Experimental methods for immunization and challenge in antigenicity studies in guinea pigs.

Optimal experimental methods for antigenicity studies in guinea pigs were investigated on: (1) the effects of different immunizing methods using complete or incomplete Freund's adjuvants (CFA or IFA), and various injection sites, the number of immunizations, the immunizing doses, and the immunizing periods, (2) the relationship between the severity of active systemic anaphylaxis (ASA) reactions and passive cutaneous anaphylaxis (PCA) titers, (3) positive control for oral administration, and (4) the effects of incubation mixture of drug and serum protein as the challenge for the ASA assay. The following results provided useful information for designing more appropriate methods for antigenicity studies: (1) The optimal immunization method for benzylpenicillin (PcG), cephaloridine, 2,4,6-trinitrobenzene sulfonic acid and adriamycin, which were selected as positive controls for low molecular medicines in this experiment, involved subcutaneous administration of 1 ml of a test substance in CFA (1st immunization) or IFA (2nd and 3rd immunizations) at two doses, 1 and 10 mg/animal, 3 times at 2-week intervals on the back of a guinea pig. Blood collection for PCA assay was needed 2 weeks after the last immunization, and ASA assay, 1 or 2 days after the blood collection. (2) The insensitivity of ASA reactions in bovine serum albumin-immunized animals with very high PCA titers was overcome by increasing the challenge antigen dose from 1 to 10 mg/animal. (3) Most animals administered lysozyme at 0.1, 1 or 10 mg/animal by gavage for 2 weeks or more showed ASA and PCA reactions. (4) Incubation of a mixture of 20 mg/ml of PcG and 2 mg/ml of guinea pig serum albumin for 4 hr was the most effective as challenge for the induction of ASA reaction in PcG-immunized guinea pigs.

Quantitative techniques in neuropathology.

In the future, quantitative techniques will probably be used in industry as part of Tier II studies for the evaluation of chemicals and drugs for their neurotoxic potential. Movement towards quantifying some structures or neuropathological changes will be made possible by advances in tissue preparation and computer technology. Emphasis will need to be placed on standardized techniques, good quality samples and sampling techniques in order to produce good quantitative data in a reasonable time. In this paper, different sampling techniques are evaluated using a cross section of rat sural nerve as the tissue for quantitative investigation.

Quantitative immunocytochemistry by digital image analysis: application to toxicologic pathology.

Recent advances in immunocytochemical techniques allow the localization of specific antigens in tissue sections. The work reported here attempts to evaluate the application of antibody-labeled, disease-related protein, followed by microscopy and computerized image analysis. Using an experimental anti-tumor, polyclonal antibody (anti-oncomodulin) as a model, various tissues were prepared for light microscope immunocytochemistry. Sections were incubated with primary antibody, then biotinylated secondary antibody. This was followed by incubation with avidin-biotin-peroxidase (ABC method). Marker was visualized by the presence of precipitated diaminobenzidine. Samples were evaluated using a Zeiss/Kontron IBAS I & II semi-automatic digital image analysis system. Statistical analyses were performed on output data. Results demonstrated the localization and determined optical density of immunolabel. Statistical comparisons showed significant differences between control and experimental sections. The practical application of these combined techniques provides the toxicologic pathologist with a powerful tool for accurate and objective determination of the location and relative amount of selected proteins in normal and abnormal tissues.

Comparison of central and peripheral nervous system lesions caused by high-dose short-term and low-dose subchronic acrylamide treatment in rats.

The effects of high-dose subacute acrylamide treatment of up to 50 mg/kg/day for 4 or 10 d were compared to those of subchronic exposure, up to 12 mg/kg/day for 90 d. In the subacute study, Purkinje cells, long ascending tracts of the spinal cord, optic tract terminal or preterminal regions in superior colliculus, sensory ganglion cells, and distal large-caliber peripheral axons were severely affected. Purkinje cells and fasciculus gracilis changes were the earliest lesions. In the subchronic study, the dominant lesion was confined to the distal peripheral axon, with only minor changes occurring in spinal cord and medulla. Paranodal swellings with the characteristic appearance of neurofilament aggregations were not seen. This morphological study suggests a significant difference between high- and low-dose acrylamide-induced lesions. If there is a reduced tendency for long-term low-dose acrylamide exposure to produce CNS lesions, the risk of irreversible nervous system damage would be less than that predicted from subacute studies.

