Larval <Emphasis Type="Italic">Hysterothylacium</Emphasis> sp. (Nematoda,Anisakidae) and trematode metacercariae from the amphipod <Emphasis Type="Italic">Paracorophium excavatum</Emphasis> (Corphiidae) in New Zealand

Previously undescribed fourth-stage larvae of anisakid nematodes were found in the haemocoel of the amphipod Paracorophium excavatum (Thomson, 1884) (Corophiidae) in New Zealand. Morphological examination by light microscopy showed that the worms belonged to a species of Hysterothylacium Ward et Magath, 1917, based on several characters including the presence of interlabia, the location of the excretory pore posterior to the nerve ring, and the characteristics of the intestinal caecum and ventricular appendix. Interestingly, several male specimens showed precocious sexual development. This is the first record of fourth larval stage and precocious adult male specimens of Hysterothylacium in an invertebrate host, as well as the first record of anisakid larvae in New Zealand crustaceans. In addition, metacercariae of two trematode species, Coitocaecum parvum and Microphallus sp., are recorded for the first time from the amphipod P. excavatum.     

Experimental life-cycle studies ofRaillietiella Sambon, 1910 (Pentastomida: Cephalobaenida): the fourth-stage larva is infective for the definitive host

The larval development in the intermediate host of threeRaillietiella species which utilize lizards as definitive hosts and insects as intermediate hosts has been studied. In experimentally infected cockroaches (Periplaneta americana) early larval development involves three moults to an infective fourth-stage larva which appears from six weeks post-infection. The three successive larval stages in the intermediate host that develop in the haemocoel encysted by cells of the arthropod host differ in size of the body, length of the hooks, and number of chitinized spines in the penetration apparatus. Their metamorphosis occurs progressively, comparable to the development in hemimetabolic insects where the successive larval stages differ but gradually, too. The infective fourth-stage larvae of lizard raillietiellids which develop in arthropods are so similar in size and overall morphology to the known larves infectantes of snake raillietiellids found in lizard intermediate hosts (Fain 1961, 1964) that a fourth-stage larva is to be expected as infective larva not only of lizard raillietiellids but also of snake raillietiellids.Dedicated to Prof. Dr. W. Frank on the occasion of his sixtieth birtday     

Surface antigens of Obeliscoides cuniculi inducing local and systemic humoral responses in infected rabbits

¹²⁵I-radiolabelled surface proteins of various developmental stages of Obeliscoides cuniculi were used to analyze serum and gastrointestinal immunoglobulin responses of the host to primary infection with 10000 larvae of the nematode, using a radioimmunoprecipitation method. Serum IgM, IgA, and IgG antibodies reacted mostly with infective larval and adult nematodal surface proteins. The highest binding activity was shown by IgG against the surface of infective larvae on days 56 and 86 after infection. Local (gastric mucosal) antibodies were also predominantly directed against surface antigens of infective larvae and adults, although considerable binding of the fourth-stage larval surface proteins by gastric mucosal IgG occurred 35 days after infection. Physicochemical analysis of the radiolabelled surface antigens revealed that the strongest antigenicity was found in proteins with molecular weights of 40000 and 145000. Nonspecific binding demonstrated in our tests by infective larval surface proteins was associated with particles with a molecular weight of 550000.     

Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from Angiostrongylus cantonensis

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, a novel cystatin molecule (AcCystatin) was cloned from a cDNA library of Angiostrongylus cantonensis fourth-stage larvae. The putative 14-kDa protein contained 120 residues with cystatin-conserved motifs known to interact with the active site of cysteine peptidases and showed high identities with cystatins from other nematodes. RT-PCR analysis revealed that the expression pattern of AcCystatin was equal at the time points of third-stage larvae, fourth-stage larvae, and adults of the parasite life cycle. The recombinant AcCystatin (rAcCystatin) expressed and purified from Escherichia coli has been demonstrated to possess an obvious inhibitory activity against cathepsin B and could significantly upregulate nitric oxide production from IFN-γ activated RAW 264.7 macrophages. Sera from mice (non-permissive host) infected with A. cantonensis detected rAcCystatin by Western blot, while the sera from infected rats (permissive host) could not. The results implied that AcCystatin might be an immunoregulator in A. cantonensis infection.     

