Resistance status against natural infection to Haemonchus contortus as well as influence

of season, sex, body weight, and haemoglobin type on resistance levels were evaluated in 309 numbers

of Garole sheep. In adult Garole sheep, egg per gram(EPG) of faeces for Haemonchus contortus was

varied from 300 to 1600, but overall EPG in Garole have been recorded as 829.96 ± 20.60. The effects

of season, sex, and body weight and haemoglobin type on EPG were all found to be highly significant

(P < 0.01). EPG count was highest during monsoon (986.27 ± 28.26), followed by summer (832.88 ±

28.26) and lowest during winter (670.74 ± 28.26) which indicated the existence of a seasonal variation

of EPG. Rams had higher EPG (954.32 ± 57.93) than ewes (705.60 ± 45.79) which reflected that males

appeared to be more susceptible to Haemonchus contortus infection compared to females. Animals

with lower body weight (upto 10 kg) showed higher EPG (1017.20 ± 54.82), then the infection level

decreased as body weight increased (886.79 ± 56.23 for 10 kg to 12 kg and 737.18 ± 50.29 for 12 kg to

14 kg) and lowest EPG was recorded in animals with above 14 kg body weight (678.68 ± 54.49). This

study reveals Hb-BB type animals had higher EPG count (983.81 ± 18.22) in comparison to Hb-AB

type animals (676.12 ± 33.96) indicating that Haemoglobin-A locus has some relation with resistance.

From our study it can be concluded that resistant level of Garole sheep against Haemonchus contortus

is influenced by some intrinsic factors like sex, body weight, and haemoglobin type of the sheep and

