The Malaria Research and Reference Reagent Resource (MR4) Center--creating African opportunities.

The Malaria Research and Reference Reagent Resource Center (MR4) is a program to support the international malaria research community. The primary goal is to improve access to well-characterized quality-controlled parasite, host and vector reagents for standardization of assays and other general research purposes. MR4 is open to all scientists worldwide, however many of its inaugural activities will have a focus in Africa, where the concept for the MR4 was born. Reagents are donated to the MR4 Center by malaria researchers and their institutions for distribution to other investigators special arrangements are also possible for developing reagents in-house, or for coordinating reagents for multi-site studies. In addition to providing reagents, the MR4 also aims to promotoe technology transfer and to foster scientific exchange between new and established malaria researchers. This will be done via workshops, training programs, and dissemination of information (electronic and hard copy). Suggestions for reagents, information resources, or training opportunities are welcome from the research community to make this a responsive, service-oriented program.

Managing genetic material to protect intellectual property rights.

One of the most important policy instruments for the promotion of further biotechnology development is intellectual property right (IPR) protection. However, one cannot improve upon a biotechnological invention without physical access to the germplasm, making exchanges of genetic material necessary. A formal transfer agreement, which addresses the key issues of ownership, access, use, and equitable benefit-sharing, is a powerful legal instrument for intellectual property. Other restrictions are generally imposed as a result of national and international safety regulations. Forming strategic alliances, such as joint ventures, collaborative research agreements, joint research and development agreements, and manufacturing and distribution alliances to exploit the economic value of genetic material, provides scientists with the mechanisms they need to bring their research material and products to the marketplace.

Nematospiroides dubius: two H-2-linked genes influence levels of resistance to infection in mice.

Strains of mice sharing common H-2 haplotypes but different genetic backgrounds, and H-2 congenic strains of mice differing only at H-2 genes were studied to assess the role of H-2 and non-H-2 genes in immunity to challenge infections with the nematode parasite Nematospiroides dubius. Strains of mice sharing the H-2k haplotype were uniformly more susceptible to challenge than strains expressing H-2q alleles, regardless of genetic background. However, in some cases strains of mice sharing the k or q haplotypes differed significantly in levels of resistance. Therefore, non-H-2 genes must influence the response observed. H-2 cogenic strains of mice differed markedly in their ability to resist challenge infections. Mice sharing the C57BL/10 background but expressing k alleles were very susceptible to challenge, while the H-2q, H-2f, and H-2s, haplotypes were associated with resistance. Studies of H-2 congenic recombinant strains of mice suggested that two H-2 genes influence the antiparasite response. One of these genes maps to the left of E alpha and the other to the D-end of the H-2 complex. It is concluded also that the unique configuration of H-2 genes in F1 hybrids contributes to increased resistance to challange.

Nematospiroides dubius: genetic control of immunity to infections of mice.

Inbred strains of mice differ in their susceptibility and resistance to challenge infections with Nematospiroides dubius. In our studies, F1 hybrid mice from resistant SJL and susceptible CBA parents were resistant to N. dubius challenge infections. Only 22% of backcrosses to SJL were susceptible while backcrosses to CBA had a wide range of susceptibility. Male mice were more susceptible than female mice. In another experiment, inbred strains of mice were compared in their ability to resist N. dubius challenge infection: SJL and A.SW (H-2s) mice became resistant after one immunizing infection, A, A/He (both H-2a), as well as BALB/c and DBA/2 (both H-2d) mice became resistant after two immunizing infections, while C57BL/6 (H-2b), C3H/He, CBA, and AKR (H-2k) mice remained susceptible. The resistance to reinfections was characterized by reduction of worm burdens between Days 6 and 14 postinfection. It was concluded that (1) resistance to N. dubius challenge infections is inherited in a dominant fashion and that multiple genes may influence such response, which in turn might be modulated by the Y chromosome; (2) both MHC and non-MHC genes may influence, in conjunction with the number of exposures to parasite antigens, the resistance to challenge infections.

Influence of immunizing dose and presence or absence of adult worms on the development of resistance to Nematospiroides dubius challenge infections of mice.