Immunisation of mice againstStrongyloides ratti

Experimental infections of C57B1/6 mice withStrongyloides ratti produced marked resistance to challenge. Resistance to reinfection was induced by as few as six filariform larvae. The degree of resistance produced by soluble somatic antigens prepared from filariform larvae ofS. ratti was comparable to that seen after a primary infection. The addition of Freund's adjuvants to the antigen did not increase resistance significantly. Intraperitoneal implantation of micropore chambers containing filariform larvae failed to induce resistance to reinfection. Considerable resistance to a challenge infection withS. ratti was found after infection withS. stercoralis.     

Post-embryonic development and ultrastructural characteristics of the polycephalic larva of <Emphasis Type="Italic">Taenia parva</Emphasis> Baer, 1926 (Cyclophyllidea,Taeniidae)

Post-embryonic development and fully-formed polycephalic larvae of Taenia parva Baer, 1926 were examined by light (LM) and transmission electron microscopy (TEM). Three developmental stages were recognised: (1) an early stage of exogenous budding at the surface of the central vesicle; (2) a stage of polycephalic cyst development accompanied by segmentation of the growing larval strobile and an obvious decrease in the size of the central vesicle; (3) fully-formed larval strobile and invaginated scoleces. In fully-developed encysted polycephalic larvae, there are usually 14–24 segmented larval strobilae, each terminating with an invaginated scolex; larval strobilae arise from a common central vesicle and remain attached posterior to it during the entire development. The number of segments varies between 109 and 120 per larval strobila. The polycephalic larvae examined closely resemble the strobilocercus type of taeniid larvae. The structure of developing and fully-formed larvae was examined by TEM. The tegument, scolex, subtegumental musculature of the strobilar segments, protonephridial system, calcareous corpuscles and medullary parenchyma of larvae exhibit general similarity with the same structures in adults at both LM and TEM levels. The morphogenesis of the larva of T. parva is compared with that of the polycephalic larvae of other Taenia spp. (T. krepkogorski, T. twitchelli and T. endothoracica) and with other asexually-multiplying cestode larvae (mesocestoidids, hymenolepidids and dilepidids). Dedicated to the memory of Professor Konstanty Janicki, on the occasion of the 75th anniversary of his death     

Nematospiroides dubius: response of the late fourth-stage larva to anthelmintics in vitro

Approximately 60% of fourth-stage larvae ofNematospiroides dubius recovered from mice 6 days after infection, developed to the young adult stage when cultured over a 7-day period in a complex medium in vitro. Larvae at the late fourth stage of development were found to be highly susceptible to most broad spectrum anthelmintics under in vitro conditions, the benzimidazole, imidazothiazole, pyrimidine, isothiocyanate and macrocyclic lactone compounds all being active at very low concentrations. Narrow spectrum anthelmintics active only against ascarids, pinworms, filariae, cestodes or trematodes had little or no effect on these larvae. Ineffective also were those chlorinated hydrocarbon, substituted phenol and salicylanilide compounds known to affectHaemonchus but not trichostrongylid worms in general. It is concluded that in vitro assays employing larvae ofN. dubius are useful for the stringent screening of compounds for broad spectrum antitrichostrongyle activity. Used in conjunction with in vivo screens employingN. dubius in mice, they also afford means for detecting intrinsic activity against the parasite in a system free from any complicating host pharmacokinetics.     

Molecular cloning and characterization of a cathepsin B from Angiostrongylus cantonensis

Cysteine proteases, a superfamily of hydrolytic enzymes, have numerous functions in parasites. Here, we reported the cloning and characterization of a cDNA encoding a cathepsin B (AcCPB) from Angiostrongylus cantonensis fourth-stage larvae cDNA library. The deduced amino acid sequence analysis indicated AcCPB is related to other cathepsin B family members with an overall conserved architecture. AcCPB is evolutionarily more close to other parasitic nematode cathepsin B than the ones from hosts, sharing 43–53% similarities to the homologues from other organisms. Real-time quantitative PCR analysis revealed that AcCPB was expressed significantly higher in the fourth-stage larvae (L4) and the fifth-stage larvae (L5) than that in the third-stage larvae (L3) and adult worms (Aw). Unexpectedly, AcCPB was expressed at a higher level in L4 and L5 derived from mice than the larvae at the same stages derived from rats. The protease activity of recombinant AcCPB (rAcCPB) expressed in Escherichia coli showed high thermostability and acidic pH optima. The role in ovalbumin digestion and enzyme activity of rAcCPB could be evidently inhibited by cystatin from A.cantonensis. Furthermore, we found rAcCPB increased the expression levels of CD40, MHC II, and CD80 on LPS-stimulated dendritic cells (DCs). In this study, we provided the first experimental evidence for the expression of cathepsin B in A.cantonensis. Besides its highly specific expression in the stages of L4 and L5 when the worms cause dysfunction of the blood–brain barrier of hosts, AcCPB displayed different expression profiles in non-permissive host- and permissive host-derived larval stages and was involved in the maturation of DCs, suggesting a potential role in the central nervous system invasion and the immunoregulation during parasite–host interactions.     

