Interaction of bacterial Fc receptors with goat immunoglobulins.

The interaction of type I (staphylococcal protein A) and type III (streptococcal FcRc) bacterial Fc receptors with goat immunoglobulins has been studied. Staphylococcal protein A bound poorly to the majority of goat immunoglobulins at all pHs tested. There was some evidence that protein A bound IgG2 better than IgG1, particularly at pH 8 and above. One of 10 sera tested demonstrated a high level of reactivity with protein A and this was shown to correlate with the presence of a natural antibody to protein A. The streptococcal Fc receptors, FcRc, bound efficiently to all goat IgG and goat sera tested. Both goat IgG subclasses reacted efficiently with the FcRc between pH 6 and 8. Inhibition of binding of 125I-FcRc to immobilized goat IgG enabled levels of IgG in goat serum to be estimated. These results suggest the streptococcal FcRc will be of value as an immunochemical reagent in studies involving isolation and quantitation of goat immunoglobulins.

A rapid technique for measuring leukocyte chemotaxis in vivo.

A modification of connective tissue air bleb technique was used to develop a model system in inbred strains of mice for the study of chemoattractants in vivo. The method was developed using the well characterized n-formylated chemotactic peptide, f-Met-Leu-Phe, as the positive control. Injection of 0.1 ml of f-Met-Leu-Phe solutions from 10(-7) to 10(-10) M resulted in an influx of polymorphonuclear leukocytes within 2 h. Study of the kinetics of the response showed that the number of infiltrating cells reached a peak within 8 h and slowly declined over a 2-day period. The predominant infiltrating cell type during the first 24 h was the polymorphonuclear leukocyte. Between 24 and 48 h the polymorphonuclear leukocytes were replaced by monocytes. By utilizing an inbred mouse strain (DBA-1J and 2J) sufficient or deficient in C5 it was possible to distinguish compounds that were directly chemotactic from those that worked indirectly, or whose chemotactic potential could be enhanced by generation of the chemotactic complement split product C5a. The method was found to be technically simple, reproducible and semi-quantitative and represents a good model system to facilitate the comparison of chemotactic responses in vivo and in vitro.

Modulation of porcine peripheral blood-derived macrophage functions by in vitro infection with African swine fever virus (ASFV) isolates of different virulence.

Macrophages play an important role in defense against invading pathogens and neoplastic diseases. Their ability to maintain their functional characteristics is also important in the survival of the host. Loss of macrophage function during infection may be important in determining resistance or susceptibility of a host species. In this communication, we describe the effect of ASFV replication, in macrophages in vitro, on the functional characteristics of these cells. Infection with ASFV did not alter the expression of Fc receptors nor their ability to mediate ADCC. However, African swine fever virus was shown to modulate antibody mediated phagocytosis, chemiluminescence and chemotaxis. Similar modulation of certain macrophage functions was observed with ASFV isolates that differed in their virulence. We were therefore, unable to correlate the virulence of ASFV with the ability to alter macrophage function.

Bovine peripheral blood leukocyte subpopulation dynamics following a primary bovine herpesvirus-1 infection.

Population dynamics of bovine peripheral blood leukocyte subpopulations were quantitated following a primary bovine herpesvirus-1 (BHV-1) infection. Percoll isolated peripheral blood mononuclear cell (PBMC) subpopulations were analyzed using flow cytometry (FC) and cytochemical stains. Between days two to eight post-infection (PI) there was a significant decrease in the percentage of T-cells and nonT/nonB cells which was accompanied by an increased percentage of B-cells and monocytes. These percentages were extrapolated to the number of Percoll isolated PBMC during this period. A decrease in the T-cell population was the primary cause of the observed lymphopenia and a relative increase in the percentage of B-cells. The increased percentage of monocytes was caused by an increased number of circulating monocytes. These monocytes were characterized by an increase in Fc receptor expression, a decrease in plastic and Sephadex-G10 adherence and no apparent change in the level of class II MHC antigen (Ia) expression. Serum cortisol was significantly elevated on day 2 PI and may have been responsible for both the reduction in circulating T-cells and a decrease in the in vitro viability of peripheral blood lymphocytes. The percentage of Ia positive PBMC was increased significantly on day 4 PI. However, on days 4 and 6 PI the summated percentages of monocytes and B-cells (total Ia expressing population) exceeded significantly the actual percentage of Ia positive cells. This apparent suppression of Ia expression did not coincide with the elevated serum prostaglandin E2 concentrations on days 8 and 10 PI.

