Herpesvirus saimiri-induced malignant lymphoma in rabbits.

Of 9 New Zealand White rabbits inoculated at multiple sc and im sites with a single dose of Herpesvirus saimiri (HVS), 2 developed malignant lymphomas 40-50 days post inoculation. At least 1 animal developed a terminal leukemic phase of the disease. HVS was isolated from the oral and conjunctival swabs and blood and tissue lymphocytes, but not from monolayer cell cultures derived from kidney or lung tissues of the diseased animals. The inoculated rabbits developed low titers of neutralizing antibodies against the virus. Antibodies against HVS specific early and late antigens were not detected in the sera of 7 animals that failed to develop clinical disease, but were detected in the serum of the 1 rabbit with lymphoma. The immunologic response of rabbits to HVS infection was compared to similar responses in infected nonhuman primates.

Herpesvirus saimiri-induced lymphoproliferative disease in howler monkeys.

Four of 5 howler monkeys (Alouatta caraya) experimentally infected with Herpesvirus saimiri (HVS) developed a rapidly fatal malignant lymphoma accompanied by peripheral T-cell lymphocytosis. HVS was isolated from fresh and tissue cultured blood and tissue lymphocytes and from cell cultures derived from nonlymphoid organs. Humoral antibodies against HVS-induced antigens were detected in the sera of the animals. The in vitro response of the peripheral blood lymphocytes to mitogenic stimulants was depressed following HVS infection.

Effects of presensitization on the development of lymphatic lesions in Brugia pahangi-infected jirds.

Experiments were conducted to measure the degree of lymphatic pathology which develops in mongolian jirds (Meriones unguiculatus) sensitized to Brugia pahangi antigens prior to subcutaneous infections. Two protocols were used to sensitize jirds. One group of animals received three intravenous (IV) inoculations of 5,000 frozen, washed, B. pahangi-microfilariae at 10-day intervals. A second group received three inoculations of 150 micrograms of soluble somatic adult B. pahangi antigen (Ag) in Freund's complete adjuvant (FCA) at 10-day intervals. Groups of animals receiving saline in FCA and animals receiving no treatment served as controls. Following immunizations, animals from each group were tested for circulating antibody by the indirect hemagglutination assay, and for immediate and delayed hypersensitivity responses by the footpad swelling assay. All sensitized animals tested showed positive reaction to these assays. Observations at necropsy 90 days after inoculation with infective larvae showed that: 1) the percent recoveries of adult worms were the same in all treatment groups; 2) the numbers of patent infections which developed in the Ag in FCA-treated animals was greatly reduced; 3) level of microfilaremia which developed in animals sensitized with microfilariae was markedly lower; and 4) the degree of lymphatic pathology as judged by lesion score, numbers of intralymphatic thrombi, and lymphatic vessel size was significantly greater in presensitized animals than in nonsensitized infected controls. The increased lymphatic pathology seen in presensitized animals was most marked in jirds with occult infections and high antibody titers. These observations indicated that the B. pahangi-jird model is a useful semiquantitative system for the study of filarial-associated lymphatic pathology and strongly supports the hypothesis that the host immune response is involved in the pathogenesis of lymphatic filariasis.

Specific hypo-responsive granulomatous tissue reactions in Brugia pahangi-infected jirds.

Two types of experiments were used to study the degree of tissue responsiveness which occurred in Brugia pahangi-infected jirds. In experiment 1, the severity of lymphatic lesions which developed following subcutaneous infection of jirds which has existing intraperitoneal infections was compared to the severity of lymphatic lesions that developed following subcutaneous infection of jirds without previous infections. In experiment 2, comparisons were made of the sizes of granulomas which formed in the lungs around cyanogen bromide-activated Sepharose 4B beads coupled with B. pahangi antigen, which were inoculated intravenously into jirds which: 1. had existing lymphatic infections; 2. were uninfected. Results of both experiments indicated that jirds with intraperitoneal or lymphatic infections of B. pahangi of 120 to 160 days duration were significantly less responsive to B. pahangi or B. pahangi antigens as measured by pathologic tissue reactions than were uninfected jirds. The sizes of granulomas which formed around beads coupled to B. pahangi antigen in noninfected and nonsensitized jirds suggest that this worm material may contain factors which nonspecifically enhance the granulomatous inflammatory reaction.