Protective immune responses of the jird to larval Dipetalonema viteae.

In vivo and in vitro experiments were performed to study immune protective mechanisms against larval Dipetalonema viteae. Jirds infected with 30 third-stage larvae (L3) of D. viteae for 1, 3 or 5 weeks showed significant killing of challenge larvae implanted for 2 weeks in diffusion chambers. A retardation of larval growth was seen 7 days after larval implantation, and larval death was observed beginning at 10 days. When L3 were placed in vitro with peritoneal exudate cells (PEC) from normal jirds, cellular adherence was seen starting on Day 4, and larval death was seen on Day 10. It was concluded that larvae had to undergo some development in vitro, that would allow cellular adherence to larval surface. Larvae, recovered after 7 days in vivo or in vitro, were placed in culture with normal PEC; cell adherence and worm death occurred at equal rates for both groups of worms. Larvae which had been in culture for 7 days were implanted in immunized jirds for 7 days. Significant killing of these worms was observed, whereas larvae recovered from ticks prior to implantation were not killed. In vivo and in vitro results therefore show that larval development is required for generating susceptibility to specific and/or non-specific immune reactions. A hypothesis is suggested for the function of larval retardation.     

TEM studies on morphological “junctions” between Trichinella spiralis larvae and mouse skeletal muscle cells with particular emphasis on the early changes in host muscles

Larvae of the parasitic nematode Trichinella spiralis migrate via the bloodstream or the lymphatic system to the skeletal muscle cells where they induce multiple alterations in the intracellular environment leading to the formation of nurse cells. The “nurse cell-T. spiralis larva” complex is composed of a transformed fragment of a skeletal muscle cell and the wall of the larva. The pathological process responsible for the formation of this complex, known as basophilic transformation, is essential for the development of T. spiralis larvae, but it still not known how newborn larvae penetrate the transformed fragment of the muscle cell. In this study, we aimed to characterize the ultrastructure of the region of the nurse cell in direct contact with the larval wall, after one and two weeks of T. spiralis infection in mice. For this purpose, a transmission electron microscope fitted with a goniometer was used to make observations of samples tilted at an angle of ±40° relative to the axis of the electron beam. Examination of electron micrographs revealed the continuity of the nurse cell membrane adjacent to the larval surface and the presence of a large quantity of glycogen particles close to the inner surface of this membrane. Our results showed that death of the T. spiralis larvae was associated with destruction of the contact region between the larval wall and the adjacent surface of the nurse cell. We conclude that the T. spiralis larva does not penetrate the nurse cell, but a morphological “junction” is formed between the larval wall and the cell membrane.     

Protective immune responses with trickle infections of third-stage filarial larvae of Wuchereria bancrofti in mice

Groups of inbred BALB/c mice were immunized with trickle doses of 20 live third-stage larvae (L3) of Wuchereria bancrofti each subcutaneously or with 150 microg of sonicated microfilarial antigens emulsified in Freund's adjuvant intramuscularly. An antibody response was distinctly seen after seven trickle doses of L3 and following with the sonicated microfilarial immunization. Due to the non-permissive nature of inbred mice to W. bancrofti infections, a novel immunization approach was adopted using appropriate age- and sex-matched controls. The anti-L3 response in terms of antibody-dependent cell-mediated adhesion and killing was assessed in the immunized animals by implanting live L3 in micropore chambers subcutaneously. About 75% L3 W. bancrofti were affected in animals sensitized with seven trickle doses of L3. When sensitizations were continued, as high as 92% of L3 were seen affected with ten trickle doses compared with 27% in age-matched controls. Immunization with sonicated microfilarial antigen affected about 70% of L3 as opposed to only 12% in controls. A positive correlation was observed in the antibody response with protectivity. This method of induction and assessment of the anti-L3 response involving a small set of animals has not only allowed quantification of affected L3 but has also enabled us to visualize larval conditions in immunologically activated animals. The micropore chamber system, would be useful in monitoring the induction of protective immune response against W. bancrofti in inbred mice. Experimentation on large numbers of animals is required to elucidate further the response of mice towards L3 and also to pinpoint the putative protective antigens.     

