Vaccination against the nematode Trichostrongylus colubriformis. II. Attempts to protect guinea-pigs with worm acetylcholinesterase.

Soluble material extracted from T. colubriformis fourth stage larvae was fractionated by membrane ultrafiltration, gel filtration and ion-exchange chromatography. The acetylcholinesterase (AChE) peak obtained by gel filtration protected guinea-pigs against infection but it was contaminated by worm allergen. However, there was no relationship between the AChE content of fractions obtained by membrane ultrafiltration and ion-exchange chromatography and their ability to stimulate protective immunity. Purified T. colubriformis AChE, prepared by ion-exchange chromatography and free from demonstrable allergen did not stimulate protective immunity whereas another fraction, containing less than one-thousandth the amount of AChE, was effective in doing so.     

Mechanism of 2-acetyl-1-pyrroline biosynthesis in Bassia latifolia Roxb. flowers

The flowers of Bassia latifolia are known to contain 2-acetyl-1-pyrroline (2AP), the compound responsible for pleasant aroma in basmati and other scented rice. Four growth stages of Bassia flowers were identified and 2AP contents were analysed in each stage. It was found that 2AP (3.30 ppm) gets synthesized only in fleshy corolla of mature flowers (fourth stage). The activity of γ-aminobutyraldehyde dehydrogenase (AADH); an enzyme responsible for synthesis of γ-aminobutyricacid (GABA) from γ-aminobutyraldehyde (GABald) was assessed in these four stages. The AADH activity was absent in the fourth stage. It was concluded that ceased activity of AADH in fourth stage flowers leads to the accumulation of γ-aminobutyraldehyde which is cyclised spontaneously to Δ¹-pyrroline, the key precursor of 2AP. Δ¹-pyrroline further reacts unenzymatically with methylglyoxal to form 2AP.     

Brugia malayi and B. pahangi: Cultivation in vitro of infective larvae to the fourth and fifth stages

Cultivation of fourth stage Brugia pahangi and B. malayi larvae from infective larvae (stage 3) were obtained in culture medium RPMI 1640 supplemented with 10% human AB serum and an LCC-MK₂ rhesus monkey kidney continuous cell line feeder layer. This culture system kept larvae alive in excess of 7 weeks, and served as a source for collection of the worms' secretory, excretory, and moulting antigens.     

Phocanema decipiens: Molting in seals

Infective larvae of the anisakine nematode Phocanema decipiens from the muscle of Atlantic cod (Gadus morhua) were fed to harbor seals (Phoca vitulina) and gray seals (Halichoerus grypus). During maturation in the stomach of seal hosts, P. decipiens molted twice; these molts are the third and fourth of its life cycle. The third molt occurred between the second and fifth days of infection. The third stage, i.e., infective larva entering the third molt, had a cuticular tooth ventral to the mouth; the fourth stage larva emerging from the third molt had three bilobed lips with dentigerous ridges. The fourth molt occurred between the 5th and 15th days in seals. A female nematode emerging from the fourth molt possesses a vulva and a vagina; a male possesses caudal alae, pre- and postanal papillae. Significant morphometric changes in nematodes were associated with both molts. Females and males of P. decipiens reached maturity after 15 to 25 days in seals. Ova were detected in the feces of the seal hosts as early as the 16th day.     

Distribution of trichostrongylid nematodes in the abomasum of sheep

The distributions of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus axei during their development in lambs were studied by freezing and sectioning the abomasum and then recovering the worms from the contents and mucosa of each section. In previously worm-free lambs, the distribution of fourth stage H. contortus was very regular, the frequency of larvae being highest in the anterior fundic area and then decreasing in each section towards the pylorus. The distributions of the early larval stages of O. circumcincta and T. axei were variable but tended to be biomodal, with one peak of larvae in the fundic area and a second in the pyloric area. It is suggested that the difference in distribution between H. contortus and the other two species may be due to a difference in the rate of response of exsheathed third stage larvae to stimuli in the abomasum. In the later stages of development, H. contortus and O. circumcincta tended to migrate towards the pyloric area. In previously infected lambs, the distribution of inhibited early-fourth stage H. contortus was similar to that of normally developing fourth stage larvae in previously worm-free lambs and appeared to be unaffected by the host response which caused an altered distribution and rejection of late-fourth stage and adult worms. The rate of rejection of adult O. circumcincta differed between the fundic and pyloric areas of the abomasum, which suggests that the host response in these two areas may be different.     

