Characterization of Brucella abortus infection of bovine monocyte-derived dendritic cells.

Brucella abortus is a Gram negative facultative intracellular pathogen of cattle, and an important zoonosis in humans worldwide. Previous studies have shown that dendritic cells (DC) from humans and mice are highly permissive for Brucella survival and proliferation. Impairment of DC activation and maturation by Brucella infection has also been reported in these two species. The aim of this study was to characterize infection of bovine DC with B. abortus. Monocyte-derived DC (mdDC) were cultured from bovine peripheral blood mononuclear cells (PBMC) using the recombinant bovine cytokines IL-4 and GM-CSF. The resulting mdDC were DEC205(+), MHC class II(hi). Approximately 70% of the cultured cells were DEC205(+), MHC II(+). MdDC were infected with B. abortus strain 2308 at an MOI of 1 and 100. Parallel infection experiments were performed in monocyte derived macrophages (mdM) isolated from the same subjects. Bacteria were successfully killed by mdDC by 24 hours post infection (PI) with high and low dose of B. abortus, bacteria persisted in mdM infected with a high dose. Expression of IL-1b, IL-6, IL-10, IL-12p40, IFNγ, iNOS and TNFα in B. abortus infected and LPS stimulated mdDC at 6 and 24 hours PI were evaluated using RT-qPCR. At 6 hours PI all transcripts were increased over control cells and significantly less IL-10, IL-12p40, and IFNγ were expressed in mdDC infected with B. abortus compared to LPS stimulation. Evaluation of mdDC cultures by flow cytometry was performed. Flow cytometric analysis of infected and LPS stimulated mdDC 24 hours PI showed expression of CD80 and CD86 was impaired in two of the three animals analyzed. MHC class II expression was equivocal between the groups. From these results we conclude that cultured bovine mdDC are not permissive for intracellular proliferation of B. abortus, and infected mdDC exhibit some signs of maturational and activational impairment.

Immunophysiological responses of horses to a 12-hour rest during 24 hours of road transport.

Thirty-eight mature horses were assigned to one of two equal groups to evaluate two treatments consisting of either 24 hours of continuous road transport (24T) or two 12-hour periods of transport separated by off-loading, resting and feeding the horses for 12 hours (12/12T). A subset of six horses from each group served as controls for the other group. The horses were loaded into a commercial straight-deck trailer and travelled loose in one of two standard-sized compartments. After the journeys the horses were put back into their paddocks for a 24-hour recovery period. Venous blood samples were collected before loading, after unloading and after the 24-hour recovery period. Transport significantly increased the horses' cortisol concentrations, neutrophil counts and neutrophil:lymphocyte (nl) ratios, and decreased the numbers of all the lymphocyte subpopulation cell types. Collectively, no significant differences were observed between the two treatments in the horses' cortisol concentrations, total leucocyte counts, neutrophil and lymphocyte counts, nl ratios, and the cd8a+ and cd21+ lymphocyte subpopulations, but there were differences in the numbers of cd3+, cd4+, and cd8b+ subpopulations. The inclusion of a 12-hour rest-stop interrupted the transport-related decline in the lymphocyte subpopulations and allowed them to recover towards their resting levels.

Otarine Herpesvirus-1, not papillomavirus, is associated with endemic tumours in California sea lions (Zalophus californianus).

The purpose of this study was to determine if Otarine Herpesvirus-1 (OtHV-1) is associated with the presence of urogenital carcinomas in California sea lions. Polymerase chain reaction (PCR) analysis with primers specific for OtHV-1 was used to compare the prevalence of OtHV-1 infection in 15 sea lions affected by urogenital carcinoma with that of age-matched and juvenile tumour-free animals, and animals with tumours of non-urogenital origin. The herpesvirus was more prevalent (100%) and more widespread in the 15 animals with urogenital carcinoma than in 25 control animals, and was most often found in the urogenital tissue (vagina and prostate) and in the draining lymph nodes. Moreover, OtHV-1 DNA was not found in any juvenile animal, or in the neoplastic tissues of animals with non-urogenital tumours. Papillomavirus-specific PCR analysis of urogenital carcinoma tissues detected papillomavirus sequences in only one carcinomatous tissue. Further studies are needed to determine if OtHV-1 contributes to oncogenesis in the California sea lion; these data show, however, that OtHV-1 is associated with urogenital carcinomas, is preferentially present in urogenital tissues, and may be sexually transmitted. Papillomaviruses, which are known to contribute to urogenital tumours in other species, did not appear to be associated with the sea lion carcinomas.