Role of eosinophils and neutrophils in innate and adaptive protective immunity to larval strongyloides stercoralis in mice

The goal of this study was to determine the roles of eosinophils and neutrophils in innate and adaptive protective immunity to larval Strongyloides stercoralis in mice. The experimental approach used was to treat mice with an anti-CCR3 monoclonal antibody to eliminate eosinophils or to use CXCR2-/- mice, which have a severe neutrophil recruitment defect, and then determine the effect of the reduction or elimination of the particular cell type on larval killing. It was determined that eosinophils killed the S. stercoralis larvae in na?ve mice, whereas these cells were not required for the accelerated killing of larvae in immunized mice. Experiments using CXCR2-/- mice demonstrated that the reduction in recruitment of neutrophils resulted in significantly reduced innate and adaptive protective immunity. Protective antibody developed in the immunized CXCR2-/- mice, thereby demonstrating that neutrophils were not required for the induction of the adaptive protective immune response. Moreover, transfer of neutrophil-enriched cell populations recovered from either wild-type or CXCR2-/- mice into diffusion chambers containing larvae demonstrated that larval killing occurred with both cell populations when the diffusion chambers were implanted in immunized wild-type mice. Thus, the defect in the CXCR2-/- mice was a defect in the recruitment of the neutrophils and not a defect in the ability of these cells to kill larvae. This study therefore demonstrated that both eosinophils and neutrophils are required in the protective innate immune response, whereas only neutrophils are necessary for the protective adaptive immune response to larval S. stercoralis in mice.     

Pupation height inDrosophila: Sex differences and influence of larval developmental time

Several lines ofDrosophila simulans andD. melanogaster of different origin were examined for pupation height. In all lines male larvae pupated, on average, higher than females. The pupation heights of early-, intermediate-, and late-pupating larvae were also recorded. As pupation progressed in the vials, larvae tended to pupate lower and lower, possibly as a response to diminishing levels of humidity inside the vials, which suggests a strong negative correlation between larval developmental time and pupation height. Thus, selection experiments for pupation height may also select for developmental rate. Since females generally pupate later than males, larval sex differences in pupation height may reflect sex differences in duration of development. The joint effects of sex and duration of development upon pupation height are discussed in relation to the lack of response previously reported in some experiments selecting for pupation height.     

Comparison of faecal techniques including FLOTAC for copromicroscopic detection of first stage larvae of Angiostrongylus vasorum

Angiostrongylus vasorum is a metastrongylid nematode that resides in the pulmonary arteries and the right heart chambers. In dogs, infection results in respiratory, bleeding and neurological disorders and further clinical signs. In the present study, FLOTAC was evaluated for the detection of first-stage larvae (L1) of A. vasorum in canine faecal samples. This technique is based on the counting of parasitic stages (eggs, larvae, oocysts and cysts) in chambers after spinning of faecal samples onto a surface. In a first step, nine flotation solutions were evaluated using faeces of two experimentally infected dogs. Zinc sulphate (specific gravity (s.g.) 1.2) and zinc sulphate plus potassium iodomercurate (s.g. 1.45) gave good results. However, with the latter technique, the larvae were slightly deformed. Subsequently, FLOTAC, using zinc sulphate, was compared through a randomisation technique with McMaster, flotation in tube and Baermann–Wetzel technique. The mean larvae per gramme (LPG) obtained by the FLOTAC for both dogs was significantly higher (P < 0.05) than those obtained by the other three techniques (the means of the other techniques all lie below the 95% CI of the mean LPG of the FLOTAC technique). In addition, the FLOTAC results were consistent across replicates with only Poisson (or random) variation between individual replicates. The other techniques appear to be less consistent with evidence of extra-Poisson variation in at least one of the two dogs across the replicates within each technique. The FLOTAC technique may contribute to an improvement of the ability to diagnose canine lungworm infections and represent a valuable alternative for larval counting of A. vasorum in faecal samples, especially following transport or storage where there may be limited larvae viability, and larval migration techniques cannot be used.     