Peripheral and central nervous system lesions caused by triethyl- and trimethyltin salts in rats.

Both trimethyltin and triethyltin salts are known to produce toxic lesions in the central nervous system. Triethyltin intoxication has been associated with central intramyelin edema, while trimethyltin has been shown to produce neuronal necrosis in selected limbic and sensory regions of the brain. Only scant attention has been paid to peripheral nerves of animals treated with alkyltins. In this study, we have treated rats with 6 or 8 mg/kg trimethyltin, and 1, 2, 4, 6, or 8 mg/kg triethyltin (single or multiple exposure), and evaluated in detail at the light microscope level both central and peripheral nervous system lesions. In addition to the central neuron necrosis or myelin edema described previously, both compounds produced peripheral axon degeneration and chromatolysis of large spinal cord and brain stem neurons. Chromatolysis was seen in reticular neurons of the brain stem and ventral horn or spinal cord in rats receiving high doses (6 or 8 mg/kg) of triethyltin, and in these same areas plus mesencephalic trigeminal nucleus in animals treated with trimethyltin. Wallerian-like degeneration of peripheral axons was seen in sciatic and tibial nerve and ventral roots of animals receiving 3 injections of 4 mg/kg or single or multiple injections of 6 or 8 mg/kg triethyltin. Axon degeneration was also seen in sciatic and tibial nerves 21 days after a single exposure to 8 mg/kg trimethyltin. Since myelin edema is believed to be reversible, the axonal changes described here may be of greater clinical significance in relation to human exposure.

Quantitative methods in the study of the pathogenesis of haemoprotozoal diseases.

The study of pathogenicity of haemoprotozoa and the pathogenesis of the diseases they cause requires quantitative descriptions. Statistical and mathematical methods are introduced to describe infectivity, parasitaemia, total body parasitosis and the severity of diseases.

Experimental bovine trypanosomiasis (Trypanosoma vivax and T. congolense). III. Serum levels of immunoglobulins, heterophile antibodies, and antibodies to T. vivax.

Twenty-five steers were infected with T. vivax (EATRO 1721), 25 steers with T. congolense (EATRO 1800) and 25 steers kept as controls. Serum levels of immunoglobulins (IgG1, IgG2, IgM), natural antibodies to erythrocytes of chicken and sheep, and complement-fixing antibodies to T. vivax were measured. A significant decrease of serum IgM and natural antibodies to chicken erythrocytes occurred in the T. vivax group at day 9, i.e. at the decline of the first parasitemic wave. This was followed by a transient moderate increase of IgM accompanied by a transient decrease of IgG2. The T. congolense group had moderate decreases (less than 30%) of the mean IgG1 levels and moderate increases (less than or equal to 50%) of the mean IgG2, levels. It was concluded that there was little evidence for polyclonal activation of lymphocytes and that the decreased IgG1 levels in the T. congolense group might have been a reflection of immunosuppression. The complement-fixation test proved to be a sensitive tool for monitoring the antibody response to T. vivax.

Experimental Bovine Trypanosomiasis (Trypanosoma congolense). Lack of relation of the level of Parasitemia to the J blood group.

Twenty-five Holstein-Friesian steers were experimentally infected with Trypanosoma congolense EATRO 1721. They were tested for their J blood group status. Twelve steers were found to belong to Ja blood group and thirteen steers were either Js or Jcs. The level of parasitemia did not significantly differ between these two groups of cattle. It was concluded that the level of parasitemia is not influenced by J blood group antigens.

The effect of Trypanosoma congolense and T vivax infections on the antibody response of cattle to live rinderpest virus vaccine.

Infections with Trypanosoma congolense or T vivax did not significantly depress the neutralising antibody response of cattle to live rinderpest vaccine when vaccination was carried out eight or 25 days after infection.

