The morphogenesis of Ascaris suum to the infective third-stage larvae within the egg.

Studies of the morphology of Ascaris suum larvae developing in the egg during embryonation in vitro at room temperature showed that 2 molts take place within the egg. The first larval stage (L1) appeared in the egg after 17-22 days of cultivation, the first molt to the second larval stage (L2) took place from day 22 to day 27, and the second molt to the third larval stage (L3) started on day 27 and continued during the 60-day observation period. Infectivity of the eggs was studied by oral egg inoculation in mice and showed that the L3 are the infective stage for mice. Molting to the L3 stage occurs gradually over a period of 2-6 wk, and it is recommended to have an additional maturation period so the infectivity of an egg batch may reach maximum level.     

A preliminary study of the effect of microclimate on third-stage larvae of Haemonchus contortus and Haemonchus placei on irrigated pasture.

Assessments were made on the influence of several microclimatic variables on the availability of third-stage larvae of Haemonchus contortus and Haemonchus placei on four strata of irrigated Kikuyu pasture. Three replicates of these pasture samples were collected on 18 sample days over 12 months and the log10 mean counts of the larvae recovered were analysed by a step-wise regression model. Predictors for the log counts of the four strata for the two nematode species included relative humidity, illumination, air temperature and windspeed. The effect of air temperature on larvae of both Haemonchus species was similar; as air temperature increased, the number of larvae on pasture increased. The inverse was true for windspeed; as windspeed increased larval counts decreased. For H. contortus, relative humidity increased as the number of larvae increased on all strata except upper herbage. The R2 values ranged from 0.11 to 0.21 for H. contortus and from 0.04 to 0.12 for H. placei. Under the conditions of this study, only 21% of the effect on H. contortus and 12% on H. placei third-stage larvae on pasture can be explained by microclimatic conditions.     

The effect of weather in modifying the pattern of pasture larval contamination withOstertagia circumcincta

Groups of 3 ewes and 6 lambs were run on each of three paddocks carrying residual pasture larval contamination ofOstertagia circumcincta, pasture larval contamination following the simulated spring rise on both types of infection. On all paddocks a rise in pasture larval contamination occurred in early August. The absence of the usual peak in June on the paddocks contaminated from the spring rise was attributed to the cold weather during April and May which slowed up development of the eggs ofOstertagia circumcincta     

Experimental infections with Cooperia oncophora (Railliet, 1918) in calves. Results of single infections with two graded dose levels of larvae.

Two experiments were carried out in which calves reared parasite-free were infected with a single dose of 3rd-stage larvae of Cooperia oncophora. In the first experiment the calves received 20 000 or 200 000 infective larvae and they were autopsied 28 or 56 days after the infection. In the second experiment the doses were the same but the animals were killed 14, 84 or 140 days after infection. If a dose of 20 000 was given, clinical signs were never observed, while at a dose level of 200 000 the weight gain was less on 56 and 84 days after the infection compared with the low-infected groups or the control animals. After 170 days the differences in weight gain were compensated. Faecal egg output was higher in the 200 000 groups only in the first period of patency, thereafter the calves in the 20 000 groups produced more. No obvious differences between the two infection levels were observed with regard to the haematological data. In the low-infected groups worm counts were only slightly lower when the results after 28 days were compared with those after 56 days. Also, the worm numbers after 14 days were almost equal to those after 84 days, while at 140 days 1 animal still had the same number, the other one had lost its worm burden. In the high-infected groups the worm loss was much quicker. After 28 days a great part of the population had already been lost. Obviously, at the 200 000 level the reaction of the host against the parasite was much stronger. Adult worms were expelled at a higher rate than early 4th-larval stages. In the first experiment worm measurements revealed differences between the length of females, males and spicules of males, these being significantly longer in the low-infected groups. Analysis of the distribution of worms over the small intestine showed that in the low-infected groups worms were mainly restricted to the first 6 metres. In the high-infected groups the worm population was distributed more evenly over the whole small intestine.     

