Experimental hybridization between Oesophagostomum dentatum and O. quadrispinulatum in pigs

To investigate eventual hybridization between two nodular worm species of pigs, Oesophagostomum dentatum and O. quadrispinulatum, we used either mature, adult worms or 10-day-old fourth-stage larvae (L₄) as starting material, employing a nonsurgical transplantation technique. Following the transfer of adult worms the ensuing first generation of larvae gave rise to adult worms that were found by morphological examination to be purely O. dentatum. Therefore, we decided to use the immature L₄ as starting material. After the transfer of L₄ to recipient pigs, fecal cultures were established and the L₃ derived from the O. dentatum male/O. quadrispinulatum female cross gave rise to adult but infertile worms, which morphologically had the sexual characters of their parent generation, whereas other characteristics were intermediate between the two species. Attempts to reproduce the hybrid worms or the reciprocal cross were unsuccessful, indicating that hybridization between the two species is a rarely occurring phenomenon. Received: 6 April 1997 / Accepted: 15 May 1997     

Development and infectivity of eggs and larvae derived from pigs trickle-infected with Oesophagostomum dentatum at different dose levels

We examined the impact of different Oesophagostomum dentatum dose levels and durations of infection on the development and infectivity of the following generation. Pigs were trickle-infected with 200, 2,000 or 20,000 L₃/week over 20 weeks. Egg hatch assays were performed at monthly intervals; however, no consistent differences were found between any of the dose groups in the development of eggs into first-stage larvae. To compare larval infectivity, larvae were derived from faecal cultures set up from the low- and the high-dose groups in the early and the late part of the experiment, and were inoculated into helminth-free pigs (5,000 L₃/pig). Worm establishments were significantly higher (P < 0.05) in the group of pigs receiving larvae derived early in the experiment from the low-dose group compared with the two groups receiving larvae from high-dose groups, thus indicating an adverse effect of high doses of trickle infection on the later infectivity of L₃ larvae derived from excreted eggs. Received: 16 March 1998 / Accepted: 15 June 1998     

In vitro development of histotropic larvae of Oesophagostomum dentatum under various conditions of cultivation

We performed a total of 14 trials to evaluate the culture conditions suited best for the in vitro production of fourth-stage larvae (L4) of Oesophagostomum dentatum. Chicken embryo extract was shown to be redundant, whereas pig blood serum, trypticase, liver extract, and yeast extract proved to be important medium ingredients. Development to L4 could be moderately accelerated by increases of temperature (40 °C) and of CO₂ concentration (20% in air), but the total yield of L4 could not be improved. Thus, conditions of 38.5 °C and 10% CO₂ are recommended for routine application. Received: 15 June 1998 / Accepted: 9 July 1998     

Growth and fecundity of Oesophagostomum dentatum in high-level infections and after transplantation into naive pigs

 This experiment was designed to examine the growth, proportion of stages, and fecundity of an Oesophagostomum dentatum population by transplantation of a known small number of worms from a high-density population into helminth-naive recipient pigs. Approximately 1,500 4-week-old worms [69% fourth-stage larvae (L₄), 31% adult worms] were transplanted into each of 5 recipient pigs (group B), and these pigs, along with a group of 5 high-level-infection control pigs (group C), were killed at 4 weeks after transplantation to determine and compare the worm burdens. By 2 weeks after transplantation and throughout the experiment, fecal egg counts of group B exceeded those of group C and the fecundity of the worms was higher, though not statistically significantly so, in the transplanted worms. In the recipient pigs, all worms (approx. 70% establishment) had developed to the adult stage and were significantly longer than worms recovered from the group C pigs. Received: 17 October 1995 / Accepted: 31 October 1995     

Control of Oesophagostomum dentatum and Hyostrongylus rubidus in outdoor-reared pigs by daily feeding with the microfungus Duddingtonia flagrans

 An investigation of pasture-reared pigs experimentally infected with Oesophagostomum dentatum and Hyostrongylus rubidus showed that daily doses in the feed with the microfungus Duddingtonia flagrans over a 2-month period led to lowered herbage larval infectivity of both species. This was further substantiated by low worm recoveries in initially parasite-naive tracer pigs that were later introduced to the pasture plot. The control setup comprised the release of similarly infected but nondosed pigs on a plot of the same area, followed by a group of tracer pigs. This paper discusses the potentials for using this biological control principle in the pig industry and emphasizes the research required, primarily regarding production technology and elaboration of feasible epidemiology-based dosing regimens, before such control can be implemented in practice. Received: 20 December 1995 / Accepted: 19 March 1996     

Comparative studies on the development of Oesophagostomum dentatum in vitro and in vivo

