The ovicidal effect of Metarhizium brunneum on Ascaridia galli eggs on solid media and activity of its crude extract

The aim of this study was to test the ovicidal effect of Metarhizium brunneum on Ascaridia galli eggs and the protease activity of M. brunneum grown on standard media enriched with A. galli eggs. Ascaridia galli eggs were transferred to water agar plates and the fungus, M. brunneum, was added as a spore suspension. The viability of the eggs was assessed at 7, 14, 21 and 28 days post-inoculation. M. brunneum was grown on three types of growth media: SDA, SDA + chitin and SDA + A. galli eggs. Crude extract of the fungus was produced by mincing and centrifuging biomass taken from the growth plate. The supernatant was added to Azurine cross-linked-casein plates to test for protease activity. M. brunneum showed a significant impact on the viability of A. galli eggs, decreasing the percentage of viable eggs to 5.1%, indicating that it is a potential candidate for biological control of this nematode. Crude extract of M. brunneum grown on SDA + A. galli eggs showed a significantly higher protease activity than the crude extract of M. brunneum grown on SDA alone and M. brunneum grown on SDA + chitin, indicating a possible ovicidal effect of these proteases. Such an extract may have potential as a safe and reliable method for nematode control.     

Ascaridia galli (Nematoda): development and survival in gnotobiotic chickens

Infection and growth rates of the fowl nematode Ascaridia galli were compared in germ-free, conventional, and other gnotobiotic chickens. Uniform experimental infections induced in chickens at 7–12 days of age showed that significantly fewer (P < 0.01) worms established themselves in germ-free chickens than in chickens with conventional flora. Numbers of worms established in monoassociated hosts (Bacillus subtilis, B. cereus, and Penicillium sp.) were intermediate between those in grossly contaminated and germ-free hosts. Mean worm lengths were shorter from germ-free than conventional hosts but differences were not statistically significant. These experiments demonstrate that some A. galli larvae may establish themselves and grow for 14 or 21 days postinoculation in a germ-free environment.     

Oesophagostomum radiatum in calves: intestinal hemorrhage associated with larval emergence

Intestinal blood loss in calves infected with various numbers of Oesophagostomum radiatum was measured using ⁵¹Cr-labeled erythrocytes, and compared with the subsequent changes in the packed cell volume of jugular blood. Emergence of histotropic fourth-stage larvae from the submucosal cysts was associated with intestinal hemorrhage in all infected calves but only in the calves given a near lethal dose of larvae was the bleeding sufficient to cause anemia. At the lower infection rates the hemorrhage caused by larvae was considerably less than that produced by the ensuing adult population. The proportion of adult worms recovered from the infecting dose diminished as the dose increased.     

Establishment of a serodiagnosis system for the detection of Toxocara spp. and Ascaris suum infection in chickens

Chickens are considered to act as paratenic hosts for agents, Toxocara canis, T. cati and Ascaris suum; which cause ascarid larva migrans syndrome (ascarid LMS) in humans. In addition, they are the definitive host for Ascaridia galli, considered not to be infective for humans. All ascarid parasites can have a high homology of antigenicity, leading to cross-reactivity in serodiagnostic assays. This study was conducted to establish a procedure for the serological detection of those roundworm infections in chickens.Twenty-five male Julia chickens were divided into five groups (n = 5); T. canis-, T. cati-, Ascaris suum- and Ascaridia galli-infected, and an uninfected control group. In Ascaris suum-soluble worm antigen preparation (As-SWAP) ELISA, all infected groups showed an elevation of anti-ascarid antibodies, indicating the usefulness of As-SWAP as a screening antigen for the detection of ascarid infections. For infecting species identification, T. canis-excretory/secretory (Tc-ES) and Ascaris suum-ES (As-ES) antigen ELISA were conducted by serial dilution sera. Toxocara spp.-infected sera showed stronger binding to Tc-ES than As-ES, while Ascaris suum and Ascaridia galli-infected sera bound to As-ES more strongly than Tc-ES. To discriminate between Ascaris suum and Ascaridia galli infection, sera were pre-incubated with Ascaridia galli-SWAP antigen and applied to Tc-ES and As-ES ELISAs. In this pre-adsorbed ES antigen ELISAs, only the Ascaris suum infected group showed positive binding to As-ES, resulting from the adsorption of cross-reactive antibodies in Ascaridia galli-infected sera. Finally, anti-Toxocara specific antibodies were confirmed by Tc-ES western blot (WB). Toxocara spp.-infected sera showed toxocariasis-specific band pattern in Tc-ES WB, while no specific band appeared on any strip incubated with Ascaris suum, Ascaridia galli-infected and uninfected sera.In conclusion, the serodiagnostic assays evaluated in this study are useful for the detection of ascarid infections in chickens.     

