Parenterally administered gastrointestinal nematode infective larvae viable after more than 15 years in liquid nitrogen.

After cryopreservation for 13.3-15.8 years, the viability of the infective larvae (L3) of Trichostrongylus axei, T. colubriformis, Oesophagostomum columbianum, Haemonchus contortus, Ostertagia circumcincta, T. falculatus, Nematodirus spathiger, Chabertia ovina and Dictyocaulus filaria was assessed in sheep, by being deposited at their predilection sites. D. filaria was, however, an exception, in that the L3 were injected into the jugular vein. The mean development of all the species was 22.8%, but if three species (O. columbianum, C. ovina and D. filaria), that developed poorly are disregarded, then the mean development was 33.4%, similar to previous tests after shorter periods of cryopreservation. The L3 of some of the species appeared sluggish when examined 10-15 min after being thawed, and in the case of H. contortus practically all the larvae of the original batch tested in the previous trials of the series appeared dead when thawed for use in the present trial, and were replaced by another batch of L3 of the same species. When re-examined after about 8 h, however, a high percentage of the L3 of the original batch appeared to have become revitalised, and their viability was tested in a trial reported elsewhere. The intestinal cells of the majority of the L3 of N. spathiger, O. circumcincta and C. ovina were vesiculated when they were thawed. Nevertheless, the degree of development of the former two species was of the highest in the trial, and it can be concluded that this phenomenon does not necessarily impede the viability of larvae.

Recovery of nematodes from ruminants by migration from gastro-intestinal ingesta and mucosa gelled in agar: preliminary report.

When gastro-intestinal ingesta and mucosa containing larvae or adult Haemonchus contortus, Ostertagia circumcincta. Trichostrongylus colubriformis, Strongyloides papillosus, Nematodirus spathiger, Gaigeria pachyscelis, Oesophagostomum columbianum or Chabertia ovina were suspended in 0,75--1,00% agar gel and incubated in physiological saline, a mean of 93,6% of the helminths migrated from the agar-ingesta or agar-mucosa into the physiological saline. The lowest suitable concentration of agar was 0,85--0,90% for one batch of agar and 0,65% for another. Although most of the worms had migrated from the agar gel after 3--4 h of incubation, migration usually continued for longer than 7 h. While low concentrations of formalin partially inhibited migration from the gel, slightly more worms were recovered from agar containing 1% bile than from agar alone.

Survival and development of larvae of the common nematodes of ruminants after long-term cryopreservation and investigation of different routes of infestation.

Exsheathed infective larvae (L3) of 16 species of nematodes were tested for infectivity in either sheep or cattle after they had been frozen and stored in 0,09% NaCl solution in the gas phase of liquid nitrogen for periods of up to 59 months. A mean of greater than 90% of the L3 of Haemonchus contortus, Ostertagia circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Nematodirus spathiger, Oesophagostomum columbianum and Chabertia ovina of sheep and Haemonchus placei, Ostertagia ostertagi, Cooperia spp. (C. pectinata and C. punctata), Nematodirus helvetianus and Oesophagostomum radiatum of cattle was alive when they were thawed, after having been frozen for 52-59 months. These L3 as well as those of Marshallagia marshalli and Trichostrongylus falculatus, which had been frozen for 30-33 months, were infective to sheep or cattle when dosed per os or inoculated into the abomasum or the duodenum. Thawed Dictyocaulus filaria L3, frozen for 31 months, developed poorly when injected intravenously into sheep. This appears to be the first report showing infectivity of L3 of O. circumcincta, T. colubriformis, N. spathiger and, possibly, of O. radiatum by the oral route after cryopreservation in liquid nitrogen.

A field strain of Haemonchus contortus showing slight resistance to rafoxanide.

A field strain of H. contortus, already resistant to benzimidazole anthelmintics, was also found to be slightly resistant to rafoxanide. This is apparently the first report of resistance to rafoxanide in a field strain of H. contortus.

Benzimidazole-resistant Haemonchus contortus--the effect of cryopreservation on the resistance of successive generations.

Infective larvae (L3) of a strain of benzimidazole-resistant Haemonchus contortus were stored in the gas phase of liquid nitrogen, thawed and used to infest worm-free sheep from which, in turn, larvae were cultured, frozen and thawed. This cycle was repeated 5 times. Thereafter, the progeny of the cryopreserved larvae were compared with the progeny of the original untreated larvae for susceptibility to benzimidazole anthelmintics. Repeated freezing of consecutive generations of L3 in liquid nitrogen did not appear to affect their relative resistance to benzimidazoles and, although other strains must also be tested, it would seem that cryopreservation may be used for storing resistant strains.