Expressed MHC class II genes in sea otters (Enhydra lutris) from geographically disparate populations.

The major histocompatibility complex (MHC) is central to maintaining the immunologic vigor of individuals and populations. Classical MHC class II genes were targeted for partial sequencing in sea otters (Enhydra lutris) from populations in California, Washington, and Alaska. Sequences derived from sea otter peripheral blood leukocyte mRNAs were similar to those classified as DQA, DQB, DRA, and DRB in other species. Comparisons of the derived amino acid compositions supported the classification of these as functional molecules from at least one DQA, DQB, and DRA locus and at least two DRB loci. While limited in scope, phylogenetic analysis of the DRB peptide-binding region suggested the possible existence of distinct clades demarcated by geographic region. These preliminary findings support the need for additional MHC gene sequencing and expansion to a comprehensive study targeting additional otters.

Histochemical and immunohistochemical evidence of a bacterium associated with lesions of epizootic bovine abortion.

Epizootic bovine abortion (EBA), a tick-transmitted disease of pregnant cattle grazing foothill pastures, is a major cause of reproductive failure in California and adjacent states. Affected fetuses develop a chronic disease, resulting in late-term abortion or premature calving. Despite investigations spanning 50 years, to the authors' knowledge, the etiologic agent of EBA has not yet been isolated from affected fetuses or the tick vector. The diagnosis of EBA is based on gross and microscopic lesions. Recently, documentation that the etiologic agent is susceptible to antibiotics and identification of a unique 16S deltaproteobacterial rDNA gene sequence in 90% of thymus tissues from aborted fetuses have supported the role of a bacterial infection as the cause of EBA. To determine whether bacteria could be detected in the tissues, histochemical staining and immunohistochemical procedures were used on formalin-fixed, paraffin-embedded tissues. Use of a modified Steiner silver stain revealed small numbers of intracytoplasmic bacterial rods in 37 of 42 thymic samples from EBA-affected fetuses. Improved detection was achieved by use of immunohistochemical staining with serum from EBA-affected fetuses that resulted in detection of numerous bacterial rods in the cytoplasm of histiocytic cells in the thymus from all 42 EBA-affected fetuses. Immunohistochemical examination of additional tissues from 21 field and experimental EBA cases revealed positively stained intracytoplasmic bacterial rods in many organs with inflammatory lesions. Use of the modified Steiner stain and immunohistochemical staining of tissues from negative-control fetuses failed to reveal organisms. To the authors' knowledge, this is the first report to document morphologic evidence of a bacterium associated with the lesions of EBA.

MHC gene configuration variation in geographically disparate populations of California sea lions (Zalophus californianus).

Major histocompatibility complex (MHC) class II DRB genotypes were examined in two geographically isolated populations of California sea lions (Zalophus californianus) (Gulf of California and California coastal Pacific Ocean). Genomic DNA from 227 California sea lions was examined using eight sequence-specific primer (SSP) pairs flanking the putative peptide-binding site. A total of 40 different Zaca-DRB genotype configurations were identified among the 227 individuals. Using SSP-PCR, significant differences were found between coastal California and Gulf of California Zalophus populations in numbers of DRB sequences per individual and configuration of sequences within individuals. Additionally, unique local patterns of MHC diversity were identified among the Midriff Island animals. These population differences are consistent with either ecologically distinct patterns of selection pressures and/or geographical isolation. The consequences of these partitioned MHC configurations at the population level are as yet unknown; however, the worldwide increase in emerging marine diseases lends urgency to their examination.

Tissue distribution of phocine herpesvirus-1 (PhHV-1) in infected harbour seals (Phoca vitulina) from the central Californian coast and a comparison of diagnostic methods.

