Serum zinc levels and their relationship with diseases in racehorses.

Zinc is one of the essential microelements involved in the regulation of enzyme activity, as well as metabolism of nucleic acid and proteins. There have been few reports on equine serum zinc concentrations during the training period, and little is known about the relationship between zinc levels and diseases in horses. In this study, we measured serum zinc levels in healthy Thoroughbred racehorses, as well as in other horses, under general disease or training conditions. The reference value for serum zinc levels in Thoroughbred horses was 41-79 µg/dl. There were no differences in serum zinc levels due to sex or age. Significant decreases in serum zinc levels were observed after training, but serum zinc levels did not vary with intensity of sweating. Serum zinc levels were lower in horses clinically diagnosed as having shipping fever (36.3 ± 2.7 µg/dl), fever (45.3 ± 3.0 µg/dl) and cellulitis (44.0 ± 3.4 µg/dl), as compared to control values (59.7 ± 9.7 µg/dl). They also tended to decrease in experimentally infected horses one day after inoculation. Changes in serum zinc levels reached nadir one day after surgical invasion, except for a horse that experienced complicating shock. These results suggest that zinc is a serological indicator of inflammatory status in Thoroughbred horses.

Serum IgG response in calves to the putative pneumonic virulence factor Gs60 of Mannheimia haemolytica A1.

Bovine pneumonic pasteurellosis vaccines incorporate various antigens of Mannheimia haemolytica, including the acknowledged virulence factor leukotoxin (Lkt), and Gs60, a surface lipoprotein. To examine the role of antibodies to Gs60 in protection, an enzyme-linked immunosorbent assay (ELISA) was developed for retrospective analysis of serum samples from previous trials in which vaccines containing native or recombinant Gs60 were administered parenterally. The analysis revealed a positive correlation between the titer of antibodies to Gs60 and protection against experimental challenge in both vaccinates and naturally exposed controls. There was a strong correlation between production of IgG antibodies to Gs60 and Lkt neutralizing antibodies. Analysis of the relationship between the serum antibody titers and resistance to experimental challenge using linear statistical models revealed a significant association between prechallenge titers of serum antibodies to Lkt and protection. Further analysis suggested that antibodies against Gs60 were beneficial when Lkt neutralizing antibody titers were low.

Les vaccins pour la pneumonie bovine à Pasteurella contiennent divers antigènes de Mannheimia haemolytica incluant la leucotoxine (Lkt), facteur de virulence reconnu, ainsi que Gs60, une lipoprotéine de surface. Afin d'examiner le rôle des anticorps contre Gs60 dans la protection, une épreuve immunoenzymatique (ELISA) a été développée pour analyse rétrospective d'échantillons de sérum provenant d'études antérieures au cours desquelles des vaccins contenant la Gs60 native ou recombinante étaient administrés par voie parentérale. L'analyse a révélé une corrélation positive entre le titre d'anticorps contre Gs60 et la protection contre une infection expérimentale autant chez des animaux vaccinés que des témoins exposés naturellement. Il y avait une forte corrélation entre la production d'anticorps de type IgG contre Gs60 et des anticorps neutralisants Lkt. Une analyse de la relation entre les titres d'anticorps sériques et la résistance à une infection expérimentale utilisant des modèles statistiques linéaires a révélé une association significative entre les titres d'anticorps sériques pré-infection avec Lkt et la protection. Des analyses supplémentaires ont suggéré que les anticorps contre Gs60 étaient bénéfiques lorsque les titres d'anticorps neutralisants anti-Lkt étaient bas.(Traduit par Docteur Serge Messier).

Fatal pneumonia epizootic in musk ox (Ovibos moschatus) in a period of extraordinary weather conditions.

The musk ox is adapted to extreme cold and regarded as vulnerable to the impacts of climate change. Population decline is proposed to occur due to changes in forage availability, insect harassment, parasite load, and habitat availability, while the possible role of infectious diseases has not been emphasized. The goal of the present article is to describe an outbreak of fatal pasteurellosis that occurred in the introduced musk ox population of Dovrefjell, Norway in 2006, causing the death of a large proportion of the animals. The epizootic coincided with extraordinary warm and humid weather, conditions that often are associated with outbreaks of pasteurellosis. The description is based on long series of data from the surveillance of the musk ox population, weather data from a closely located meteorological station, and pathoanatomical investigation of the diseased animals. It is concluded that the weather conditions likely were the decisive factors for the outbreak. It is suggested that such epizootics may occur increasingly among cold-adapted animals if global warming results in increased occurrence of heat waves and associated extreme weather events, thereby causing population declines and possibly extinctions.

