Bovine respiratory disease (BRD) cause-specific and overall mortality in preweaned calves on California dairies: The BRD 10K study.

Mortality in preweaned dairy calves is a significant source of economic loss for dairy producers. In particular, bovine respiratory disease (BRD) is a leading cause of death in preweaned dairy calves. The objectives of this study were to investigate management practices and their effects on mortality, both that specifically attributed to BRD and overall mortality due to all causes, in preweaned dairy calves. Rates of failure of passive transfer of immunity (FPT) are also reported. The study consisted of a convenience sample of 5 dairies across California, selected based on management practices, calf records, location, and size. Trained study personnel performed comprehensive calf management surveys on every dairy at least once every season. Calves were enrolled in the study at birth and followed until weaning. Mixed-effect logistic regression models were specified for the outcomes all-cause mortality (any death before weaning) and mortality attributed to BRD. The 2 final models included a total of 11,470 calves that were born on the study dairies and followed until weaning. The study cohort's overall crude mortality was 2.8%, with crude mortality of individual dairies ranging from 1.7 to 7.2%. The proportion of mortality attributed to BRD was 19.3%, with a range of 0 to 27.1% on the study dairies. Increasing the frequency of changing maternity pen bedding was associated with a decreased risk of mortality due to BRD. Calves diagnosed with BRD in the spring had an increased risk of mortality compared with calves born in the summer; mortality in calves with fall and winter BRD diagnoses did not different significantly from that in summer. Season of mortality was not significant in either model. Feeding ≥5.7 L of milk per day per calf (vs. ≤3.7 L/d) decreased the risk of mortality in calves over 21 d of age. Twins had a 68% increased risk of all-cause mortality compared with calves born as singletons. Both mortality models showed an association between administration of a modified live vaccine in dams (targeting BRD pathogens) and a decreased risk of mortality in calves. Using a serum total protein cut-off of 5.2 g/dL, 16.8% of calves had FPT, with a mean serum total protein concentration of 5.94 ± 0.06 g/dL across all calves sampled.

Epidemiology of bovine respiratory disease (BRD) in preweaned calves on California dairies: The BRD 10K study.

Bovine respiratory disease (BRD) is one of the leading causes of death in dairy heifers. The objective of this prospective cohort study was to characterize the epidemiology of BRD in preweaned dairy calves and to identify management practices associated with decreased risk of BRD. Dairies were chosen for the study based on management practices, location, size, and willingness to participate. A total of 6 dairies, ranging in size from 700 to 3,200 milking cows, in 6 counties across California's Central Valley, were enrolled in the study for at least 1 year. A total of 11,945 calves were born on the study dairies and followed until weaning. Incidence of BRD was estimated using treatment records. Trained study personnel performed comprehensive calf management surveys and estimated BRD prevalence on every dairy at least once every season. A shared frailty model was used to model the associations between management practices and BRD hazard. The final models included data from complete records of 11,470 calves. The overall BRD study period prevalence across the study herds was 22.8%. The mean BRD incidence density rate on all the study dairies was 0.17 BRD cases [95% confidence interval (CI) = 0.16-1.74] per calf-month at risk. The shared frailty model identified that feeding only waste or saleable milk (compared with use of milk replacer), feeding over 3.8 L of milk per day to calves under 21 d of age, frequent changing of maternity pen bedding, and administration of modified live or killed BRD vaccines to dams before calving significantly reduced the risk of BRD. Risk factors for BRD included housing calves in wooden hutches with metal roofs, compared with all-wood hutches, twin births, and perception of dust occurring "regularly," as reported by calf managers, compared with a perception of "no dust" in the calf-raising area. All 4 seasons were analyzed, and both summer (hazard ratio = 1.15; 95% CI = 1.01 to 1.32) and spring (hazard ratio = 1.26; 95% CI = 1.11 to 1.44) were associated with a higher risk of BRD compared with winter. The current longitudinal study identified specific housing and feeding practices that could be modified to decrease risk of BRD. In addition, season was observed to have a strong effect on calves' risk of developing BRD on California dairies.