also by extrinsic factor like season

Debraj Nandi

Manoranjan Roy

P.K.Senapati

Sukanta Roy

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57FACTORS  AFFECTING  VARIABILITY  OF  RESISTANCE  INGAROLE  SHEEP  NATURALLY  INFECTED  WITHHAEMONCHUS CONTORTUSManoranjan Roy*1, P.K.Senapati2, Sukanta Roy3 and Debraj Nandi4ABSTRACT:  Resistance status against natural infection to Haemonchus contortus as well as influenceof season, sex, body weight, and haemoglobin type on resistance levels were evaluated in 309 numbersof Garole sheep. In adult Garole sheep, egg per gram(EPG) of faeces for Haemonchus contortus wasvaried from 300 to 1600, but overall EPG in Garole have been recorded as 829.96 ± 20.60. The effectsof season, sex, and body weight and haemoglobin type on EPG were all found to be highly significant(P < 0.01). EPG count was highest during monsoon (986.27 ± 28.26), followed by summer (832.88 ±28.26) and lowest during winter (670.74 ± 28.26) which indicated the existence of a seasonal variationof EPG. Rams had higher EPG (954.32 ± 57.93) than ewes (705.60 ± 45.79) which reflected that malesappeared to be more susceptible to Haemonchus contortus infection compared to females. Animalswith lower body weight (upto 10 kg) showed higher EPG (1017.20 ± 54.82), then the infection leveldecreased as body weight increased (886.79 ± 56.23 for 10 kg to 12 kg and 737.18 ± 50.29 for 12 kg to14 kg) and lowest EPG was recorded in animals with above 14 kg body weight (678.68 ± 54.49). Thisstudy reveals Hb-BB type animals had higher EPG count (983.81 ± 18.22) in comparison to Hb-ABtype animals (676.12 ± 33.96) indicating that Haemoglobin-A locus has some relation with resistance.From our study it can be concluded that resistant level of Garole sheep against Haemonchus contortusis influenced by some intrinsic factors like sex, body weight, and haemoglobin type of the sheep andalso by extrinsic factor like season.Key words: Garole sheep, Haemonchus contortus, EPG, Resistance, Haemoglobin type.1Assistant Professor, Department of Animal Genetics & Breeding,West Bengal University of Animal &Fishery Sciences, 37, Kshudiram Bose Sarani,  Kolkata – 700037, West Bengal, India.2Professor, Department of Animal Genetics & Breeding, West Bengal University of Animal & FisherySciences, 37, Kshudiram Bose Sarani,  Kolkata – 700037, West Bengal, India.3Assistant Director (ARD-Administration), Bankura, West Bengal, India.4Veterinary Officer, ABAHC, Matha (under Bagmundi Block), Purulia, West Bengal, India.*Corresponding authorINTRODUCTIONGastro-intestinal parasite infections are themajor constraint to the sheep industry causingproduction losses, increased costs ofmanagement and treatment, and even mortalityin severe cases (Barger and Cox 1984, Larsenet al. 1995). Most of the genera (of parasites)found in ruminants stimulate a relativelyeffective level of immunity in most animalsafter several months on pasture. This immunityExplor. Anim. Med. Res.,Vol.3, Issue - 1, 2013, p. 57-64ISSN  2277- 470X (Print), ISSN 2319-247X (Online)Website: www.animalmedicalresearch.org58Exploratory Animal and Medical Research, Vol.3, Issue -1, June, 2013significantly reduces the number of worms thatbecome established in the grazing animals, theexception to this is Haemonchus contortus insheep. In sheep, lambs below the age of8 months do not develop significant protectiveimmune responses to infection or immunization(Urquhart et al. 1996). This leads to prolongedsusceptibility to reinfection is a major reasonin lambs and therefore H. contortus remains themost economically important GI nematodes insheep.Faecal egg counts have been shown to havemoderate (0.61) to high (0.91) correlation withnematode burdens (Baker et al. 1991, Stear etal. 1995, Bisset et al. 1996) and in essence mightbe considered a direct measure of infection. Themost effective and only, practical way toestimate potential pasture contamination is fromFEC and in one sense, this is also directselection.Several factors affect resistance to internalparasites, including intrinsic factors like sex ofthe sheep and extrinsic factors like season (Stearand Wakelin, 1998). Haemoglobin type alsofound to be associated with resistance level toH. contortus (Allonby and Urquhart 1976,Altaif  and Dargie 1978, Preston and Allonby1979,  Kassai et al. 1990).The main habitat of Garole sheep is the salinebelt of Sunderban Delta of South 24 Parganas,some parts of North 24 Parganas and somesaline zone of Purba Medinipur district of WestBengal, India. The climate is hot and humid.These sheep are indigenous and is popular forits biannual lambing with multiple births andtheir ability to graze on aquatic weeds andgrasses standing even in knee-deep water forhours together (Kar and Prasad 1992).The research in connection to variability ofresistance against natural infection ofHaemonchus contortus in Garole sheep is verylimited although infections exist and arewidespread. Realizing the importance of thesubject, a research programme has been takenup in Garole sheep to study the variability ofresistance against natural infection ofHaemonchus contortus in Garole sheep as wellas to evaluate the relationship