Sarcocystis muris--a rare case in laboratory mice.

In a lifespan transgeneration study under standard laboratory conditions using a total of 4682 CBA/J mice, unusual intramuscular inclusions were found in the diaphragm, heart and skeletal muscle of one mouse using light microscopy. Located within the myocytes, they caused no visible tissue reaction. Cross-sections of these spherical and cystic lesions showed numerous banana-shaped structures, identified as permanent parasitic bradyzoites, which permitted these infections to be diagnosed microscopically as sarcocystosis.

Helminth parasites of conventionally maintained laboratory mice - II. Inbred strains with an adaptation of the anal swab technique

Worm burdens recovered from inbred mice strains, namely C57B1/6, C57B1/10, CBA, BALB/c, DBA/2 and C3H/He, conventionnally maintained in two institutional animal houses in the State of Rio de Janeiro, RJ, Brazil, were analysed and compared, regarding their prevalences and mean intensities. Three parasite species were observed: the nematodes Aspiculuris tetraptera, Syphacia obvelata and the cestode Vampirolepis nana. A modification of the anal swab technique is also proposed for the first time as an auxiliary tool for the detection of oxyurid eggs in mice.

A protocol for the production of Neospora caninum tissue cysts in mice.

Identification of a definitive host for Neospora caninum has been inhibited by lack of an efficient method for producing bradyzoites, needed for oral infectivity trials. An improved protocol for producing bradyzoite-containing tissue cysts in mouse brains is described. Six variables, including mouse strain (Balb/C, CBA/Ca, and ICR), sex, N. caninum isolate (NC-2 and NC-Liverpool), tachyzoite inoculum dose, immunosuppression with methylprednisolone acetate (MPA), and sulfadiazine treatment were tested. Tissue cyst numbers were estimated using an immunohistologic staining procedure specific for bradyzoites. Male ICR mice (> or = 30 g) that were immunosuppressed with 2 mg MPA 7 days prior to and 2.5 mg MPA at the time of subcutaneous inoculation with 400,000 N. caninum tachyzoites produced the highest numbers of tissue cysts. Significant numbers were produced by methods using the NC-2 strain of N. caninum; however, protocols using NC-Liverpool produced greater numbers of tissue cysts. Sulfadiazine treatment did not appear to contribute to tissue cyst production. The procedure described is superior to previously described methods with regard to numbers of tissue cysts produced, protocol reproducibility, and survival of mice until tissue cyst formation.

Effects of qinghaosu (artemisinin) and its derivatives on experimental cutaneous leishmaniasis.

We have investigated the effect of qinghaosu (QHS, artemisinin) and its derivatives on Leishmania major replication in vitro and on the disease development in mice infected with L. major. Artemisinin is effective against promastigotes in vitro, with an ED50 (50% effective dose) at 7.5 x 10(-7) M. Both artemisinin and artemether are leishmanicidal for amastigotes in infected murine macrophages in vitro, with ED50 at 3 x 10(-5) M and 3 x 10(-6) M respectively. These compounds have no effect on the viability of macrophages or on the phytohaemaglutinin-induced proliferation of normal spleen cells, even at 10(-4) M. BALB/c mice infected in the footpad with L. major developed significantly smaller lesions and parasite loads when treated with the compounds. Intra-lesion injection of the compounds was the most effective route. The intramuscular and oral routes were also effective; however, intravenous injection with artesunate was not effective.

The population dynamics of acquired immunity to Heligmosomoides polygyrus in the laboratory mouse: strain, diet and exposure.

An experiment was designed to investigate aspects of the population dynamics of acquired immunity to Heligmosomoides polygyrus in laboratory mice. The influence of host strain (CBA or NIH), rate of exposure (5 or 40 L3/mouse/2 weeks) and diet (3 or 16% protein w/w) on the population dynamics of repeated infection and the response to a standard challenge infection were investigated. The time delay between the end of the period of repeated infection and the subsequent challenge (between 1 and 24 weeks) had no effect on worm recovery. The effects of both exposure and diet were significant and similar whether assessed on the basis of the dynamics of repeated infection or response to challenge: low rates of exposure and low dietary protein were both associated with low levels of acquired immunity. Mouse strain was the most important determinant of worm recovery after challenge, but had no significant effect on the degree to which parasite population growth was constrained by acquired immunity during repeated infection. It is suggested that both CBA and NIH mice raise immune responses which act on parasite survival, but that only NIH mice raise responses operative against larval establishment.

Early expression in mice of genetic resistance to intestinal amebiasis.