Immunosuppression in bovine trypanosomiasis. The establishment of "memory" in cattle infected with T. congolense and the effect of post infection serum on in vitro (3H)-thymidine uptake by lymphocytes and on leucocyte migration.

Cattle infected with Trypanosoma congolense were intravenously immunized with Leptospira biflexa 15 days after trypanosomal infection. The primary immune response to L. biflexa was considerably reduced as compared to uninfected controls. The infected cattle mounted a secondary response when they were cured of trypanosomes by treatment with Berenil 25 days after infection and re-immunized 8 days later. The mean secondary response in these previously infected animals was lower tha, but not significantly different from that of the uninfected control animals. Serum collected 15, 20 and 25 days after infection was inhibitory to the migration of both autologous and isologous (control) peripheral blood leucocytes. The migration inhibitory activity was abolished by heating the serum at 56 degrees C for 30 minutes implying the involvement of a heat labile serum component(s). The same serum did not modify the mitogenic effect of PHA on autologous peripheral lymphocytes.

Experimental bovine trypanosomiasis (Trypanosoma vivax and T. congolense). II. Serum levels of total protein, albumin, hemolytic complement, and complement component C3.

Serum levels of total protein, albumin, activity of hemolytic complement, and complement component C3 were decreased in cattle infected with either T. congolense or T. vivax. The hemolytic complement activity was reduced most, i.e. to 20% (T. congolense) or 5% (T. vivax) of control levels. The development of hypocomplementemia was closely associated with the first peak of parasitemia.

Suppression of antibody response to Leptospira biflexa and Brucella abortus and recovery from immunosuppression after Berenil treatment.

Zebu cattle infected with either Trypanosoma congolense EATRO 1800 or Trypanosoma vivax EATRO 1721 had suppressed humoral immune responses to Leptospira biflexa injected intravenously and to attenuated Brucella abortus injected subcutaneously. T. congolense infections were more suppressive than T. vivax infections. In cattle infected with T. vivax, the suppression of immune responses to both bacterial immunogens was abrogated when the animals were treated with Berenil at the time of antigen administration. In cattle infected with T. congolense, simultaneous Berenil treatment at the time of vaccination abolished the suppression of immune response to L. biflexa, and lessened but did not abrogate the suppression of immune response to B. abortus.

Bovine granulocyte/macrophage and erythroid colony culture: characteristics of the colonies and the assay systems.

Bovine bone marrow granulocyte/macrophage colonies were cultured in vitro in methyl cellulose and in plasma clots using bovine endotoxin-stimulated serum as a source of colony stimulating activity. The endotoxin-stimulated serum was four times as potent as the control serum in the methyl cellulose cultures. No significant increase in the number of colony forming units was observed when bovine marrow cells were maintained in suspension cultures for various periods prior to plating in methyl cellulose. The percentage of glass/plastic adherent cells in bovine marrow cells was observed to be 43% +/- 12 (SD). Benzidine positive erythroid colonies appeared in plasma clot cultures on day 4 and disappeared by day 9. No second population of erythroid colonies appeared either as a function of time or as a function of erythropoietin concentration. The optimum erythropoietin concentration for bovine erythroid cultures was found to be 1.0 unit/mL. A significant difference was observed between animals in their marrow capacity to produce erythroid colonies in culture but no significant difference was observed within individual animals over a period of three months.

Experimental bovine trypanosomiasis (Trypanosoma vivax and T. congolense). I. Sumptomatology and clinical pathology.

Twenty five cattle were infected with T. vivax, 25 with T. congolense, and 25 served as controls. Pathogenic clinical signs of trypanosomal infection were not observed. Secondary bacterial infections were common. Fever, increased heart and respiratory rates, anorexia, and emaciation developed. Elevations in parasitemai and body temperature were positively correlated in the T. vivax group. Infected groups were affected non-uniformly, with some animals in each group remaining asymptomatic and tending to have lower parasitemias. The T. vivax parasitemia was cyclic and the organisms had a genaration time of 7.9 SD 2.5 hours. The first peak of parasitemia in both infections was closely associated with the development of pancytopenia, i.e. anemia, leukopenia, and thrombocytopenia. The bone marrow erythroid response in the T. congolense group was significantly greater thn that in either the T. vivax or control groups. Leukopenia was due to concomitant neutropenia and lymphopenia.

