Experimental infection of domestic cats with Bartonella henselae by inoculation of Ctenocephalides felis (Siphonaptera: Pulicidae) feces.

Caged cat fleas, Ctenocephalides felis (Bouché), were fed on 6 cats; 3 cats were injected with 5 x 10(7) colony forming units of Bartonella henselae intradermally and 3 cats were injected with an equal volume of saline. After the fleas fed for 4 d, 5 groups of 50 B. henselae-exposed fleas were caged and allowed to feed on 5 cats for 6 d. Five cats each were injected intradermally with 1 ml of saline containing 45 mg of feces from B. henselae-exposed fleas. Five cats were fed 50 B. henselae-exposed fleas and 45 mg of fresh feces from B. henselae-exposed fleas. Five cats received all 3 treatments by using fleas and feces collected from cats inoculated with saline (controls). Cats were bled weekly and tested by culture and serology. The cats that were injected with feces from infected fleas were positive by culture for B. henselae at 1 or 2 wk after exposure and were the only cats to become bacteremic or seropositive by week 20.

Analysis of workshop antibodies to null cells on a non-T/non-B lymphocyte population.

Non-T and non-B lymphocytes (null cells) were obtained by Sephadex G-10 depletion followed by treatment with mAbs to CD2 and MHC class II and complement. The enriched cells were principally CD5dim and contained greatly increased numbers of null lymphocytes. This methodology will be useful for null lymphocyte enrichment in examining cell surface molecules and functional attributes of null lymphocytes.

The bovine p150/95 molecule (CD11c/CD18) functions in primary cell-cell interaction.

The monoclonal antibody (MAb), C5B6, recognizes the CD11c/CD18 molecule on the surface of bovine peripheral blood monocytes. C5B6 was reactive with 69-83% monocytes, all granulocytes, and less than 5% of lymphocytes from cattle. Of the lymphocyte series, the antibody had specificity for large lymphocytes and two lines expressing T cell markers, but was not reactive with small lymphocytes, thymocytes, a tumor cell line of B-cell lineage, an interleukin 2 (IL2)-dependent T cell line, fibroblasts, or human, sheep, goat or pig peripheral blood mononuclear cells. No dual fluorescence was seen using C5B6 and antibodies to bovine IgM, CD2, CD4 or CD8. Immunoprecipitation of 125I labeled peripheral blood mononuclear cells with C5B6 antibody defined two bands: 150,000 and 95,000 Da. Antibody to the beta chain (CD18) of the leukocyte adhesion receptor family precipitates the 95 kDa beta subunit and the three associated alpha subunits (180, 165 and 150). The bands obtained using MAb C5B6 correlated with the p150/95 bands observed using an antibody that precipitated the alpha and beta chains of the leukocyte adhesion receptor family. Functionally, the primary but not the secondary proliferative response to alloantigens was inhibited by C5B6 MAb. No effect was seen using C5B6 MAb in cytotoxicity assays or in the secondary proliferative response to Brucella abortus or bovine herpes virus type 1.

Effect of monoclonal antibodies on in vitro function of T-cell subsets.

We have examined the functional heterogeneity of 43 monoclonal antibodies which recognised CD1, CD2, CD4, CD5, CD6 or CD8. Blocking of the proliferative response to alloantigen, virus, and phytoheamagglutinin (PHA) was studied. The majority of anti-CD2 and CD4 antibodies could inhibit an alloantigen response. Some of the anti-CD5 and CD8 antibodies could also block this response. Only a few antibodies to CD2 and CD4 could prevent proliferation of lymphocytes to PHA or virus.

Biochemical analysis of bovine T cell antigens with workshop monoclonal antibodies.

The relative molecular weight (Mr) of the CD1, CD2, CD4, CD5, CD6, CD8 and additional molecules identified by monoclonal antibodies (mAb) submitted to the workshop was determined using 125I surface-labelled leukocytes. Of 37 mAb precipitating lymphocyte surface molecules, 33 had Mr correlating with their clusters of differentiation. Further, we were able to determine Mr for three "undefined T lymphocyte" antibodies. Of the 20 mAb that did not precipitate a molecule from the surface of leukocytes, one was non-reactive as determined by flow microfluorimetry. The inability to precipitate a molecule from the remaining cells was probably due to lower affinities of those antibodies and conformational changes in the antigen(s) following lysis of the leukocytes.

A novel population of cells in the peripheral blood of a cow with a neurofibrosarcoma.

