Infective Larvae Of Ancylostoma Caninum Research Articles

English

Three groups (10 in each) (6 to 8 weeks old) of experimental mice were orally infected each with 500 (group A) 1000 (group B) and 2000 (group C) infective larvae of Ancylostoma caninum . Group (D) was kept as uninfected control for comparison. Two mice from each of the experimental groups were sacrificed on day 1, 4, 9, 16 and 30 after infection. Two mice from control group were also sacrificed on the same designated days. Spleen was separated and the content of DNA and RNA was estimated following standard methods. DNA and RNA levels in spleen of experimental groups (A, B and C) are lower and higher than control group during 1 to 30 days of infection respectively. The synthesis of DNA and RNA was influenced by three varied doses of infection; the occurrence of abnormal physiological changes in spleen indicates the influence of infection in the lymphoid organ.

Molecular characterisation of the Ancylostoma-secreted protein family from the adult stage of Ancylostoma caninum

The Ancylostoma-secreted proteins are a family of nematode-specific cysteine-rich secreted proteins belonging to the pathogenesis-related protein superfamily. Previously we reported that third stage infective larvae of Ancylostoma caninum produce two different Ancylostoma-secreted proteins, a single and double-domain Ancylostoma-secreted protein, designated as Ancylostoma-secreted protein-1 and Ancylostoma-secreted protein-2, respectively. Here we report that adult A. caninum hookworms produce and release four additional Ancylostoma-secreted proteins ( Ancylostoma-secreted protein-3–6). Using antiserum against adult excretory/secretory products, Ancylostoma-secreted protein cDNAs were isolated from cDNA expression libraries. Immunolocalisation experiments using specific antisera indicated that the single-domain Ac- Ancylostoma-secreted protein-3 is located in the adult pharyngeal and oesophageal glands. Ac- Ancylostoma-secreted protein-4, Ancylostoma-secreted protein-5 and Ancylostoma-secreted protein-6 are composed of two pathogenesis-related protein domains linked in tandem as a heterodimorphic repeat. Ac- Ancylostoma-secreted protein-4 is localised to the cuticular surface of the adult hookworm, whereas Ac- Ancylostoma-secreted protein-5 was found in the intestinal brush border membrane, and Ancylostoma-secreted protein-6 in the cephalic and excretory glands. All of the adult Ancylostoma-secreted proteins were identified in excretory/secretory products of adult hookworms by Western blotting and are presumably released by the parasite. None of the adult Ancylostoma-secreted proteins were detected by immunoblotting in L3 extracts, although mRNAs of Ac- Ancylostoma-secreted protein-3 and Ac- Ancylostoma-secreted protein-4 were present in the larval stage. The functions of the adult Ancylostoma-secreted proteins are unknown, although the secretion of multiple family members by the adult suggests an important role in the establishment or maintenance of the parasitic relationship.

Natural History of Primary Canine Hookworm Infections After Three Different Oral Doses of Third-Stage Infective Larvae of Ancylostoma caninum

Abstract The clinical courses of 6 canine hookworm infections were examined in 19–20-wk-old dogs infected with 400, 600, or 800 third-stage infective larvae (L3) of Ancylostoma caninum. Dogs infected with 600 L3 exhibited the greatest adult hookworm burden (289 ± 44), fecal quantitative egg counts, leukocyte counts, and eosinophilia, as well as the lowest overall hematocrit and hemoglobin concentrations. Dogs infected with 800 L3 harbored fewer adult hookworms in their intestines (226 ± 20) than dogs infected with 600 L3 (289 ± 44). Dogs infected with 400 L3 exhibited the lowest hookworm burden (133.5 ± 12.5). All of the dogs developed anti-A. caninum antibody responses. The adult hookworm recovery data were used to calculate a sample size for future canine vaccine trials that will employ recombinant hookworm antigens formulated with adjuvant.

Pulmonary superoxide dismutase activity in the mouse infected with Ancylostoma caninum larvae.

Albino mice were infected per os with 1000 infective larvae of Ancylostoma caninum and pulmonary superoxide dismutase activity was studied after completion of lung migration. The total superoxide dismutase activity per mg of lung protein was elevated by 15% in infected mice. Of the two forms of superoxide dismutases studied, that containing Cu-Zn was elevated by 44%. This suggests that A. caninum in mice may result in histopathological changes leading to transient pulmonary oxygen toxicity.

IgG antibody binding to the outer surface of infective larvae ofAncylostoma caninum

Applying the indirect fluorescent antibody technique to the infective stages of the hookworm Ancylostoma caninum, it appeared that they do not show IgG antibody binding when serum from dogs infected with A. caninum was used in the test (antiserum). However, inhibiting these stages metabolically with azide or with low temperatures, IgG antibody binding to the outer surface was observed. When the inhibitory factors were removed, shedding of fluorescent substances was seen, which were obviously coming from the outer surface of the larvae. This suggests that shedding of the antigen might occur.

Survival of Ancylostoma caninum on Bluegrass Pasture

The survival of infective larvae of Ancylostoma caninum on outdoor grass plots was studied in 40 experiments over 1 year. Weather data were collected over the period. Mean larval survival from August to early November was 24 days (range 1 to 49), from December through February was 0 days, and from March to mid-August was 6.6 days (range 0 to 21). Moderate to high temperatures and substantial rainfall favored larval survival; low temperatures and rainfall favored larval destruction.