of response tonatural infection of H. contortus withhaemoglobin genotypes / phenovariants.MATERIALS  AND  METHODSSite of experiment and animal :The research work was carried out with 103nos. of adult Garole sheep (Ovis aries) of which45 were male and 58 were female, maintainedin a farm under National AgriculturalTechnology project on ‘Animal GeneticResource Biodiversity’ of West BengalUniversity of Animal and Fishery Sciences,Mohanpur Campus, Nadia, West Bengal, India.The farm is situated at 88032¢E longitude and22056¢N latitude with an altitude of 9.75 mabove mean sea level and having sub-tropicalhumid climate. On the basis of temperature,relative humidity and rainfall, the year has beendivided into three seasons viz. winter (fromNovember to February), summer (from Marchto June) and monsoon (from July to October).During the experiment period temperaturevaried within the range of 7.27ºC to 39.98ºCand relative humidity varied from 47.18% to97.69%.General  management  practice:All the experimental animals were allowedfor grazing on natural pasture from 8 A.M. to 3P.M. in winter months. During summer andmonsoon animals were grazed in two shifts viz.from 7 A.M. to 10.30 A.M. and 2.30 P.M. to5.30 P.M. The pasture comprised mainly ofdoob grass (Cynodon dactylon) with other59grasses and leaves viz. goose grass (Eleusineindica), horse purseane (Trianthema monogyna)and anjan grass (Cenchrus ciliaris). In additionto grazing all the animals were offered about50g concentrates after returning from grazingin the evening. Adlibitum water was providedto all the animals in all seasons. All the ewesand rams were dewormed for 3 times in a yearwith Fenbendazole or Albendazole andRafoxanide (7.5 mg to 10 mg/kg body weightorally) in three different seasons (summer,monsoon and winter). Animals were given dipsfrom time to time to protect them fromectoparasites.Collection of faecal sample and estimationof parasitic load:Since sheep were maintained in a commonnatural pasture throughout the year, it wasassumed that they got equal dose ofHaemonchus  infection from the grazing field.The faecal samples from each animal werecollected rectally after one week of dewormingand examined for Haemonchus contortus eggs.During each season two faecal samples werecollected from each animal at five days intervaland accordingly eggs per gram of faces (EPG)were determined following Stoll’s DilutionMethod as described by Soulsby (1982).Collection of blood samples anddetermination of hemoglobin pheno-variants:About 5 ml of fresh blood was asepticallycollected from each animal by puncturingjugular vein with 0.5 ml of anticoagulant(Sodium citrate) and accordingly hemoglobinpolymorphism was conducted following theStarch gel electrophoresis method described bySmithies (1955).Grouping of animals according to bodyweight:Body weights of adult sheep (rams and ewes)were taken in the morning with the help ofspring balance (SALTER), which ranges from8.5 kg to 18.0 kg. Thus sheep were groupedaccording to their body weight (Table 1).Statistical  Analysis:All the data were then subjected to Least-Squares analysis (1966) for studying the effectsof season, sex and body weight andhaemoglobin type on EPG of adult sheep. Themodel used for analysis was-Yijklm = m + Ai +Bj +Ck +Dl +eijklmWhere Yijkl is the observation on the mthindividual in  ith season  of  jth sex of  kth bodyweight group and  lth haemoglobin type.m = General effect (Overall mean commonto all observations);Ai = Effect of the  ith season  (i=1,2,3);Bj = Effect of the  jth sex (j=1,2);Ck = Effect of the  kth  body weight group(k=1,2,3,4);Dl = Effect of the  lth haemoglobin type(l=1,2)  andeijkl = random error assumed to be normallydistributed with zero mean and variance,ó2e .Duncan’s Multiple Range Test (Kramer Body weight of sheep  Body weight group Up to 10 Kgs Group-I< 10 Kgs to 12 Kgs Group-II< 12 Kgs to 14 Kgs Group-III< 14 Kgs Group-IVTable 1: Body weight groups of adult sheep.Factors  affecting  variability  of  resistance  in  garole  sheep naturally  infected...60Exploratory Animal and Medical Research, Vol.3, Issue -1, June, 20131957) was performed to examine thesignificant differences between meanswhenever the analysis of varianceshowed significant differences fordifferent factors.RESULTS  AND DISCUSSIONThe resistance status was evaluatedby measuring the level of Haemonchuscontortus infections through eggs pergram in faeces (EPG). In the presentstudy the EPG count in the Garolesheep was varied from 300 to 1600.In Garole sheep the overall EPG forHaemonchus contortus have beenrecorded as 829.96 ± 20.60 (Table 2).The effect of season, sex, body weightand haemoglobin type was all foundto be highly significant (P < 0.01) onEPG.SEASON: EPG count was highestduring monsoon (986.27 ± 28.26),followed by summer (832.88 ± 28.26)and lowest during winter (670.74 ±28.26). This indicates the existence ofa seasonal variation in patterns of FEC(Table 2 & Fig.1). The findings ofTembely et al. (1998) and Doligalskaet al. (1997) were in agreement withthe results obtained in this study withrespect to the natural infection onpasture. Seasonal variation in FEC ismost likely to be dependent onvariation