Effects of UV irradiation in a continuous turbulent flow UV reactor on microbiological and sensory characteristics of cow's milk.

The dairy industry under current pasteurization conditions (15 s at 72°C) and sanitary standards achieves a safe product with excellent quality. In an ever-competitive market there is still a need to improve product quality and extend shelf life of dairy products to increase competitiveness and open up new markets. In an attempt to test the effect of UV irradiation on microbiota of fluid milk, a continuous flow UV system at 254 nm was used to treat 3.5 and 2% fat milk at two UV doses (880 and 1,760 J liter(-1)). Milk was obtained from three processors, and two lots from each processor were assessed. To assess the impact on the most descriptive native microbiota in pasteurized milk after UV illumination, the product was held at two storage temperatures (4 and 7°C) and tested weekly for 5 weeks for aerobic plate counts (psychrotrophic and mesophilic bacteria), laboratory pasteurization counts, aerobic sporeformers, coliform organisms, and titratable acidity. Microbial counts for all tested microorganisms were lower in UV-treated milk when compared with control throughout storage at 4 and 7°C in both 3.5 and 2% fat milk. Sensory analysis indicated that there is a sensory defect associated with UV treatment at the wavelength used.

Effect of selected dairy starter cultures on microbiological, chemical and sensory characteristics of swine and venison (Dama dama) nitrite-free dry-cured sausages.

The aim of this study was the evaluation of selected lactic acid bacteria (LAB) starter culture of dairy origin in the production of nitrite-free low-acid fermented venison (Dama dama) sausage (salame di daino) produced in a small-scale plant in Umbria (Italy), and their effect on microbiological, physico-chemical and sensorial properties of the products. Salame di daino was obtained with two different processes: with and without the addition of selected LAB starter cultures. Microbial counts of Enterobacteriaceae, coliform organisms and Pseudomonas spp. were lower in salami made with the addition of starter cultures. Staphylococcus aureus, Salmonella spp, and Listeria monocytogenes after the first week of ripening were only detected from control salami. Control salami were paler and harder, whereas those made with the addition of starter cultures were slightly saltier, juicier and in general more acceptable. Selected dairy-origin starter (SDS) cultures did prevent the growth of both indicators of food safety and of process hygiene and increased the acceptability of full-ripened salami.

Enterotoxin production by Staphylococcus aureus isolated from mastitic cows.

Staphylococcus aureus is an important cause of mastitis in cows. The ability of S. aureus strains to produce one or more enterotoxins in milk and dairy products is linked to staphylococcal food poisoning. To determine whether staphylococci causing bovine mastitis could cause human foodborne intoxication, the production of staphylococcal enterotoxins A through D (SEA, SEB, SEC, and SED) by 160 S. aureus isolates was evaluated with the use of a reverse passive latex agglutination enterotoxin kit. All S. aureus strains were isolated over a 9-month period from 2,343 routine submissions of a composite quarter collection of individual mastitic cows at 18 dairy farms in the San Joaquin Valley in California. Prior to enterotoxin detection, isolates were grown by a method that enhances the in vitro synthesis of enterotoxin. Twenty-two of 160 S. aureus isolates produced enterotoxin. Seven produced SEC, 12 produced SED, and 3 produced both SEC and SED. None of the isolates produced SEA or SEB.

The cytokine markers in Staphylococcus aureus mastitis of bovine mammary gland.

TaqMan real time PCR was used to study the transcriptional activity of the bovine IL-2, IL-6, IL-12p40, IFN-gamma, TNF-alpha and granulocyte-monocyte colony stimulating factor of whole milk cells in bovine mammary gland experimentally infected with Staphylococcus aureus. Cytokine transcriptional activity was monitored at 7, 24 and 32 h Post-infection (Pi). IL-12 and TNF-alpha levels were significantly elevated at 24 h Pi followed by sharp decrease at 32 h pi. IL-2 level was decreased at 32 h pi. IL-12 and IFN-gamma showed a significant interaction at 24 h pi. The significant elevations of the IL-12 and TNF-alpha transcriptional level most likely indicate their important role in regulation of the immune responses of bovine mammary gland in S. aureus infection. Depression of IL-2 could reflect the suppressive nature of the S. aureus mastitis.