Some strains of mice are known to be relatively resistant to hepatic or intestinal amebic infections. In order to know if the intestinal resistance is expressed few hours after infection, we inoculated axenic amebae in three inbred strains of mice either by direct intracecal injection or by infection of a washed-closed cecal loop. We found that amebae do not survive in conventional animals but they colonize longer in animals with the cecal loop. However, the survival was low after 24 hours postinfection. Balb/c mice were more susceptible and CBA mice more resistant. Our results suggest that genetic resistance to intestinal amebiasis is expressed in mice in the early phases of infection.

Host diet in experimental rodent malaria: a variable which can compromise experimental design and interpretation.

Over the past few years several experienced groups studying malaria have encountered significant problems with their particular rodent malaria-host system. This has involved, in some cases, periods during which the recovery of cryopreserved parasite stocks and growth of bloodstream parasites was markedly inhibited and, in other cases, periods of drastically increased mortality rates. The common factor linking these incidents was that they coincided with alterations in the experimental animal diet used. The inhibition of growth of cryopreserved stabilates or bloodstream parasites was abolished by supplementation with p-aminobenzoic acid (PABA) or by changing the diet used. Although the suppressive effects of diets lacking PABA on parasite growth have been known for over 30 years, the variation of PABA levels in modern laboratory animal feed concentrates is not well recognized. We have not established the exact cause of increased mortality, but it has been overcome by changing the diet used. We are documenting our experiences with this potential variable to forewarn workers in other laboratories of possible problems inherent in the use of different diets.

The host-parasite relationship of Schistosoma haematobium in CBA mice.

The host-parasite relationships of two geographical isolates of Schistosoma haematobium in CBA mice are described and compared to previous reports on this parasite in other experimental hosts and in man. The mean percentage establishment of worms in mice was 17% and was not affected by the age or sex of the host. Adult worm burdens remained constant over 20 weeks, but were reduced after 18 months of infection. Male and female worms reached mean maximum lengths of 4.78 and 5.9 mm respectively. Egg laying commenced 9.5 weeks after infection and eggs accumulated in the tissues throughout the period of infection. A large increase in the rate of egg accumulation occurred coincidental with the appearance of eggs in the bladder of some mice. Faecal eggs were first observed in some mice at 12.5 weeks and most mice excreted a few eggs by 17 weeks p.i. (post-infection). Eggs were not found in the urine of infected mice. Excreted eggs and eggs isolated from the livers of infected mice hatched, but the resulting miracidia were unable to infect appropriate snail hosts. The development of hepatic granulomas and egg-induced pathology in the bladder of mice is described.

Parasitological observations of Schistosoma bovis in normal and T-cell deprived mice.

A laboratory life-cycle of Schistosoma bovis was established in order to study the host-parasite relationship in immunologically intact and T-cell deprived mice. Normal mice were found to have 'self-cured' their S. bovis infections almost completely by 10 weeks after cercarial administration, and there was no evidence of self-cure by day 79 in T-cell deprived animals. Thus, groups of deprived mice autopsied between 9 and 11 weeks after infection were invariably found to have greater worm burdens and a greater total number of eggs in the liver than comparably-infected normal mice. However, liver egg counts/worm pair were similar in the two types of host, and differences between normal and deprived mice with respect to total S. bovis egg counts in the intestine were also not consistently in the same direction in all experiments. Faecal egg counts were always less in deprived mice than in normal mice, even in an experiment in which the deprived mice had a significantly higher intestinal tissue egg count than the normal control group. The results are discussed in relation to the better known S. mansoni/mouse host-parasite relationship.

The response to Schistosoma bovis in normal and T-cell deprived mice.

Normal and T-cell deprived mice have been compared in their response to infection with Schistosoma bovis. The deprived mice survived longer than comparably infected, immunologically intact controls, despite an increased longevity of the adult S. bovis worms in the former animals giving rise to higher tissue egg densities. The reduced pathology in deprived mice was due to inhibition of T-cell dependent granuloma formation around tissue-bound schistosome eggs, with concomitantly decreased tissue disruption as evidenced by smaller spleens and lower circulating transaminase concentrations. These observations on S. bovis contrast with the greater morbidity and earlier mortality induced by S. mansoni in T-cell deprived mice, the latter due to an hepatotoxic potential of S. mansoni eggs that is expressed in the absence of the host immune response. The absence of hepatocyte damage around S. bovis eggs in deprived mice indicates that this schistosome lacks such a toxin, and this could explain why during S. bovis infection synthesis of the two acute-phase proteins, complement C3 and serum amyloid P-component (SAP) here seemed less T-dependent than has previously been found during S. mansoni infection of mice. In a time-course experiment the hypergammaglobulinaemia induced by S. bovis, and the specific IgG antibody response against egg antigens were significantly T-cell dependent during the early stages of patency. Similarly, in most experiments assayed once between 9 and 11 weeks after S. bovis infection, deprived mice had significantly reduced hypergammaglobulinaemias, and reduced specific IgM and IgG antibody responses against both worm and egg antigens.

