De novo pathway is an active metabolic pathway of cysteine synthesis in Haemonchus contortus.

Haemonchus contortus, commonly known as Barber's pole worm, is an economically important gastrointestinal nematode of sheep and goats especially in tropical and sub-tropical regions of the world. Cysteine synthesis is a very important metabolic pathway for the parasite, however the functional aspects of cysteine synthesis in parasite are largely unknown. The key question which we have investigated in the study is; whether the parasite uses a de novo pathway of cysteine synthesis, which is unknown in multicellular organisms of the animal kingdom and known to be absent in mammals. Directional cloning of the cysteine synthase (CS) gene was done in pET303 champion vector using restriction sites XbaI and XhoI. The CS gene of the H.contortus was closely related to CS-A protein of Oesophagostomum dentatum and a hypothetical protein of Ancylostoma ceylanicum. Recombinant protein of the H contortus CS (rHC-CS) gene was expressed using pET303 vector in pLysS BL21 strain of E.coli and subsequently purified by Ni-NTA affinity chromatography. Western blot using anti-His tag antibody confirmed the presence of rHC-CS. Biochemical assay, FTIR and enzyme kinetics studies revealed that rHC-CS used O-acetyl serine as substrate to produce cysteine using de novo pathway and CS activity was also confirmed with the homogenate of H.contortus. Upregulation of CS transcripts in the adult and its downregulation in the L3 larval stage suggests that de novo pathway contributes to the cysteine requirement of mature H.contortus. It is concluded that de novo pathway is an active metabolic pathway in H.contortus.

Selecting sheep for Haemonchus contortus resistance and susceptibility: Flock dynamics and genetic architecture.

The study was carried out in the selected strains of Malpura and Avikalin sheep for nematode resistance versus susceptibility (Malpura resistant: MR, Malpura susceptible: MS, Avikalin resistant: AR and Avikalin susceptible: AS). Strains were selected since 2004 and inter-se breeding followed since year 2007. The objective was to decipher the genetic structure of strains and to estimate genetic parameters for fecal egg count (FEC). The population size was small (107-279); however, all the four flocks had sizable effective population size (fe) for founders. The ratio for fe to effective number of ancestors (fa) was nearly 1 indicating genetic stability. Inbreeding was under control. The log transformed FEC (LFEC) was significantly (P < .05) affected by sex, year and month of recording for all 4 strains. Generation had desired genetic trend. There was low heritability for LFEC in MR(0.02), MS(0.06), AR(0.07) and AS(0.02). The estimate of the permanent environment effect (c2) was low. Repeatability (ρ) for LFEC were 0.05, 0.11, 0.07 and 0.06 for MR, MS, AR and AS, respectively. Pruning the data for repeated records in peak infection period (1: "Aug-Sep" and 2: only "Sep") resulted in considerable increase in ρ for MR (Aug-Sep: 0.07 ± 0.03 and Sep: 0.12 ± 0.07) and AR (Aug-Sep: 0.05 ± 0.04 and Sep: 0.09 ± 0.10). Results revealed genotype x environment interaction for repeated measures across months. Low flock size and low genetic variability are hurdles for improvement in R strains. It is recommended to incorporate new candidates post selection from the other breeding flocks and admit them in the R strains for selective breeding.

Genetic parameter estimates for fecal egg counts and their relationship with growth in Avikalin and Malpura sheep.

Breeding for resistance against nematodes has become the need of the hour due to emergence of anthelmintic resistant strains of major pathogenic nematodes of economic importance and rising demand for chemical residue free food by consumers. ICAR-Central Sheep and Wool Research Institute Avikanagar (Rajasthan) has developed Haemonchus contortus resistant lines of sheep in Avikalin and Malpura breeds by harvesting benefits of over-dispersion in fecal egg counts (FEC) through executing a breeding program since year 2004. Aim of the present study was to assess the genetic parameters for nematode resistance in these lines and also to develop suitable criteria for selection targeting resistance as well as growth improvement in these two lines. The data on 1240 Avikalin and 2172 Malpura sheep generated over 13 years (2004-16) for FEC along with deep pedigree and growth records for live weight at 6 (6WT) and 12 month were used for study. Data were analyzed using Average Information Restricted Maximum Likelihood (AIREML) approach. Results revealed moderate heritability (h2) for pre-drench log transformed fecal egg count (LFEC) in Avikalin (0.21±0.06) and Malpura (0.18±0.04) sheep. The post-drench h2 for LFEC was low in Avikalin (0.04±0.03) and Malpura (0.11±0.03) sheep. Effective selection program can be carried out for further improving the resistance against H. contortus in both the breeds using pre-drench LFEC estimates. The genetic correlation between the pre-drench LFEC and growth traits was not in the desired direction. Existence of substantial genotype × environment (G×E) interaction was seen in Malpura sheep, where major shift in ranks of sheep based on pre-drench LFEC as that of post-drench LFEC was observed owing to genetic correlation of 0.65±0.15. The G×E was absent in Avikalin sheep. Unreliable genetic correlation between growth and LFEC does not warrant a multi trait selection index development and its utilization in breeding program. The independent selection for LFEC followed by corrected 6WT can precisely help in achieving the goal of improving growth in nematode resistant sheep.