in availability of infectivelarvae on pastures. Two peaks in FECcan be seen: one during early summerdue to development of over-winteredlarvae; and another in monsoondepending on weather condition(Gregory et al. 1940; Whittier et al.1997). During monsoon animals were exposed to higherinfection levels on pasture, owing to favourable conditionsfor larval survival leading to increase in FEC. But in winterthe phenomenon is just reverse leading to lowest FEC.SEX: Rams had higher EPG (954.32 ± 57.93) than ewes(705.60 ± 45.79) as presented in Table 2 & Fig. 2. Malesheep appeared to be more susceptible to H. contortusEffects Number EPG  (Mean ± SE)Overall 309 829.96 ± 20.60Season:Summer 103 832.87 ± 28.26 bMonsoon 103 986.27 ± 28.26 aWinter 103 670.74 ± 28.26 cSex:Male 135 954.32 ± 57.93 aFemale 174 705.60 ± 45.79 bBody weight group:Group-I 87 1017.20 ± 54.82 aGroup-II 78 886.79 ± 56.23 bGroup-III 30 737.18 ± 50.30 bGroup-IV 114 678.68 ± 54.49 bHaemoglobin type:Hb-BB 252 983.80  ± 18.22 aHb-AB 57 676.12 ± 33.96 bTable 2 :  Least Squares means of EPG in adult sheep.Means under a particular effect in a column havingdifferent superscripts differed significantly.61infections compared to female sheep. Courtneyet al. (1985a) Barger (1993), Woolaston andPiper (1996) also found that females were moreresistance to natural as well as experimentalinfection with T. colubriformis and H.contortus. Females had lower FEC than malesafter puberty, although there were no differencesbefore puberty. Our results with adult sheep arethus consistent with earlier reports. Thesedifferences may be due to a stimulatory effectof estrogens on immune responses and thatandrogens may actually have an opposite effect(Woolaston and Piper 1996).BODY WEIGHT: Animals with lower bodyweight i.e. group I animals showed higher EPG(107.20 ± 54.82), then the infection leveldecreases as body weight increases (i.e. forgroup II EPG was 886.79 ± 56.23 and for group-III EPG was 737.18 ± 50.29) and lowest EPGwas recorded in group IV animals (678.68 ±54.49). The appreciable differences amongst thegroups as encountered in the present study(Table 2 & Fig. 3) may be attributed due todifferences in immuno-competent resistancestatus against natural infection of H. contortus.Hb Type : It has also been observed that Hb-BB type animals (Fig. 4) showed higher EPGcount (983.81 ± 18.22) in comparison to Hb-AB type animals (676.12 ± 33.96) as presentedin Table 2 & Fig. 5. The reports of Allonby andUrquhart (1976), Altaif and Dargie (1978),Preston and Allonby (1979b) were in agreementwith the results of our study. They reported thatanimals with haemoglobin type-AA (HbAA)were more resistant than Hb-AB, which againmore resistant than Hb-BB to infection with H.contortus and its effects. This study alsorevealed that haemoglobin-A locus was relatedwith resistance and/ susceptibilityFig. 1:  EPG of adult Garole sheep in differentseasons.Fig. 2:  EPG of adult Garole sheep in differentsexes.CONCLUSIONThe variability of resistance to naturalinfection of H. contortus in Garole sheep wasstudied for some intrinsic factor like age, sexand body weight of sheep and extrinsic factorlike season. This study recorded significantvariation (p< 0.01) of response to naturalinfection for all these intrinsic and extrinsicfactors. Lowest faecal egg count (EPG) wasobserved in winter and highest in monsoon.Male was found to be more susceptible toFactors  affecting  variability  of  resistance  in  garole  sheep naturally  infected ...62Exploratory Animal and Medical Research, Vol.3, Issue -1, June, 2013infection rather than female. In adult sheep thehigher body weight sheep possessedsignificantly higher resistance than the lowerbody weight group.  Haemoglobin AB typeanimals showed significantly (P<0.01) betterresistance than the Haemoglobin-BB typeanimals. On the basis of the results obtainedand discussion it is concluded that variabilityof resistance to infection with H. contortus isexisted in Garole sheep and there is ample scopefor increasing the resistance through selectivebreeding / assortive mating. During selectioninclusion of haemoglobin type as an indicatortrait may be helpful to achieve the goal early.Further investigation on resistance status andvariation in the artificial infection as well asidentification of specific marker gene forresistance to H. contortus may be carried outas these are beyond the purview of the presentstudy. This may be give us a clear picture aboutthe genetic basis of resistance to H. contortusinfection in Garole sheep, which may be helpfulto develop a resistant population.Fig. 3:  EPG of adult Garole sheep in differentbody weight groups.Fig. 4:  Starchgel electrophoresis pattern of different haemoglobin.63REFERENCESAllonby EW and Urquhart GM.(1976). Apossible relationship between haemonchosis andhaemoglobin polymorphism in Merino heep inKenya.  Research  Vet. Sci.  20: 212-214.Altaif KI and Dargie JD.( 1978). Geneticresistance to helminthes. 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FACTORS AFFECTING VARIABILITY OF RESISTANCE IN GAROLE SHEEP NATURALLY INFECTED WITH HAEMONCHUS CONTORTUS

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