Collagen isotypes, laminin, and fibronectin in granulomas of the liver and intestines of Schistosoma mansoni-infected mice.

Patterns of fibrosis within hepatic and intestinal granulomas of Schistosoma mansoni-infected mice were analyzed by indirect immunofluorescence. Deposition of collagen isotypes, laminin, and fibronectin was evaluated semiquantitatively between 8 and 20 weeks of the infection. Liver granulomas were the largest at 8 weeks and contained low amounts of type I and higher amounts of type III collagen and fibronectin. Collagen deposition became pronounced as infection progressed. The relative amounts of type I collagen deposits rose and equalled that of type III. In the smaller immunomodulated granulomas at 20 weeks both types I and III were high, and type IV collagen deposition was observed. Fibronectin and laminin deposits were also detected. The small ileal granulomas did not change their size during the course of the infection. At 8 weeks, connective tissue matrix deposition was barely detectable within these lesions. Gradually, small deposits of types I and III appeared in equal amounts and attained highest levels by 20 weeks of the infection. Fibronectin deposits at that time were very prominent but laminin and type IV collagen were absent. Colon granulomas at 8 weeks of the infection were only somewhat smaller than those of the liver, yet contained very sparse deposits of types I and III collagen. During the ensuing weeks collagen deposits rose only slightly. By 20 weeks the granulomas diminished in size and within those lesions type III collagen was predominant. Whereas the presence of fibronectin was pronounced, type IV collagen and laminin were detectable only in trace amounts. These observations indicate the existence of important organ-related differences in the intragranulomatous deposition of connective tissue matrix.

Protective immunity to malaria: studies with cloned lines of Plasmodium chabaudi and P. berghei in CBA/Ca mice. I. The effectiveness and inter- and intra-species specificity of immunity induced by infection.

CBA/Ca mice were immunized by infection with cloned lines of Plasmodium berghei (isolates ANKA, KSP-11). Plasmodium chabaudi chabaudi (AS, CB) or Plasmodium chabaudi adami (DS) and then challenged with either homologous or heterologous parasites. Protective responses were assessed in immune mice relative to the controls by their ability to (i) extend the time taken for the mean parasitaemia to reach a predetermined level (1% or 0.1%) (ii) reduce peak parasitaemia (iii) resolve the parasitaemia sooner and/or (iv) control or eliminate recrudescences. At both the inter- and intra-species level, immunity appeared largely specific for the cloned line inducing it. At the interspecies level marginally effective cross-immunity was sometimes evident, thus P. berghei KSP-11 immune mice displayed some immunity against P.c. chabaudi AS, although immunity to this parasite was relatively ineffective against P. berghei ANKA or KSP-11. Cross-immunity was more apparent between the subspecies P.c. adami and P.c. chabaudi and between cloned lines of the latter parasite derived from the AS and CB isolates. These data reflect considerable inter- and intra-species structural and immunogenic differences in certain antigens of parasitized erythrocytes and merozoites, which have been identified in a number of murine malarias and associated with protective immunity. Similar differences recently identified in the equivalent antigens of the human parasite P. falciparum may therefore have important implications for protective immunity in man.

Serodiagnosis of mansonian schistosomiasis with CEF6, a cationic antigen fraction of Schistosoma mansoni eggs.

Studies of the host-parasite relationship of Schistosoma mansoni in mice revealed that the parasite's eggs were toxic to liver cells of immunosuppressed hosts. Attempts to identify the toxic egg constituent led to the isolation of a fraction CEF6 from crude egg homogenate by cation exchange chromatography. CEF6 contains two cationic antigens, omega 1 and alpha 1, the former being recognized as the putative hepatotoxin. In an initial assessment of its serodiagnostic potential in enzyme immunoassay (ELISA), CEF6 yielded an enhanced degree of sensitivity and specificity when compared with crude egg homogenate, and a subsequent 'blind' inter-laboratory collaborative study confirmed that in certain circumstances the use of a purified fraction such as CEF6 may be advantageous. We here review the experimental work leading up to the isolation of CEF6, and summarize the results so far obtained from its use as a diagnostic aid in human schistosomiasis.

Metabolism of Babesia parasites in vitro. Change in adenylate energy charge and infectivity of Babesia rodhaini-infected erythrocytes.

Studies were undertaken to investigate a possible correlation between the change, during experimental in vitro treatments, in adenylate energy charge (AEC) of B. rodhaini-infected erythrocytes and the infectivity of the parasites for mice. When the AEC and infectivity of B. rodhaini-infected erythrocytes were modified by incubation in basal salts media containing various substrates, parasite infectivity correlated significantly (r2 = 0.83, p less than 0.01) with change in AEC. However, B. rodhaini frozen with and without cryoprotectant demonstrated large changes in infectivity but relatively small changes in AEC. The final AEC values were similar to those of unfrozen organisms, although infectivity was greatly depressed. The results suggest that, when in vitro treatment changed the adenine nucleotide pool (ATP + ADP + AMP) of infected erythrocytes, change in AEC correlated with infectivity. When the adenylate pool was unchanged, no such correlation was evident. Thus, the AEC can be a useful index of parasite infectivity in some, but not necessarily in all, in vitro systems. Data on the relative utilization by B. rodhaini of various substrates, obtained when AEC and infectivity of infected erythrocytes were modified, are also reported and discussed.