Prevalence of Cooperia punctata, C. pectinata and C. oncophora infections in dairy calves in Brazil

Cooperia is the most prevalent helminth parasitizing calves in Brazil. Three species of this genus occur most often: C. punctata, C. pectinata and C. oncophora. Six calves from dairy farms in the south of the State of Minas Gerais aged six to 15 months were killed and necropsied each month over a period of two years. The Cooperia species were identified, counted, and the numbers related to the calves' age. The worm burdens due to the three species of Cooperia were statistically different. C. punctata was the most prevalent species and had a positive correlation with the age of the calves; C. pectinata appeared with lower intensity but was always present, and C. oncophora was not found in calves older than 11 months. These results show the existence of different degrees of resistance to Cooperia species among calves as the three species did not behave similarly. It seems to be an acquired resistance. C. punctata appears to be less immunogenic than C. pectinata and C. oncophora. As C. punctata and C. pectinata are more pathogenic than C. oncophora, it seems that this pathogenicity can be related to immunogenic aspects associated with the species.     

Immune expulsion of the trichostrongylid Cooperia oncophora is associated with increased eosinophilia and mucosal IgA

Previous experiments have shown that a primary infection with 100000 infective larvae of the trichostrongylid Cooperia oncophora allows discrimination between different type of responder animals based on the speed by which the parasite is expelled from the host. In most of the animals (intermediate responders) the expulsion occurs 35-42 days after infection. This experiment was carried out to investigate which mechanisms contribute to the clearance of the parasite from the intestine. Sequential necropsy of the animals 14, 28 and 42 days after infection together with a segmental division of the small intestine, allowed us to characterise essential components associated with development of immunity and expulsion of the parasite from its niche. The results show that during the patent phase of the infection the parasite preferentially resides in the proximal gut. Forty-two days after infection ongoing expulsion is characterised by a migration of the worms to the more distal part of the intestine. Expulsion of the adult worm population appears to be mast-cell independent and is associated with a significant increase in parasite-specific mucous IgA and IgG1 as well as with an influx of eosinophils in the intestinal lamina propria. Although we did not observe a specific lymphocyte recruitment into the intestinal mucosa, the accumulation of eosinophils seems to be mediated by CD4+ cells. We measured significant negative correlations between the number of eosinophils and the expulsion rate of the parasite expressed by sex ratio and ratio eggs per gram faeces. Parasite-specific mucosal IgA levels were negatively correlated to the fecundity of the worms, expressed as number of eggs per female worm. Our results describe the involvement of both eosinophils and mucosal IgA in the regulation of C. oncophora expulsion and suggest the development of a Th2 effector immune response.     

Effects of egg size on the development time of non-feeding larvae

The evolution of egg size in marine invertebrates remains a topic of central importance for life-history biologists, and the pioneering work of Vance has strongly influenced our current views. Vance's model and most models developed since have assumed that increases in egg size result in an increase in the prefeeding period of marine invertebrate larvae. For lecithotrophic species, this means that the entire development period should be correlated with egg size. Despite the importance of this assumption, it has not been tested at the appropriate scale-within species. We investigated the effects of egg size on development time for three lecithotrophic species from two phyla: the ascidians Phallusia obesa and Ciona intestinalis, and the echinoid Heliocidaris erythrogramma. We found that within individual broods of eggs, larger eggs took longer than smaller eggs to develop or become metamorphically competent larvae. It has long been recognized that producing larger eggs decreases fecundity, but our results show that increasing egg size also carries the extra cost of an extended planktonic period during which mortality can occur. The substantial variation in egg sizes observed within broods may represent a bet-hedging strategy by which offspring with variable dispersal potentials are produced.     

Cryopreservation of Dirofilaria immitis microfilariae and third-stage larvae

Microfilariae of Dirofilaria immitis retained their infectivity for susceptible mosquitoes after cooling to -196 degrees C in the presence of 5% dimethylsulphoxide (Me2SO) using a two-step cooling sequence. Motility and in vitro development of cryopreserved microfilariae also compared favourably with unfrozen controls. Third-stage larvae frozen by the same cooling sequence in the presence of either 5% Me2SO or 16% hydroxyethyl starch were motile upon thawing. Thawed larvae completed the third- to fourth-stage moult in vitro at a frequency approximately 5 to 10% of that seen in unfrozen controls.     