Nematode-Induced Interference with Vaccination Efficacy Targets Follicular T Helper Cell Induction and Is Preserved after Termination of Infection

One-third of the human population is infected with parasitic worms. To avoid being eliminated, these parasites actively dampen the immune response of their hosts. This immune modulation also suppresses immune responses to third-party antigens such as vaccines. Here, we used Litomosoides sigmodontis-infected BALB/c mice to analyse nematode-induced interference with vaccination. Chronic nematode infection led to complete suppression of the humoral response to thymus-dependent vaccination. Thereby the numbers of antigen-specific B cells as well as the serum immunoglobulin (Ig) G titres were reduced. T_H2-associated IgG1 and T_H1-associated IgG2 responses were both suppressed. Thus, nematode infection did not bias responses towards a T_H2 response, but interfered with Ig responses in general. We provide evidence that this suppression indirectly targeted B cells via accessory T cells as number and frequency of vaccine-induced follicular B helper T cells were reduced. Moreover, vaccination using model antigens that stimulate Ig response independently of T helper cells was functional in nematode-infected mice. Using depletion experiments, we show that CD4⁺Foxp3⁺ regulatory T cells did not mediate the suppression of Ig response during chronic nematode infection. Suppression was induced by fourth stage larvae, immature adults and mature adults, and increased with the duration of the infection. By contrast, isolated microfilariae increased IgG2a responses to vaccination. This pro-inflammatory effect of microfilariae was overruled by the simultaneous presence of adults. Strikingly, a reduced humoral response was still observed if vaccination was performed more than 16 weeks after termination of L. sigmodontis infection. In summary, our results suggest that vaccination may not only fail in helminth-infected individuals, but also in individuals with a history of previous helminth infections.     

Acetylcholinesterase secretion by parasitic nematodes. 3. Oesophagostomum spp

Acetylcholinesterase (AChE) activity in Oesophagostomum radiatum increased markedly during the fourth and early fifth stages of parasitic development and thereafter remained relatively constant in the mature parasites. Fourth stage Oe. radiatum maintained in vitro in a saline medium released AChE steadily for 4 h. Whereas the excretory glands of Oe. radiatum appeared to be the major site of AChE secretion, the highest concentration of the enzyme in Oe. venulosum was found in the cephalic tissues.Antibodies to Oe. radiatum AChE appeared in the serum of calves three weeks after primary infection with the parasite and were also found in the serum of neonatal calves and in the colostrum of their dams.Several soluble non-specific esterases were present in homogenates of adult Oe. radiatum and Oe. venulosum. In Oe. radiatum these esterases occurred both in gut tissue and excretory glands, and were present in secretions released in vitro by fourth-stage larvae. However, no antibodies against the esterases were detected in host serum.     

The isolation and partial characterization of a protective antigen from developing larvae of Ascaris suum.

An antigen (ACF antigen) obtained by in vitro cultivation of third-stage larvae of Ascaris suum to the fourth stage in defined medium induced significant protection in guinea pigs. The antigen was further characterized by ultrafiltration and gel filtration and was shown to be a single component by gel precipition and immunoelectrophoresis. It induced active cutaneous anaphylaxis in sensitized animals. The ACF antigen is estimated to contain about 79% protein and 22% carbohydrate and to have a molecular weight of approx 67,000.     

