Partial protection of lambs against Haemonchus contortus by vaccination with a fractionated preparation of the parasite.

Florida Native lambs, less than 6 months of age, were successfully vaccinated against Haemonchus contortus with a high mol. wt fraction (greater than 30,000 daltons) derived from a somatic extract of H. contortus larvae (SEL) and excretions and secretions (ES) of larvae isolated during in vitro development from the infective 3rd to 4th stage. A 59% reduction in adult worm numbers was obtained in vaccinates compared to naive lambs following challenge. The protection in vaccinated lambs was similar to that seen in lambs exposed to a primary infection of H. contortus larvae which had been cleared with anthelmintic prior to the challenge infection. The unfractionated SEL/ES preparation and a low mol. wt fraction gave no significant protection against challenge infection.

Helminth immunity and vaccines in sheep.

Vaccination of domestic animal species against various parasitic helminths using attenuated parasites or nonliving parasitic material is possible. Improved prospects for vaccines composed of somatic and metabolic parasite components hinge on the isolation and characterization of helminth protective antigens and their synthesis by modern bioengineering techniques. Vaccination strategies beg an understanding of the host's immune effector mechanisms for their most efficient prolonged stimulation. Parameters of importance are antigen dose, frequency of and interval between doses, use of liposomes or other antigen delivery vehicles, and the use and choice of adjuvants.

Humoral immune responses to soluble somatic extracts of Dipetalonema viteae adult worms infected hamsters.

The present study examined the serum IgG1 and IgM antibody responses of infected hamsters to unfractionated and fractionated soluble somatic (SS) antigens of adult Dipetalonema viteae. Male and female adult worms were homogenized in a French pressure cell and the SS proteins extracted with 0.375 M Tris-glycine buffer. Serum IgG1 and IgM antibody responses in D. viteae infected with LVG, PD4 and CB hamster strains to the male and female unfractionated SS proteins were measured by an indirect enzyme linked immunosorbent assay (ELISA). Serum IgG1 antibody responses to the SS proteins were similar in all three strains of hamsters during the course of infection. There was no correlation between the level of IgG1 antibody and the onset of microfilariae clearance. The serum IgM antibody response was similar in both outbred LVG and inbred PD4 hamster strains. A lower IgM antibody response was found in CB hamsters and could be related to the failure of this hamster strain to eliminate microfilariae. The SS proteins of male and female adult worms were fractionated by preparative flat-bed isoelectric focusing on granulated gels (PIEF) to yield 8 and 7 fractions, respectively. The comparative antigenicity of the PIEF fractions from the SS proteins was measured by ELISA, using hyperimmune serum from LVG hamsters and rabbit antihamster IgG1-and IgM-alkaline phosphatase conjugates. No difference in ELISA reactivity was noted among the 8 and 7 PIEF fractions from male and female SS proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody responses to experimental Brugia malayi infections in patas and rhesus monkeys.

Humoral antibody responses in experimental infections with Brugia malayi (subperiodic strain) were compared in two primate species. Erythrocebus patas and Macaca mulatta. Antibody responses were related to the infection protocol and the duration and magnitude of microfilaremia. Patas monkeys were uniformly susceptible to infection and characteristically exhibited prolonged microfilaremia; infections in Rhesus monkeys produced low and usually microfilaremia. Antibody, measured by enzyme linked immunosorbent assay with extracts of adult Brugia and microfilariae as antigens, declined at patency in Patas monkeys and there was an inverse relationship between serum antibody concentration and the number of circulating microfilariae. Rhesus monkeys generally had high, sustained antibody levels relative to Patas monkeys, but antibody levels were comparable in the two species when the numbers of circulating microfilariae were similar. By fluorescent antibody technique, antibodies reactive with somatic antigens of microfilariae were detected in all infected monkeys; antibodies reactive with the cuticle of infective larvae were also present in both primates and were consistently detected in monkeys receiving multiple infections. Antibodies (IgG, IgM) reactive with the sheath of microfilariae were detected only in certain Rhesus monkeys which were essentially amicrofilaremic and sera with antibodies specific for microfilarial sheath promoted in vitro microfilarial agglutination and leukocyte adherence.

The ferret (Mustela putorius furo) as an experimental host for Brugia malayi and Brugia pahangi.