Interrelationship between late gestational ewe factor and early life lamb factors in semi-arid tropical region.

The limitation in feed availability in the semi-arid region during the lean period can result in a variation of the body condition, body weight of pregnant ewe which in turn may affect the lamb birth weight, colostrum immunoglobulin, growth hormone (GH), and insulin-like growth factor-1 (IGF-1). Therefore, the present study was initiated to assess the interrelationship between late gestational ewe factor and early life lamb factors in the semi-arid tropical region. For this purpose, 83 Malpura and 45 Avikaline pregnant ewes were selected and their body condition score (BCS) at late gestation, body weight at lambing, and birth weight of lambs was recorded. The BCS of ewes in late gestation had significant (P < 0.001) positive correlation (r2 = 0.465) with the birth weight of lambs. The body weight at lambing was significantly (P < 0.05) higher in single-lamber ewes as compared to twin-bearing ewes. The plasma IGF-1 of lamb increased significantly (P < 0.05) with the increase of BCS of ewe at the late gestation as well as body weight after lambing. The colostrum of twin-lamb producing ewes had higher (P < 0.05) IgG content than single-lamb producing ewes. The concentration of IGF-1 was significantly (P < 0.05) higher in single-born lamb as compared to twin-born lamb. Based on the results of the present study, it is to conclude that higher BCS at late gestation and higher body weight of ewes at lambing is desirable for producing lambs with a higher birth weight having higher growth potential as well as chances of survival.

Estimates of genetic parameters for faecal egg count of Haemonchus contortus infection and relationship with growth traits in Avikalin sheep.

Genetic parameters for faecal egg count were estimated in naturally challenged Avikalin sheep developed and maintained at Central Sheep & Wool Research Institute, Avikanagar, India, over a period of 4 years (2004-2007). The data on faecal egg count for 433 animals descended from 41 sires, and 151 dams were used for the study. Genetic analyses were carried out using restricted maximum likelihood, fitting an animal model and ignoring or including maternal genetic or permanent environmental effects. Direct heritability for the trait was 0.149 +/- 0.096 when maternal effects were ignored. In the model which takes in to account direct genetic, maternal genetic and maternal permanent environment effect together, it was observed that maternal heritability (m(2)) accounts for 0.6% of total variation whereas maternal permanent environmental effect (c(2)) accounts for 6.14% of total phenotypic variation. Effect of faecal egg count on the growth characteristics was observed to be significant. It was seen that wherever FEC was high, body weight or average daily gain declined in active infective stage. After termination of the infection, these effects were found to be non-significant. Result suggests that direct genetic and maternal permanent environmental effects were important for this trait; thus, they need to be considered for improvement in the trait.

Detection of benzimidazole resistance in Haemonchus contortus using RFLP-PCR technique.

Benzimidazole (BZ) resistance in Haemonchus contortus is linked primarily with the mutation in the beta-tubulin isotype 1 gene that substitute phenylalanine (Phe) to tyrosine (Tyr) at 200 codon of the gene. In the present study, a new restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) technique has been developed for detection of BZ resistance in the beta-tubulin isotype 1 gene of H. contortus. The technique utilizes two primers viz. AvikaF and AvikaR to amplify the region containing mutation in the beta-tubulin gene followed by restriction digestion. After digestion, the 'rr' individuals (homozygous resistant) revealed 257 and 48 bp bands, the 'rS' individuals (heterozygous) showed 305, 257 and 48 bp bands, while 'SS' individuals (homozygous susceptible) revealed uncut 305 bp band. A total of 162 adult male H. contortus collected from Avikanagar, Jaipur and Bikaner regions (54 from each region) were genotyped for analyzing BZ resistance in the beta-tubulin gene. Out of which, 130 adults were 'rr' types, 20 'rS' types and 12 'SS' types. The results showed that genotypic frequencies of different genotypes (rr, rS and SS) were highly significant difference among the three regions (P<0.001). The 'rr' individuals were higher (98%) in Jaipur followed by Avikanagar (93%) and Bikaner (50%) regions. Overall, the prevalence of BZ resistant allele (r) was higher (86%) as compared to BZ susceptible allele (S) (14%). The technique was also found suitable for genotyping of larvae of H. contortus and yielded reproducible results. The study indicated that RFLP-PCR is an easy, reproducible and less expensive than allele specific PCR. This technique will be helpful in establishing the prevalence rate of BZ resistance in H. contortus and can also be utilized for existing worm control programme.

Anthelmintic resistance on an organized sheep farm in India.