An unusual population of leukocytes was observed in the peripheral blood of a cow with a large tumor burden, using flow microfluorimetry. This new population accounted for 50% of the total cells in the peripheral blood of this animal. These cells expressed the p150,95 molecule (bovine CD11c equivalent), identified by the monoclonal antibody C5B6, a molecule found on myeloid cells and activated lymphocytes. The new population did not express the pan T molecules BoCD2 (the bovine T11 equivalent), BoCD5 (the bovine CD5 equivalent) or surface IgM. Isolated peripheral blood mononuclear cells maintained in bulk culture were able to kill autologous tumor cells and BHV-1 infected A549 in an NK-like assay. In vitro cytotoxicity by cells cultured from the peripheral blood of this animal was augmented 2- to 4-fold by the addition of IL-2.

Bovine major histocompatibility complex class I specific monoclonal antibodies characterized by flow cytometry, one- and two-dimensional electrophoresis, western blot and inhibition of cytotoxic T lymphocyte function.

To identify and characterize the bovine major histocompatibility complex (MHC) class I molecules, a panel of 11 monoclonal antibodies (mAbs) were analyzed. The mAbs reacted with bovine MHC class I antigens, as assessed by flow cytometry and immunoprecipitation followed by one- and two-dimensional gel electrophoresis. Analysis by flow cytometry revealed that class I molecules were expressed less on a class I mutant B-lymphoblastoid cell line than on the parent cell line. The relative molecular weights of the proteins identified by these mAbs were similar to those reported previously for cattle and humans. Nonequilibrium pH gradient two-dimensional gel electrophoresis showed that RH16C recognized four different class I gene products, indicating this mAb reacts with a conserved epitope present on different class I molecules. These mAbs effectively blocked cytotoxic T lymphocyte killing of allogeneic lymphoblasts, suggesting the functional importance of beta-2m in this process. These mAbs should be useful reagents for studying bovine MHC class I molecules.

Sensitivity comparison for detection of respiratory bovine coronaviruses in nasal samples from feedlot cattle by ELISA and isolation with the G clone of HRT-18 cells.

A monoclonal antibody-based capture enzyme-linked immunosorbent assay (ELISA) was developed to detect respiratory bovine coronavirus (RBCV) antigens in nasal swabs collected from cattle showing signs of respiratory tract disease following shipping. These samples had been previously tested for RBCV by inoculation of G clone cultures of human rectal tumor cells (HRT-18G) and for bovine herpes virus 1, parainfluenza virus 3, bovine adenovirus, bovine respiratory syncytial virus, and bovine viral diarrhea virus on other specifically permissive cell cultures. RBCV has not previously been recognized as an important etiological factor in the bovine respiratory disease complex of feedlot cattle. Thirty of 100 samples tested positive for RBCV antigen by capture ELISA in contrast to 38 of 100 samples that yielded RBCV isolates in G clone cells. Samples yielding other bovine respiratory viruses in the absence of RBCV were negative in the capture ELISA, which was based on the use of a single monoclonal antibody that recognizes one RBCV epitope on the S glycoprotein with the broadest reactivity with different strains of RBCV tested. Some RBCV strains may not be detected by this ELISA, which may account for the higher percentage of RBCV-infected cattle detected by RBCV isolation. However, the ELISA was simple to perform, sensitive, and specific and was more rapid than virus isolation. This assay will be useful for processing large numbers of field samples in future epidemiologic and diagnostic studies of RBCV infections of cattle.

Estimation of sensitivity and specificity of two diagnostics tests for bovine immunodeficiency virus using Bayesian techniques.

The validation of assays for bovine immunodeficiency virus (BIV) in cattle is hampered by the absence of a gold standard. Two tests that often are used to detect BIV are the indirect fluorescent-antibody assay (IFA) and the nested-set polymerase chain-reaction assay (PCR). IFA detects an antibody response whereas PCR detects the provirus in white blood cells. Using Bayesian techniques performed simultaneously on animals from two different dairy herds, we estimated the performance of the IFA and PCR assays and infection prevalence. Bayesian techniques also were used to derive posterior distributions of sensitivities, specificities, and prevalences. The Bayesian estimates were IFA sensitivity=60%, IFA specificity=88%, PCR sensitivity=80%, PCR specificity=86%, Herd A prevalence=20%, and Herd B prevalence=71%. Although PCR was the more sensitive assay, substantial misclassification of infection would be expected in epidemiological studies of BIV regardless of which assay was used.