Light-microscopic studies of 3T3 cell plasma membrane alterations mediated by melittin.

Various light microscopic techniques were used to study the effect of melittin, a major toxic constituent of honey bee venom, on plasma membranes of 3T3 mouse fibroblasts. Bright-field light microscopy and Trypan Blue dye exclusion were used to demonstrate changes in membrane permeability after exposure to melittin. Differential interference contrast (DIC) microscopy showed that membrane vesiculation induced by melittin was dose dependent. Using both fluorescent lipid and glycoprotein markers, we found that membrane vesicles were primarily composed of lipids. A sequence of events associated with vesicle formation was depicted by DIC and fluorescence microscopy. Confocal laser scanning fluorescence microscopy demonstrated a translocation of membrane glycoproteins from the plasma membrane to the cytosol following melittin treatment. The significance of membrane vesiculation and translocation of membrane glycoproteins in damaged cells is discussed.

Characterization of the caprine model for ruminant brucellosis.

The relationship between man, the goat, and brucellosis is historical. Today Brucella melitensis and Brucella abortus pose a serious economic and public health threat in many countries throughout the world. Infection of pregnant goats and sheep with B. melitensis results in abortion during the third trimester of pregnancy. Although nearly eradicated in the US, bovine brucellosis is still a problem in many countries and the potential for re-infection of domestic stock from wildlife reservoirs in this country is a regulatory nightmare. Humans infected with this pathogen develop undulant fever, which is characterized by pyrexia, arthritis, osteomyelitis, and spondylitis. Although available for both organisms, currently available vaccines have problems ranging from false positive serological reactions to limited efficacy in different animal species. With the continued need for new and better vaccines, we have further developed a goat model system to test new genetically derived strains of B. melitensis and B. abortus for virulence as measured by colonization of maternal and fetal tissues, vaccine safety, and vaccine efficacy.

Brucella abortus differs in the multiplication within bovine chorioallantoic membrane explants from early and late gestation.

The ability of Brucella to infect and grow within extraplacentomal chorioallantoic explants (CAMs) derived from early and late gestational cattle was compared. Following inoculation of CAMs with equal numbers of strain 2308 B. abortus, the infectivity was approximately the same in CAMs from both ages, however, bacterial replication was significantly greater in late gestational CAMs than in early gestational CAMs. Co-culture of both early and late gestation CAMs or culture of both types of CAMs in the presence of tissue culture media collected from either early or late B. abortus inoculated CAMs failed to alter B. abortus growth rates and/or cytopathic effects.

Pathogenesis of abortion of bovine brucellosis.

Bovine brucellosis is a major disease of cattle characterized by abortion during the last trimester of gestation. During many years important pieces of research have been done looking for a better understanding of this particular phenomenon. Yet, the fact that the abortion takes place in the last period of gestation result in a fascinating interrogant for such a unique event. The present review includes most of the information available regarding to this matter. Emphasis is done in the interaction of Brucella abortus with the trophoblastic cells of the bovine placenta.

Characterization of bovine pulmonary and serum antibody responses after parenteral or intrapulmonary vaccination with live Pasteurella haemolytica.

Pulmonary and serum antibody responses were evaluated in eight calves vaccinated [four intrapulmonary-right diaphragmatic lobe (IP) and four subcutaneous (SC)] with Pasteurella haemolytica A1 (Ph-1) impregnated agar beads and eight respective sham-vaccinated calves. Experimental and sham groups were challenged in both diaphragmatic lobes with Ph-1 34-37 d after vaccination (DAV) and necropsied 6 d after challenge (DAC; 40-43 DAV). IgG antibodies contained in fluids from the diaphragmatic lobes of vaccinated calves had different patterns of antigen specificity compared with IgG antibodies in analogous sera. Using ELISA, anti-Ph-1 IgA and IgG antibody concentrations were significantly higher (P < 0.05) in lung lavage fluids from the IP group before and after challenge compared to the SC and sham groups. The IP and SC groups developed IgA, IgG and IgM antibody titers in nonvaccinated lung lobes after vaccination and challenge. The IP and SC groups exhibited significantly (P < 0.05) smaller pulmonary lesions than the sham groups and pulmonary IgG and IgA antibodies were associated with increased protection.