H-2 congenic strains expressing resistant (H-2q, H-2f) or susceptible (H-2k) haplotypes were compared for their ability to resist challenge infection with N. dubius following a 6- or 14-day ivermectin-abbreviated immunizing infection. B10.BR mice (H-2k) were considerably more resistant to infection when the priming interval was shortened from 14 to 6 days. B10.Q (H-2q) and B10.M (H-2f) mice resisted challenge regardless of which immunization regimen was used. The influence of parasite numbers on the response to challenge was studied by comparing infections in resistant DBA/1 (H-2q) and susceptible CBA/J (H-2k) mice that differ at both H-2 and non-H-2 genes. DBA/1 mice, immunized with 50 or 150 L3 of N. dubius for 14 days, resisted challenge, whereas mice receiving 300 worms did not. In contrast, CBA/J mice failed to resist challenge at all priming doses tested. When the immunizing infection was shortened from 14 to 6 days, DBA/1 mice resisted challenge regardless of priming dose and CBA/J mice resisted challenge only when the highest dose of 300 worms was used for priming. The data suggest that susceptible strains of mice may be preferentially immunosuppressed, particularly at low infective doses, and that suppression is associated with adult worms present in the lumen of the small intestine.

In vivo and in vitro egg production by Nematospiroides dubius during primary and challenge infections in resistant and susceptible strains of mice.

Experiments were conducted to examine adult worm burdens, fecal egg output, and in vitro fecundity of Nematospiroides dubius in resistant LAF1 and susceptible CBA mice 12, 15, 18, and 21 days following primary and challenge infections. A strong correlation was obtained on the number of eggs produced by worms cultured in vitro and the egg production as assessed by fecal egg count. Worm counts, fecal egg counts, and in vitro fecundity were similar on all days studied following a primary infection in both mouse strains. However, after challenge infection, LAF1 mice showed lower worm burdens, fecal egg output, and in vitro egg production when compared to CBA mice. Although the egg production of surviving female worms from immune LAF1 mice was decreased, it never fell below a threshold of 100 eggs/day. The reduced fecundity may be a manifestation of a general anti-worm response rather than responses directed specifically at worm reproduction.

The tissue reactions of mice to infection with Heligmosmoides polygyrus.

The intestines of normal and resistant LAF1 mice were subjected to histologic study to determine the timing and mechanisms of resistance to reinfection by Heligmosmoides polygyrus. During reinfection third-stage larvae are less able to penetrate the intestinal wall. Larvae which are able to encyst develop at a slower rate and provoke an increase in nonspecific inflammation around their cysts. After emergence from intestinal cysts, preadults are rapidly lost, but at no time were injured or destroyed larvae or adults noted. Exsheathed larvae were injected via tail vein into control, sensitized and resistant BALB/c mice. The inflammatory response around entrapped larvae in the lung was measured at 1, 2, 4, and 8 days. A heightened inflammatory response, consisting primarily of polymorphonuclear cells with some round cells which peaked in size on day 2, was observed in both sensitized and resistant mice. A similar heightened inflammatory response was also observed in both AKR (non-resistant) BALB/c (resistant) mice vaccinated subcutaneously with exsheathed larvae.

Adoptive transfer of resistance to Nematospiroides dubius infections of mice and an assay to measure the in vitro proliferation of lymphocytes reactive with N. dubius antigen.

Spleen and mesenteric lymph node cells from Nematospiroides dubius-infected and normal control mice were cultured in vitro with N. dubius antigen. Proliferation of these cells in response to antigen was measured by the uptake of [3H]TdR. Cells harvested from mice during a primary infection did not proliferate in vitro; however, low levels of specific proliferation could be demonstrated if these mice were treated on Day 5 post-infection with 20 mg/kg of cyclophosphamide i.p. A strong cell proliferative response was measured 6 days following a challenge infection; spleen cells responded more strongly than cells from the mesenteric lymph nodes (MLN), but the addition of lymph node cells to spleen cell cultures did not suppress the latter response. Responsiveness of spleen cells to concanavalin A (Con A) was two-fold higher in infected mice than in normal controls, but the proliferation of MLN cells to Con A was similar in infected and uninfected mice. When N. dubius-resistant B10.M (H-2f) mice were compared to the susceptible B10.BR (H-2k) mice, no differences were observed in the spleen cell response to N. dubius adult antigen following challenge infections. However, after a tertiary infection, MLN cells from the resistant strain proliferated strongly in comparison to cells from susceptible mice. Spleen or MLN cells from resistant mice transferred immunity to naive recipients provided that the recipients had received a prior injection i.p. with adult N. dubius antigen. The injection alone, or cells in the absence of the injection, failed to protect the recipients from N. dubius challenge.

The impact of single versus repeated exposure of mice to Toxocara canis.

