John R. Gorham

The development and analysis of species specific and cross reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the major histocompatibility complex for use in the study of the immune system in cattle and other species

We examined the potential of developing a set of species specific and cross reactive monoclonal antibodies (MoAbs) for use in the study of the phylogenetic and functional relation of class I and class II antigens of the major histocompatibility complex (MHC) and leukocyte differentiation antigens in cattle and other species. Comparing immunization strategies demonstrated the number of hybrids producing cross reactive antibodies can be increased by hyperimmunization of mice with lymphoid cells from multiple species. Comparing various methods of assay (antibody-complement mediated cytotoxicity [CT], enzyme linked immunosorbent assay [ELISA] and flow microfluorimetry [FMF]), revealed FMF is the most useful technique for the primary assay of hybridomas producing MoAbs of potential interest. By using dual parameter and dual fluorescence analysis, we could determine whether a given MoAb reacted with mononuclear cells (lymphocytes and monocytes) and/or granulocytes, and also whether any two MoAbs of different isotype and specificity recognized antigens present on identical or separate populations of leukocytes. Comparing the patterns of MoAb reactivity with leukocytes obtained from cows, goats, sheep, pigs, horses and humans, as well as comparing the patterns of reactivity with a panel of lymphoid cell lines derived from cattle (with enzootic bovine leukemia) and humans (with various forms of leukemia), revealed sets of MoAbs reactive with unique antigenic determinants present on BoLA class I (15 MoAbs) and class II (9 MoAbs) antigens, and also MoAbs reactive with determinants present on leukocyte differentiation antigens (36 MoAbs). Dual fluorescence analysis demonstrated the antigens detected by some MoAbs are predominantly expressed on one lineage of leukocytes while others are expressed on two or more lineages of leukocytes. Dual and single fluorescence analysis also demonstrated the PNA receptor(s) is: expressed on T cells, granulocytes and class II antigen monocytes and absent or expressed in low amount on sIgM+ B cells and a newly defined Non T/Non B population of cells. The strategies described for identifying and analyzing the specificity of MoAbs demonstrate the feasibility of developing a set of cross reactive MoAbs for identifying homologous molecules in multiple species and delineating their functional and phylogenetic relation.

An Epizootic of Equine Sarcoid

JACKSON1 first recognized and described equine sarcoids, which are locally aggressive, fibroblastic tumours of equine skin. They are often multiple, frequently recur following surgical excision or radiotherapy, and generally have fibropapillomatous features. Horses, donkeys and mules are susceptible. Neither a seasonal incidence nor an age distribution of equine sarcoids has been detected2. Jackson pointed out that the gross and microscopic appearance of the tumours, the predilection sites, and the pattern of spread to secondary sites on affected animals were suggestive of viral aetiology. Auto- and homo-transplantation have been reported3–6. Voss recently reported that several tumours were produced in horses with centrifuged sarcoid extracts which probably were cell-free although they had not been filtered5. Intrader-mal inoculation of bovine papilloma virus in horses produced localized, fibroblastic growths similar to equine sarcoid which suggests the interesting possibility of a causal relationship to equine sarcoid7. Equine sarcoid has no known relationship to Boecks sarcoid in man.

Etiologic Studies of Old Dog Encephalitis I. Demonstration of Canine Distemper Viral Antigen in the Brain in two Cases

In 2 cases of old dog encephalitis (ODE), viral antigen to canine distemper was demonstrated in frozen brain tissue by means of the direct FA test. The distemper viral antigen was most abundant in cells of the gray matter of the cerebral cortex, thalamus, mesencephalon, and medulla. Also, in the 1 dog in which serum was available, there was a significantly elevated serum antibody titer to the distemper agent. Attempts to isolate a viral agent in tissue culture or to transmit disease to distemper-susceptible ferrets or distemper-immune adult dogs were unsuccessful. Results of this study indicate that the virus of distemper play a role in the pathogenesis of ODE. Also, the progressive disease with inflammatory reaction suggests that a slow viral infection rather than a totally masked or latent virus be involved.

PREVALENCE OF ANTIBODY TO MALIGNANT CATARRHAL FEVER VIRUS IN WILD AND DOMESTIC RUMINANTS BY COMPETITIVE-INHIBITION ELISA

A competitive-inhibition ELISA (CI-ELISA), based on a monoclonal antibody to an epitope conserved among malignant catarrhal fever virus (MCFV) strains of both wildebeest and sheep origin, was used to determine the prevalence of antibody to MCFV in selected domestic and wild ruminants, both free-ranging and captive, from the USA. We evaluated 2528 sera from 14 species between 1990 and 1995, including 80 pronghorn antelope (Antilocapra americana), 339 bighorn sheep (Ovis canadensis), 103 bison (Bison bison), 17 black-tailed deer (Odocoileus hemionus columbianus), 395 domestic cattle (Bos taunts), 291 domestic goats (Capra hircus), 680 domestic sheep (Ovis ammon), 323 elk (Cervus elaphus), 41 llamas (Lama glama), 21 mouflon sheep (Ovis musimon), 54 mountain goats (Oreamnos americanus), 101 mule deer (Odocoileus hemionus), 20 muskox (Ovibos moschatus), and 63 white-tailed deer (Odocoileus virginianus). A high seroprevalence (37 to 62%) was observed in domestic sheep, domestic goats, muskox, and some bighorn sheep populations. Seroprevalence in these species was generally age-related: a very low seroprevalence was present in these animals under one year of age. A low seroprevalence (2% to 13%) was found in clinically-susceptible species such as domestic cattle, deer, elk and bison, supporting the concept that significant numbers of non-lethal infections occur among clinically susceptible ruminants.

