Cryopreservation of the infective larvae of the common nematodes of ruminants

Abstract

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% NaCI 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 > 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, > 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, 0. circumcincta, T. colubnformis, T. falculatus, N. spathiger, C. ovina, H. placei, 0. ostertagi, Cooperia spp. and N. helvetianus were recovered from the faeces of animals infested with cryopreserved L 3. No ova of 0. columbianum or 0 . 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. ovina, D. filaria, H. placei, 0. ostertagi, Cooperia spp., N. helvetianus and 0. radiatum have been shown to be infective after freezing in liquid nitrogen (gas phase), and that G. pachyscelis, S. papillosus and B. phlebotomum have been found to survive similar freezing and thawing, even though they do not appear to be infective thereafter.