Infection with T. canis can alter dramatically the brain and behavior of the host. Previous results suggest that if the mammalian host is exposed either simultaneously to lead, or has a history of prior exposure to that toxic substance, the magnitude of the behavioral reaction to T. canis may be modified or even reduced. The present data suggest that the magnitude of both the behavioral and tissue/immune reactions may be less if the organism has multiple, instead of a single exposure, to T. canis. Lead, and perhaps other environmental toxicants may alter neurotropic products of the parasite, the behavior of the parasite, and/or reactivity of the host in the presence of the parasite. Such considerations may help explain, in part, the relative rarity of reported toxocariasis in humans, despite the fact that serological indices suggest that exposure to T. canis may be as high as 7% of the world population.

Behavioral effects of early lead exposure and subsequent toxocariasis in mice.

Mice were maintained under standard conditions, or treated with lead from birth, until they were approximately 40 d of age. At that time the lead solution was replaced with tap water and all animals were gastrointestinally intubated with either the parasite Toxocara canis or normal saline. At 2-5 wk following intubation the behavioral effects of T. canis in combination with the prior history of lead ingestion were compared to those produced by administration of either T. canis or lead alone or a single saline intubation. Differential group activity scores (cage crosses and standups) in response to changes in the home-cage environment during testing sessions were observed. While the addition of a second tier to the home cage during testing resulted in increased activity scores for all groups, changing the home-cage bedding produced increased activity only in the T. canis alone or prior lead ingestion alone groups. However, there were no differences in the number of parasite larvae found in fresh brain tissue preparations in either the T. canis alone or T. canis plus lead-history combination groups.

A survey of susceptibility to infection with Trichinella spiralis of inbred mouse strains sharing common H-2 alleles but different genetic backgrounds.

Twenty-eight different inbred strains of mice representing five different H-2 haplotypes were compared for degree of susceptibility to a primary infection with Trichinella spiralis. Marked differences in susceptibility, measured by the average number of muscle larvae per host, were seen among strains of mice sharing common H-2 alleles. The genes controlling these differences must therefore map at loci outside the major histocompatibility complex. Strains of mice sharing the H-2k haplotype were generally more susceptible than strains expressing other haplotypes and strains expressing H-2q alleles were most resistant. Strains of mice were ranked in order of decreasing susceptibility. Knowledge of these ranking may be of value to researchers wishing to select strains of mice appropriate for studies on T. spiralis.

Trichinella spiralis: role of non-H-2 genes in resistance to primary infection in mice.

Two strains of mice which share identical H-2 genes but differ in their genetic backgrounds were compared for their ability to resist infection with Trichinella spiralis. The two strains of mice, C3HeB/FeJ and AKR/J, share the H-2k haplotype which is associated with susceptibility to primary infection with T. spiralis in H-2 congenic strains of mice. AKR/J mice, infected with 150 infective muscle larvae, harbored significantly fewer muscle larvae 30 days postinfection than did mice of the strain C3HeB/FeJ. Approximately equal numbers of worms establish in the small intestine of AKR and C3H mice, but the AKR mice expelled adult worms from the gut more rapidly than did mice of the C3H strain. By Day 9 postinfection, 50% of the worms had been expelled by the AKR mice whereas expulsion of worms from C3H mice was delayed beyond Day 9 and occurred primarily between Days 10 and 12. Over this same experimental period (Days 6-12), fecundity of female worms from AKR mice, measured as the mean newborn larvae/female/hour, was approximately one-half that of worms taken from C3H mice. These results support the conclusion that genes outside of the mouse H-2 complex regulate expulsion of adult worms from the gut. These background genes also markedly influence the fecundity of female worms.

Neuropathological studies of experimental toxocariasis in lead exposed mice.

Binghamton Heterogeneous stock mice intubated with embryonated eggs from the common parasite of dogs, Toxocara canis, display dramatic alterations in behavior. Many of these behavioral changes are markedly attenuated if mice are maintained also on a drinking solution containing lead acetate. The data presented here, however, show that the extensive neural degeneration of the CNS occurs after T. canis infection and that the nature and extent of this degeneration is similar, regardless of whether mice had been administered parasites alone or in combination with lead. Thus the neuropathological data reported in detail here cannot account for the striking interactive influence of T. canis and lead on behavior.

Immunity to Nematospiroides dubius: parasite stages responsible for and subject to resistance in high responder (LAF1/J) mice.