Oral infectivity of cryopreserved Haemonchus contortus infective larvae that recovered relatively slowly when thawed after more than 15 years in liquid nitrogen.

Infective larvae (L3) of two different batches of anthelmintic-susceptible Haemonchus contortus, cryopreserved for 15,6 and 15,7 years in liquid nitrogen appeared dead when examined 10-20 min after thawing. However, having become motile later in the day, their viability was tested the next day in two separate groups of sheep. The mean development of 41,8% and 66,3% of the H. contortus compared favourably with previous trials with this strain, in which the L3 were injected into the abomasa of sheep after various periods of cryopreservation. It is speculated that the relatively slower recovery rates after thawing of the L3 used in this trial, when compared with those in previous trials in which one of the two batches of susceptible H. contortus was used after shorter periods of cryopreservation, may be an indication that these L3 were gradually approaching the limits of survival after almost 16 years in liquid nitrogen.

A technique for the recovery of nematodes from ruminants by migration from gastro-intestinal ingesta gelled in agar: large-scale application.

A gelled-agar technique for worm recovery was adapted to facilitate the recovery of larval and adult nematodes from the total ingesta of large numbers of sheep. The technique was also used to recover nematodes from 4 calves. In one trial involving 120 sheep, 100% of 2013 4th stage larvae (L4) and 92,1% of 134,205 adult Haemonchus contortus migrated from the agar preparations. Highly significantly more male than female worms failed to migrate. Using 1 x 1/10 aliquot to estimate the numbers of worms that failed to migrate from the agar, the mean error in the total worm count (worms that migrated plus those that failed to migrate) per sheep was 2,2%; with an examination of 2 x 1/10 aliquot the error was 1,7%. We concluded from this that the gelled-agar method may be of value for quantitative worm recovery, for example, in anthelmintic tests. In a second trial, 98,5% of 17,056 L4 and adult nematodes of 5 genera migrated from the ingesta of 4 calves and 96,4% of 62,597 L4 and adult nematodes of 9 species from the ingesta of 15 sheep. In general, L4 migrated slightly more efficiently than adult worms. In sheep and, to a lesser extent, in calves, Haemonchus spp. did not migrate as efficiently as the other genera such as Ostertagia, Trichostrongylus, Nematodirus, Oesophagostomum, Marshallagia and Chabertia.

Slight resistance to the residual effect of closantel in a field strain of Haemonchus contortus which showed an increased resistance after one selection in the laboratory.

On the strength of a complaint of suspected resistance to closantel, a field strain of Haemonchus contortus was isolated from sheep on a farm near Pretoria and tested for resistance to the remedy, both without exposure to closantel in the laboratory (designated the "unselected" substrain of the parasite) and after a single selection with closantel (called the "selected" substrain). For comparative purpose a few sheep were treated with rafoxanide to ascertain whether the efficacy of this drug was unchanged. While the unselected substrain appeared to show only slight increased resistance to the residual effect of closantel drenched at a dosage rate of 5 mg/kg, there was a dramatic increase in resistance after one selection. The residual efficacy (determined by the modified NPM test of Groeneveld & Reinecke, 1969, as outlined by Reinecke, 1973), 13-14 days after treatment with closantel was reduced from the registered claim of the remedy in South Africa of greater than 80% effective in greater than 80% of the treated flock to "ineffective", or less than 50% effective in less than 50% of the treated flock. Furthermore, there also appeared to be a slight increase in the resistance of this substrain to rafoxanide, as its efficacy was reduced from 99-100% to about 92%. This sharp reduction in efficacy after a single selection with closantel seemed to indicate that the slight increase in resistance of the unselected substrain was probably due not to possible variation in the repeatability of the trail method but to repeated treatment with closantel on the farm.

Cryopreservation of the infective larvae of the common nematodes of ruminants.