Ferrets inoculated subcutaneously with 150--200 infective larvae of Brugia malayi (subperiodic strain) usually developed patent infection during the 3rd month post inoculation. Microfilaremia was transient, and most animals became amicrofilaremic after the 6th month of infection. Ferrets developed a persistent eosinophilia at the time of patency. At necropsy, 5--8 months post infection, adult worms were recovered principally from lymphatic vessels and recovery ranged from 0.5--13% of the inoculated larvae. The inflammatory response of ferrets to microfilariae was characterized by nodules 1--5 mm in diameter in the liver, lungs, spleen, and submucosa of the gastrointestinal tract. The center of these lesions contained a degenerated microfilaria or the cast of a microfilaria embedded in Splendore-Hoeppli substance. The Splendore-Hoeppli substance was surrounded by eosinophils and/or foreign body giant cells. Identical lesions were observed in ferrets experimentally infected with Brugia pahangi. Sera from ferrets infected with B. malayi demonstrated a 3- to 5-fold increase in IgG by the 4th month of infection and these sera produced 2--3 precipitin bands in double gel diffusion assays with an extract of B. malayi microfilarial antigen. Skin tests with B. malayi microfilarial antigen showed that the majority of the infected ferrets had immediate hypersensitivity responses, but none had Arthus or delayed hypersensitivity responses.

Serum immunoglobulin and antibody levels and the passive transfer of resistance in hamsters infected with Dipetalonema viteae.

Clearance of microfilariae from the circulation of hamsters infected with Dipetalonema viteae was demonstrated following passive transfer of serum obtained from hyperinfected hamsters. The exclusion fraction after gel filtration of this serum on Sephacryl S 200 also cleared microfilariae whereas the other fractions did not. There was no clearance with serum taken during the pre-patent, patent or latent periods of singly infected hamsters. IgM antibody to the microfilaria cuticle measured by the fluorescent antibody technique was increased 2 to 4 times in the protective serum over the other sera. Suppression of microfilaremia was also adoptively transferred by cells from infected hamsters. Serum Ig and antibody levels to microfilaria cuticle were measured in 3 strains of hamsters differing in their ability to clear microfilariae. IgM antibody to microfilaria cuticle correlated with the ability to clear. No IgG antibody to microfilaria cuticle was detected and the major Ig response to infection was in IgG1, which increased 10 to 20 fold.

Isolation and partial characterization of prolactin from equine pituitary gland (hypophysis).

Highly purified equine prolactin was prepared from equine pituitary glands (hypophysis) by serial extractions with water at pH 5.5, 0.1 M (NH4)2SO4 at pH 4.0, and 0.25 M (NH4)2SO4 at pH 5.5 to remove other hormones, and then finally with 70% ethanol at pH 9.3 to 10.0 to extract prolactin. Preliminary purification of the extract involved salting out other substances with 0.1% NaCl at pH 9.0. Prolactin was precipitated out by adding three times the volume of 95% ethanol at 4 C. This prolactin preparation had a biological potency of 24 IU/mg. Further purification by isoelectric focusing on a pH gradient of 5 to 7 gave three prolactin components with the following characteristics: isoelectric point 5.8, 5.7, and 5.25; biological potencies (IU/mg) 35.6, 19.6, and 11.3. The major component had a molecular weight of 25,000, an isoelectric point of 5.8, and a biological potency of 35.6 IU/mg. Antiserum produced against this component did not cross-react with equine follicular stimulating hormone, luteinizing hormone, and growth hormone, but did cross-react with ovine and bovine prolactin. Human and murine prolactin had little cross-reactivity with the equine prolactin antiserum.

Kinetics of Dipetalonema viteae infections established by surgical implantation of adult worms into hamsters.

Native LVG strain hamsters were infected with Dipetalonema viteae by the surgical implantation of adult worms. Groups of hamsters received either 50 male, 50 female, 50 male plus 50 female or 25 male plus 25 female worms per hamster. Approximately 50% of the transferred worms became established in the recipient hosts regardless of the number or sex of the worms implanted. Microfilaremia occurred in recipient hamsters within 1 week after the transfer of female or male plus female worms. This microfilaremia became negative on week 9 post-transfer and no microfilaremia developed in these hamsters following a secondary challenge infection of male plus female worms. Hamsters whose primary infection consisted solely of male worms developed a microfilaremia when challenged with male plus female worms.