The susceptibility of BALB/C and other inbred mouse strains to Brugia pahangi.

The susceptibility of several strains of inbred mice to infection with the filarial worm Brugia pahangi has been examined. BALB/C, C57BL/10, C3H/He, 101, CBA/Ca mice, congenitally asplenic (DH/+) mice and their normal litter-mates (+/+) were each challenged by the intraperitoneal inoculation of 50 infective larvae. During the first four weeks of infection high (19-42%) larval recoveries were obtained from the CBA/Ca, BALB/C and Dh/+ mice but fewer than 10% of inoculated larvae were recovered from C3H/He, 101; C57BL/10 and +/+ mice. Larval growth rates in all mice were similar. BALB/C and Dh/+ mice only were examined later than four weeks after infection. The yield of adult worms from BALB/C was 7.5% at 16 weeks and from Dh/+ 4.2% at 21 weeks. Microfilariae were present in the peritoneal fluids but not the blood of some mice harbouring both adult male and female worms.

Intraerythrocytic development and antigenicity of Plasmodium falciparum and comparison with simian and rodent malaria parasites.

Using sorbitol-synchronised cultures and metabolic labelling with [35S]methionine, the stage specificity of polypeptides synthesised by the intraerythrocytic stages of Plasmodium falciparum was studied. We confirmed that the synthesis of many polypeptides is restricted to defined morphological stages of parasite development, while other polypeptides are synthesised more or less throughout the cycle. The synthesis of at least 6 polypeptides was confined to the period of differentiation of mature trophozoites to schizonts and merozoites. Polypeptides synthesised by a cloned long-term passage isolate were very similar to those of a recently cultured uncloned isolate. Comparison of polypeptides synthesized during differentiation of mature trophozoites to schizonts and merozoites by P. falciparum with those of P. chabaudi and P. knowlesi showed that while P. chabaudi and P. knowlesi synthesised a 250 000 molecular weight polypeptide at this stage the apparently equivalent polypeptide of P. falciparum was of significantly lower molecular weight being 200 000. Using a surface immunoprecipitation technique, it was shown that this 200 000 mol. wt. polypeptide was accessible to antibodies on the surface of erythrocytes infected with mature trophozoites and schizonts. A 150 000 mol. wt. polypeptide was also accessible to antibodies. By comparing polypeptides synthesised during the differentiation of mature trophozoites to schizonts and merozoites with those recovered in the ring stage parasites after schizogony and erythrocyte invasion, it was shown that this 200 000 mol. wt. polypeptide and 140 000 and 120 000 mol. wt. polypeptides were not taken into the erythrocyte by the invading merozoite. The importance of these polypeptides in terms of the parasite biology and in the induction and expression of immunity to malaria is discussed.

The effects of selective immunosuppression on resistance to Mesocestoides corti in strains of mice showing high and low initial susceptibility.

Previous work has shown that C57BL/6 mice had the lowest initial susceptibility to Mesocestoides corti of six strains of mice examined. Parasite burdens in this strain and in CBA/H mice, a strain showing a higher initial susceptibility to M. corti, were compared following selective immunosuppressive treatments. Irradiation, splenectomy and the administration of cyclophosphamide and methyl prednisolone all resulted in higher parasite burdens in C57BL/6 mice. In contrast these treatments had a minimal effect on parasite burdens in CBA/H mice. In the light of these results the role of antibody in controlling parasite proliferation is discussed.

The use of host strain variation to assess the significance of mucosal mast cells in the spontaneous cure response of mice to the nematode Trichuris muris.

A proportion of DBA/2 mice do not reject the large intestinal nematode parasite Trichuris muris. In this strain and the early rejecting NIH and the late rejecting CBA/Ca strains the kinetics of the mast cell accumulation in a primary infection were similar with peak mast cell numbers being recorded on day 20 post-infection. Comparisons between rejector and non-rejector DBA/2 mice showed no differences in the mast cell accumulation. There was no rise in mast cell numbers in response to a secondary infection, in either the NIH or CBA/Ca strains, for at least 3 days after the infection had been expelled. It is suggested that mucosal mast cell accumulation is not induced by a simple response to parasite factors, that the cells are not directly involved in the expulsion of T. muris and that any role they play in the spontaneous cure response is subject to more complex control.