Failure to vaccinate lambs against Haemonchus contortus with functional metabolic antigens identified by immunoelectrophoresis.

Metabolic products from Haemonchus contortus larvae cultured in vitro from the infective third to fourth stage were collected and concentrated. Chromatographic and immunoelectrophoretic analyses were made to study the numbers and activity of antigens in the metabolic products derived from the in vitro cultured larvae. Three-month-old lambs were given a series of injections of metabolic antigens with and without adjuvant at dose rates of 0·05, 0·5 and 5·0 mg antigen protein per injection. These animals and saline injected controls were each challenged with 3000 H. contortus infective larvae after the last antigen injection and killed 35 days later. No difference was seen in the faecal worm egg counts or the differential worm counts among the vaccinated and control animals. The antigen preparation of worm metabolic products conferred no resistance to challenge infection with the parasite.     

The life history of Sulcascaris sulcata (Nematoda: Ascaridoidea), a parasite of marine molluscs and turtles

Berry G. N. and Cannon L. R. G. 1981. The life history of Sulcascaris sulcata (Nematoda: Ascaridoidea), a parasite of marine molluscs and turtles. International Journal for Parasitoiogy11: 43–54. The morphology, development and hatching of Sulcascaris sulcata eggs are described. Two moults occurred in the egg. Third stage larvae spontaneously hatched and were found to develop in marine bivalves and gastropods. Larvae grew steadily and after three to four months, when about 5 mm long, they moulted to fourth stage larvae characteristic of natural infections in bivalves from commercial catches. Experimentally, when fed to laboratory-reared Caretta caretta, the fourth stage larvae first attached at the oesophago-gastric junction where they moulted to adults in 7–21 days. Subsequent growth to mature adults was obtained by at least 5 months after infection. It is suggested that under natural conditions the life history may take up to 2 years to complete. These findings are discussed in relation to the predatory mode of feeding and the breeding habits of C. caretta and the significance of a possible health hazard to man.     

The ontogeny of multiple hemoglobins in Chironomus thummi (Diptera): The effects of a compound with juvenile hormone activity

The multiple hemoglobins (Hbs) of Chironomus thummi show distinct and significant ontogenetic changes during development from the third instar through the fourth instar and metamorphosis into the pupa. A total of nine Hbs are resolved by 12.7% acrylamide gel electrophoresis (pH 8.65). Hbs 2 and 3, which are stage specific for the fourth instar, are first detected on the fourth day of this stage by electrophoresis and immunoprecipitation. Hb 4 is the predominant Hb species in the early and middle fourth instar, but during the late fourth instar and prepupa, Hb 1 predominates. The concentrations of Hbs 5–9 remain relatively constant in middle instars and decrease during later development. The Hb content of larval hemolymph exhibits changes that coincide with developmental stages; molting is characterized by low Hb content, whereas, the hemolymph of intermolt animals contains relatively high levels of Hbs. Treatment of fourth instars with a juvenile hormone analog, Altosid, prolongs this stage and inhibits the progress of normal development resulting in the formation of larval-pupal intermediates. Altosid also appears specifically to inhibit the accumulation of soluble hemolymph proteins related to pupation and metamorphosis, without affecting the concentration of Hb. Most significantly, it induces the precocious appearance of Hbs 2 and 3, which remain elevated above control levels in the late larval and prepupal stages. The present results strongly suggest that Altosid stimulates the appearance and accumulation of larval-specific proteins in vivo, while it inhibits the appearance of pupation-related proteins.     