Natural transplacental infection of dairy calves with bovine immunodeficiency virus and estimation of effect on neonatal health.

Many experimental infection studies with bovine immunodeficiency virus (BIV) have been conducted, but neither virus transmission under natural conditions nor longitudinal clinical effects of naturally occurring infections in non-experimental populations are well explored. We tested the hypotheses that BIV is transmitted across the placenta during gestation and that intragestionally infected calves are at increased risk of neonatal disease. A cohort of 59 dairy cows on one farm were enrolled at parturition and the BIV serostatus of the cows and their pre-colostral calves determined with an indirect fluorescent-antibody assay. Moreover, the enrolled calves were monitored thrice weekly for specific clinical signs through the duration of the 30 day neonatal period and the occurrence of clinical signs analyzed for association with calf pre-colostral BIV serostatus and dam BIV serostatus. Confounding due to calf passive immunity and season of birth were also explored. Forty percent of seropositive cows (14/35) gave birth to seropositive calves but no seropositive calves (0/19) were born to seronegative dams (estimated relative risk 16, 95% exact confidence interval 2.6-5.8 x 10(29)). Calf pre-colostral BIV serostatus was not associated with the occurrence or frequency of clinical signs--but dam BIV serostatus was associated with the odds of occurrence of calf hyperthermia and with the frequency of occurrence of calf hyperthermia and hyperventilatory events. This study is inconclusive about the effects of prenatal BIV infection on neonatal health--but it does provide evidence for the natural occurrence of transplacental BIV infection.

Clinical disease in kittens inoculated with a pathogenic strain of Bartonella henselae.

OBJECTIVE: To evaluate disease in kittens inoculated with Bartonella henselae strain LSU16. ANIMALS: Eighteen 12-week-old specific-pathogen-free kittens. PROCEDURE: Kittens were inoculated with B henselae strain LSU16 or saline (0.9% NaCl) solution. Blood samples were collected from kittens on alternate weeks, and bacteremia, clinical signs, and antibody concentrations were monitored for 6 months after inoculation. RESULTS: Kittens developed raised, erythematous areas at the site of inoculation within 72 hours. Swelling peaked at 14 days and resolved by 28 days after inoculation. Fever had a biphasic pattern, with an episode of 1- to 3-days' duration beginning 6 to 7 days after inoculation followed by an episode of 3- to 8-days' duration beginning 11 to 13 days after inoculation. Kittens were bacteremic by day 14 with peak bacteremia at days 14 to 28. Strong antibody responses to B henselae were detected. Clinical disease resolved before bacteremia became undetectable, but signs of disease correlated with the highest degree of bacteremia. Regional lymphadenopathy also was evident. CONCLUSION AND CLINICAL RELEVANCE: Clinical disease in kittens was similar to that in adult cats infected with B henselae strain LSU16, except that lethargy and anorexia were less severe in kittens, and a biphasic pattern of fever was detected in kittens. Clinical disease after inoculation with B henselae may be strain-dependent. To limit transmission of Bartonella organisms, appropriate flea prevention should be instituted. IMPACT FOR HUMAN MEDICINE: Kittens that are febrile, anorectic, lethargic, and that have lymphadenopathy should be tested for Bartonella organisms, and contact with immunocompromised owners should be discouraged.

Infection of polarized epithelial cells with enteric and respiratory tract bovine coronaviruses and release of virus progeny.

OBJECTIVE: To investigate the susceptibility of polarized epithelioid human rectal tumor (HRT-18G) cells to bovine coronaviruses (BCV) isolated from enteric (EBCV) and respiratory (RBCV) tract infections. PROCEDURE: Cells of the G clone of HRT-18 were grown to confluent monolayers on permeable supports, and were directionally infected at the apical and basolateral domains with 3 wild-type BCV strains, RBCV-LSU-94LSS-051-2, RBCV-OK-0514-3, and EBCV-LY138-2, and 1 cell culture-adapted strain, EBCV-L9-80. Sequential cytopathic changes were microscopically monitored. Medium samples for titration of hemagglutinins and viral infectivity were collected directionally from both domains of the infected cell cultures at various intervals. RESULTS: Polarized epithelioid HRT-18G cells from apical domains had maximal susceptibility to infection with the EBCV and RBCV strains, and those from basolateral surfaces had minimal susceptibility. Titers of hemagglutinins and infective progeny BCV reached 1,280 hemagglutinin units and 4.2 x 10(8) plaque-forming units/ml for apical samples, but were minimal for basolateral samples. Asymmetric virus release occurred through the apical surfaces of the HRT-18G cells by 12 hours after infection when cell fusion as a sign of cytopathic changes began. When cells were infected basolaterally, progeny virions released from apical surfaces reinfected the target cells from the apical domains and induced cytopathic changes were delayed about 12 hours, compared with changes detectable in apically exposed cultures. CONCLUSIONS: EBCV and RBCV, isolated from cattle, had marked tropism for polarized epithelioid HRT-18G cells. Entry of BCV into the polarized HRT-18G cells was effected maximally through the apical domains and minimally through the basolateral domains. Release of progeny BCV occurred preferentially from the apical domains.