Monitoring anthelmintic resistance in strongyle nematodes by the faecal egg count reduction test and a commercial larval development assay on an organized sheep farm in the semi-arid area of Rajasthan revealed the emergence of resistance to benzimidazoles and rafoxanide and a potential risk of the development of levamisole/tetramisole resistance. A benzimidazole/levamisole combination, avermectins and closantel were each found to be efficacious.

Comparative anthelmintic activity of strategic sustained low-level administration of albendazole in feed pellets compared to single doses of closantel and tetramisole against natural ovine parasitic gastroenteritis.

The strategic use of single therapeutic doses of closantel, tetramisole or sustained low-level administration of albendazole in feed pellets in controlling naturally acquired parasitic gastroenteritis in sheep was investigated on a farm in semi-arid Rajasthan, India. A total of 303 5- to 6-month-old sheep were divided into three groups. Two groups were dosed with single therapeutic doses of closantel and tetramisole and the third group was given a low-level medication with albendazole through feed pellets for 30 days. Faecal egg counts revealed significantly lower counts (p<0.001) in the group treated with closantel compared to the other two groups. The faecal egg counts in the group receiving sustained low-level albendazole rose after withdrawal of the medication but remained significantly lower than those in the group treated with tetramisole up to 7 weeks after treatment (p<0.05). On the other hand, in the group treated with tetramisole, the mean faecal egg count rose from 3 weeks after treatment and remained continuously higher than those in any other group up to 12 weeks after treatment. The closantel-treated group gained more body weight but the first six-monthly greasy fleece yield was greater in the group treated with medicated pellets. During the first 3 months of the experiment, three animals in the group treated with tetramisole died of parasitic gastroenteritis. Following sustained low-level administration of albendazole in feed pellets, the plasma disposition curve of both the sulphoxide and sulphone metabolites reached its plateau level by day 5 and remained almost constant thereafter. The comparative cost-effectiveness of the three treatment regimes during the first 3 months of treatment was best for the group treated with closantel followed by the group treated with medicated feed pellets.

Influence of diet type and pretreatment fasting on the disposition kinetics of albendazole in sheep.

The influence of the quality and quantity of diets on the disposition kinetics of albendazole were studied in sheep in two different experiments. The plasma concentration profiles of albendazole sulphoxide and albendazole sulphone were measured following intraruminal administration of albendazole at 5.0 mg/ kg body weight in weaner sheep offered three different diets: 100% green Sorghum spp., 100% dry mature Cenchrus ciliaris hay and a 50:50 mix of these two diets. The peak plasma concentrations and the availability of the albendazole metabolites, as measured by the area under the concentration time curve, were significantly higher (p < 0.01) in the animals offered exclusively dry fodder compared to other diets. Changing the diet from dry to green fodder resulted in a significantly lower systemic availability of the drug metabolites. It is suggested that a decreased transit time of the digesta in the bowel on the green diet, with its high water content, limited the systemic availability of the drug by reducing the time available for gastrointestinal absorption. An experiment on the influence of different levels of pretreatment fasting on the pharmacokinetics of albendazole revealed significantly higher (p < 0.05) plasma concentrations of the anthelmintically active sulphoxide metabolite from 12 h onwards following administration of the drug in animals subjected to 24 h of pretreatment fasting compared to other groups with pretreatment fasting of 8, 12 or 18 h. The area under the concentration time curve and the minimum residence time of the drug metabolites were significantly greater (p < 0.05) in animals that had been fasted for 24 h. It is suggested that fasting induces a decrease in the flow of digesta through the gastrointestinal tract of ruminants and prolongs the duration of dissolution of the drug, resulting in enhancement of the absorption of albendazole and of the systemic availability of its metabolites.

Further studies on anthelmintic resistance in sheep at an organised farm in arid region of Rajasthan.

Faecal egg count reduction test (FECRT) and in vitro egg hatch assay (EHA) was performed on Indian Karakul (3/4 crosses of Karakul with Malpura, Marwari and Sonadi) transported from arid region campus, Bikaner, where resistance was reported in 1996. FECRT revealed that fenbendazole and levamisole were 100% effective in reducing the egg counts. LC50 value on EHA was 0.074 +/- 0.015 microg thiabendazole ml(-1). The faecal culture examination revealed the presence of Haemonchus contortus only. It was concluded that H. contortus was fully susceptible to both benzimidazole and levamisole.

Comparative disposition kinetics of albendazole in sheep following oral and intraruminal administration.

The pharmacokinetics of albendazole was studied in sheep following single oral and intraruminal administration at nematocidal dose rates. The disposition curves of its metabolites indicated increased uptake of the drug in sheep following intraruminal as compared to oral dosing (p < 0.05). The increased bioavailability of benzimidazole anthelmintics given by the intraruminal route could be exploited for optimizing the use of anthelmintic for sustained parasite control in small ruminants.