Although in vitro cultivation of Oesophagostomum dentatum provides defined material of third- (L3) and fourth- (L4) stage larvae, these are morphologically and biochemically different from larvae recovered ex vivo. The development of pre-cultivated larvae was investigated by rectal transplantation into worm-free pigs with subsequent recovery of worms from intestinal contents after different time periods and determination of worm burdens and sizes. Additionally, the in vitro maintenance of L4 and adults recovered from intestinal contents of orally infected pigs in different media was investigated. Although growth and development rates of cultivated L4 are lower than those of larvae recovered from intestinal contents after oral infection, pre-cultured L4 are able to develop into egg-laying adults in the large intestines (without nodule formation) within 9–14 days in rates comparable with those after oral infection. In contrast, rectally transplanted L3 only establish in low numbers without egg excretion. L4 and adult worms recovered from intestinal contents cannot be maintained in cultivation medium for more than 1 week, although most L4 grow and moult during the first 3 days. Although the standard cultivation conditions for mass production of L4 are not suitable for development or maintenance of preadult and adult stages, L4 recovered from cultures have the ability to establish in vivo as fertile adults, indicating that the basic biological functions are retained in vitro. Received: 29 July 2000 / Accepted: 7 August 2000     

Oesophagostomum species in pigs in england: 2. Oesophagostomum quadrispinulatum: Resistance to reinfection

Under experimental conditions, repeated infection with 500 larvae of Oesophagostomum quadrispinulatum induced a solid resistance in about 50 days, this being manifested by a failure of third-stage larvae to develop. An apparently stable population of a few hundred adult worms remained in the pigs for at least 200 days.     

Investigations into the production and function of leukotrienes during histotropic development of Oesophagostomum dentatum

 The dynamics of production and excretion of leukotrienes by parasitic larvae of Oesophagostomum dentatum and their role for the development of the larvae were studied. Larvae were cultured in vitro to the fourth stage (L4). Leukotriene B4 (LTB4) was detected in homogenates and in supernatant of larvae, with the homogenate-protein-based values steadily decreasing during development. The homogenate-protein-based values of peptidyl leukotrienes (pepLT) remained fairly stable in both homogenates and supernatants, whereas the worm-count-based pepLT values increased significantly. The addition of diethylcarbamazine (DEC) to the culture medium straight from the beginning of culturing (12.8 or 25.5 mmol/l) reversibly hampered growth and development to L4. Application of DEC at 12.8 mmol/l beginning on day 13 of in vitro cultivation exerted no significant effect on further development to L4. LTB4 appeared to counteract the inhibition of development by DEC. The results of this study indicate that endogenous LTs participate in regulation of the growth and development of O. dentatum. Received: 7 September 1995 / Accepted: 12 December 1995     

Oesophagostomum spp. in pigs: resistance to reinfection

For a study on the occurrence of resistance to reinfection with porcine nodular worm species, pigs were infected twice weekly with 1,000 infective larvae (L₃) of Oesophagostomumquadrispinulatum for 8 weeks. All pigs, including noninfected controls, were then treated with fenbendazole. At 10 days after treatment, all pigs received a single challenge inoculation of 5,000 L₃ of either O. dentatum or O. quadrispinulatum, respectively. Pigs were slaughtered at 6 weeks after the challenge infection for determination of their worm burdens. The pigs trickle- and challenge-infected with O. quadrispinulatum had significantly lower egg excretion levels (P < 0.01) and worm burdens (P < 0.05) than challenge control pigs, thus indicating some degree of host immunity against the homologous challenge infection. No resistance to reinfection was evident for the heterologous challenge infection. This study elucidates further aspects of the interaction between nodular worm species in the pig. Received: 17 April 1998 / Accepted: 8 June 1998     

Sequence variations in the introns of the triosephosphate isomerase genes of Oesophagostomum dentatum and O. quadrispinulatum

Degenerated primers were used to amplify DNA fragments of the triosephosphate isomerase (TPI) gene from complementary DNA (cDNA) and from genomic DNA of two species of porcine gastrointestinal nematodes, Oesophagostomum dentatum and O.quadrispinulatum. Polymerase chain reaction (PCR) fragments amplified from cDNA were 520 bp in size for both species, while genomic fragments were 1,035 bp for O. dentatum (GC-content: 45%) and 1,331 bp for O. quadrispinulatum (44%). Sequence analyses revealed blocks of high homology in the exons interrupted by more variable parts in the intron regions. Five exons were predicted from the genomic sequences in the conserved regions which corresponded to the respective cDNA sequences with 6% interspecific differences. The predicted protein sequences (161 amino acids) were 98% similar between the species and showed 71% similarity to the putative protein of Caenorhabditis elegans. As a housekeeping gene, TPI could be amplified from cDNA of both infectious third-stage larvae and adults. Interspecific variations in the non-coding regions allow the PCR-based differentiation of the two Oesophagostomum spp.     

Early third-stage larvae of Gnathostoma hispidum in cyclops

Surface structures of the early third-stage larae were examined by scanning electron microscopy. There were hemispherical head-bulbs at the apical ends. These bulbs could be clearly distinguished from the bodies. The head-bulbs (52 × 29 m) had four transverse rows of sharp booklets. The number of booklets in each row was 37, 36, 38, and 43, posteriorly. The larvae possessed a pair of lateral lips in the head-bulb. Each of the lips had two labial papillae and an amphid between the papillae. Small unidentate cuticular spines were present along the entire length of larvae on their transverse striations. The number of these cuticular striations was 175–217. A pair of cervical papillae and an excretory pore were present on the anterior part of the body. Another pair of papillae was detected laterally on the posterior one-third of the body. The shape of the posterior body papillae resembled that of cervical papillae. The cuticular spines were absent around the anus.     