Expression of a modified Mannheimia haemolytica GS60 outer membrane lipoprotein in transgenic alfalfa for the development of an edible vaccine against bovine pneumonic pasteurellosis.

The GS60 antigen is one of the protective antigens of Mannheimia haemolytica A1. GS60 contains conserved domains belonging to the LppC family of bacterial outer membrane lipoproteins. A high antibody titer to GS60 has been shown to be significantly correlated with resistance to pneumonic pasteurellosis. Calves vaccinated with a commercial vaccine (Presponse) and demonstrating protection against M. haemolytica A1 produced antibodies directed against GS60. Alfalfa was chosen as the platform for an edible vaccine. Agrobacterium tumefaciens was used to mediate the transformation of alfalfa with sequences encoding a slightly shortened derivative of the GS60 antigen (GS60(54)). Stable transgenic alfalfa lines were recovered and production of GS60(54) was examined by Western immunoblot analysis. The antigen is stable in dried transgenic plant material stored at ambient temperature for more than a year. The plant-produced GS60(54) protein was shown to be immunogenic when injected into rabbits. Feeding of the dried transgenic alfalfa expressing the GS60(54) to rabbits is capable of inducing seroconversion, suggesting that GS60(54) could be an effective oral antigen for stimulating mucosal immune responses.

Mannheimia haemolytica leukotoxin-induced cytolysis of ovine (Ovis aries) leukocytes is mediated by CD18, the beta subunit of beta2-integrins.

Mannheimia (Pasteurella) haemolytica causes severe pneumonia in cattle, sheep and goats. Leukotoxin (Lkt) is the most important virulence determinant produced by this organism. Previously, we identified CD18, the beta subunit of beta(2)-integrins, as the receptor for Lkt on bovine leukocytes. Since Lkt is specific for leukocytes of cattle, sheep and goats, we hypothesized that Lkt utilizes CD18 as its receptor on ovine leukocytes as well. Therefore, the objective of this study was to transfect an Lkt-resistant murine cell line (P815) with cDNA encoding ovine CD18, and to determine the susceptibility of the transfectants to Lkt-induced cytolysis. cDNA for ovine CD18 cloned from polymorphonuclear leukocytes was transfected into P815 cells. Flow cytometric analysis of the transfectants revealed surface expression of ovine CD18, and Lkt binding. In a cytotoxicity assay, the transfectants were lysed by Lkt in a concentration-dependent manner, whereas the parent cells were not. Pre-incubation of Lkt with an anti-Lkt neutralizing antibody and pre-incubation of transfectants with an anti-CD18 antibody resulted in inhibition of cytolysis confirming the interaction between Lkt and CD18. Taken together, these results indicate that CD18 on ovine leukocytes serves as a receptor for Lkt, and that CD18 is sufficient to mediate Lkt-induced cytolysis of ovine leukocytes.

Minimum inhibitory concentrations of tulathromycin against respiratory bacterial pathogens isolated from clinical cases in European cattle and swine and variability arising from changes in in vitro methodology.

The in vitro activity of tulathromycin was evaluated against common bovine and porcine respiratory pathogens collected from outbreaks of clinical disease across eight European countries from 1998 to 2001. Minimum inhibitory concentrations (MICs) for one isolate of each bacterial species from each outbreak were determined using a broth microdilution technique. The lowest concentrations inhibiting the growth of 90% of isolates (MIC90) for tulathromycin were 2 microg/ml for Mannheimia (Pasteurella) haemolytica, 1 microg/ml for Pasteurella multocida (bovine), and 2 microg/ml for Pasteurella multocida (porcine) and ranged from 0.5 to 4 microg/ml for Histophilus somni (Haemophilus somnus) and from 4 to 16 microg/ml for Actinobacillus pleuropneumoniae. Isolates were retested in the presence of serum. The activity of tulathromycin against fastidious organisms was affected by culture conditions, and MICs were reduced in the presence of serum.