Evaluation of a diagnostic monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of a 26- to 28-kd Fasciola hepatica coproantigen in cattle.

OBJECTIVE: To develop a monoclonal antibody-based capture ELISA for detection of a 26- to 28-kd coproantigen of Fasciola hepatica in the feces of infected cattle. ANIMALS: 27 crossbred yearling calves, 2 New Zealand White rabbits. PROCEDURE: A capture ELISA that uses a previously described monoclonal antibody (MAB) M2D5/D5F10 was developed. The MAB was used to capture the antigen from the feces, and hyperimmune rabbit serum raised against the purified 26- to 28-kd glycoprotein was used to detect the coproantigen. This test was used for the detection of the antigen in the feces of 27 experimentally infected calves with known numbers of flukes. Fecal specimens obtained before infection from the same calves were used as negative controls. RESULTS: The assay results identified all calves infected with more than 10 flukes at necropsy, and as little as 300 pg of coproantigen/ml of fecal supernatant was detected. The assay results correlated well with the number of flukes, suggesting that it is possible to estimate fluke burden. Infections as early as 6 weeks duration were detected, before flukes mature to adults and start to shed eggs. CONCLUSIONS: In experimentally infected calves, the coproantigen capture ELISA was more sensitive and easier to perform than microscopic examination for the diagnosis of F hepatica infection; moreover, 6-week-old prepatent infections were detectable. CLINICAL RELEVANCE: This capture ELISA containing an F hepatica 26- to 28-kd coproantigen is a quantitative assay that is more sensitive than fecal egg counting. In addition, the assay is rapid, easy to perform and lends itself well to large numbers of samples. Because it is antigen based, the ELISA may be useful for diagnosis of F hepatica infection in other species, including human beings.

Detection of strain-specific antigenic epitopes on the lipo-oligosaccharide of Haemophilus parasuis by use of monoclonal and polyclonal antibodies.

OBJECTIVE: To investigate the antigenic diversity of lipo-oligosaccharides of Haemophilus parasuis. PROCEDURES: Immunoblot assays were done with monoclonal and polyclonal antibodies on whole-cell lysates. Individual colonies of H parasuis strains H 54, H 53, and H 128 were tested for reactivity with lipo-oligosaccharide-specific monoclonal antibodies after a single passage on chocolate agar, and colonies of strain H 54 were analyzed after 10 passages. Colony blot tests were used to screen H parasuis strains for spontaneously occurring antigenic variation in their lipo-oligosaccharides. RESULTS: Eight H parasuis strains were separated into 4 lipo-oligosaccharide serovars on the basis of immunoblot reactions with 3 polyclonal rabbit antisera. Nine monoclonal antibodies against lipo-oligosaccharides of a lipo-oligosaccharide-serovar I strain reacted with all tested serovar I strains but failed to react with other H parasuis strains. CONCLUSIONS: Variations in the antigenic reactivity after 1 or 10 passages on chocolate agar were not observed. The serovar I lipo-oligosaccharide strains included virulent as well as avirulent H parasuis strains, indicating that these epitopes do not correlate directly with virulence properties of H parasuis.

Increased interleukin-10 mRNA expression in tumor-bearing or persistently lymphocytotic animals infected with bovine leukemia virus.