Antibiotic susceptibility patterns for environmental streptococci isolated from bovine mastitis in central California dairies.

Environmental streptococci are frequently isolated from bovine mastitis in dairy cows with only limited information available on the antimicrobial susceptibility of these organisms. A total of 362 environmental streptococci isolated from cases of bovine mastitis from the central San Joaquin Valley of California over a 3-yr period were used in the study. Overall, 39.9% of the strains tested were Streptococcus uberis, 42.2% were Streptococcus dysgalactiae, and 11.1% were Enterococcus spp. The antimicrobial susceptibility for these organisms was determined for the following antimicrobial agents: penicillin, ampicillin, cephalothin, ceftiofur, penicillin + novobiocin, erythromycin, pirlimycin, tetracycline, and sulfadimethoxine. Results demonstrate substantial differences in the susceptibility patterns for the various organisms collectively referred to as the environmental streptococci. The MIC90 for penicillin was 0.06 microg/ml for 152 strains of S. dysgalactiae compared with 0.25 microg/ml for 133 strains of S. uberis. However, the Enterococcus spp. were the most resistant organisms tested. These data also indicate that the use of interpretive criteria based on human data may provide misleading results. In conclusion, these data confirm that the environmental streptococci are a diverse group of organisms comprised of several different genera and species and that identification of environmental streptococci to the species level is needed to appropriately modify control methods. Moreover, the use of the agar disk diffusion (Kirby-Bauer) susceptibility test for agents with human-based interpretive criteria is contraindicated, and these tests should only be performed with agents with mastitis specific interpretive criteria.

Prevention of diseases caused by Staphylococcus aureus using the peptide RIP.

Staphylococcus aureus causes many diseases including cellulitis, keratitis, osteomyelitis, septic arthritis and mastitis. The heptapeptide RIP has been shown to prevent cellulitis in mice, which was induced by S. aureus strain Smith diffuse. Here we show that RIP can also significantly reduce the overall pathology and delay the onset of disease symptoms in several other models of S. aureus infections, including: keratitis (tested in rabbits against S. aureus 8325-4), osteomyelitis (tested in rabbits against S. aureus MS), mastitis (tested in cows against S. aureus Newbould 305, AE-1, and environmental infections) and septic arthritis (tested in mice against S. aureus LS-1). These findings substantiate that RIP is not strain specific in its inhibitory activity and that RIP is an effective inhibitor of bacterial pathology at multiple body sites following diverse routes and doses of administration. These findings strongly evidence the potential value of RIP as a chemotherapeutic agent.

Serologic detection of adenoviral hemorrhagic disease in black-tailed deer in California.

An enzyme-linked immunosorbent assay (ELISA) and a serum neutralization (SN) test were developed to measure serum antibodies against the adenovirus causing hemorrhagic disease in free-ranging and captive experimentally-infected black-tailed deer (Odocoilenus hemionus columbianus) in California (USA). There was a strong (rho = 0.874) and significant (P < 0.0001) correlation between ELISA and SN titers, although the SN assay was more sensitive than the ELISA.

Passive transfer, rate of decay, and protein specificity of antibodies against equine arteritis virus in horses from a Standardbred herd with high seroprevalence.