Conglutinin and immunoconglutinin titers in stressed calves in a feedlot.

OBJECTIVE: To determine whether increased conglutinin titers are evident in stressed calves that do not develop respiratory tract disease in feedlots, compared with respiratory tract disease, and to determine the increase in immunoconglutinin titers. ANIMALS: 101 mixed-breed beef calves. PROCEDURE: Calves were processed at 4 farms of origin and allowed to remain with their dams for another 100 days. Calves from each farm were brought to a centrally located order-buyer barn. In a feedlot, 101 calves were assigned to pens and observed daily for clinical signs of acute respiratory tract disease. When sick calves were detected, they were treated with antibiotics and isolated in a pen for 4 days. Conglutinin and immunoconglutinin titers were determined for all calves. RESULTS: During the 28-day study, 73 calves developed respiratory tract disease, whereas 28 calves remained healthy. Mean conglutinin titers differed significantly among calves from the 4 farms. Significant differences were not detected in conglutinin titers among calves on the basis of sex, morbidity, or vaccination status against Mannheimia haemolytica at each farm, the order-buyer barn, or the feedlot on days 8, 15, and 28 after arrival. Immunoconglutinin titers in calves differed significantly among farms and morbidity status. CONCLUSIONS AND CLINICAL RELEVANCE: Mean conglutinin titers in calves do not appear to be associated with the incidence of acute respiratory tract disease; however, increased immunoconglutinin titers appear to be associated with recovery of stressed calves from respiratory tract disease during the first 15 days after arrival in a feedlot.

Decreased apolipoprotein C-III concentration in the high-density lipoprotein fraction from calves inoculated with Pasteurella haemolytica and bovine herpes virus-1.

Lipoprotein lipid and apoprotein concentrations are known to be altered during the acute-phase response. We have previously shown that the serum activity of lecithin:cholesterol acyltransferase (LCAT) and concentration of cholesteryl esters, both constituents of the high-density lipoprotein (HDL) fraction, are reduced in calves inoculated with Pasteurella haemolytica and bovine herpes virus-1, the two major pathogens for calf pneumonia. The concentration of apolipoprotein C-III (apoC-III), a low molecular mass protein component distributed mainly in the HDL fraction, was therefore examined in bacteria- and virus-inoculated calves. An enzyme-linked immunosorbent assay demonstrated that it was decreased by inoculations of Pasteurella haemolytica and bovine herpes virus-1. The decrease was detected as early as 1 day after inoculation in both groups. A decreased serum apoC-III concentration was also observed by immunoblot analysis. It was detected in the HDL fractions from the bacteria- and virus-inoculated calves, and HDL apoC-III concentrations in the inoculated calves were decreased compared with controls. These results, coupled with the previous findings on LCAT activity and the cholesteryl ester concentration, indicate that a decreased HDL concentration is one of the early events occurring during the acute-phase response evoked by infections with Pasteurella haemolytica and bovine herpes virus-1.

Detection of haptoglobin in the high-density lipoprotein and the very high-density lipoprotein fractions from sera of calves with experimental pneumonia and cows with naturally occurring fatty liver.

In addition to the lipoprotein-deficient d > 1.25 fraction, haptoglobin was detected in the high-density lipoprotein (HDL) and the very high-density lipoprotein (VHDL) fractions from sera of calves with experimental pneumonia and cows with naturally occurring fatty liver. It was not found in the chylomicrons, very low-density lipoprotein and low-density lipoprotein fractions. Washing of the HDL fraction did not decrease the haptoglobin concentration. Transferrin and immunoglobulin G were immunoblotted to examine the possibility of contamination of the lipoprotein fractions by the d > 1.25 fraction. The two serum proteins were detected only in the d > 1.25 fraction, not in any lipoprotein fractions. The distribution pattern of haptoglobin in the lipoprotein fractions was distinct from that of serum albumin. Concentrations of haptoglobin in the HDL fractions from pneumonic sera were largely proportional to those in whole sera. Cholesteryl ester concentrations were decreased in sera from calves with pneumonia, as in cows with fatty liver. A protein immunologically related to hemoglobin was also detected in particular in the VHDL fractions from sera of both groups. These results suggest that haptoglobin or a complex with the hemoglobin-like protein may have a role or roles related to the lipid metabolism.