The polymerase chain reaction (PCR) was used to determine the tissue distribution of phocine herpesvirus-1 (PhHV-1) DNA in 20 stranded Pacific harbour seals (17 pups and three seals older than one year) that died during rehabilitation. The aim was to begin to define stages of infection and to investigate the relation between the presence of PhHV-1 in tissues, histological lesions and serology. PhHV-1 DNA was detected in a wide range of tissues from 10/17 pups and 3/3 subadults or adults. Different clinical patterns emerged from the examination of ante- and post-mortem samples. These patterns probably represented pups with active PhHV-1 infection, pups recovering from infection, and older harbour seals with chronic, reactivated infection. As PhHV-1 DNA was detected in tissues in the absence of typical histological lesions in seven seals and in the absence of PhHV-1 specific antibodies in four seals, it is clear that both histological examination and serology underestimate the presence of infection. These results showed that infection can occur in the absence of obvious disease and that seroconversion may be associated with clinical recovery.

Immunological response to long-term transport stress in mature horses and effects of adaptogenic dietary supplementation as an immunomodulator.

REASONS FOR PERFORMING STUDY: Little information exists on the immunological effects of transport or the use of supplements to minimise transport stress. OBJECTIVES: To establish baseline ranges and evaluate immunophenotypic and functional changes associated with transport and a nutritional 'adaptogen' supplement. METHODS: Horses received either supplement (n = 10) or placebos (n = 9) during the 30 day study. After 28 days in stalls, 12 horses (6 supplement; 6 placebo) were transported for 24 h, then unloaded and recovered. Venous blood samples were collected on Days 1, 14 and 28 to establish baselines, and on Days 28, 29 and 30 to examine changes during transport and recovery. RESULTS: Transport prompted elevations (P<0.05) in cortisol concentration, neutrophil count and white blood cell counts, while lymphocyte subpopulation counts (CD3+, CD4+, CD8+, CD21+) decreased (P<0.05). Normal phenotypic lymphocyte profiles returned within 24 h of recovery. Supplement effects on immunophenotype (CD21+ and CD8+) were observed in stabled horses (P<0.05), but not in transported horses. CONCLUSIONS: These results provide insights into the immunological mechanisms associated with long-term transport. POTENTIAL RELEVANCE: The existence of a small window of immunological uncertainty follows long-term transportation, enhancing the potential risk of infectious disease in susceptible individuals.

Characterization of F21.A, a monoclonal antibody that recognize a leucocyte surface antigen for killer whale homologue to beta-2 integrin.

The specificity of F21.A, a monoclonal antibody raised against bottlenose dolphin leucocytes, was characterized in killer whale on the basis of immunoprecipitation of a protein of 94 kDa, as well as flow cytometric analysis. While minimally expressed on resting cells, F21.A labeled a homologue to beta-2 integrin in 89-97% of PMA-activated neutrophils, 53-66% of activated monocytes, and activated B cells but not T cells. Activation of neutrophils reached its maximum 10 min after PMA stimulation. F21.A did not label intracellular stores as did both cross-reacting anti-canine CD11b and CD18, suggesting that an activation-induced conformational change would expose a neoepitope recognized by F21.A. F21.A labeling was largely inhibited by pre-incubation with plasma, suggesting a binding site closely related to that for fibrinogen. In vitro phagocytosis and respiratory burst were almost fully inhibited upon pre-incubation with F21.A, demonstrating its functional importance. This antibody is foreseen as a possible valuable diagnostic and research tool in cetacean immunology.

Genomic characterization of equine interleukin-4 receptor alpha-chain (IL4R).

Three overlapping fragments of the equine interleukin-4 receptor alpha chain gene (IL4R) were cloned and sequenced. The resulting 3553 bp cDNA sequence exhibited homology to human, murine and bovine IL4R. The equine IL4R exhibits many conserved features when compared to other species, including intron-exon boundary positions and amino acid sequence motifs characteristic of type I cytokine receptors. The IL4R gene was localized to horse chromosome ECA13 by synteny mapping on a somatic cell hybrid panel. Evidence for an alternative splice variant of IL4R was found in the genomic sequence and subsequently verified using RT-PCR on equine monocyte RNA. A polymorphism screen of the largest exon, homologous to exon 12 of the human IL4R gene, was performed using DNA from 60 horses of various breeds which yielded 11 coding-region single nucleotide polymorphisms (SNPs), 7 synonymous and 4 non-synonymous. Three of the four non-synonymous SNPs occur at high frequencies and are found very near a conserved tyrosine residue.

