (Silvestre and Humbert, 2002). In Central and Southern France goat farms, the variety in sequence of isotype-1 beta-tubulin alleles can be used in identification of resistant by the F200Y mutation.
Migration of resistance alleles
The flow of parasites depend upon host movement and direct correlation has been seen reported and it is not ignoring to say that such single resistant allele are because of such movement across the whole regions. On the other hand, countries like UK, extensively uses the anthelmintic as quarantine drenching before movement across the borders. So, it means migration of host from one place to another place is directly related with transfer of nematode parasite along with resistant allele from one region to another region. T. circumcincta having small population structures but very valuable in using genetic markers (Braisher et al., 2004; Grillo et al., 2006, 2007). So it is very difficult to determine migration of parasitic genotype but it can be achived by characterization of resistence alleles in parasite population. It is reported for T. circumcincta isolates that are exposed to seven different types of isotype-1 beta-tubulin reisitant haplotypes shows mutation in F200Y region within a single population (Grillo et al., 2006)
So it can be concluded that anthelmintic resistance is dependent on conditions and a lot of research for both resistant and susceptible alleles from different populations is required which include sequencing and detail phylogenetic analysis.
GENETIC FOOTPRINTS OF SELECTION
Genetic changes and variations are studied on a selected single locus area and its effects on resistance alleles under specific parasite population are known as genetic foot prints of selection. It can be certified by genetic hitchhiking and meiotic recombination (Gilleard and Beech., 2007)
Genetic hitchhiking is basically positive mutation on linked loci and occuannly on restricted sense. It involves in selection of alleles which are favourable and also reduce harmful mutations. It will lead to change in genetic system. It may be due to linkage disequilibria means association between genes statistically. It point out new mutation in old gene (Barton., 2000).
Selection of gene which is associated with resistance must have relation between polymorphism and resistant. For determination of this relationship specific genes should be selected which have direct role in expression of resistance. So genes involved in specific mutation of DNA or protein can be isolated (Beech., 2008). Mechanisum of resistance does not involve markers but Test for SNP markers on any position of same chromosome as the resistance mutation observed for finalization of resistance. If genetic recombination not present, a phenomenon of decrease in polymorphism together with linkage disequilibrium enlarges throughout the chromosome having resistant mutant (Gilleard and Beech., 2007).
Effect of meiotic recombination
Offspring's of nematodes are diploid and genetic recombination is main requirement for meiosis. Every parent transfer single copy of chromosome to offspring. But during meiotic recombination DNA fragment is changed within homologous pair of chromosome. It will result into existence of linked markers on same chromosome. As a result it mutation is increased in offspring than parents.
Candidate Gene Studies in Nematodes
Differentiation in gene expression was studied between genetically resistant isolates and isolates from lambs infected from field isolates of larval nematodes by using 10,204 bovine cDNA microarray. This study is further confirmed by using Northern blot analysis. Out comes were further explored for identification of promoter motifs responsible for gene expression. Restriction is found in upregulated gene promoter regions. Motifs of downregulated gene promoters were also present in upregulator gene promoters and unchanged expression was observed. Technique of gene ontology was applied for studying cell signalling process involved in parasite resistence. From this two path ways are determined in resistant animals including acquired immune response and structure of intestine smooth muscle. Main responsibility of parasite resistance lies on these gene (Diez-Tasco´n et al., 2004)
Frequent use of benzenidazole (BZ) results into development of resistance. This resistance is hereditary transferred to offspring. It change the genetics of parasite population and treatment of BZ is ineffective. Its basic mechanisum involves depolarization of microtubules. BZ has high affinity towards β-tubulin. Drug resistance is due to decrease binding affinity of BZ to β-tubulin (Beech et al., 1994). β-tubulin is involved in subcellular gene expression (DriscoU et al ., 1989). Beta-tubulin study is key to studying candidate gene in nematodes because it contain genes encoding for BZ resistance. BZ reduces metabolic activity of enzyme, fumarate reductase in nematodes (Prichard, 1970). Concepts moves from metabolism to beta-tubulin for study of BZ resistance (Davidse and Flach, 1977). In BZ resistance populations, replacement of Beta-tubulin with Y200F has also been observed (Silvestre and Humbert, 2002).
Ivermectin (IVM) is extensively used and its first reported against sheep. IVM resistance against H.contartus is about 99% when used in low doses (Molento et al., 1999). IVM is released through bile and faces as parent drug. Invert studies shows that IVM is inhibitor of P-GP. IVP excretion modes change when it is administrated with loperamide (a P-gp modulator) and its availability increase in tissues produce synergistic effect (Ballent et al., 2006). IVM cause paralysis of nematode muscles (Geary et al. 1993). Studies on cDNA libraries shows, IVM effects on the glutamate-gated chloride channels (Cully et al. 1994). IVM resistance directly related with GABA-gated chloride channels and glutamate calcium channels in H. contortus (Blackhall et al. 2003; Njue and Prichard, 2004).
PROCEDURES TO VERIFY THE CANDIDATE GENE
There are two main ways to identify and test the association of candidate genes with an anthelmintic resistance.
Largely used technique for identification of alteration in genes of parasite is to study the parasite genetic changes after treatment with anthelmintic drug. In this technique lines of parasites are developed from both resistant and susceptible single isolates. These lines can be developed from single parent parasite nematode. By giving treatment with anthelmintic drugs to one line of parasite for many generations resistance can be produced against drug. This is treated line. Similarly a second line of parasite is developed without treatment. By analysis of these two lines candidate gene can be studied (Molento et al., 1999). Lines are developed from common genetic pool, different anthelmintic drugs produces different effects on genetic change which can be used in identification of candidate gene involved in resistance. This technique is mainly used in macrocyclic lactones resistence study (Njue and Prichard, 2004).
Comparison of field isolates
This is direct approach for study anthelmintic resistance and susceptibility in population of parasites. In this technique we directly take samples from field (Ardelli et al., 2006a, b). Difficulty in this technique is the separation of parasite population that are resistant from susceptible parasites. Candidate gene, allele frequency is only strong indicator for resistant population. Detail study of differences in allele of many loci in whole genome is required for proper identification of resistant population from susceptible population (Varady et al., 1995).
This review article elaborate gastrointestinal tract parasites, genetics of GIT parasites, economic impact of GIT parasites, drugs used against GIT parasites and development of resistance against drugs in parasite population pole along with annual loss due to development of resistance against drugs. Furthermore this article highlighted different characteristics of parasite genetics in relation with drug resistance. It includes development of drug resistance in coordination with pre-existing alleles, mutation and migration. After that a detail discussion on substitutions on the genetic footprints in nematodes which elaborate the candidate gene studies, experimental selection and analysis of field resistance. It is necessary to understand genetic variations and procedures to detect candidate gene for knowing drug resistance and its handling on grass root level. But up till now a lot of research is needed to handle the issue of anthelmintic resistance at molecular and population level. There is need to develop better genetic tools and procedures for elaboration and easy understanding of complex parasite genomic structure. After that we will be able to solve the issue of parasitic drug resistance.