Effect of temperature on activity of third-stage larvae of Angiostrongylus vasorum

To evaluate the effect of temperature on the activity and mortality of the L3 of Angiostrongylus vasorum, 1,500 L3 were isolated from experimentally infected snails and distributed into five equal groups. Three groups were incubated at 37°C, 27°C, and 5°C. The remaining two groups were incubated at 27°C and 5°C for 10 days, at which time the temperature for the 27°C group was reduced to 5°C and the 5°C group increased to 27°C. Larva activity was observed daily and inactive larvae were removed. At 37°C, larvae survived up to 8 days. At 27°C, larvae were active until day 6. When subjected to a reduction in temperature from 27°C to 5°C beginning on day 10, the number of active larvae increased until day 13. Only on day 17 did the number of active larvae decline to zero. At 5°C, larvae remained active until day 15, surviving to 24 days. When temperature was increased from 5°C to 27°C beginning on day 10, larvae were found active until day 12 and maintained an intermediate level of activity to day 21. Survival of larvae was greater at lower temperatures, while high temperatures were associated with higher mortality.     

A multiplex PCR tool for the specific identification of Oesophagostomum spp. from pigs

Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal deoxyribonucleic acid (DNA) of the porcine nodule worms Oesophagostomum dentatum and O. quadrispinulatum, a pair of specific primers (OdspF/OdspR2) for O. dentatum and a pair of specific primers (OqspF/OqspR) for O. quadrispinulatum were designed and used to develop a multiplex polymerase chain reaction (PCR) assay for the identification and differentiation of the two porcine nodule worms. This approach allowed the specific identification and differentiation of O. dentatum and O. quadrispinulatum, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the sequences amplified. The minimum amount of DNA detectable using this multiplex PCR assay was 0.1 ng for both O. dentatum and O. quadrispinulatum. The identity of 53 porcine nodule worms collected from pigs from different geographical localities in mainland China was ascertained as O. dentatum or O. quadrispinulatum, respectively, by this multiplex PCR method. This multiplex PCR assay is useful for the simultaneous identification of eggs of O. dentatum and O. quadrispinulatum and should provide a useful tool for the diagnosis and molecular epidemiological investigation of Oesophagostomum spp. infection in pigs.     

Improvements in the infectivity of cryopreserved third-stage larvae of Angiostrongyluscantonensis using a programmable freezer

Although there have been some advances in the cryopreservation of Angiostrongyluscantonensis, the degrees of viability and infectivity of the cryopreserved developmental stages have not been high. A two-step freezing protocol using a programmable freezer was determined to be effective in improving the infectivity of the cryopreserved third-stage larvae of this parasite. After washing steps and suspension in 10% (v/v) dimethylsulfoxide and equilibrium at room temperature the larvae were placed into the freezer. The temperature was lowered first at 0.8 °C/min from room temperature to −40 °C and then at 10 °C/min to −70 °C. The samples were plunged into liquid nitrogen. After storage in liquid nitrogen for 7–15 days the larvae were thawed rapidly in 37 °C water and 27.6% were found to show vigorous “S-shape” movement without significant changes in appearance. These larvae (50/rodent) could develop to the fifth stage in mice (42.6%) and establish patent infection in rats (40.4%). Moreover, there was no significant difference in the recovery rates of cryopreserved worms and their unfrozen counterparts. These findings indicate that steady precooling conditions may decrease damage with regard to the infectivity of cryptopreserved third-stage larvae of A. cantonensis. Received: 5 July 1998 / Accepted: 5 August 1998     

Induction of micronuclei in peripheral erythrocytes of axolotl larvae following in vivo exposure to mutagenic agents

Previous work from this laboratory demonstrated the presenceof micronuclei in erythrocytes from larvae of the urodele amphibianPleurodeles waltl reared in water containing clastogenic substances.In order to investigate the generality of this finding, larvaefrom another urodele Ambystoma mexicanum (axolotl) were rearedin water containing one of the two following compounds: benzo[a]pyrene(BaP) or ethylmethane sulphonate (EMS). The level of mironucleatederythrocytes on blood smears was compared with control samplesfrom larvae reared in fresh water. The optimum larval stagefor this test system was determined. The effects of the indirectmutagen (BaP), and the direct mutagen (EMS) were found to dependon both dose and exposure to the clastogen. Positive resultswere obtained for BaP after 8 days of treatment at a concentrationof 0.025 p.p.m. After 10 days of treatment at a concentrationof 0.1 p.p.m. numerous micronuclei were seen (> 250% ). Positiveresults were also obtained with EMS after 8 days of treatmentat a concentration of 24 p.p.m. At 62 p.p.m., positive resultswere found after 6 days of treatment, while at 124 p.p.m. positiveresults were found after only 4 days. The results with boththese agents show that the axolotl holds promise as an in vivotest system for the detection of low concentrations of clastogensin an aquatic environment. Address for reprint requests: Unité Associée no. 675 (CNRS) and Université de Toulouse — Le Mirail, Toulouse, France