Humoral immune responses to phocine herpesvirus-1 in Pacific harbor seals (Phoca vitulina richardsii) during an outbreak of clinical disease.

Infection with phocine herpesvirus type-1 (PHV-1) has been associated with morbidity and high mortality in neonatal harbor seals (Phoca vitulina). A PHV-1 specific indirect enzyme linked immunosorbent assay (ELISA) was developed to sequentially measure the serological status of 106 harbor seal neonates admitted to a Pacific coast rehabilitation center (total number of sera tested was 371). Early in the season (February-April), the majority of pups had low serum levels of PHV-1 specific antibody. A dramatic increase in PHV-1 specific antibody, involving the majority of hospitalized pups, was observed during a 4-week period in May. This coincided with a high incidence of PHV-1 associated adrenal lesions and mortality. Although there was overall agreement between the timing of seroconversion to PHV-1 and histological evidence of PHV-1 infection, 82.4% of individual pups with adrenalitis had no evidence of a humoral response to PHV-1 at the time of their death. This suggests either a rapid disease course, or an inability to develop a humoral response in some neonatal seals.

Immunohistochemical investigation of the cross-reactivity of selected cell markers from various species for characterization of lymphatic tissues in the harbour porpoise (Phocoena phocoena).

To facilitate a detailed investigation of cetacean lymphoid organs, 13 canine-, six bovine-, one equine-, one human- and four killer whale-specific monoclonal antibodies (mAbs) directed against cell surface antigens of the haematopoietic system (including CD2, CD4, CD8, CD45R, MHC class II, granulocyte, thrombocyte, pan-T cell and B-cell antigen), as well as a mAb and a polyclonal antibody (pAb) directed against the -peptide of the human CD3 complex, were tested for immunohistochemical cross-reactivity on frozen or formalin-fixed, paraffin wax-embedded lymphatic tissues of harbour porpoises. Eight of 26 mAbs and the pAb showed a specific reaction with harbour porpoise cells. Lymphocytes in T-cell compartments were labelled by the mAb and the pAb directed against the CD3 complex and by two killer whale mAbs specific for CD2 antigen. CD45R, labelled by a killer whale-specific mAb, was strongly expressed on B and weakly on T cells. MHC class II antigen, recognized by killer whale- and bovine-specific mAbs, was expressed on B and T cells. A canine MHC class II-specific mAb recognized an epitope on the surface of antigen-presenting cells and B lymphocytes. An anti-equine-pan-leucocyte marker labelled the majority of cells in B- and T-cell compartments. Thus, with leucocyte antigen markers from various species, it is now possible to determine the phenotype of lymphocytes in normal and diseased lymphoid organs of harbour porpoises.

Molecular cloning and sequencing of the low-affinity IgE receptor (CD23) for horse and cattle.

Expression of the low-affinity IgE receptor (CD23) on the surface of mononuclear cells is a critical event in the development of IgE-mediated immunologic responses. Using PCR and cDNA library screening, a 2.7kb cDNA encoding equine CD23 and a 627bp PCR fragment of cattle CD23 were sequenced and characterized. The equine CD23 sequence encodes a complete and continuous open reading frame of 327 amino acids, while the shorter cattle fragment encodes 209 amino acids corresponding to nucleotides 325-1098 of the equine CD23 transcript. In addition to high identities in their nucleotides and translated amino acids with CD23 sequences published for other species, the translated equine CD23 protein also shares many of the structural features of this molecule described for human and rodents. Interestingly, an additional repetitive element of possible functional significance consisting of 18 amino acids, located between the transmembrane region and the carbohydrate-binding lectin domain of horse CD23, was also identified. Based on these sequences, molecular assays will be developed to measure CD23 mRNA in tissues and expression of recombinant proteins will be essential to the production of species-specific antibody reagents. These assays and reagents will be useful in future studies of allergic and lymphoproliferative diseases in horses and cattle.

