Survival and infectivity studies of in-vitro cultivated larvae of Haemonchus contortus in Sheep and Goats in Nigeria

The survival and infectivity of indigenous isolates of Haemonchus contortus and in vitro cultivated infective larvae (L ) were studied. Active motile gravid females, adult and immature parasites were morphologically and 3 morphometrically characterized and isolated from Haemonchus infected goats. These were subsequently inoculated into the abomasum of parasite free lambs through left sided laparatomy for production of fertile eggs to be used for 0 coproculture. Harvested faeces from these lambs were cultured in a nutritive medium and kept in the incubator at 32 C 0 and room temperature 24-28 C to obtain in vitro cultivated L of H. contortus. The infectivity and survival of in vitro 3 0 0 cultivated L stored in refrigerator at 4-8 C and under room temperature 24-28 C were evaluated. Incubation period of 3 o 0 coproculture was observed to be shorter in medium at 32 C than under room temperature 24-28 C. The storage of in vitro cultivated L under different temperature range showed that the quantity of active larvae in a given volume 3 th 0 0 th decreased at p<0.05 from the 5 day under 24-28 C while at 4-8 Cbecame significantly lower from 20 day. Infectivity 0 0 of isolate kept at 4-8 C was sustained for 18-20 days while at 24-28 C it was maintained for 10-12 days. Laboratory invitro cultivated L of H. contortus had limited infectivity and larvae mortality increase with time under different 3 temperatures.


Introduction
cultivated larvae from local strain are to be assessed as a prelude to determining their immunogenicity and Gastrointestinal parasitism had been identified usefulness as candidate vaccine in the future.as a complex disease constituting a major impediment to efficient and profitable livestock production Materials and Methods particularly among sheep and goats all over the world Experimental animals and their management: (Baker et al., 1999, Chiezey et al., 2008).
Two (2) male lambs aged 6-7 months old and five (5) Among various intestinal parasites H. contortus West African Dwarf (WAD) kids (6-8 months) were is the most important and it is responsible for most sourced from markets and house-hold keepers in rural field outbreaks of acute and sub acute parasitic areas in the outskirts of Abeokuta.The ethics committee gastroenteritis of small ruminants (Fakae et al., 1999, of the college of veterinary medicine university of 2004).The potential use of vaccines as a means of Abeokuta approved the study.The experiment was controlling gastro intestinal parasitic infection appears carried out in a goat house belonging to the College of to be a promising and viable option (Bowels et   both storage methods were tested every two days to were fed with grasses, harvested from fields not ascertain for how long the pathogenic strength can be previously grazed by any livestock and after harvest sustained.grasses were washed with 1% salt solution and sun Five hundred (500) L obtained from culture 3 dried before feeding (to take care of wild life helminth medium was inoculated per os into 5 worm free WAD larvae).Tsetse fly traps were strategically located kids.Incubation period, clinical manifestation, around the pen house.Spp.Gravid adult and immature H. contortus were 0.02mgkg ) to ascertain that the Haemonchus worms obtained from these abomasa and worms identified isolated were not antihelmintic resistant.according to Soulsby, 1982, Hasen and Perry 1991 and graphical representation of rate of decrease of larvae and antihelmintic susceptibility of isolate: The quantity showed an intersection at a point of storage 12 number of infective larvae in a given volume was temperatures (Figure II).The interactions between the concentration of toxic metabolites.These factors may two storage temperatures revealed the same quantity be responsible for the longevity of larvae and of larvae at this intersection (16 active larvae at day 3 prolonged infectivity at lower temperature.The sharp rd 0 post storage).The storage of motile 3 stage larvae of decrease of 57% in larvae count stored under 24-28 C 0 0

Zajac
Haemonchus contortus in the refrigerator at 4-8 C compared with 6% of 4-8 C stored larvae 5 days post showed that larvae infectivity was maintained for 18-storage (Figure II).The reasons for this drastic 0 0 20 days.However storage at 24-28 C showed the decrease at this 24-28 C temperature may also be in infectivity was sustained for 10-12 days.l i n e w i t h t h e e x p l a n a t i o n above owing to the fact that the more metabolically active parasites will exhaust