Generation of IL-2 dependent bovine cytotoxic T lymphocyte clones reactive against BHV-1 infected target cells: loss of genetic restriction and virus specificity.

To gain insight into the mechanisms of immunity to bovine herpesvirus type-1 (BHV-1) in particular the importance of the T-cell response, we attempted to clone bovine cytotoxic T lymphocytes that were specific for BHV-1 infected autologous target cells. A number of bovine T cell clones were generated by limiting dilution in the presence of bovine recombinant IL-2 and BHV-1 infected target cells as feeder layers. These clones were maintained in culture on crude IL-2 containing supernatants. In functional studies, 4 of the 16 T cell clones were shown to have high levels of cytotoxic activity specific for autolgous BHV-1 infected target cells with significantly lower cytotoxic activity against uninfected target cells and heterologous BHV-1 infected target cells. Continuous culturing of these 4 T cell clones, using either the crude IL-2 or high concentrations of recombinant bovine IL-2, resulted in the loss of both MHC restricted and BHV-1 specific cytotoxic activity. These clones now exhibit promiscuous type cytotoxic activity with the ability to lyse a variety of target cells. Using flow cytometric analysis, the phenotype of the T cell clones were shown to have bovine T lymphocyte characteristics including expression of the BoT8 marker. This is the first report of cloned bovine cytotoxic T lymphocytes reactive against BHV-1 and the generation from these clones of promiscuous cytotoxic activity against both virus-infected and non-infected bovine target cells.

T lymphocyte population dynamics and function following a primary bovine herpesvirus type-1 infection.

Following a primary bovine herpesvirus-1 (BHV-1) infection the concanavalin A (Con A) induced proliferative responses of peripheral blood T lymphocytes were suppressed. This suppression occurred in the absence of detectible serum suppressor factors, suppressor cell activity or decreased accessory cell function. However, regression analysis demonstrated a significant correlation between the percentage of T lymphocytes present within the peripheral blood mononuclear cell (PBMC) population and the amplitude of Con-A-induced lymphocyte proliferative responses (LPR). Direct evidence that a numerical deficit of responder T lymphocytes was limiting LPR was obtained by using an immunomagnetic microsphere (IMM) negative enrichment protocol to produce a PBMC population with a constant percentage (75 +/- 6%) of T lymphocytes. The Con-A-induced LPR of these enriched T lymphocytes remained constant following BHV-1 infection. Flow cytometric (FC) analysis of PBMC indicated that the decreased percentage of circulating T lymphocytes, associated with BHV-1 infection, was caused primarily by a selective depletion of the BoT8 subset. These FC data were consistent with the indirect evidence of increased TH activity, as indicated by elevated Con A-induced IL-2 production. Thus, 2 to 5 days following viral infection, the circulating T lymphocytes were activated as shown by elevated IL-2 production, increased recombinant bovine IL-2 (rBo

Bovine interferon: its biology and application in veterinary medicine.

Investigations of the production and potential use of bovine interferons against viral infections have occurred since the first descriptions of interferons in other systems. The recent advent of recombinant DNA-technology has facilitated such studies and furthered our knowledge about the bovine interferon system in general. This review gives an overview of the biology, antiviral and immunomodulatory activities of bovine interferons. Areas in which the interferons are now applied or have potential application in viral diseases in cattle are described. Finally, the value of studies of the bovine interferon system with respect to comparative interferon research is discussed.

Studies on the in vitro biological activities of recombinant bovine tumor necrosis factor (rBoTNF) alpha. I. Synergistic antiviral efficacy of rBoTNF alpha, recombinant bovine interferon gamma (rBoIFN gamma) and their combination.