Antigenic and nucleotide characterization of a herpesvirus isolated from Pacific harbor seals (Phoca vitulina richardsii).

This report describes the antigenic and nucleotide characterization of a herpes-like virus that has been isolated from the adrenal tissues of neonatal Pacific harbor seals. Infection with this virus has been previously implicated as a major cause of death of animals undergoing rehabilitation. Comparison and phylogenetic analysis of sequenced fragments of the DNA polymerase, glycoprotein B and glycoprotein D genes, and immunofluorescence assay using herpesvirus-specific monoclonal antibodies, demonstrated close similarity of the Pacific harbor seal herpesvirus to European isolates of phocid herpesvirus-1 (PHV-1) and other alpha-herpesviruses affecting terrestrial carnivores.

Morbillivirus infection in stranded common dolphins from the Pacific Ocean.

From August 1995 to August 1997, six of 18 common dolphins (Delphinus delphis) that stranded along beaches of southern California (USA) tested antibody positive for dolphin morbillivirus (DMV). Titers ascertained by virus neutralization ranged from 1:50 to 1:910 while those determined by ELISA ranged from 1:80 to 1:195. The first individual to strand survived and was released back into the Pacific Ocean 14 mo later. Histopathologic examination of tissues from the other five dolphins did not reveal lesions characteristic of morbilliviral disease; however, morbilliviral RNA was detected in three of the five by reverse transcriptase-polymerase chain reaction testing. This is the first report of morbilliviral infection in any marine mammal species in the northern hemisphere of the Pacific Ocean. These data indicate that DMV, or a closely related morbillivirus, is present in the Pacific Ocean and infection of common dolphins may not be associated with morbillivirus disease.

Characterization of a monoclonal antibody that recognizes a lymphocyte surface antigen for the cetacean homologue to CD45R.

As part of our current efforts to develop assays and reagents to study the immune system of marine mammals, and in view of the effort currently made to develop monoclonal antibodies to cell surface proteins of lymphocyte subsets in different species, the present paper reports on the characterization of a monoclonal antibody against the homologue of CD45R on cetacean lymphocytes. The specificity of this antibody has been characterized on the basis of immunoprecipitation of the antigen it recognized, immunoperoxidase staining on cetacean lymph node and thymus sections, as well as one and two-colour flow cytometric analysis of cetacean peripheral blood mononuclear cells and single-cell suspensions of thymus, lymph node and spleen. Anticetacean CD45R (F21.H) immunoprecipitated proteins of 180, 200 and 220 x 10(3) MW, with the 180 x 10(3) MW from being predominantly expressed on T cells and the 220 x 10(3) MW form expressed predominantly on B cells and thymocytes F21.H labelled all B cells and a proportion of T cells on single-cell suspensions of spleen cells. CD45R- killer whale peripheral blood lymphocytes expressed a higher density of CD2 than CD45R+, a characteristic of memory T cells. Killer whale T lymphocytes also lost the expression of CD45R upon activation with concanavalin A (Con A) and phytohaemagglutinin (PHA). This is the first report of a monoclonal antibody to CD45R in cetaceans, and this antibody is foreseen as a possible valuable diagnostic and research tool to assess immune functions of captive and wild cetaceans as part of the evaluation of their health status.

Report of the Second Equine Leucocyte Antigen Workshop, Squaw valley, California, July 1995.