OBJECTIVE: To determine rate of decay of passively acquired antibodies in Standardbred foals on a farm with a high seroprevalence to equine arteritis virus (EAV) and to determine whether vertical or horizontal transmission of the virus was responsible for infection on the farm. DESIGN: Repeated-measures study. ANIMALS: 46 Standardbred horses (15 brood mares and their foals, 5 stallions, and 11 young horses). PROCEDURE: Serum samples obtained from horses on the farm were evaluated by serum neutralization and western immunoblot analysis to detect EAV-specific antibodies. The half-life of passively acquired antibodies in foals was estimated by use of regression analysis. RESULTS: Most (14/15) of the mares evaluated were seropositive to EAV. After suckling, their foals were also seropositive. Mean biological half-life for passively acquired antibodies in serum samples obtained from foals was 32 days (r2 = 0.61). The foal born to a seronegative dam and all 11 young horses from the farm were seronegative to EAV. At least 2 of 5 stallions on the farm were persistently infected carriers that were shedding virus in their semen. Immunoblot analysis of seropositive serum samples most consistently recognized the M protein of EAV. CLINICAL IMPLICATIONS: Analysis of these data indicated that a modified-live EAV vaccine can be administered to foals after they are 8 months old without risk of interference from maternal antibodies, regardless of serologic status of the foal's dam. Horizontal transmission of EAV via the respiratory tract apparently was uncommon on the farm, indicating that mares primarily were infected by venereal transmission of virus from carrier stallions.

Neutralization determinants of laboratory strains and field isolates of equine arteritis virus: identification of four neutralization sites in the amino-terminal ectodomain of the G(L) envelope glycoprotein.

The N-terminal hydrophilic ectodomain of the G(L) envelope glycoprotein of equine arteritis virus (EAV) contains neutralization determinants of the virus. We developed a panel of 17 neutralizing murine monoclonal antibodies (MAbs) to further characterize the neutralization determinants of EAV. Included were 6 MAbs previously raised against a laboratory strain (EAVUCD) of the original Bucyrus strain of EAV, as well as 11 additional MAbs that were raised against a neutralization-resistant variant [escape mutant (EM)] virus (EM6D10) that was derived from EAVUCD. All MAbs raised against EAVUCD and 4 of the MAbs raised against EM6D10 (2B3, 5F8, 8D4, and 10B4) reacted with the corresponding G(L) envelope glycoprotein in a Western immunoblotting assay, whereas the remaining 7 MAbs raised against EM6D10 did not react with any viral protein in the immunoblotting assay but competitively inhibited the binding of MAbs 2B3, 5F8, 8D4, and 10B4, indicating that they also recognize epitopes on the G(L) protein. A panel of 18 EM viruses raised to the MAb panel, 19 field isolates of EAV from North America and Europe, the modified-live virus vaccine (ARVAC), and 3 other laboratory strains of EAV were characterized by microneutralization assay with the panel of neutralizing MAbs and polyclonal rabbit and horse antisera. Comparative analysis of the nucleotide sequences of ORF5 and the deduced amino acid sequences of the G(L) protein of individual EM viruses and field isolates of EAV identified four distinct neutralization sites. These sites include amino acids 49 (site A), 61 (site B), 67 through 90 (site C), and 99 through 106 (site D). With the notable exception of site A, the sites were all located in the V1 variable region (amino acids 61-121) within the second half of the N-terminal hydrophilic ectodomain of the G(L) protein. Site D includes several overlapping linear epitopes which appear to interact with amino acids in the other three sites to form conformationally dependent epitopes. Amino acid substitutions within any of these four sites can alter the neutralization phenotype of individual strains of EAV.

A novel canine leukointegrin, alpha d beta 2, is expressed by specific macrophage subpopulations in tissue and a minor CD8+ lymphocyte subpopulation in peripheral blood.

The beta 2 or leukointegrin family is comprised of three structurally related leukocyte surface heterodimers: LFA-1 (CD11a/CD18), Mac-1/Mo-1 (CD11b/CD18), and p150,95 (CD11c/CD18). In this work, we describe a novel canine beta 2 (CD18)-associated leukointegrin, designated alpha d. Expression of alpha d in tissues was prominent in macrophages in splenic red pulp, lymph node medullary regions, and bone marrow. In peripheral blood, alpha d expression was limited to a minor subpopulation of CD8+ T cells, which included small lymphocytes and large granular lymphocytes. A minor subpopulation of either CD8+ or CD4-CD8- splenic red pulp lymphocytes also expressed alpha d. Immunoprecipitation of alpha d from canine splenocytes revealed a heterodimer of 155 kDa and 95 kDa. Prior clearance of splenocyte extracts with an anti-CD18 mAb resulted in complete removal of alpha d. In addition, prior clearance of canine splenocyte extracts with anti-CD11a, anti-CD11b, or anti-CD11c mAb failed to clear alpha d. These immunoclearance data indicated that canine alpha d was antigenically distinct from the three known CD11 molecules, and occurred as an alpha d beta 2 heterodimer. Amino acid sequencing of canine alpha d affinity isolated from spleen further suggested that canine alpha d beta 2 probably represented a fourth member of the canine leukointegrin family via its homology to a subsequently discovered, novel human leukointegrin, alpha d beta 2, which further supported the uniqueness of the canine protein. The discovery of canine alpha d, and the demonstration of its highly restricted cell and tissue distribution, support a re-evaluation of leukointegrin-dependent inflammatory and immunologic interactions that involve cells now known to express alpha d.