In vitro function of canine neutrophils during experimental inflammatory disease.

The effect of acute inflammation on neutrophil function in the dog was studied by measuring in vitro phagocytosis and killing of Staphylococcus aureus. Phagocytosis was not impaired after 30 or 60 minutes and bactericidal activity was not impaired after 60 minutes incubation. However, average bactericidal activity after 30 minutes incubation was diminished significantly (P less than 0.01). Wide variations in bactericidal activity after 30 minutes incubation during the course of the inflammation did not correlate with neutrophil count, number of toxic neutrophils, or clinical course of the inflammation. These results indicate that a defect in bactericidal activity can occur in dogs with severe inflammatory disease, and that repeated assays, rather than single determinations, may be needed to detect this dysfunction.

Phagocytosis, killing, and oxidant production by bovine monocyte-derived macrophages upon exposure to Brucella abortus strain 2308.

Phagocytosis and intracellular survival of Brucella abortus, and oxidant production by monocyte-derived macrophages from ten B. abortus-naive cows were studied. Phagocytosis of bacteria opsonized with naive-autologous sera or reactor serum was significantly less than phagocytosis of bacteria opsonized with fetal bovine serum. After phagocytosis, intracellular survival of bacteria opsonized with naive-autologous or reactor sera was significantly less than survival of bacteria opsonized with fetal bovine serum. Production of oxidant by macrophages stimulated with B. abortus opsonized with naive-autologous, reactor, or fetal bovine sera was not significantly different. Although macrophages from one animal showed significantly less phagocytic activity, intracellular killing and oxidant production by macrophages from the ten individual cows toward B. abortus opsonized with naive-autologous, reactor, and fetal calf sera were homogeneous. The abilities of the macrophages to phagocytize and to kill B. abortus were not associated with each other or with oxidant production. Innate resistance or sensitivity to B. abortus was not identified in the cows based on macrophage function.

Comparative histopathology in BALB/c mice infected with virulent and attenuated strains of Brucella abortus.

The course of infection in BALB/c mice of virulent Brucella abortus strain 2308 (S-2308) and attenuated strain 19 (S-19) varies markedly. Whereas S-19 is eliminated at an exponential rate beginning at 2 weeks post infection (p.i.), strain 2308 assumes a steady state or plateau during the first 6 weeks p.i. and thereafter is eliminated very slowly over a period exceeding 6 months. Here we compared the initiation and maintenance of inflammatory reactions in spleens and livers of mice infected with either of the two strains of B. abortus for the first 6 weeks p.i. Histological changes in the liver were similar in response to either strain and were characterized by the development of small granulomas and an influx of polymorphonuclear leukocytes (PMN) and monocytes. Tissue reactions in the spleen were similar at weeks 1 and 2 p.i. At 3 weeks p.i. and thereafter, focal granulomatous responses in S-2308-infected mice exceeded those in mice infected with S-19. Numbers of nonspecific esterase (NSE) positive mononuclear leukocytes in S-19-infected spleens had increased by 3 weeks p.i. and remained elevated. No comparable increase in NSE positive cells occurred in mice infected with S-2308, and numbers were significantly lower. At 4 weeks p.i. the influx of mature neutrophils and the intensity of extramedullary hematopoiesis were significantly greater in S-19-infected spleens. A profound depletion of periarteriolar lymphoid tissue was noted in both infections for the first 3 weeks p.i. However, repopulation of lymphoid sheaths in S-19-infected spleens became significantly greater by 4 weeks p.i. and continued to increase at significantly higher levels during the next 2 weeks. This study demonstrates quantitative differences in splenic inflammatory responses which are temporally related to the more rapid elimination of S-19. Based upon the lower susceptibility of strain 2308 to the protective effects of immune serum it is hypothesized that the different patterns of infection and inflammation displayed by the 2 strains may related to the differential capacities of antibody opsonized S-19 and S-2308 to survive in activated macrophages.