Host sex as a factor in development of Ascaridia galli

In a series of experiments involving 1745 immature chicks given known numbers of infective Ascaridia galli eggs, more worms were found to develop in male chicks than in female chicks. Statistically significant differences in worm numbers did not occur, however, until the birds in these experiments were 5 to 9 weeks of age.     

Differentiation of cuticular structures during the growth of the third-stage larva of Ascaris suum (Nematoda, Ascaridoidea) after emerging from the egg

In order to monitor the early phases of the development of Ascaris suum from domestic pigs, third-stage larvae, retrieved from the liver and the lungs, were studied by analyzing worm growth and length increase of individual transverse annuli in the cuticle. Material for study using light and scanning electron microscopy was obtained from experimental infections. The results show that the third-stage larva (not the second-stage) after emergence from the egg grows continuously, without an ecdysis in the liver. During growth, each annulus is split into a complex of 2 subannuli, each of which attains a bimodal appearance and is a prominent feature during a late phase of the third-stage larva. The results suggest that the first 2 molts occur inside the egg, a synapomorphic feature of the Ascaridoidea. The third-stage larvae of ascaridoids, with some functional similarities of the dauer-larva stage of Caenorhabditis sp., facilitate transmission of these parasitic worms to the digestive tract of the vertebrate final host (utilizing the tracheal route in A. suum), where the third and the fourth molts take place.     

Reproductive Development of Verutus volvingentis (Tylenchida: Heteroderidae)

Verutus volvingentis Esser, 1981 deposits eggs in the rhizosphere without a gelatinous matrix. Ecdysis was not observed to occur in the egg. Spicular primordia in the rectal area of a second-stage larva were well defined. One larva increased in width from 28.2 μm to a maximum of 51.7 μm after 176.5 hours of feeding, prior to the second ecdysis. It then decreased steadily in width to 33.3 μm, at which time it had molted to a fully developed male. Males leave the third-stage larval integument embedded in the root following final ecdysis. The unique feature of female development was the occurrence of large vaginal primordial cells. Male and female development took from 6 to 15 days and 17 days, respectively.     

Nematospiroides dubius: arrested development of larvae in immune mice.

When immune NIH mice were killed 10 days after a challenge infection with Nematospiroides dubius, approximately 10% of the inoculated larvae were recovered from the intestinal lumen, irrespective of the dose administered. When such mice were treated with cortisone from Day 10 for a period of 8 to 14 days and were subsequently killed for worm counts, it was found that they had significantly more worms than the immune control mice killed on Day 10. During the week following the beginning of treatment with cortisone there was little change in the low worm burdens in immune mice. However, 9 to 11 days after this treatment worm counts indicated that worms were accumulating in the intestinal lumen, and concurrently eggs were recorded in the feces of the mice. These observations indicated that a period of 9 to 11 days was required after the initiation of cortisone treatment on Day 10 for the worms in immune mice to complete their development to the adult lumen-dwelling stage. It is suggested that the larvae in the challenge infection became arrested early in their development in the intestinal wall and that growth resumed only after cortisone treatment. When treatment with cortisone was initiated later after challenge, it was still effective in reactivating arrested worms, but the lower worm recoveries in these mice indicated that the arrested larvae were being slowly rejected by the host. In subsequent experiments it was established that the arrested larvae of N. dubius were insusceptible to the activity of pyrantel embonate, an anthelmintic which is 99% effective against adult worms in the intestinal lumen. The mechanism whereby the larvae of N. dubius became arrested in immune mice and subsequently resumed their development after cortisone treatment is discussed.     