Evaluation of structural and functional alterations of circulating neutrophils in calves with experimentally induced pneumonic pasteurellosis.

OBJECTIVES: To determine the structural and functional alterations in circulating neutrophils that may lead to sequestration in lung microvasculature and endothelial injury in calves with experimentally induced pneumonic pasteurellosis. ANIMALS: 10 healthy, 2- to 4-week-old male Holstein calves. PROCEDURES: Holstein calves were anesthetized and inoculated intrabronchially with Dulbecco phosphate buffered saline (0.9% NaCl) solution (DPBSS; 5 control calves) or 1 x 10(9) Pasteurella haemolytica organisms (5 infected calves). Blood samples were collected before and 1, 2, 4, and 6 hours after inoculation. Total and differential WBC count, dilute whole blood leukocyte deformability, neutrophil size distribution, and neutrophil surface CD11b expression were measured in blood samples. RESULTS: A progressive decrease in leukocyte deformability and increase in neutrophil size was detected 1, 2, 4, and 6 hours after inoculation of P haemolytica. Neutrophil surface CD11b expression was greater than baseline values at 6 hours after inoculation of P haemolytica. Two populations of neutrophils with an increase in size were detected in P haemolytica-infected calves. Both subpopulations had increased CD11b expression, compared with neutrophils that were typical in size. CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophils circulate in an activated and nondeformable state in calves with experimentally induced pneumonic pasteurellosis. A decrease in neutrophil deformability and neutrophil aggregation may contribute to neutrophil trapping in the lung microvasculature during pneumonic pasteurellosis in calves.

Role of tissue factor in intra-alveolar fibrin deposition and coagulopathy associated with pneumonic pasteurellosis in cattle.

OBJECTIVE: To determine the role of tissue factor (TF) in the coagulation events leading to intra-alveolar fibrin deposition and intravascular thrombosis associated with pneumonic pasteurellosis in cattle. ANIMALS: Healthy 2- to 4-week-old male Holstein calves. PROCEDURES: Blood and bronchoalveolar lavage samples were collected before and at 1, 2, 4, and 6 hours after inoculation of saline solution or Pasteurella haemolytica. Total leukocyte count, platelet count, plasma total protein concentration, prothrombin time, and partial thromboplastin time were measured in blood samples. Total nucleated cell count, total protein concentration, and procoagulant activity were measured in bronchoalveolar lavage samples. Additionally, platelet survival in blood platelet accumulation in affected lung tissue, and gross and microscopic lung lesions were determined. RESULTS: Administration of TF monoclonal antibodies (MAB) TF1-1F7 prevented the decrease in platelet survival and the increase in bronchoalveolar lavage fluid TF-dependent procoagulant activity observed in calves not treated with MAB TF1-1F7 antibody, but did not attenuate the increase in lavage fluid neutrophil numbers and total protein concentration, MAB TF1-1F7 administration reduced the percentage of lung affected by pneumonic lesions from 51.81% to 10.40% and attenuated intra-alveolar deposition of fibrin, neutrophils, and erythrocytes. CONCLUSION: Intra-alveolar fibrin deposition and activation of coagulation in cattle with pneumonic pasteurellosis is, at least in part, mediated by TF. CLINICAL RELEVANCE: Treatments that neutralize TF activity may attenuate lung injury in cattle with pneumonic pasteurellosis.

Comparative evaluation of antibodies induced by commercial Pasteurella haemolytica vaccines using solid phase immunoassays.