Effects of reproduction,genotype and anthelmintic treatment of ewes on Ostertagia spp. populations

Donald A. D., Morley F. H. W., Waller P. J., Axelsen A., Dobson R. J. and Donnelly J. R. 1982. Effects of reproduction, genotype and anthelmintic treatment of ewes on Ostertagia spp. populations. International Journal for Parasitology12: 403–411. Merino and Border Leicester × Merino (BL × M) ewes, nearly all of the same age and reared at the same site, were either unmated or mated to Border Leicester rams. Ewes of each genotype and reproductive status were untreated or were given a single pre-lambing drench with thiabendazole at 50 or 100 mg/kg a week before the start of lambing in spring on pastures at Canberra which had been contaminated during autumn and winter by adult sheep. The two genotypes grazed together within each combination of reproductive status and anthelmintic treatment which grazed separately. Thiabendazole was highly effective in removing both fourth stage larvae and adults of Ostertagia spp., the most abundant genus. Eight weeks after the pre-lambing drench lactating ewes carried larger Ostertagia spp. populations than did unmated ewes of both genotypes, but as a result of reinfection after treatment, differences between drenched and undrenched ewes were not significant. At this time lactating as well as unmated ewes harboured large populations of arrested early fourth stage larvae of Ostertagia spp. acquired during the last 8 weeks, showing that arrest of development is not prevented by lactation. There was strong evidence that some ingested larvae which became arrested in lactating ewes were rejected by unmated ewes. At all stages of the reproductive cycle studied, BL × M ewes were substantially more resistant to Ostertagia spp. infection than Merinos. No persistent benefits in parasite control or in animal production were detected from the pre-lambing drench.     

Effect of exogenous JHI on imaginal determination in Aeshna cyanea

Last instar larvae of Aeshna cyanea injected with 8 μg or more JHI before the end of the fourth ocular stage (day 7 or 8 of the instar) became perfect supernumerary larvae which indicates that adult determination does not occur before this time. When JHI is applied later (days 9–13) larval-adult intermediates are formed. It seems, therefore, that imaginal programming takes place at the end of the fourth ocular stage and might be correlated with the first ecdysone peak which occurs at the same time.     

Development of larvae of Ascaris suum from the third to the fourth stage in a chemically defined medium

Third stage larvae of Ascaris suum, recovered from the lungs of rabbits 7 days after oral infection with eggs, were cultured to the fourth larval stage in chemically defined, low molecular weight medium. The medium consisted of tissue culture medium 199, supplemented with glucose and glycyl-hystidyl-lysine and gassed with a mixture of N₂-CO₂-O₂ (90:5:5). Growth and development in this medium were similar to that in media supplemented with whole serum or with a serum dialysate.     

Vaccination against schistosomes and other systemic helminths

Helminth parasites, compared to other infectious agents are extremely complex. A comparison of genome sizes (e.g. mammals, 5.5 × 10⁹ base pairs; Schistosoma, 3 × 10⁸ b.p.; Plasmodium 1 × 10⁷ b.p.; bacteria 2 × 10⁶ b.p.) shows clearly that helminths are about midway between protozoa and mammals in their complexity; in fact, they lie closer to the higher animals than they do to bacteria and viruses. This complexity has been the main barrier to progress in understanding immune responses to helminth infections. Large multicellular parasites are killed slowly by host immunity and it may be that several different effector mechanisms participate in immune killing; and, the complex mixture of molecules that compose the whole worm has increased the difficulty of isolating single antigens and recognising those that play a significant part in the host-parasite relationship.With modern developments in molecular biology however, the identification of host protective antigens and their production for testing as molecular vaccines is no longer a pipe dream (Mitchell, 1984). Monoclonal antibodies have provided a tool for studying defined antigens and recombinant DNA technology has enabled parasite genes to be cloned, sequenced, and their corresponding antigens produced in the quantities required for animal experimentation (Young, Hockmeyer, Gross, Ballou, Wirtz, Trosper, Beaudoin, Hollingdale, Miller, Biggs & Rosenberg, 1985). Whilst a molecular approach to helminth immunity remains more difficult than comparable studies in micro-organisms, the recent revolution in molecular biology has created the necessary environment and means for a steady advancement towards the creation of defined helminth vaccines.High technology however must be applied in conjunction with good parasitology. An understanding of immune mechanisms, the recognition of the immune-susceptible stages of the parasite and the identification of host protective antigens and their stage and species specificity will provide information that will guide future rational strategy. An alternative approach is to assay the immunogenicity of all recombinant molecules for there may be several reasons why parasite products not normally immunogenic during infection could, when presented with the appropriate adjuvant induce an immunity qualitatively and quantitatively superior to that developed under normal conditions. Whatever the strategy however, a relevant experimental animal model is an essential prerequisite before defined recombinant molecules can be tested as vaccines, since In vitro protection assays of parasites do not always correlate with resistance in vivo.     