The recent demonstration of the antiviral activity of recombinant human TNF has launched an interest in the use of TNF alpha for antiviral therapy in veterinary medicine. In the precent report, we demonstrate that pretreatment of bovine cells with rBoTNF alpha reduces the yield of bovine herpesvirus type-1 (BHV-1) from infected cells. Reduction in yield was similar to that observed in the presence of rBoIFN gamma. Similarly, rBoTNF alpha was able to protect bovine cells from virus-induced cytopathology. Enhanced antiviral activity was demonstrated when rBoTNF alpha was administered in combination with rBoIFN gamma. Studies on the induction of 2',5'-oligoadenylate synthetase (2'-5' AS) production by cytokine-treated cells showed that although rBoTNF alpha by itself did not induce 2'-5' AS activity it was capable of enhancing the production of 2'-5' AS by rBoIFN gamma-treated cells. Combination of these two cytokines was also evident in the inhibition of proliferation of treated cells. In contrast, the cytotoxic effect of rBoTNF alpha towards actinomycin D-treated cells was not affected by the combination of rBoTNF alpha with rBoIFN gamma.

Activity of polymorphonuclear (PMN) leukocytes during bovine herpes virus-1 induced respiratory disease: effect of recombinant bovine interferon alpha I1.

Following infection of cattle with bovine herpes virus-1 there is a state of generalized immunosuppression involving various leukocytes including polymorphonuclear (PMN) leukocytes. Since the PMN is considered to be pivotal in recovery from secondary bacterial infections during bovine respiratory disease, investigations were initiated to determine PMN activity in this disease and whether interferon could modulate PMN activity. In this study, the in vivo administration of recombinant interferon alpha-I1 was shown to increase PMN functions as measured by migration/chemotaxis and generation of reactive oxygen species. This augmented activity of PMN appeared to correlate with the reduction of overall clinical disease, that is, number of sick days, lung lesions and weight loss. In the study administration of interferon by the intranasal or intramuscular route were as effective in stimulating PMN function. Based on these studies it was concluded that the reason for improved performance of calves treated with interferon would be due to its immunomodulatory effects on leukocytes. Although interferon did not alter the initial suppression of PMN functions, these functions returned to normal and exceeded normal activities by 7-9 days post-infection, the time when maximal bacterial activity normally is present.

Production of Brucella abortus-specific protein A-reactive antibodies (IgG2) in infected and vaccinated cattle.

The IgG2 anti-Brucella antibody response of cattle to Brucella vaccination and infection was measured. Three groups of animals were studied; Group 1 contained 11 non-vaccinated cows, Group 2, 17 cows vaccinated with a low dose of Strain 19 vaccine and Group 3, 17 cows vaccinated with a high dose of Strain 19 vaccine. All animals were challenged at Week 33 with an infectious isolate of B. abortus (Strain 2308). Studies of the IgG2 antibodies response indicated an absolute correlation between anti-Brucella IgG2 levels and infection of the animal. All animals showing reciprocal titers of greater than or equal to 3000 (16 of 45 tested) were found to be positive for the challenge organism at slaughter. Animals with reciprocal IgG2 titers less than or equal to 1000 (29 of 45 tested) were found to be negative for the challenge organism at the time of slaughter. The predictive value of IgG2 antibody levels for infection held for animals in all three groups and consequently this suggests that monitoring of specific IgG2 anti-Brucella antibody levels may be of value in detection of Brucella-infected cattle.

Interaction of bovine immunoglobulin gamma 2 (IgG2) with staphylococcal protein A: evidence for IgG2a and IgG2b sub-subclasses in the bovine.

The reactivity of bovine IgG with protein A is confusing with respect to which of the bovine IgG class and subclasses are reactive. We have, therefore, re-examined the interaction of bovine immunoglobulins with protein A. The results presented in this paper indicated that at pH 8.0 protein A binds only immunoglobulin of the IgG2 subclass. The bound IgG2 can be readily recovered from an immobilized protein A column at pH 5.0. Furthermore, the antigenic IgG2 eluted demonstrated two charged species which could readily be separated by ion-exchange chromatography. These results indicate that IgG2 in the bovine exists in two sub-subclasses, IgG2a and IgG2b. The two sub-subclasses of IgG2 could be rapidly isolated with a good yield in two-steps namely protein A affinity chromatography followed by ion exchange chromatography.