B cells and antibody response in calves primary-infected or re-infected with Cooperia oncophora: influence of priming dose and host responder types

We investigated whether the generation of protective memory humoral immunity in Cooperia oncophora infected calves occurs in a dose-dependent way and whether it depends on the animal responder types. To this end, serum and mucus antibody responses were measured in animals primary-infected with 30000 or 100000 L3, treated with anthelmintics and subsequently challenged with 100000 L3. A detailed phenotypic and functional analysis of B cells was done in animals infected once or twice with 100,000 L3. Based on the similarity in parasitological variables of animals primed with 30000 or 100000 L3, we concluded that with these doses priming conferred protection in a dose-independent way. Upon challenge significant increases in Cooperia-specific serum and mucus IgG1 and IgA and total serum IgE titres were induced in primed animals in a dose-independent way. In contrast, intermediate and low responders differed in the onset of the production of Cooperia-specific serum IgG1. Furthermore, not only the onset but also the level of total serum IgE significantly differed between intermediate and low responders. Phenotypic and functional analysis of B lymphocytes revealed that (i). priming induced the generation of memory B cells which upon challenge readily differentiated into antibody secreting cells; (ii). sensitised B cells were more efficiently recruited to the intestinal effector sites; (iii). based on the expression of CD62L and CD86 two distinct B cell subpopulation could be differentiated. CD62L(+)CD86(-) B cells that were likely lymphocytes not yet activated and with an enhanced recirculation capacity, and CD62L(-)CD86(+) B cells that were activated B cells with a reduced recirculation ability; and finally (iv). the increased expression of CD86 and subsequent correlations with parameters of the T helper 2 immune response induced by C. oncophora, suggested that CD86- interactions are involved in the generation of protective immunity against Cooperia.     

Field experiments on the ability of Arthrobotrys oligospora (Hyphomycetales) to reduce the number of larvae of Cooperia oncophora (Trichostrongylidae) in cow pats and surrounding grass

In field experiments, conducted on parasite-free grass plots in two consecutive summers, artificially prepared cow pats containing Cooperia oncophora eggs were inoculated with the nematode-trapping fungus Arthrobotrys oligospora. Numbers of infective C. oncophora larvae isolated from the pats as well as from the surrounding herbage were subject to an approximately ten-fold reduction as compared with numbers in fungus-free pats and herbage surrounding these. This reduction was undoubtedly a result of entrapment of the parasite larvae within the faecal pats.     

Determinants of the longevity of third-stage infective larvae of Ancylostoma tubaeforme.

The effects of some extrinsic factors on the lipid (energy) reserves and longevity of third-stage larvae of the cat hookworm Ancylostoma tubaeforme, were investigated under controlled laboratory conditions. In nonstressful microenvironmental conditions, larval longevity was directly related to the rate of utilisation of the lipid reserves. The effects of the various environmental stresses on longevity could also be explained largely on the basis of their deleterious effects on the lipid metabolism of the larvae.     

Field experiment on the ability of earthworms (Lumbricidae) to reduce the transmission of infective larvae of Cooperia oncophora (Trichostrongylidae) from cow pats to grass

In Denmark earthworms play an important role in the disappearance of cow pats on pastures. Field investigations were designed to study the influence of earthworms on the transmission of infective Cooperia oncophora larvae from experimental cow pats to grass. Results from the present experiment showed that cow pats, protected from attack by earthworms, disappeared at a much lower rate than unprotected cow pats. Development in faeces and transmission to grass of C. oncophora larvae were disturbed by the disintegration of cow pats. The rapid disintegration of unprotected cow pats resulted in an approximately 50% reduction of infective C. oncophora larvae on grass in the vicinity of these cow pats, as compared with larval contamination of grass around protected cow pats. How earthworms may cause a reduction in the transmission of infective C. oncophora larvae to grass by creating adverse conditions for the survival of parasite larvae in the cow pats, by eating and killing the parasite larvae, and by depositing parasite larvae in the soil is discussed.     