Comparison of a DNA probe, complement-fixation and indirect immunofluorescence tests for diagnosing Anaplasma marginale in suspected carrier cattle.

Most estimates of the prevalence of anaplasmosis have been based on serologic data using the complement-fixation (CF) and/or card agglutination tests. Since these tests are considered to be only about 50 percent reliable for detecting carrier cattle in enzootically stable herds, the need for more sensitive diagnostic tests is widely recognized. The objective in the present study was to compare the sensitivity of the CF test with that of the indirect immunofluorescence (IIF) test and a recently developed DNA probe in determining the prevalence of Anaplasma marginale infection in cattle from an enzootic area. The study herd consisted of 52 8-month-old steers and 13 3-year-old cows of mixed beef breed. All cattle were initially tested for this comparative purpose. All but one animal (one that was a positive reactor as assessed by all three tests, and served as a positive control), were treated with long-acting oxytetracycline in an attempt to clear any carrier infections. Each animal was then retested at 1 month and 2 months post-treatment (PT), in an effort to determine if the DNA probe could be used to evaluate the effectiveness of the drug. Six of the 65 (9.2%) initial serum samples were CF positive. In contrast, 60 (92.3%) and 64 (98.5%) of the samples were positive as assessed with the IIF test and the DNA probe, respectively. The DNA hybridization reactions varied in intensity within the sample population indicating different individual levels of infection. The DNA probe hybridized with two samples taken at 1 month PT, and with two different samples taken at 2 months PT. The mean IIF titers were reduced at both the 1 month and 2 month sampling times. These results suggest that the drug did not eliminate infections in all cattle. Some may have been cleared, but, in any event, the drug did reduce the level of infections below the sensitivity of the DNA probe and interrupted continuity of stimulation of antibody. Therefore, the DNA probe and the IIF test appear to be considerably more sensitive in detecting carrier infections than the CF test, and should be considered in future epidemiologic studies.

Studies of Old Dog Encephalitis. II. Electron Microscopic and Immunohistologie Findings

Intranuclear inclusions in neurons and glial cells of a dog with old dog encephalitis had paramyxovirus nucleocapsid structures identical to those observed in distemperinfected CNS tissue. This finding further suggests that the distemper virus is involved in the pathogenesis of old dog encephalitis. Canine γ-globulin (IgG) was demonstrated in frozen brain sections by means of the direct fluorescent antibody test. Although IgG occurred in the cyptoplasm of mononuclear cells and in the neural parenchyma in those areas where viral antigen was most abundant, it was not established if it was antibody to distemper virus.

The Isolation and Partial Characterization of A Babesia Sp. From Desert Bighorn Sheep (Ovis Canadensis Nelsoni)

ABSTRACT. A novel Babesia parasite of desert bighorn sheep was isolated. Its taxonomic description, host range, pathogenicity and antigenic relatedness were in vestigated. the parasite was infective for black‐tailed and white‐tailed deer, but with host‐specific differences compared to that of bighorn sheep. A splenectomized calf and domestic sheep were refractory to infection. A comparative immunofluorescence assay detected antigens cross‐reactive with Babesia odocoilei, B. divergens, B. equi and B. caballi, but not with B. bovis or, B. bigemina. Babesia odocoilei was also infective for bighorn sheep, allowing comparison by a cross‐challenge experiment, the results of which supported the conclusion that this parasite was not B. odocoilei. However, the bighorn sheep Babesia cannot currently be distinguished from B. capreoli described from roe deer in northern Germany. Data indicate that, while this parasite may not present a problem for domestic animals, it may cause disease in bighorn sheep and deer populations.

The Sequential Development of Lesions in Spontaneous Aleutian Disease of Mink

Normal and Aleutian disease affected mink from ranches with a high prevalence of Aleutian disease were killed at approximately monthly intervals from July to November. Serum was subjected to zone electrophoresis and the serum protein changes were compared to the histologic stage of development of the renal, hepatic, and arterial changes. The initial renal lesions were interstitial infiltrations of plasma cells and lymphocytes with later development of glomerular changes. The arterial lesions were early medial necrosis followed by the appearance of fibrinoid and later by healing with concentric fibrillar proliferation. There seemed to be a direct relation between the severity of renal, hepatic, and arterial changes and the magnitude of hypergammaglobulinemia. Possible pathogenetic mechanisms are discussed.

Hypergammaglobulinemia in Mink.

Summary The electrophoretic patterns of normal and Aleutian disease affected mink have been determined. The serum from diseased animals had increased total serum proteins, increased gamma globulin, and decreased albumin.

A study by the agar diffusion technique of precipitating antibody directed against blue tongue virus and its relation to homotypic neutralizing antibody.

The technique of agar diffusion was applied to a study of blue tongue virus antigens prepared from infected mouse brains, chicken embryos, and cell culture fluid. Antigens from these different sources, contributing to precipitate formation, appeared to be virus-specific, noninfectious and serologically indistinguishable one from another in the systems tested. The onset and production of circulating ovine precipitating antibody was correlated to corresponding data for homotypic virus infectivity neutralizing antibody. The onset of circulating precipitating antibody formation was detected about the same time as for neutralizing antibody but the precipitating antibody persisted longer. An anamnestic response was observed for neutralizing antibody but not for the precipitating antibody. An antigen-antibody system containing one reagent in low concentration precipitated if adjacent to a precipitating control system but not if placed by itself or an angle >90° to the control system. This phenomenon, termed the “recruiting effect”, was found to influence quantitation of precipitating antibody.