Discussion
the available nutrients and accumulate wastes more rapidly than the larvae at refrigeration temperature.Our observation of shorter incubation period at higher ambient temperature is consistent with earlier The time length of infectivity of 10-12 days observations of Hansen and Perry 1991, Zajac et al., observed in this study can be used to determine 2006 who reported variation in incubation periods of intervals of deworming animals in the field during in-vitro cultivated larvae under different incubating strategic control of helminthiasis.temperatures.However Graber, 1975Soulsby, 1982, This observation showed the importance of Stankiewiez et al., 1996 reported that under natural locally sourced strain of nematodes as its gives a true 0 environmental ambient temperature of 22-30 C H. reflection of infectivity as indicated in the microcontortus eggs hatched to L within 4-5 days, these 3 environment where these parasites are found.findings indicated existence of temperature dependent By this study, it is proposed that in our search for differences on the hatchability of Haemonchus eggs.It a candidate vaccine for the control of haemonchosis in is in our thinking that current global changes in the small ruminants, temperature variations may play a climate and environmental temperature may have a vital role in the modification of virulence/ pathogenicity direct bearing with the modification of the physiology of these parasites.and pathogenicity of these parasites.

2 estimated
by taking 0.02ml of cultured medium These animals were kept in individual pens (3 ½ ft ) R solution and 0.001ml of tincture iodine.The addition with slatted floor and treated with Ivomec -1 of iodine immobilizes the infective larvae to enable (Ivermectin 0.02mgkg ), Coccidiostat (200mg accuracy in the counting.This process was repeated Trimethroprim and 100mg Sulphadiazine) by thrice and the average was taken for cultured medium Chanelle Pharmaceuticals limited Spain, Provethrine 0 0 ® stored at 4-8 C and 24-28 C. The quantities of larvae in 30 Cypermethrine 3% (Panteck Holland.Berenil ® -1 a given volume were evaluated on days 0,5,15 and 20.Diaminazine Aceturate 3.5mgkg for the control of The solution containing the in-vitro cultivated infective endoparasites, ectoparasites and haemoparasities.0 larvae was kept in the refrigerator at 4-8 c and another After acclimatization, animals were vaccinated with kept on the work bench in the laboratory at room homologous Pests des Petites Ruminant vaccine 0 temperature of 24-28 C. The infectivity of larvae in (PPR) Vom, Jos, Nigeria.All experimental animals , et al., 2006.These parasites were washed in a In vitro cultivation of third stage infective larvae nutritive medium prepared as described by Hubert and 0 of H. contortus: The egg in cultured medium at 32 C kerbeouf 1992 (medium contained 5µl Amphotericine rd in the incubator hatched into 3 stage infective larvae B, which inhibits fungi growth), 1gm yeast extract in 0 within 5-8 days while on the work bench at 24-28 C in 90ml of normal saline solution 1:9 v/v (for the laboratory it took 14-19 days (figure I).The mean nourishment of hatched larvae) and Earle's balance values of hatchability under different incubating salt solution for electrolyte balance.Three hundred 0 temperatures were 11.33 and 11.88 under 32 C and 24-(300) very active gravid females, adult males and 0 28 C respectively.The comparisons of mean larvae immature H. contortus were identified microscocount between two incubating temperatures using Tpically and were transferred into the 2 male lambs test indicated no significant differences (t=0.43,df through left sided laparatomy.Thereafter the lambs =10 at p>0.05).were kept in isolation pen and fed on grasses harvested Storage interval of in vitro cultivated larvae from field where livestock do not graze.under different temperature: The quantity of In-vitro cultivation of third stage infective larvae in a given volume was represented by the larvae of H. contortus (L ) : Faeces harvested from 3 number of active larvae in field viewed under the inoculated lambs were processed using Baerman's microscope at x 400 objective.The quantity of active technique by pooling the eggs of Haemonchus spp larvae in a given volume decreased over a period of together, wrapped in Chinese cloth and submerged in time under both storage temperatures (Figure II).nutritive medium solution in a funnel and the base was 0 However larvae stored under 24-28 C became attached to a rubber tube to remove fluids.Ten (10) significantly lower at p<0.05 from 5 day post storage replica solution were made and five (5) of which were 0 0 while at 4-8 C a decrease with no significant kept in an incubator at 32 C and remaining 5 (five) on 0 difference (p>0.05) from day 0 to day 15 but became the work bench at 24-28 C in the laboratory.significantly decreased from day 20 post storage.The Estimation of larvae number, storage interval

FigFig . 2 Effe c t o f s to r a g e te m p e r a tu r e o n in vitr o c u ltiva te d la r va l c o u n ts o f H a e m o n c h u s c o n to r tu s
Fig.1 Hatchability count of in vitro cultivated larval of Haemonchus contortious al., Veterinary Medicine, University of Agriculture 1995, Chiejina et al., 2002, Meeusen and Piedrafita Alabata, Abeokuta, Ogun state, Nigeria.Alabata lies at 2003, Newton and Meeusen, 2003).Previous studies