Toxocara canis larvae viability after disinfectant—exposition

The effect of three routinely used disinfectants on the embryonary development of Toxocara canis eggs was evaluated both in vivo and in vitro. In the in vitro experiment, T. canis eggs were treated with the ethanol, sodium hypochlorite, and one commercial mix of benzalconium chloride and formaldehyde, and the embryonary development was assessed. After a period of 24 days incubation, ethanol was the best disinfectant because it prevented the development of the T. canis larvae 2 in the eggs, and sodium hypochlorite caused degeneration in 50% eggs. By using the commercial mix, 25% T. canis eggs developed to 2nd stage larvae. In the in vivo experiment, the embryonated eggs treated with the disinfectants were inoculated to mice, and their brain tissues were examined for larval presence on the 24th day postinfection. In addition, a control group was set up for comparison with the infected groups. No injury or T. canis larvae were observed in mice infected with sodium hypochlorite-treated eggs, opposite to that recorded in the animals infected with the commercial disinfectant-treated eggs. These results showed that both ethanol and sodium hypochlorite are very appropriate because of their full efficacy against infective T. canis eggs. Disinfection of kennels, animal shelters, cages, and veterinary clinics with one of these products to eliminate T. canis eggs and to avoid contamination is strongly recommended.     

In vitro culture methods for transition stages of filarial parasites: Evidence that host factors are important for parasite development

Simulium vittatum is a black fly that can serve as a surrogate intermediate host for the filarial parasite,Onchocerca lienalis. Use of media conditioned by excised thoraces ofS. vittatum led to the identification of a soluble chemical cue(s) used by the microfilariae to migrate to fly thoracic muscles, the site where continued differentiation occurs. Medium conditioned by a vertebrate cell line (LLCMK₂) supported vigorous culture of third-stage (L3) and fourth-stage larvae (L4), and provided an environment for uncomplicated screening of therapeutic drugs such as ivermectin.Abbreviations mf microfilaria - L1 first-stage larva - L2 second-stage larva - L3 third-stage larva - L4 fourth-stage larva - CCM cell-conditioned medium - TCM thorax-conditioned medium - ACM abdomen-conditioned medium - FBS fetal bovine serum - BSA bovine serum albumin - PBS phosphate buffered saline     

Relation between the genetic variability of digging behavior ofDrosophila larvae and their susceptibility to a parasitic wasp

TwoDrosophila strains were compared with respect to the behavior of their larvae on food substrate: a wild-type strain (D) from the West Indies exhibited digging behavior, while a laboratory strain (S), bearing theebony mutation, remained on the surface. Chromosome transfers showed this difference to be due mainly to autosomes. There was a significant difference between the two strains in the proportions of larvae parasitized by a cynipid wasp. This was not due to theebony mutation or to other traits such as larval size, cuticle thickness, and speed of development. Chromosome transfers demonstrated a significant role of the three major chromosomes in the susceptibility to the parasite. A clear parallelism was found between the susceptibility to parasitization and the proportion of surface larvae. The depth of concealment of the larvae in the food matter appears to be a favorable behavioral strategy for escaping parasite attacks. The possible adaptive significance of genetic variations in larval behavior is discussed.     

Apolipophorin-III in Galleria mellonella potentiates hemolymph lytic activity

Heat-inactivated serum of non-immune Galleria mellonella larvae enhanced the lytic activity of larval cell-free hemolymph against Micrococcus lysodeikticus. The increase in bacterial lysis was due to a 17.2 kDa protein known previously to bind to bacterial lipopolysaccharides. The protein enhanced the lytic activity of insect cell-free hemolymph and hen lysozyme in vitro and insect hemolymph in vivo. The hydrophobic protein, which adhered to M. lysodeikticus, was identified by its amino acid sequence homology as apolipophorin-III. The titer of apolipophorin-III in 200–250 mg last instar larvae was 8.7 mg/ml of hemolymph. Apolipophorin-III did not bind to lysozyme. A possible mode of action of apolipophorin-III with lysozyme in the insect is proposed.     

Pseudoleptobothrium aptychotremae Young, 1967 (Monogenea, Microbothriidae) redescribed from a new host, Trygonorrhina fasciata (Rhinobatidae) in South Australia with a description of the larva and post-larval development

The adult of Pseudoleptobothrium aptychotremae Young, 1967 (Monogenea, Microbothriidae) is redescribed from the dermal denticles of the southern fiddler ray, Trygonorrhina fasciata (Rhinobatidae) collected off Adelaide, South Australia. This is a new host and locality record. The anatomy of the larva is described from observations of live larvae and the presence of six needle-like spicules in the larval haptor is confirmed. The development of P. aptychotremae is also described.