Alteration of amicrofilaremia in Dipetalonema viteae infected hamsters with immunosuppressive drugs.

Dipetalonema viteae-infected hamsters with amicrofilaremic infections were subjected to immunosuppressive therapy. Methyl prednisolone acetate caused the most severe recrudescence of microfilariae while cyclophosphamide caused a low level, transient microfilaremia. Saline injected control hamsters remained amicrofilaremic. Neither drug influenced the number of adult worms recovered at necropsy in the treated hamsters compared with control hamsters.

The pathology associated with single and quadruple infections of hamsters with Dipetalonema viteae.

Two groups of five hamsters were each infected subcutaneously with infective larvae of Dipetalonema viteae; one group received single infections, and the second group received quadruple infections. A third group of five hamsters served as controls. Hamsters with primary and quadruple infections had cellular infiltrates in the liver and glomerular basement membrane thickening; these lesions were more extensive in the multiple than single infections. Hyperinfected hamsters also developed subcutaneous nodules. By histologic examination, it was seen that nodules were encapsulated abscesses which had a central, necrotic core, and were confined by a fibrous connective tissue capsule. The central portion of some nodules contained nondegenerated worms. The same area of other nodules contained dead worms, while still other nodules contained calcified worms or necrotic and calcified debris.

A comparison of the acquired resistance to Dipetalonema viteae stimulated in hamsters by trickle versus tertiary infections.

Two groups of hamsters were hyperinfected with Dipetalonema viteae. Each of the 15 hamsters in the first group received a total of 900 larvae given in three equal doses on days 0, 150 and 250 from the start of the experiment (tertiary-infection group). Each of the 20 hamsters in the second group received a total of 900 larvae given in 18 equal doses (50 larvae per dose) at 14 day intervals. Thus the final dose was given on day 238 from the start of the experiment (trickle-infection group). About half of the hamsters in each group were killed 100 days after the last sensitizing infection and their adult worm burdens and subcutaneous nodules were counted. The tertiary-infection group had a higher average number of adult worms per hamster, but fewer subcutaneous nodules than the trickle infection group. However, when the number of adult worms and subcutaneous nodules were added together, the sums from both groups were similar. The remaining hamsters of the above two groups, along with a group of nine previously uninfected hamsters, were given a challenge infection of 500 larvae per animal. Necropsy data taken 70-80 days after the challenge infection indicated inhibition and/or destruction of developing larval stages in the trickle + challenge-infection group, but no such acquired resistance phenomenon in the tertiary + challenge-infection or challenge-infection groups. While the mechanism remains unknown, it is clear that prior exposure to repeated small infections over an extended period stimulated a protective response to D. viteae in hamsters. This response was not seen in animals given three large infections over a similar time period.

Fractionation of a soluble somatic extract and solubilized cuticular extracts of Dipetalonema viteae adult worms.

A soluble somatic preparation (SSP) of adult Dipetalonema viteae was prepared. Aliquots of the aqueous insoluble debris (cuticles and membranes) left after the extraction of SSP were solubilized separately with Triton X-100 and lithium diiodosalicylate to yield a Triton solubilized preparation (TSP) and lithium diiodosalicylate solubilized preparation (LSP), respectively. SSP was sequentially chromatographed on a series of Sephadex columns, G50; then fraction 1 from G50 on G100 and finally fraction 1 from G100 on G200 yielding 5, 4, and 5 fractions on G50, G100, and G200, respectively. TSP and LSP were each sequentially chromatographed on Sephadex G50 and G200 to yield 5 and 4 fractions for TSP and 6 and 3 fractions for LSP, respectively. Each Sephadex fraction from each separation was further resolved by polyacrylamide gel electrophoresis (PAGE). Immunodiffusion studies with a rabbit antiserum to SSP gave 4 precipitin bands with SSP, 1 with TSP, and none with LSP. The single precipitin arc produced with TSP showed partial identity with an SSP arc. Disc immunoelectrophoresis allowed the identification of the proteins separated by PAGE, which precipitated with the rabbit anti-SSP serum.