Exsheathed infective larvae (L 3) of 19 species of nematodes were tested for infectivity in either sheep or cattle after they had been frozen in 0,9% NaCl solution, stored for a relatively short time in the gas phase of liquid nitrogen and subsequently thawed. In addition, 13 of these species were tested after similar storage for up to 18 months. In sheep, Haemonchus contortus, Ostertagia circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Nematodirus spathiger and Oesophagostomum columbianum were viable after 2 years of cryopreservation, a mean of greater than 90% of the L 3 being alive when thawed after this period. Similar results were obtained with Chabertia ovina L 3 after 18 months and with Marshallagia marshalli, Trichostrongylus falculatus and Dictyocaulus filaria, after a short period of freezing. On the other hand, Gaigeria pachyscelis and Strongyloides papillosus survived freezing for up to 7 months but neither was viable at the end of this period, nor was exsheathed G. pachyscelis viable without freezing. Most of these infestations were established by inoculating the infective larvae into the abomasum and/or duodenum. M. marshalli, T. falculatus and C. ovina also proved infective after oral dosing. D. filaria, the only other species tested by this route, was not infective when dosed per os after thawing. The infective larvae of the bovine nematodes, Haemonchus placei, Ostertagia ostertagi, Nematodirus helvetianus, Oesophagostomum radiatum, Cooperia pectinata and Cooperia punctata survived freezing for a mean of 26 months, greater than 90% being alive on thawing, but infectivity was generally lower than with the same genera in sheep. Even when not frozen, exsheathed Bunostomum phlebotomum was non-infective. When Cooperia spp. after thawing were tested for infectivity by the oral route, more worms developed in one calf infested orally than in another infested by inoculation into the duodenum. Ova of H. contortus, M. marshalli, O. circumcincta, T. colubriformis, T. falculatus, N. spathiger, C. ovina, H. placei, O. ostertagi, Cooperia spp. and N. helvetianus were recovered from the faeces of animals infested with cryopreserved L 3. No ova of O. columbianum or O. radiatum were recovered from faeces, because differential larval counts were performed before they were patent. Nevertheless, gravid females were obtained post-mortem. Frozen L 3 of N. helvetianus were used to re-establish a pure strain in calves, 2,3 million ova being recovered from infestations with 10 670 L 3 frozen for 26 months. The infectivity of the progeny of frozen L 3 was tested with M. marshalli and C. ovina. In both instances infectivity was high and the worms which developed also produced ova, thus completing the cycle. This appears to be the first report of infective larvae of parasitic nematodes retaining their infectivity after being frozen in liquid nitrogen (gas phase) for longer than 2 years. This is also apparently the first time that M. marshalli T. colubriformis, T. falculatus, T. axei, N. spathiger, C...

Cryopreservation of some common nematodes of ruminants for up to 11,3 years.

Infective larvae of selected batched of the following nematode species from sheep and cattle were examined for survival and infectivity (by injection into either the abomasum, the duodenum, or the jugular vein) after having been stored in liquid nitrogen for 103-136 months: Haemonchus contortus, Haemonchus placei, Ostertagia circumcincta, Ostertagia ostertagi, Marshallagia marshalli, Cooperia spp., Trichostrongylus axei, Trichostrongylus colubriformis, Trichostrongylus falculatus, Nematodirus spathiger, Nematodirus helvetianus, Oesophagostomum columbianum, Oesophagostomum radiatum, Chabertia ovina and Dictyocaulus filaria. Excluding D. filaria, a mean of 97.7% of the ovine and 96.0% of the bovine nematode larvae were alive when thawed. Compared with previous investigations in this series, little or no reduction occurred with time in either the survival or the viability of the nematodes from cattle, or in the survival of those from sheep. In contrast, the larvae developed poorly in sheep, possibly owing to parenteral treatment of the animals with ivermectin at a dosage of 0.4 mg kg-1, either 6 or 8 days before they were infected.

Methods of infesting sheep with gastro-intestinal nematodes after cryopreservation: dosing of larvae in gelatin capsules compared to dosing of larvae in water suspension.

Cryopreservation of the infective larvae (L3) of nematodes is being used increasingly for the routine maintenance of pure strains of nematodes in the laboratory. Gelatin capsules are frequently used to administer the L3 of nematodes to sheep, but with some nematode species this method usually does not give good results with cryopreserved larvae. The development in sheep of cryopreserved L3 of Trichostrongylus spp. and other ovine nematodes was compared when the larvae were administered either in a suspension or in gelatin capsules with or without the use of CuSO4 to stimulate the oesophageal groove reflex. Significantly larger numbers of cryopreserved L3 developed when dosed per os in suspension than when the L3 were dosed in gelatin capsules. Stimulation of the oesophageal groove did not appear to affect the numbers of worms that developed from L3 dosed in suspension. It is speculated that L3 in suspension bypass the rumen to go directly into the abomasum, while those in gelatin capsules enter the rumen, thus closely approximating the natural infestation of grazing ruminants. In these trials, however, only cryopreserved L3 were used. Sufficient numbers of cryopreserved L3 of Trichostrongylus falculatus and T. colubriformis in suspension developed, so that it seems unlikely that laparotomy will be required for routine infestations in the laboratory.