Interleukin-10 (IL-10), produced by Th2 helper T cells, B cells, and macrophages, can inhibit cytokine production by Th1 cells and enhance B-cell proliferation and differentiation. Here, we show that peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus-infected animals with late-stage disease express considerably more IL-10 mRNA than animals that are not infected or that are in the early stages of disease. In contrast, the quantities of type 1 cytokines, IL-2 and gamma interferon, decrease with disease progression. In addition, we observed that IL-10 is expressed principally by monocytes/macrophages, not B lymphocytes, in persistently lymphocytotic animals. This observation supports a role for monocytes/macrophages in progression of bovine leukemia virus infection and, of importance, indicates that proliferating B cells are not the source of IL-10 expression. These findings suggest that IL-10 produced by monocytes/macrophages may influence the progression of bovine leukosis in animals that develop persistent lymphocytosis of B cells or B-cell lymphosarcoma.

Biochemical and functional characterization of 8-10C5 (gp120,90), a novel molecule on the surface of bovine T leucocytes.

An unclustered molecule, 8-10C5, present on bovine leucocytes, was characterized using a monoclonal antibody (mAb) prepared at the University of Wisconsin-Madison, and the functional and biochemical characteristics of that molecule compared to defined molecules. This molecule was present on a large percentage of peripheral blood cells (84%), including T and B lymphocytes, monocytes and neutrophils, but was absent from immature thymocytes. mAb 8-10C5 blocked proliferation to alloantigen and bovine herpesvirus-1; however, antibody to the 8-10C5 molecule was unable to stimulate proliferation alone or in combination with PMA or a cross-linking agent. These results indicate that this molecule has a role in T-cell activation, but probably not signal transduction. mAb 8-10C5 was unable to block cytotoxic T lymphocyte (CTL) or natural killer (NK)-like lysis. mAb 8-10C5 identifies a non-covalently bound heterodimer present on most peripheral lymphocytes with a molecular weight (MW) of 120,000 for the alpha chain (10% glycosylation) and 90,000 MW for the beta chain (16% glycosylation). The beta chain is an acidic molecule (pI = 4.5-5.0). Biochemical studies included the CD2 and CD5 molecules and further demonstrated their similarity to equivalent molecules in other species, confirming the phylogenetic importance of these molecules in immune responses. This functional and biochemical information will allow further characterization of these molecules at the RNA and DNA levels.

CD5dim peripheral blood lymphocytes proliferate to exogenous IL-2 in the absence of antigen and kill in an NK-like manner.

Bovine CD5 lymphocytes can be categorized into "bright" and "dim" populations on the basis of CD5-specific monoclonal antibody (mAb) binding. CD5dim lymphocytes enriched by negative selection using mAbs to CD2 and MHC class II were examined phenotypically and functionally. CD5dim-enriched lymphocytes were 50-74% T19+ and 5-36% CD8+. Fewer than 13% expressed CD4, IgM, or MHC class II on their surface, and 6-20% expressed low levels of CD2. These cells did not proliferate to alloantigen or uv-inactivated BHV-1, but did proliferate to Con A and IL-2 in the absence of antigenic or mitogenic stimulation. Following IL-2 stimulation, the T19+ CD5dim lymphocytes proliferated and CD11c expression was induced. Freshly isolated CD5dim lymphocytes failed to kill in a bovine NK-like assay; however, following culture CD5dim-enriched lymphocytes exhibited non-MHC restricted cytotoxicity. We suggest that the CD5dim-enriched lymphocyte population contains the precursors of bovine LAK killing; however, a more extensive phenotype of these precursors in the bovine is yet to be determined.

Monoclonal antibodies to ovine SBU-T8 and SBU-T6 bind analogous molecules on bovine lymphocytes.

Monoclonal antibodies (mAb) to ovine T-lymphocyte molecules SBU-T8 (Maddox, Mackay & Brandon, 1985), the cytotoxic T lymphocyte, human CD8 equivalent, and SBU-T6 (Mackay et al., 1985), the immature T lymphocyte human CD1 equivalent, were reported to cross-react with bovine cells. We have confirmed this cross-reactivity and shown that the percentage of T lymphocytes in thymus, lymph node and peripheral blood is equivalent to that reported in sheep. In addition, we have shown that the molecular weight of the molecules identified by these antibodies (T8, 34,000-38,000; T6, 44,000 and 13,000) is similar to that reported in sheep and humans. The monoclonal antibody to SBU-T8 effectively removes bovine cytotoxic cells in the presence of complement and when used alone blocks killing of allogeneic concanavalin A (Con A) lymphoblasts by mixed lymphocyte-generated cytotoxic T lymphocytes. These results confirm that the anti-SBU-T8 antibody 38-65 binds BoCD8 and anti-SBU-T6 antibody 20-27 binds BoCD1 and that these reagents will be useful in the study of the bovine immune system.