Protective immunity induced by irradiated third-stage larvae of the filaria Acanthocheilonema viteae is directed against challenge third-stage larvae before molting

Jirds (Meriones unguiculatus) were vaccinated with irradiated L3 third-stage larvae (L3) of Acanthocheilonema viteae, and the time required for killing of the challenge L3 was determined. The number of parasites recovered from vaccinated jirds was reduced to about 10% of the control values on the second day after challenge infection and later on. Histological studies revealed an eosinophil-rich infiltrate containing macrophages, neutrophils, and mast cells in the vicinity of the L3 on day 2 after challenge and destruction of the worms by day 4 after challenge. Ultrastructural studies confirmed these data and showed that eosinophils, macrophages, and mast cells were close to the L3 on day 2 after challenge. Flattening of the eosinophils onto the surface of the worms, degranulation of electron-dense material, and rupture of the L3 surface was observed on day 4 after challenge, followed by invasion of the inner of the worms by phagocytic cells. These data show that immune attack against the challenge L3 in vaccinated jirds is initiated between the first and the second day after challenge and that killing occurs around the fourth day after challenge, before the worms undergo their first molt.     

Inoculation of ferrets with ten third-stage larvae of Dracunculus insignis.

Infection with Dracunculus insignis was established in 7 of 10 ferrets experimentally inoculated intraperitoneally with 10 third-stage larvae (L3's). Worm recovery and infection rate were comparable to animals inoculated with 50 L3's. This study demonstrates that once infective larvae traverse the gut and pass into the peritoneal cavity, very few larvae are required to establish infection.     

Strategies for the storage of Ancylostoma caninum third-stage larvae

Although cryopreservation protocols for storage of hookworm larvae have been described, the circumstances under which the technique is necessary to ensure larval survival are not well defined. The motility of infective-stage larvae (as judged by observation) and their ability to migrate through canine skin in vitro were measured over a 7-mo period in worms held at room temperature and worms that had been cryopreserved at the start of the experiment. Cryopreserved worms showed motility and migration proportions of 45.6-48.0% and 26.8- 34.0%, respectively, throughout the experiment, compared with percentages of 92.7 and 84.1%, respectively, in the original fresh worms. Larvae held at room temperature showed a gradual decrease in motility and migration ability over the experimental period. Motility and migratory ability of cryopreserved larvae was only significantly higher (P < 0.01) than room temperature-stored larvae from 4 and 5 mo onward, respectively.     

One season of pasture exposure fails to induce a protective resistance to cyathostomes but increases numbers of hypobiotic third-stage larvae

The development of acquired resistance to cyathostome challenge after 1 season's exposure to a cyathostome-contaminated pasture was investigated using 17 parasite-naive ponies, which were 2-3 yr of age. These were divided into 3 groups: 1 to graze a cyathostome-contaminated pasture for 4 mo (exposed ponies), 1 to graze a "clean" pasture not previously grazed by parasitized animals (nonexposed ponies), and 1 group to remain in the barn under helminth-free conditions (parasite-free ponies). After pasture exposure all ponies were housed in stalls in the barn dewormed with ivermectin (200 micrograms/kg) and oxibendazole (100 mg/kg), a treatment that eliminated most cyathostomes encysted in the mucosa as well as all luminal parasites, on the basis of necropsies of 5 animals, after 17 days. Remaining ponies were challenged with 100,000 cyathostome-infective third-stage larvae (L3) per os 3 wk after anthelmintic treatment. Necropsies were performed 7 wk after the challenge. Total cyathostome burdens (luminal plus encysted stages) were not significantly different among any of the groups. However, a significantly higher percentage of hypobiotic early L3 (EL3) and a lower percentage of adults were found in exposed ponies. This observation supports the hypothesis that resistance acquired through exposure promotes cyathostome hypobiosis. This increase in EL3 in exposed ponies was associated with a significant increase in weight of cecum and ventral colon biopsies.