Two field strains of Haemonchus contortus resistant to rafoxanide.

Two field strains of Haemonchus contortus, one from Pretoria on the Highveld and the other from White River in the Lowveld of the Transvaal, showed pronounced resistance to rafoxanide at a dosage rate of 7.5 mg/kg live mass per os. Both of these strains originated from kikuyu pastures under irrigation. Resistance in South Africa to rafoxanide and other anthelmintics, namely, closantel, ivermectin and the benzimidazoles is reviewed. It is concluded that apart from avoiding farming practices where integrated worm control is not possible, there is at present no solution to the problem of worm resistance in the country.

The problem of escalating resistance of Haemonchus contortus to the modern anthelmintics in South Africa.

During the past decade in South Africa there has been a continual increase in sheep of strains of gastrointestinal helminths resistant to the modern anthelmintics. Five strains of Haemonchus contortus are described in this paper. Despite the fact that 2 of the 5 strains were tested for susceptibility only to ivermectin, a total of 10 instances of resistance were found. Four of the 5 strains were resistant to ivermectin, 2 to closantel, 2 to rafoxanide and 2 to the benzimidazoles. One of these strains was concurrently resistant to 3 different anthelmintic groups, namely, the ivermectins, the benzimidazoles and the salicylanilides. Resistance to ivermectin developed in 2 strains of H. contortus after a history of only 3 treatments with this compound in one instance and 11 treatments in the other. In the latter case drenching with ivermectin was well interspersed with that of other anthelmintics. This rapid development of resistance suggests that there may be cross-resistance between ivermectin and another anthelmintic group. Two of the ivermectin resistant strains were recovered from separate properties in the south-western Cape Province, where Ostertagia circumcincta, which is usually the dominant parasite in this region, was virtually eliminated by the anthelmintic treatment. On each of these properties it was apparently replaced by a resistant strain of H. contortus. A serious threat to control is the dissemination of worm strains with multiple resistance to anthelmintics. The strain of H. contortus resistant to 3 anthelmintic groups has already been widely dispersed, as the farmer concerned suddenly decided to give up farming with sheep and to sell his flock.

A field strain of Trichostrongylus colubriformis resistant to levamisole and morantel in South Africa.

A strain of Trichostrongylus colubriformis from Nottingham Road, in Natal, was found to be solidly resistant to levamisole and morantel at the recommended dose levels. Untreated control sheep in fact harboured fewer worms at slaughter than either of the 2 treated groups. In contrast, the benzimidazoles and ivermectin were more than 99.9% effective against this worm strain. The possible implications of escalating resistance to anthelmintics in the gastrointestinal nematodes of sheep are discussed.

Percutaneous infestation of calves and lambs with Oesophagostomum spp.

Oesophagostomum radiatum and Oesophagostomum columbianum became established in low numbers when 2 calves and 2 sheep respectively were exposed percutaneously to pure cultures of infective larvae of these worms. A further sheep infested in the conjunctival sac became heavily infested but this may have occurred via the lachrimal duct. As far as can be ascertained this is the first time that development of O. columbianum has been demonstrated after percutaneous exposure.

[The threat of wide dissemination of anthelmintic resistance by veld ram performance testing units]. / Die gevaar van wye verspreiding van weerstandbiedendheid teen wurmmiddels deur veldramprestasietoetssentra.

A veld ram performance testing unit consists of an association of stud or commercial ram breeders, who compare the performance of selected rams under field conditions on common pastures. The best performers are subsequently auctioned at public sales. To date, at least 10 Merino veld ram clubs have been established in various centres in South Africa. A strain of Haemonchus contortus, which was isolated from the pastures of one of these performance testing units, was found to be resistant to ivermectin, oxfendazole and rafoxanide. Closantel, levamisole and disophenol were more than 99% effective. The wide dispersal of the rams after testing, constitutes a potential danger for dissemination of helminths with resistant genes. Precautionary methods should include careful routine monitoring of drug susceptibility on the communal pastures, on the farms of individual members and thorough deworming of rams before they leave the testing unit. The prevention and control of resistance in such testing units are discussed, but it is emphasised that this is difficult, particularly if the worm strains on the communal grazing have become resistant to some of the anthelmintic groups.