Negative enrichment of bovine T lymphocytes with monoclonal antibodies and magnetic microspheres.

A highly enriched population of bovine T lymphocytes was produced from peripheral blood leukocytes following the depletion of monoclonal antibody-labelled B lymphocytes and monocytes with magnetic microspheres. This negative-enrichment protocol was simple, rapid, and specific. Also, it had a high recovery efficiency and was consistently reproducible. The enriched T lymphocytes proliferated in response to recombinant bovine interleukin 2 and, following the addition of monocytes, to concanavalin A. This methodology made it possible to determine the proliferative responses of peripheral blood lymphocytes utilizing a constant number of T lymphocytes within each assay. In this way, the in vitro T lymphocyte responses were determined independent of changes in the number of responder cells within peripheral blood.

Cloning and expression of the cDNA for bovine granulocyte-macrophage colony-stimulating factor.

A sequence encoding bovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified from a concanavalin A-stimulated bovine lymphocyte cDNA library. This sequence was isolated by hybridization with synthetic oligonucleotide probes based upon the human GM-CSF sequence. This bovine cDNA was engineered for expression and secretion of activity into the periplasmic space of E. coli. Periplasmic extracts contain a 14,500-dalton protein and stimulate colony formation of bovine bone marrow progenitor cells. The predicted protein is 70% homologous with human GM-CSF and 55% homologous with murine GM-CSF. Numerous structural features are conserved among these three proteins, such as location of cysteine residues, glycosylation sites, and overall change. The biological activity of bovine GM-CSF is species specific, since recombinant preparations do not cause proliferation of human or murine bone marrow cells. Similarly, murine GM-CSF does not exhibit activity on cells of bovine or human origin. However, human GM-CSF does stimulate colony formation of bovine bone marrow cells, although the specific activity appears reduced when compared to assays on human cells.

Solid-phase radioimmunoassay for the detection of immunoglobulins against bovine Brucella abortus.

A sensitive radioimmunoassay for the detection of Brucella abortus antibody is described. The assay, performed in flexible 96-well microplates coated with Brucella abortus antigens, utilizes 125I-labeled staphylococcal protein A to detect antibody to Brucella abortus. The parameters of the assay have been analyzed using well recognized statistical methods. Least squares analysis of antigen concentration, antiserum dilution, antigen by antiserum and replicates within antigen by antiserum, estimated an r2 of 0.98, a coefficient of variation of 3.58 and a standard deviation of 0.10. Results of regression analysis of serum dilution versus antigen concentration (ranging from 635 micrograms/ml to 6.35 ng/ml) indicated that an antigen concentration of 6.35 micrograms/ml was the most efficient for describing antibody variability (r2 = 0.98, with a coefficient of variation = 3.28). Regression analysis also revealed a closer correlation between the radioimmunoassay with complement fixation test (r2 = 0.98) than with the standard tube test (r2 = 0.84). Detection of specific antibody assessed by radioimmunoassay was 4 to 64 fold more sensitive than the standard tube test titer and 16 to 32 fold more sensitive than the complement fixation test titer. The results described here indicate that this radioimmunoassay is very sensitive and may be capable of discriminating between false positives and false negatives, thereby, improving the diagnostic efficiency of Brucella serologic tests.

Preliminary observations on transplacental infection of bluetongue virus in sheep-a possible overwintering mechanism.

Sheep infected mid-gestation with bluetongue virus type 4 and type 16 produced clinically normal lambs that were viraemic at birth. Viraemia persisted for two months in some lambs even though they received colostrum. It is suggested that transplacental infection of bluetongue virus in sheep may be an overwintering mechanism for the virus in some areas of the world.

The isolation of adenoviruses from goats affected with peste des petits ruminants in Nigeria.

Mucosal scrapings from the large intestine of two goats that had died from peste des petits ruminants (PPR) in separate outbreaks in Nigeria were examined for viruses. A mixed viral infection of PPR virus (morbillivirus) and adenovirus was confirmed in both goats. The adenoviruses did not conform to any of the ovine and bovine serotypes recognised; the two isolates were considered different serotypes. It is concluded that, although the role of adenoviruses in the epizootiology of PPR in Nigeria is difficult to appraise, they are probably commensals. This is believed to be the first report of the isolation of adenoviruses from goats.