Effect of Route of Administration of Vaccine and Challenge on the Immunogenic Efficiency of Double Vaccination with Irradiated Ancylostoma caninum Larvae

Double subcutaneous vaccination of pups aged 3 and 4 months with 1,000 40-kr-irradiated Ancylostoma caninum larvae is more effective than double oral vaccination with the same preparation when resistance is measured by the establishment of adult hookworms resulting from an experimental challenge of normal larvae. In terms of resistance to the pathogenic effects of the challenge, as demonstrated by hematologic and coprologic findings and by clinical observation, both methods of vaccination are equally effective in protecting the vaccinates when compared with the severely affected challenge control dogs. The results suggest that the arrest of X-irradiated larvae in some somatic location on their migratory route following subcutaneous inoculation produces a better immunogenic stimulation of the host. Previous experiments have shown that treatment of the infective larvae of Ancylostoma caninum with X rays produces a characteristic attenuation (Miller, 1964). This attenuation was demonstrated by a reduction in the infectivity of the larvae, as measured by adult hookworm establishment in the intestine of the host, by a reduction in pathogenicity of the resultant burden, and by a sterilizing effect on the female worm at levels of larval irradiation of 40 kr or greater. A single subcutaneous infection with 1,000 40-kr-irradiated larvae was shown to confer a highly significant resistance to a subsequent challenge of normal larvae. Double subcutaneous vaccination with 40-krirradiated larvae is more than twice as effective as single vaccination (Miller, 1965a). A highly significant and satisfactory immunity can be produced in dogs of any age, even when the first dose of vaccine is administered as early as 72 hr after birth (Miller, 1965b). The present report describes experiments designed to investigate the immunogenic efficiency of a 40-kr-irradiated vaccine when the route of administration of vaccine and challenge larvae was varied. Received for publication 13 October 1964. MATERIALS AND METHODS These have been described elsewhere (Miller, 1964). This report comprises two separate experiments. The plan of experiment is illustrated in Table I. In each experiment, groups of pups without prior exposure to hookworm were vaccinated at 3 and at 4 months of age with 1,000 40-kr-irradiated A. caninum infective larvae, followed by a challenge of 1,000 normal infective larvae at the age of 5 months. At the time of challenge of the vaccinates, further groups of dogs aged 5 months, similar to the vaccinates in every respect except for the absence of prior exposure to hookworm, were infected with 1,000 normal larvae as challenge controls. On each occasion on which vaccine was prepared two further groups of dogs were infected, one group with irradiated larvae and the other with normal larvae. The necropsy worm burdens in these groups served as controls of normal larval infectivity and radiation attenuation for each batch of vaccine. Hematologic and fecal examinations were performed at suitable intervals throughout the experiments and the dogs were observed for clinical signs of ancylostomiasis.

Effect of X-Irradiation upon the Infective Larvae of Ancylostoma caninum and the Immunogenic Effect in Dogs of a Single Infection with 40 kr-Irradiated Larvae

X-irradiation of the infective larvae of Ancylostoma caninum reduces the infectivity of the larvae as measured by subsequent intestinal establishment of adult hookworms. As the dose of radiation is increased the infectivity of the larvae is decreased and the pathogenicity to the host of the resulting burden of hookworms is reduced. Male larvae are more sensitive to the effects of X-irradiation than are female larvae, particularly at the higher levels of irradiation. At radiation doses of 40 kr and greater the female worms in the resulting population are invariably sterile. A single subcutaneous vaccination of dogs aged 3 months with 1,000 40 kr-irradiated larvae confers a highly significant resistance. This vaccination confers highly satisfactory resistance to the pathogenic effects of such a challenge, when measured in terms of hematological, clinical, and coprological changes. In a number of host-helminth systems it has been shown that the previous administration of X-irradiated infective larvae will protect the host against a potentially pathogenic challenge of normal larvae. The present state of these helminth vaccines has been surveyed by Urquhart et al. (1962). The work of Herrick (1928), McCoy (1931), Foster (1935), Otto and Kerr (1939), and Otto (1941) has demonstrated that dogs can be immunized against subsequent severe challenge of Ancylostoma caninum by the administration of normal infective larvae. This resistance to challenge was manifested by reduced worm establishment from the challenge of normal larvae, and in some cases also by a reduction in worm egg output. Cort and Otto (1940) and Otto (1948) have surveyed the literature on the subject of experimental vaccination against ancylostomiasis in dogs. Many of the recorded results describe experiments in which stray or pound dogs of unknown age and originating from hookworm enzootic areas were used. Resistance shown to challenge infection in these often uncontrolled experiments, ascribed by the author to Received for publication 27 April 1964. be a result of either age or acquired immunity, appears retrospectively and in the light of current knowledge to have been, in many cases, a combination of both manifestations of resistance. This paper describes the effects of X-irradiation on the parasite and an attempt to immunize dogs by a single vaccination regime using such irradiated larvae. MATERIALS AND METHODS Since this is the first of such reports, the details of technique are described in full. Acquisition and preparation of experimental animals Dogs of mixed breeding were purchased from dealers as newly weaned litters at the age of 6 weeks and were isolated in small groups until approximately 3 months of age. During this period they were vaccinated against canine distemper, contagious canine hepatitis, and leptospiroses canicola and icterohaemorrhagiae (Canilep-Glaxo Laboratories, Ltd., Greenford, England) and were treated twice with piperazine adipate for ascarids (Coopane-Cooper, MacDougall and Robertson, Ltd., Berkhamsted, England). Repeated fecal examination of all dogs after purchase and prior to any anthelminthic medication has demonstrated the absence of strongyle eggs and hence precludes the possibility of prior exposure to hookworm (including Uncinaria stenocephal7).

The Physiological Ageing of Ancylostome Larvae

The activity, infectivity and fat content of ageing infective larvae of Ancylostoma caninum were investigated at regular intervals.The reduction in activity and fat content was found to occur at a similar rate.