Comparison of oxidant production by bovine neutrophils and monocyte-derived macrophages stimulated with Brucella abortus strain 2308.

Oxidant production by bovine monocyte-derived macrophages and neutrophils was compared after stimulation with phorbol myristate acetate (PMA), opsonized zymosan (OZ), and B. abortus opsonized with naive-autologous, reactor, or fetal bovine sera. Neutrophils responded more rapidly to all stimuli and produced up to 100-fold greater oxidant than did equal numbers of bovine monocyte-derived macrophages. Macrophages and neutrophils stimulated with PMA, OZ, and reactor-opsonized B. abortus had higher mean oxidant production than phagocytes exposed to B. abortus opsonized with autologous sera, fetal bovine serum, or nonopsonized bacteria. Stimulation of macrophages by opsonized zymosan, buffer, and B. abortus opsonized with autologous sera, reactor serum, or fetal bovine serum resulted in low levels of oxidant production that were not significantly different. Only PMA caused a significantly higher level of oxidant production by macrophages.

Effect of the antimicrobial peptide, D-hecate, on trichomonads.

Tritrichomonas foetus and Trichomonas vaginalis are protozoan parasites that cause sexually transmitted diseases in cattle and humans, respectively. There is a need for new antimicrobial agents to treat or prevent trichomoniasis because there are currently no approved chemotherapeutic agents against T. foetus and resistance of T. vaginalis to metronidazole does occur. Therefore, we evaluated the effect of a novel antimicrobial peptide, D-hecate, on the viability of 6 isolates of T. foetus and T. vaginalis in vitro. Tritrichomonas foetus and T. vaginalis were grown to mid log phase (24 hr) or late log/stationary phase (48 hr). Parasites at 10(6)/ml were mixed with equal volumes of D-hecate to final concentrations of 10 microM, 20 microM. and 40 microM of D-hecate. Controls had minimal essential medium (MEM) alone. The numbers of viable parasites were determined microscopically after 10, 20, and 30 min of incubation at 37 C with D-hecate or MEM. Our results show that D-hecate killed all 6 isolates of T. foetus and T. vaginalis evaluated. The killing effect was dependent on the concentration of the peptide, incubation time, and phase of growth of the parasites. Ultrastructural studies of parasites treated with 10 microM of D-hecate revealed extensive damage to the plasma membrane of most T. foetus and T. vaginalis cells, while a few cells were distorted but remained intact. D-Hecate may be a useful chemotherapeutic agent for the treatment of trichomoniasis.

Induction of lymphatic lesions by Brugia pahangi in jirds with large and small preexisting homologous intraperitoneal infections.

The effect of intraperitoneal (i.p.) infections induced by inoculations of 30 or 150 Brugia pahangi third-stage larvae (L3) on the development of infections and lymphatic lesions induced by subsequent homologous subcutaneous (s.c.) inoculations were compared in the present study. Lymphatic lesion severity, as judged by the numbers of lymph thrombi present, and lymphatic lesion scores were significantly reduced in both groups of jirds with existing i.p. infections. The numbers of adult worms that developed, locations of these worms, and the subsequent microfilaremias did not differ significantly between groups. All jirds with i.p. infections developed similar antibody titers to crude somatic adult antigen as measured by ELISA. These levels did not change following s.c. infections. Immediate and delayed footpad swelling responses were also similar in all groups. Results of these experiments support and extend previous studies indicating that i.p. infections of B. pahangi induce a hyporesponsive state in jirds to subsequent s.c. infections without significantly affecting the subsequent parasite burden. This effect appears to be independent of the numbers of L3 inoculated i.p. prior to lymphatic-induced infection. Circulating antibody titers and footpad swelling responses to B. pahangi antigen were not reduced in jirds with the hyporesponsive lymphatic inflammatory response and do not correlate with this condition.