Ultrastructure of schizonts in the liver of cats with experimentally induced cytauxzoonosis.

Schizonts in the liver of 2 cats with cytauxzoonosis were studied by both light and electron microscopies. By light microscopy, the cytoplasm of macrophages in the sinusoids and small vascular channels contained schizonts with cytomeres or both cytomeres and mature merozoites. By electron microscopy, it was determined that schizogony occurred in 4 stages. The earliest stage was the presence of a multilobed structure containing finely granular protoplasm in the cytoplasm of the macrophage. The 2nd stage was an increase in height and number of the lobulations on the surface of the schizont. The 3rd stage involved the development of cytomeres and the appearance of a polar ring and rhoptries in everted sacculations on the cytomere membrane. Nuclei and mitochondria were incorporated into the sacculations before the release of mature merozoites into the host cell cytoplasm. In the last stage of schizogony, following massive merozoite formation and reduction in size of the schizont, residual nuclei divided by multiple fission. Each nuclear division became incorporated into a developing merozoite having preformed rhoptries, mitochondria, and a polar ring.

Use of corticosteroids to isolate IBR virus from cattle in Cyprus after respiratory disease and ataxia.

An outbreak of ataxia, blindness, respiratory disease and kerato-conjunctivitis occurred in October 1972 in a beef feedlot in Cyprus. Fifteen animals died and 10 that were severely ataxic were slaughtered; many animals became blind. There was no opportunity to isolate virus when the disease was active but in March and October 1973 infectious bovine rhinotracheitis (IBR) virus was isolated from cattle after they had been treated corticosteroids to stimulate virus excretion. It is probable that IBR virus caused the disease. This is the first report of the isolation of IBR virus from cattle in Cyprus.

African swine fever in Malta, 1978.

In March 1978 an outbreak of African swine fever (ASF) occurred in Malta. The disease spread rapidly and by April 13, ASF had been found on 304 premises involving 25,100 pigs. A census carried out on April 15/16 showed that there were at least 1440 premises containing 70,700 pigs on the island. A slaughter policy was implemented and depopulation of known infected premises started on April 15. Pigs which appeared normal on these premises were stored in freezers for subsequent processing for human consumption and by the end of June more than 4500 carcases were in cold store. The most consistent clinical signs were fever, anorexia and reluctance to move. Haemorrhagic lymph nodes and petechial haemorrhages in the kidneys were the predominant macroscopic lesions. A serum survey, using the immunoelectroosmophoresis technique, was carried out on 2409 sera from 200 farms collected at the Government abattoir during a four-week period. Of these sera, 308 (12.8 per cent) from 65 (32.5 per cent) of the farms contained antibodies to ASF virus. By August the original pig population had been reduced to one-third and a second census taken on August 15/16 showed that a total of 501 owners and 13,975 pigs remained. The decision was taken to slaughter all the remaining pigs and by the end of January 1979 there were no pigs in Malta. The outbreak cost an estimated 5 million pounds and provided the first occasion when any country had slaughtered all members of a species of domestic animal in order to eliminate a disease.

Foot-and-mouth disease in British deer: transmission of virus to cattle, sheep and deer.

After exposure for two hours to cattle with foot-and-mouth disease, each of the five species of deer found in the British countryside became infected. Clinical disease was typical and severe in the roe and muntjac deer, with some animals dying, less severe in the sika deer and usually subclinical in the fallow and red deer. Each species transmitted disease to its own species and to cattle and sheep. The amounts of virus present in the blood, and in oesophageal/pharyngeal samples and excreted as an aerosol during the course of the infection in the deer were similar to those recorded for the sheep and cattle in the same experiment. The fallow and sika deer commonly carried virus in the pharynx beyond 28 days after exposure; some red deer also became carriers. In epidemics of foot-and-mouth disease in the UK, it is likely that deer would have such intimate contact with farm animals as occurred in this study. The natural behavior of free-living deer in the UK suggests that, although the five species are susceptible to foot-and-mouth disease, they are unlikely to be an important factor in the maintenance and transmission of the virus during an epidemic of foot-and-mouth disease in domestic livestock.