Changes in antigen and glycoprotein patterns during the development of Oesophagostomum dentatum

During its development from free-living infectious third-stage larvae to the adult worms in the large intestines of pigs, Oesophagostomum dentatum experiences several environmental changes. Differences in protein patterns can reflect such changes. Somatic and ES antigens and glycoproteins of pre-parasitic, histotropic and intestinal stages were compared by single-dimension SDS–PAGE and stage-specific proteins were defined. Furthermore, fourth-stage larvae derived from different sources—in-vitro cultivation and intestinal contents—were compared and also found to be different. It is hypothesised that O. dentatum reacts to environmental stimuli by differential expression of specific proteins as a possible mode of adaptation to the host.     

Effects of plumbagin on development of the parasitic nematodes Haemonchus contortus and Ascaris suum

1. Plumbagin (5-hydroxy,2-methyl-l,4-napthoquinone) inhibited the motility and survival of Haemonchus contonus first-stage larvae (L1) with an ed₅₀ of 1 μg/ml, but was less effective in preventing the development of H. contortus to infective third-stage larvae in a faecal slurry assay.2. Of the structural analogs tested, plumbagin was the most potent in preventing development of L1 followed in decreasing order of potency by 1,4-napthoquinone, 5-hydroxy-1,4-napthoquinone (juglone) and 1,2-napthoquinone.3. Plumbagin had a biphasic effect on development of the fourth-stage Ascaris suum larvae that caused an increase in growth at low concentrations but was lethal at higher doses.4. Plumbagin and 1,2-napthoquinone partially inhibited embryonation of A. suum eggs.     

Associations between and development of welfare indicators in organic layers

The retail market share of organic eggs in Denmark is high, and the consumers expect high animal welfare standards in the organic production. Documentation of animal welfare is important, however, knowledge about the associations between animal-based welfare indicators is limited. The aims of the study were to investigate the associations between selected welfare indicators at two ages (peak and end of lay), and to examine the development with age of the chosen welfare indicators. The chosen welfare indicators were Ascaridia galli (roundworm) infection, Heterakis sp. (caecal worm) infection, keel bone damages, back feathering, body feathering, foot damages, comb colour and wounds on the body. An observational study with 12 organic egg farms was conducted in 2012 and 2013 with a total of 214 hens assessed individually at the peak and the end of lay. Insufficient data were obtained on helminth infection at the peak of lay. At the end of lay, all helminth infected hens were positive for A. galli, and only three of them had in addition a Heterakis sp. infection. Foot damages, pale combs and wounds on the body occurred at frequencies <5% and were therefore, together with the prevalence of Heterakis sp. infection, left out of the analysis of associations. A graphical model was used to analyse the associations between the remaining clinical welfare indicators, A. galli infection, housing systems and age of the hens at end of lay. A. galli infection was only directly associated with back feathering at end of lay (P=0.011) with an increased incidence of A. galli infection in hens with good back feathering. Between the two visits, the prevalence of hens with keel bone damages increased (P<0.001), and the plumage condition deteriorated (P<0.001), whereas the number of hens with plantar abscess (P=0.037) and pale combs (P=0.020) decreased. No significant differences were found for other foot damages or for skin damage. In conclusion, back feathering at end of lay provided information about a possible helminth infection, but this is not a useful indicator in daily on-farm management. In addition, evidence was found that the deterioration of the plumage condition with age was not only due to accumulation of damage over time.     