Homotypic and heterotypic neutralization determinants of bluetongue virus serotype 17.

Homotypic and heterotypic neutralization determinants of bluetongue virus serotype 17 (BTV-17) were investigated with a panel of five neutralizing monoclonal antibodies (MAbs). One MAb (MAb 034) was originally raised to BTV serotype 10 (BTV-10) but also neutralizes BTV-17 (P. V. Rossitto, and N. J. MacLachlan, 1992, J. Gen. Virol. 73, 1947-1952). Competitive binding studies indicate that the MAbs recognize at least two epitopes on the neutralizing outer capsid protein VP2 of BTV-17. The MAbs were used to select neutralization-resistant variant [escape mutant (EM)] viruses and to determine the phenotypic characteristics of these EM viruses by immunoprecipitation and neutralization assays. Sequencing of the L2 gene, which encodes VP2, identified mutations responsible for the altered phenotypic properties exhibited by each EM virus. Four amino acids in three regions of VP2 are critical to the expression of the epitopes recognized by the panel of neutralizing MAbs. Amino acid 199 affects the binding of MAbs 17.82, 17.83, and 17.813; amino acid 213 affects the binding of MAb 17.85; and amino acids 327 and 582 synergistically affect the binding of MAb 034. Similarly, amino acids 327 and 402 synergistically affect the binding of MAb 034 to BTV-10 (C. D. DeMaula, H. W. Heidner, P. V. Rossitto, C. M. Pierce, and N. J. MacLachlan, 1993, Virology 195, 292-296), suggesting that the neutralizing epitope common to BTV-10 and BTV-17 has a similar location in VP2 of these two antigenically distinct viruses.

Response of the regional lymph node to bluetongue virus infection in calves.

Bluetongue virus (BTV) infection of cattle is characterized by prolonged cell-associated viremia. In an effort to further evaluate the antiviral response of BTV-infected cattle, the role of the regional lymph node (LN) in the immune response of calves to BTV was characterized. Calves were inoculated with BTV in the skin of the neck in an area drained by the superficial cervical LN. Calves were euthanized at regular intervals after inoculation and both BTV-challenged and contralateral (control) superficial cervical LNs were harvested. In addition, some calves had cannulation of the superficial cervical efferent lymphatics prior to inoculation. Lymphocyte subpopulation analysis was done on LN cell suspensions and lymph cells using a panel of cell-specific monoclonal antibodies. There was a significant increase in the proportion of B cells in the challenged LN after inoculation as compared with the control LN. In addition, BTV-specific antibodies were detected in efferent lymph plasma from the challenged LN in one cannulated calf by 9 days after inoculation (DAI), as determined by competitive enzyme-linked immunosorbent assay, whereas BTV-specific antibodies were not detected in serum from this calf through 12 DAI. Analysis of lymph cells revealed a sustained increase in cell output from the challenged LN due to an increase in lymphoblasts and CD8+ T cells. In contrast, the cell output from the control LN dropped markedly by 8 DAI and there was no significant increase in any specific cell population. Double label analysis characterized lymphoblasts as activated CD8+ cells, as determined by expression of MHC Class II antigens (CD8+ MHC II+). These cells were only transiently present as CD8+ MHC II+ cells were not identified in lymph from the challenged LN at 14 DAI. Few CD8+ MHC II+ cells were identified at any time in lymph from the control LN or in lymph from a mock infected calf. The data indicate that B cell proliferation in the challenged LN and release of activated CD8+ cells from this LN were specific responses to BTV infection. The rapid expansion of activated CD8+ T cells indicates that these cells may limit early viral spread. It is concluded that the regional LN draining inoculated skin is critical to the immune response of calves to BTV infection.