The granulomatous inflammatory response in jirds, Meriones unguiculatus, to Brugia pahangi: an ultrastructural and histochemical comparison of the reaction in the lymphatics and peritoneal cavity.

A granulomatous inflammatory response develops in jirds with lymphatic or intraperitoneal infections of Brugia pahangi. Light, histochemical, and ultrastructural microscopy were used for comparative studies of the reactions in these 2 locations. The reactions observed were categorized into 3 types: (1) an initial response in which lymphocytes, monocytes, macrophages, and eosinophils were present; (2) an intermediate one which consisted of macrophages, epithelioid cells, lymphocytes, eosinophils, collagen, and mesothelial/endothelial cells with central areas of necrosis; and (3) a terminal reaction consisting of degenerating, necrotic cells. Microfilariae and adult worms were associated with these reactions. Macrophages were the predominant cell type in the lesion and were often found attached to the surface of the parasite. The inflammatory responses to B. pahangi in the lymphatics and in the peritoneal cavity appear to be similar, and thus, the peritoneal cavity may be useful in studying specific cell-parasite interactions to further define the pathogenesis of filarial disease.

Adoptive transfer of granulomatous inflammation to Brugia antigens in jirds.

Brugia pahangi infections of jirds (Meriones unguiculatus) produce a granulomatous inflammatory response within the lymphatic vessels. Granulomas that form around beads coated with soluble adult antigens embolized in the lungs have been used to measure this response. Similar lesions were observed in naive jirds receiving lymph node cells and splenocytes from animals with acute infections. This was not the case with cells from chronically infected jirds that were hyporesponsive to implanted antigen-coated beads. Passively transferred immune sera collected during the acute and chronic periods of infection did not transfer this response. Lymph node cells but not splenocytes obtained from chronically infected jirds induced a down regulation of this response in animals during the acute period. These results indicate that this inflammatory response in the jird is cell mediated and that adoptive transfer in jirds is feasible. The induction of the down-regulated state may also be mediated by cells, but not serum factors.

Experimental infection of goat fetuses in utero with a stable, rough mutant of Brucella abortus.

Fetuses of goats in their last trimester of pregnancy were experimentally infected with Brucella abortus strain RB51, a stable rough mutant deficient in the perosamine O-chain content of its lipopolysaccharide. RB51 maintained its rough phenotype in vivo and did not induce abortion. Infection with RB51 resulted in the production of significant levels of IgG type antibodies specific for B abortus cellular antigens distinct from the perosamine O-chain. These findings suggest that strain RB51 will be useful in the pregnant goat for studying the role of brucella antigens other than the lipopolysaccharide O-chain in the immune response to brucellosis.

Evaluation of a rough mutant of Brucella melitensis in pregnant goats.

Brucella melitensis strain VTRM1, a rough derivative of B melitensis strain 16M, is able to colonise the lymph nodes of goats, does not induce abortion in pregnant goats when used at doses leading to abortions with virulent strain 16M, and does not induce anti-O chain antibodies. However, strain VTRM1 as a single dose vaccine induces only partial protection against both infection and abortion following challenge.

Behaviour of a high-temperature-requirement A (HtrA) deletion mutant of Brucella abortus in goats.

Previous studies have shown that high-temperature-requirement A (HtrA) mutants of Brucella abortus are more sensitive to oxidative killing in vitro, are less able to survive in cultured murine macrophages and are attenuated in BALB/c mice. To measure the effect of an HtrA mutation on the virulence of B abortus in ruminants, pregnant goats in late gestation were exposed to infection by the conjunctival route with B abortus 2308 or an isogenic htrA mutant, PHE1. Infection with either 2308 or PHE1 resulted in abortion, but the serological responses to infection were consistent with 2308 but variable with PHE1. Strain 2308 was recovered post mortem both from aborted fetuses and infected dams, whereas PHE1 was recovered from neither. Nevertheless, short term studies revealed that PHE1 could be recovered from infected goats for up to two weeks after infection, suggesting that although the HtrA mutation may change the colonising ability of B abortus, the virulence of the mutant in pregnant goats is not reduced.