A method to evaluate relative ovicidal effects of soil microfungi on thick-shelled eggs of animal-parasitic nematodes

Thick-shelled eggs of animal-parasitic ascarid nematodes can survive and remain infective in the environment for years. The present study evaluated a simple in vitro method and evaluation scheme to assess the relative effect of two species of soil microfungi, Pochonia chlamydosporia Biotype 10 and Purpureocillium lilacinum Strain 251 (Ascomycota: Hypocreales), on the development and survival of eggs of faecal origin of three ascarid species, Ascaridia galli (chicken roundworm), Toxocara canis (canine roundworm) and Ascaris suum (pig roundworm). Ascarid eggs were embryonated on water agar with or without a fungus, and the resulting viability of the eggs was evaluated on days 7, 14, 21, 28, 35 and 42 post exposure (pe) by observing eggs in situ. On days 7–42 pe, P. chlamydosporia had reduced the viability of A. galli and T. canis eggs by 64–86% and 26–67%. Corresponding reductions for P. lilacinum Strain 251 were only 15–29% and 4–28%. In contrast, A. suum eggs were extremely resistant to both fungi (2–4% reduction). The differences in results are likely due to different morphologies and chemistry of the egg shell of the three ascarid species. The current in vitro method and evaluation criteria allow for a simple, repeatable and non-invasive evaluation of the ovicidal effects of microfungi. This study demonstrates that P. chlamydosporia Biotype 10 may be utilised as a biocontrol agent to reduce A. galli and T. canis egg contamination of the environment.     

Circadian clock and prothoracicotropic hormone secretion in relation to the larval-larval ecdysis rhythm of the saturniid Samia cynthia ricini

Observations on the timing of ecdysis and neck ligation experiments on larvae of Samia cynthia ricini under various light-dark conditions show that an endogenous circadian clock controls the timing of larval ecdysis and prothoracicotropic hormone (PTTH) secretion preceding it. The clock, upon reaching a specific phase point, causes the brain to secrete PTTH provided that the brain has acquired the secretory competence. This time may vary, in relation to a previous ecdysis, according to the light-dark conditions by which the clock phase is specifically determined, but is fixed relative to a subsequent ecdysis. Thus, in the case of the ecdysis to the 5th instar, PTTH is secreted [15+nτ] hr (τ: free-running period, slightly less than 24 hr) after the clock has started when the rhythm is free-running, and in the second and third nights of the 4th instar under a photoperiod of 12 hr light and 12 hr dark. Full secretion of ecdysone occurs 6 hr after PTTH secretion and ecdysis ensues 34 hr thereafter to complete the ultimate sequence of ecdysis.     

Epizootiology of Eustrongylides ignotus in Florida: transmission and development of larvae in intermediate hosts

Under laboratory conditions, 2 modes of transmission of Eustrongylides ignotus (Nematoda: Dioctophymatoidea) to fish were identified. Eastern mosquitofish (Gambusia holbrooki) became infected after ingestion of either eggs of E. ignotus containing first-stage larvae or aquatic oligochaetes (Limnodrilus hoffmeisteri) containing third-stage larvae of E. ignotus. After removal from the uterus of gravid E. ignotus females and incubation for 17-28 days, depending on temperature, it was found that parasite eggs contained first-stage larvae that were infective to fish and oligochaetes. Larvae developed to the third stage in oligochaetes and were infective to fish 35-77 days postinfection (PI) and when fed to fish, developed to the fourth stage between 127 and 184 days PI. Eggs containing first-stage larvae fed directly to fish developed to the fourth stage between 84 and 105 days PI. The amount of time for development from the undifferentiated egg to the fourth-stage larva was 78-156 days shorter when fish ingested eggs containing first-stage larvae than when fish ingested oligochaetes containing third-stage larvae. Three species of large piscivorous fish, including black crappie (Pomoxis nigromaculatus), largemouth bass (Micropterus salmoides), and warmouth (Lepomis gulosus), were fed mosquitofish containing fourth-stage larvae. At necropsy, live E. ignotus larvae were recovered from all 3 species. Several fish had multiple infections after ingesting > 1 larva, indicating that bioaccumulation of the parasite in the food chain may occur.     