Identification of a neutralization site in the major envelope glycoprotein (GL) of equine arteritis virus.

A panel of six neutralizing monoclonal antibodies (MAbs), neutralization-resistant variant (escape mutant [EM]) viruses, and individual viral proteins derived from a vaccinia virus expression system were used to identify the neutralizing determinants of equine arteritis virus (EAV). The neutralizing MAbs recognize a single neutralization site on the 29-kDa envelope glycoprotein of EAV (U. B. R. Balasuriya et al., 1993, J. Gen. Virol., 74, 2525-2529). Vaccinia virus recombinants which express either the GL protein or the M protein of EAV, and a GL-specific antipeptide serum were used to prove that the 29-kDa glycoprotein recognized by the MAbs is the GL protein. The MAbs were used to select a panel of seven EM viruses, whose phenotypic properties were characterized by neutralization and Western immunoblotting assays. The neutralizing MAbs segregated into three groups on the basis of these assays, indicating that they define three interactive epitopes on the GL protein. Sequencing of the entire open reading frame (ORF) 5, which encodes the GL protein, from each EM virus identified the nucleotide mutations responsible for the altered phenotypic properties exhibited by the EM viruses. Compared to the sequence of ORF 5 of the parent strain (EAV-UCD), all nucleotide changes occurred within a span of 17 nucleotides (11423 to 11439). Phenotypic alterations in the EM viruses probably were the result of amino acid substitutions within a region of six amino acids (99 to 104), all of which focally altered the predicted hydrophobicity and/or secondary structure of the GL protein. We conclude that this region constitutes an important neutralization domain of EAV.

A 29K envelope glycoprotein of equine arteritis virus expresses neutralization determinants recognized by murine monoclonal antibodies.

A panel of six neutralizing murine monoclonal antibodies (MAbs) to equine arteritis virus (EAV) was produced. The MAbs were characterized by Western immunoblotting assay and competitive ELISA. The six MAbs identify a single neutralization site on a 29K envelope glycoprotein. Deglycosylation of viral proteins prior to immunoblotting showed that the 29K protein is the glycosylated form of a 20K protein. Equine anti-EAV serum also strongly bound the 29K glycoprotein, as well as an unglycosylated protein of 17K. The equine antisera to EAV blocked the binding of a selected MAb to EAV, whereas normal equine serum did not. Two neutralization-resistant escape mutant (EM) variants of the EAV prototype were produced using MAb 6D10. The phenotypic properties of the EM viruses were characterized by neutralization and immunoblotting assays with two MAbs (6D10 and 5G11). The two MAbs failed to neutralize either EM virus, and they did not react in an immunoblot assay with any proteins of the EM viruses. In contrast, binding of the equine antiserum to viral proteins was equivalent with prototype and EM virus strains. These data clearly indicate that a 29K envelope glycoprotein expresses at least one neutralization determinant of EAV.

Neutralization determinants of United States bluetongue virus serotype ten.