In the LAF1/J mouse strain, a single prior exposure to infective larvae of N. dubius given per os resulted in greater than 90% reduction of an homologous larval challenge; in contrast, a single larval sensitizing infection had no effect on adult worms introduced directly into the duodenum via laparotomy. The LAF1/J mice given a single sensitizing infection of adult worms via laparotomy did not exhibit resistance to homologous challenge of either infective larvae or adult worms. Because all sensitizing infections were removed chemotherapeutically before administration of challenge inocula, potential effects of "overcrowding" on establishment or development of challenge infections were precluded. Worm-specific antibody determinations indicated that adult worms introduced into the gut lumen did not prime the host for a secondary response to a challenge by larval or adult worms. However, a challenge with larvae of mice previously sensitized with an homologous larval infection, did stimulate an anamnestic antibody response. Collectively, the data indicated that in a highly responsive mouse strain (LAF1/J), larval stages were requisite in stimulation of host resistance to reinfection and a larval challenge was fully susceptible to the effects of that response. Mature adult worms apparently did not stimulate nor were they susceptible to the host's immune response.

Kinetics of immunoglobulin M and G responses of nude and normal mice to Trypanosoma musculi.

A sensitive and specific enzyme-linked immunosorbent assay was designed to measure the kinetics of Trypanosoma musculi-specific immunoglobulin M (IgM) and IgG responses in mice. Serum was obtained from congenitally athymic (nude) mice, their phenotypically normal, thymus-bearing littermates (NLM), and thymus cell-repaired nude mice (Nu-TC) at 6-day intervals throughout T. musculi parasitemia. NLM mice were shown to effect an antibody response to T. musculi that included an IgM to IgG shift and was correlated in time with reduction of parasite reproduction and stabilization of parasitemia. Nude mice were shown to effect a T. musculi-specific IgM response similar in onset and magnitude to that in NLM mice; this response was correlated in time with stabilization of parasitemia. Nu-TC mice were shown to effect IgM and IgG responses to T. musculi similar in time and magnitude to those in NLM mice. In marked contrast to NLM mice, Nu-TC mice did not exhibit suppression of T. musculi-specific IgM production after the IgM to IgG shift in response to this parasite.

Epizootiologic patterns of diabetes mellitus in dogs.

A case-control study of spontaneous diabetes mellitus in dogs was undertaken, using 2 veterinary data bases. The Veterinary Medical Data Program (VMDP) contained records of 1,019 cases of canine diabetes from 14 university-affiliated veterinary hospitals. The Animal Medical Center (AMC), a private veterinary hospital which has not participated in the VMDP, contained records of 449 diabetes cases. Each data base was analyzed separately, control groups being chosen from all admissions, excluding diabetic animals. Summary odds ratios by sex adjusted for age and breed indicated significantly (P less than 0.05) elevated risks for entire females and neutered females compared with that for entire males. The VMDP data indicated a significantly elevated risk for castrated males, whereas the risk derived from AMC data was not significantly different from 1. Analysis of risks by breed adjusting for age and sex identified Poodles as being at significantly excess risk, and German Shepherd Dogs, Cocker Spaniels, Collies, and Boxers at significantly decreased risk in both data sets. The male-female risk ratio changed with age from 1 at less than 1 year of age to a predominance of females at older ages. In the AMC data base, diabetes was significantly associated with cataracts in dogs of both sexes combined. Diabetes was also significantly associated with benign mammary tumors in female dogs.

Behavioral effects of lead and Toxocara canis in mice.

Adult mice were administered the common parasite Toxocara canis or lead or both. The parasite clearly altered mouse performance on tests of exploration, activity, learning, and motor coordination; behavioral effects in mice receiving lead alone were less general. Consequence of Toxocara administration appeared attenuated in animals receiving both agents. Parasite larvae were found in the central nervous system in all infected mice.

Toxocara canis and lead alter consummatory behavior in mice.

Ingestion of palatable and unpalatable solutions was measured in adult mice in which had been administered the common parasite of the dog, Toxocara canis alone, or in combination with lead. In addition, response to hot plate and susceptibility to electroconvulsive seizure were also measured. Results from the palatability test indicated that either lead or Toxocara may alter the mouse's mode of interacting with its environment. However, the two agents in combination interacted in their effects on consummatory behavior. Results from the hot plate and ECS measures were less clear with respect to how lead and/or Toxocara influence temperature reactivity and seizure susceptibility. Histological examination of the CNS in parasite infected animals revealed Wallerian Type degeneration of fiber pathways including the corpus callosum, olfactory tract, and cerebellar penduncles.