Electron transfer between the two photosystems. I. Flash excitation under oxidizing conditions

The redox changes of cytochrome b-563 (cytochrome b), cytochrome f, plastocyanin and P-700 were measured on dark-adapted chloroplasts after illumination by a series of flashes in oxidizing conditions (0.1 mM ferricyanide). In these conditions, the plastoquinone pool is fully oxidized and the only available plastoquinol are those formed by Photosystem (PS) II reaction. According to the two-electron gate mechanism proposed by Bouges-Bocquet (Bouges-Bocquet, B. (1973) Biochim. Biophys. Acta 314, 250–256), plastoquinol is mainly formed after the second and the fourth flashes. After the second flash, the reoxidation of plastoquinol occurs by a concerted reaction which reduces most of the cytochrome b present and a fraction of PS I donors. Most of these electrons are stored on P-700, which implies a large equilibrium constant between the secondary PS I donors and P-700. One electron is stored on cytochrome b during a time ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1}\text{s}$) much longer than the dark interval between flashes. After the fourth flash, a new plastoquinol molecule is formed, which induces the reduction of PS I donors with no corresponding further reduction of cytochrome b. The number of electrons transferred after the fourth flash is larger than that transferred after the second flash although the rate of transfer is lower. To interpret these data, we assume that the plastoquinol formed after the fourth flash is reoxidized by a second concerted reaction: one electron is directly transferred to PS I donors while the other cooperates with the electron stored on cytochrome b to reduce a plastoquinone molecule localized on a site close to the outer face of the membrane. This newly formed plastoquinol crosses the membrane and transfers a second electron to PS I donors. This interpretation resembles a model proposed by Velthuys (Velthuys, B.R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2765?2769) and which belongs to the modified Q-cycle class of models.     

In vitro development of individually cultured whole mouse embryos from blastocyst to early somite stage.

The sequential processes of in vitro development of whole mouse embryos were classified by stages according to the in vivo criteria of E. Witschi (1972, “Biology Data Book,” Part II: “Rat,” L. Altman and D. S. Dittmer, eds., 2nd ed., Vol. 1, pp. 178–180, Federation of American Societies for Experimental Biology, Bethesda, Md.) and K. Theiler (1972, “The House Mouse,” Springer-Verlag, Berlin/New York). The mouse embryos which developed in vitro in each day of culture were then classified into stages according to the characteristics of mouse embryos developed in vivo. A series of 10 blastocysts were inoculated into 35-mm plastic culture dishes (30–50 blastocysts per experiment). Developing embryos were scored on the fourth, sixth, and eighth days and classified into stages. Among the total of 118 blastocysts cultured in three repeated experiments, 100 mouse embryos had attached and developed in culture dishes. Ninety-four percent of the attached mouse embryos developed to the early egg cylinder stage after 4 days of incubation, and 87% grew to the stage of late egg cylinder after 6 days of culture. An average of 62% of the embryos reached the early somite stage with heart beating after 8 days in culture with frequent medium change. In two separate experiments single mouse blastocysts were placed individually in culture dishes in 2 ml of culture medium. The development of each embryo was followed every day. Each of 10 blastocysts had attached in its respective culture dish and had developed to the early egg cylinder stage after 4 days of culture. About 50 to 70% of each of these 20 individually isolated mouse embryos developed in vitro to the early somite stage after 8 days of culture.