Inhibition of bovine bone marrow granulocyte/macrophage colony formation in vitro by serum collected from cattle infected with Trypanosoma vivax or Trypanosoma congolense.

Serum collected from cattle infected with Trypanosoma vivax or Trypanosoma congolense inhibited bovine granulocyte/macrophage colony formation in methyl cellulose cultures. Maximum inhibition was caused by serum collected two and three weeks post infection. The degree of inhibition appeared to be related to the degree of parasitemia. Inhibition of erythroid colony formation by serum collected from these animals was not observed.

Determination of volumes of Trypanosoma vivax and T. congolense separated from cattle blood.

Trypanosomes were separated on DEAE cellulose columns from blood samples taken during the first parasitemic wave in T. vivax or T. congolense infected cattle. The mean volume of T. vivax organisms in five steers increased from 16.3 fl SE 0.7 on day five to 20.7 fl SE 0.7 on day eight. Assuming an even distribution of T. vivax throughout the vasculature, the total trypanosome volume at peak parasitemia (36.400 trypanosomes per microliter on day six) was calculated to be about 0.067% of the blood volume, i.e. 8.0 ml. The mean volume of the separated T. congolense organisms was 14.0 fl SE 0.3 on day nine post infection (mean parasitemia of 3.100 trypanosomes per microliter blood). The T. congolense organisms in the jugular venous blood accounted for about 0.0043% of the jugular venous blood volume.

Safety evaluation of Aspergillus fumigatus grown on cassava for use as an animal feed.

A safety evaluation of Aspergillus fumigatus I21, grown in a cassava carbohydrate and salts medium, was undertaken. Male weanling rats were fed the fungus at 20, 30 and 40% of the diet for 90 days. A control group was given soybean oil meal as the sole source of protein. Weekly determinations of the body weights and feed consumptions were made. A few days prior to termination of the feeding study, a kidney function test was undertaken on the rats. At the end of the feeding period hematology, blood biochemistry, urine analyses and histopathology studies of various tissues were carried out, and organs were weighed. Rats fed A. fumigatus I21 gained less weight than the controls, but kidney weights were increased. Increases in serum alkaline phosphatase and glutamic-oxaloacetic transaminase were not related to dose level. The blood urea nitrogen was increased for the rats fed 40% of the fungus. Rats fed 30 and 40% of the fungus I21 showed a significant drop in albumin. Deficiency in methionine or other essential amino acids through a limited feed consumption may have caused a decrease in albumin synthesis. Rats fed the highest level of the fungus showed increases in neutrophils and monocytes concomitant with decreases in lymphocytes and eosinophils which may be a response to stress. The urine analyses did not reveal any significant differences. The test rats were capable of concentrating urine adequately when deprived of water for 24 hours. No significant differences between the control and experimental groups were found by histopathological examinations.

A comparative study of the responses of the thymus, spleen, lymph nodes and bone marrow of the albino rat to infection with Trypanosoma congolense and Trypanosoma brucei.

Using 90 albino rats, a comparison was made between the response to experimental infections of Trypanosoma brucei and T congolense of approximately three weeks duration by observation of parasitaemia, packed cell volume values, post mortem spleen and lymph weights, and histology of thymus, spleen, lymph nodes and bone marrow. In T congolense infection, phagocytosis of trypanosomes in the spleen appeared to be the main response of the host's haemopoietic tissues to the parasites, which were observed only intravascularly. In T brucei infection immunological responses occurred in the spleen and lymph nodes in addition to trypanosome phagocytosis. Trypanosomes were seen intercellularly in thymus, mediastinal tissue and lymph node sinuses and parasitaemia reached considerably higher values than in T congolense infection. Erythrophagocytosis in the spleen was the only histological feature which could account for the reduction in packed cell volume which occurred near death in both infections, medullary haemopoiesis being increased. Changes in the thymus, incorporating plasma cell production and depletion of cortical small lymphocytes, occurred in both infections.