A panel of five neutralization-resistant escape mutant (EM) viruses was used to investigate the neutralization determinants of the U.S. prototype strain of bluetongue virus serotype 10 (BTV-10). The phenotypic properties of each EM virus were characterized by neutralization and immuneprecipitation assays with a panel of four monoclonal antibodies (MAbs). These MAbs were used to select the various EM viruses and together the MAbs define four distinct neutralizing epitopes on the prototype strain of BTV-10 (Heidner, H.W., Rositto, P.V., and MacLachlan, N.J., Virology 176, 658-661 (1990)). Sequencing of the L2 gene identified mutations responsible for the altered phenotypic properties exhibited by each EM virus. The L2 gene encodes BTV outer capsid protein VP2 which is responsible for virus neutralization. Four amino acids in three distinct regions of VP2 are critical to expression of the epitopes recognized by the MAb panel. Both amino acid 208 and 211 can affect the binding of MAb 039 and MAb 045, amino acid 327 affects binding of MAb 041, and amino acids 327 and 402 cooperatively interact to affect binding by MAb 034. The location of two of these critical regions on VP2 of BTV-10 is identical to two of those which affect neutralization of Australian BTV-1, despite the fact that these two viruses are antigenically distinct and have divergent L2 gene sequences (Gould, A.R., and Eaton, B.T., Virus Res. 17, 161-172 (1990)). The four individual neutralizing epitopes on VP2 of BTV-10 are interactive (Heidner, H.W., Rositto, P.V., and MacLachlan, N.J., Virology 176, 658-661 (1990)) and at least two are conformationally dependent.

Monoclonal antibodies specific for canine CD4 and CD8 define functional T-lymphocyte subsets and high-density expression of CD4 by canine neutrophils.

The characteristics of canine homologues of CD4 and CD8, defined by murine monoclonal antibodies CA13.1E4 (IgG1) and CA9.JD3 (IgG2a) respectively, are described. These antibodies identify mutually exclusive subpopulations of non-B lymphocytes in peripheral lymphoid organs and blood. However, in thymus the antibodies defined populations of double-positive, double-negative and single-positive cells that showed a progressive maturation consistent with that described for CD4 and CD8 in other mammalian species. Furthermore, functional studies clearly associated cytotoxic effector cell function with the subpopulation reactive with CA9.JD3 (CD8). In contrast, proliferation stimulated by allogeneic cells and mitogens was more pronounced in the subpopulation reactive with CA13.1E4 (CD4). Cell and tissue distribution studies revealed that CA13.1E4 stained neutrophils with equivalent intensity to the staining of peripheral T cells. CA13.1E4 precipitated a 60 kD protein from the surface of T cells and highly purified neutrophils under both reducing and nonreducing conditions. CA9.JD3 precipitated a heterodimer of 32 kd and 36 kD under reducing conditions, and a 70 kD protein under non-reducing conditions. The expression of CD4 by canine neutrophils is without precedent in other mammalian species; the functional significance of neutrophil CD4 expression is puzzling in light of the current understanding of the functions of CD4 which include it's role as a receptor for nonpolymorphic regions of MHC class II molecules.

Flow cytometric analysis of in vitro bluetongue virus infection of bovine blood mononuclear cells.

Cultures of adherent and non-adherent bovine peripheral blood mononuclear (PBM) cells were inoculated with bluetongue virus (BTV) serotype 10. Some cultures of non-adherent cells were stimulated with interleukin 2 (IL-2) and concanavalin A for 24 h prior to virus inoculation. Cells were harvested at various intervals up to 72 h after inoculation. A panel of leukocyte differentiation antigen-specific monoclonal antibodies (MAbs), specific for bovine CD2, CD4 or CD8, monocytes and granulocytes, B cells, gamma delta T cells or the IL-2 receptor (IL-2r), was directly conjugated to fluorescein isothiocyanate, and a MAb specific for the BTV major core protein VP7 was directly conjugated to phycoerythrin. Cells were labelled with conjugated MAbs in single- and double-label immunofluorescence studies to identify specifically the BTV-infected cells in inoculated cultures. The viability of cells was determined by propidium iodide exclusion, and all analyses were done using flow cytometry. Productive infection of cultures of PBM cells was confirmed by virus titration. The data revealed a clear difference between subsets of bovine PBM cells in susceptibility to infection with BTV in vitro. Monocytes were readily infected with BTV, as were stimulated CD4+ cells, and infection was cytopathic to monocytes and stimulated lymphocytes. The proportion of infected cells decreased after 24 h and virus titres dropped markedly by 72 h in all cultures. CD4+ cells in cultures of unstimulated non-adherent cells inoculated with BTV showed increased expression of IL-2r. The possible relevance of these findings to the pathogenesis of BTV infection of cattle is discussed.