Nippostrongylus brasiliensis and Haemonchus contortus: Function of the excretory ampulla of the third-stage larva

An improved technique for measuring the water content of nematodes is described using an electronic interferometer. Changes in phase of a laser beam passing through a known pathlength of the nematode have been used to measure the refractive index and hence the water content and relative volume of the animal. Third-stage larvae of Nippostrongylus brasiliensis and Haemonchus contortus which possess an excretory ampulla, differed from second-stage larvae lacking this ampulla in requiring a greater fall in the osmolarity of artificial tap water before there was a significant increase in their water content. Increases in the pulsation frequency of the ampulla also occurred in less hypotonic solutions than those required to increase the water content of the third-stage larvae. The ampulla pulsation frequency of third-stage larvae of N. brasiliensis increased after locomotor activity in hypotonic tap water and locomotory wave frequency of third-stage larvae of N. brasiliensis was independent of the extent of hypotonicity for a range of solutions that reduced wave propogation by its second-stage larva. The results suggest that the ampulla is an adaptation to hypotonic conditions favouring a volume homeostasis that is required for optimal locomotor activity of the third-stage infective larvae of these nematodes.     

Reduced titres of ecdysone following juvenile hormone treatment in the German cockroach, Blattella germanica

Changes in ecdysone titre of the larvae of the German cockroach, Blattella germanica, exposed continuously to the juvenile hormone (JH), or to the insect growth regulator (IGR) with JH activity, can be correlated with the nature of the substance applied, its dose, and the time of application. The younger larvae exposed to the high dose of the IGR die in the next ecdysis, whereas the same treatment induces a diapause-like stage of developmental arrest in the last larval stage. The affected larvae have very little or no ecdysone, the synthesis of which takes place in the second part of the instar. The same treatment after this period has a lesser effect. The extent of the effect is correlated to the amount of ecdysone synthesized before the application of IGR. Last instar larvae exposed to the lower dose of the IGR or JH lack the peak of ecdysone normally found in the controls at the end of the second third of the instar when metamorphosis takes place. In these insects the first rise of the ecdysone titre begins towards the end of the instar, and ecdysis into the supernumerary larval stage is initiated when the ecdysone titre reached a level permitting ecdysis.A direct or indirect antagonism between these hormones, both fundamental to insect development, can explain the morphogenetic, inhibitory, and lethal effects observed in insects treated with JH or IGR with JH activity.     

Relationship between starvation and supernumerary ecdysis and recognition of the penultimate-larval instar in the common cutworm, Spodoptera litura

Supernumerary ecdysis of the common cutworm, Spodoptera litura was induced by crowded rearing and starvation. Long periods of starvation in the 3rd- and 4th-larval instars induced higher proportions of extra ecdyses, up to 100%. The pupal weights were essentially similar in spite of different instar numbers. However, starvation in the 5th (penultimate) and 6th instars hardly induced supernumerary ecdysis. Starvation caused a fluctuation in the head-capsule sizes of the larvae, but those larvae with head capsules wider than 1.65 mm were destined to become last instar at the next ecdysis. The significance of a threshold for the penultimate instar is discussed in relation to phase polymorphism.Crowded rearing also caused an increase in the proportion of extra ecdyses in the following generation, whereas isolation decreased it. It is concluded that supernumerary ecdysis is not a phase character, but is for recovery from reduced size due to food shortage.     

Regulation of Haemonchus contortus populations in sheep exposed to continuous infection

Groups of sheep were infected three times weekly for 15 weeks with infective larvae of Haemonchus contortus at four rates ranging from 600 to 4800 larvae per week. At 3-weekly intervals, sheep from each group were killed for total worm counts after receiving a dose of radiolabelled larvae which enabled a direct measurement of establishment of incoming larvae during the continuing infection. Peak H. contortus burdens were reached between 6 and 9 weeks of infection, and were related to infection rate. In the groups receiving 2400 and 4800 larvae per week, worm numbers then declined rapidly and by the end of the experiment were lower than those in the groups given 600 or 1200 larvae per week. Establishment of incoming larvae was not influenced by infection rate, and declined from 45% in the first 4 weeks of infection to insignificant levels during the final 6 weeks. The proportion of incoming larvae arrested in their development increased as the infection progressed. It was concluded that H. contortus numbers were regulated by development of resistance to infection and by a loss of established worms which was related to the current rate of larval intake, and to the host's previous experience of infection.