The final assignment of antibody clusters for leucocyte antigens and immunoglobulins, as described in detail in Sections 3 and 4, is summarized in Table 4. Together with other mAbs developed outside of ELAW II (Table 9) this pool of reagents represent a powerful array of tools for the study of equine immunity. The Second Equine Leucocyte Antigen Workshop made considerable advances in pursuing the objectives of establishing the specificities of mAbs and achieving consensus on the nomenclature for equine leucocyte and immunoglobulin molecules. Of equal importance, several productive collaborations were fostered among the participating laboratories and observers. Overall, enormous advances have been made in the past decade since mAbs specific for equine leucocyte antigens and immunoglobulins were first reported. There remains enormous scope and need for further studies of equine leucocyte antigens and immunoglobulins, both for the purposes of comparative immunology and for the good of the horse. In the future novel techniques will be required to develop reagents for specific target antigens such as the orthologues of the CD25 or CD45 isoforms. In studies of equine immunoglobulins the functional role of the IgG isotypes must be better established, reagents for IgE must be developed, and cloning of the immunoglobulin heavy chain genes will be essential if the complexities of the IgG sub-isotypes are to be elucidated. The tasks still facing the currently small group of equine immunologists throughout the world remain formidable, and will only be tackled successfully in a spirit of collaboration.

Memory and CD8+ are the predominant bovine bronchoalveolar lymphocyte phenotypes.

Bovine lymphocytes obtained by bronchoalveolar lavage (BAL) of healthy calves were simultaneously analyzed and compared to peripheral blood lymphocytes using monoclonal antibodies specific for bovine leukocyte differentiation antigens. Phenotypic differences were observed between bronchoalveolar and peripheral blood T-lymphocyte subpopulations, demonstrating selective lymphocyte migration to the bovine lung. The bronchoalveolar and peripheral blood T-lymphocyte populations, defined by expression of CD2, were similar, but bronchoalveolar T lymphocytes were predominately CD8+ while peripheral blood T cells were predominately CD4+. In addition, memory lymphocytes, characterized by low expression of CD45R and activated lymphocytes (CD25+), were found in significantly higher proportions in the bronchoalveolar compartment. The proportion of gammadelta T lymphocytes was, however, significantly higher in peripheral blood. B cells were observed in similar proportions in the bronchoalveolar compartment and peripheral blood.

Bovine cytokine expression during different phases of bovine leukemia virus infection.

The potential role of aberrant cytokine production in the pathogenesis of bovine leukemia virus (BLV) was studied by analyzing cytokine mRNA expression in pokeweed-stimulated PBMLs of cows in different phases of disease progression. To analyze the mRNA, a semi-quantitative RT-PCR assay was developed. The RT-PCR assay was developed for detection of IL-2, -4, -6, -10, -12, IFN-gamma and actin using cDNA derived from phorbol-stimulated peripheral blood mononuclear leukocytes. Using a PCR specific for BLV tax, agar gel immunodiffusion and white blood cell counts, BLV-negative, BLV-positive aleukemic (AL), and BLV-positive persistently lymphocytotic (PL) cattle were identified. Peripheral blood lymphocytes cultured in vitro for 24 h in pokeweed mitogen were analyzed for cytokine production using the RT-PCR assay. Consistently elevated levels of IL-2 and IL-12 in AL and PL cattle in pokeweed mitogen-stimulated cells was detected, while IFN-gamma was elevated in the AL but not the PL cattle.

Phenotypic characterization of lymphocyte subpopulations in horses affected with chronic obstructive pulmonary disease and in normal controls.

The alterations in lymphocyte subsets in chronic obstructive pulmonary disease (COPD) in the horse were investigated by using monoclonal antibodies to identify CD3+, CD4+, CD8+, and surface immunoglobulin positive (sIg+) lymphocytes in peripheral blood, bronchoalveolar lavage fluid (BALF), and pulmonary biopsy frozen tissue sections. COPD-affected horses (n = 5) and normal controls (n = 5) were sampled prestabling and 14 days poststabling, at which time the COPD-affected horses wee exhibiting clinical signs of COPD. The peripheral blood absolute CD4+ lymphocyte count was significantly elevated in the COPD-affected horses pre- and poststabling. The CD4:CD8 ratio in peripheral blood of COPD-affect horses was unaffected by stabling, but the same ratio in the control horses was significantly decreased. These findings support a hypothesis of deficient regulation of a systemic immune response to indoor air in the COPD-affected horses. A large population of leukocytes in pulmonary biopsy immunohistochemical sections from both groups of horses appeared to be CD3+ CD4- CD8-, an uncommon phenotype in both the peripheral blood and BALF.