The objective of this study was to evaluate the ability of four commercial vaccines to elicit antibodies against the leukotoxin (Lkt), capsular polysaccharide (CP), iron regulated outer membrane proteins (IROMPs), and whole cell (WC) antigens of Pasteurella haemolytica A1. Modified double antibody sandwich enzyme linked immunosorbent assays (ELISAs) were developed to measure antibody levels against Lkt, CP and IROMPs. An indirect ELISA was developed to measure the levels of antibody against WC antigens. The ideal cut off points for ELISAs were determined on receiver operating characteristic curves, using sera from 30 calves injected subcutaneously with a live P. haemolytica 12296 strain as positive control and sera from 30 colostrum-deprived calves as negative control. The vaccines evaluated were: 'One Shot' (SmithKline Beecham, West Chester, PA) a bacterin-toxoid, 'Presponse' (Langford Laboratories, Guelph, Ontario) a Lkt-rich culture supermatant, 'Once PMH' (BioCor Inc., Omaha, NE) a modified live vaccine, and 'Septimune' (Fort Dodge laboratories, Fort Dodge, IA) an outer membrane extract. Thirty, 4-6 week old Holstein calves were randomized into 5 groups to receive one of the four vaccines or a placebo (sterile phosphate buffered saline). The calves were vaccinated intramuscularly on day 0 and on day 14, and bled on days, 0, 14, and 28 to measure antibody levels against Lkt, CP, IROMPs, and WC antigens of P. haemolytica Al. 'One Shot', and 'Once PMH' vaccinates showed a significant (P < 0.05) increase in antibody levels against Lkt at 28 days. 'Once PMH' vaccinates also showed significant (P < 0.05) increase in antibody levels against IROMPs at 28 days compared to the other four groups but this increase was not significant over time within the 'Once PMH' group. 'Presponse', 'Once PMH' and 'One Shot' vaccinates showed a significant (P < 0.05) increase in antibody levels against CP over time. These groups also had significantly higher antibody levels against CP, compared to controls and 'Septimune' vaccinates at 14 and 28 days (P < 0.05).

Growth-condition dependent expression of Pasteurella haemolytica A1 outer membrane proteins, capsule, and leukotoxin.

Pasteurella haemolytica, strain P1148 (biotype A, serotype 1) was grown under iron-rich and iron-restricted conditions both with and without serum, and the outer membrane protein (OMP), capsule, and leukotoxin production studied. OMPs were evaluated by SDS-PAGE and examined by immunoblot to identify antigens recognized by sera from P. haemolytica A1 convalescent and vaccinated cattle. Capsule production was evaluated using fluorescent antibody staining and rapid plate agglutination reaction. Leukotoxin production was measured by neutrophil 51Cr-release assay. Expression of specific OMPs, amount and antigenic character of capsule, and quantity of leukotoxin produced by P. haemolytica A1 varied in response to alterations in the growth media. Immunoblots indicated the immune response of convalescent calves differs from vaccinated calves, and convalescent calves produce antibodies to novel OMPs induced by growth in iron-restricted conditions.

Lymphocyte subpopulations in peripheral blood of lambs experimentally infected with Pasteurella haemolytica.

The lymphocyte subpopulations in peripheral blood obtained from eleven lambs experimentally infected with Pasteurella haemolytica were compared with those obtained from eight control lambs by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with P. haemolytica was characterized by a transient but significant reduction in SBU-T1+ (CD5+) T cells and SBU-T4+ (CD4+ or helper) T lymphocytes (P less than 0.05) and a significant rise in lymphocytes which did not express the LCA p220 epitope and the pan T cell surface marker (CD5-LCA p220-) ("null"). The reductions in CD5+ and CD4+ lymphocytes occurred 24 h after experimental infection, returning to preinoculation levels 5 days post inoculation (DPI). Five to 9 days after experimental infection, there was a significant increase in the number of lymphocytes, which expresses the pan T cell surface marker (CD5+) but which were CD4-CD8-. Lymphocyte transformation responses to the mitogen phytohaemagglutinin (PHA) were significantly reduced 24 h after experimental infection with P. haemolytica (P less than 0.05).

Changes in blood and bronchoalveolar lavage fluid components in calves with experimentally induced pneumonic pasteurellosis.

Pneumonic pasteurellosis was experimentally induced in calves by inoculation of 5 x 10(8) Pasteurella haemolytica organisms into the right diaphragmatic lung lobe. Blood and bronchoalveolar lavage fluid samples were obtained prior to inoculation and at postinoculation hour (PIH) 2, 4, and 6. Calves developed acute lung injury, characteristic of pneumonic pasteurellosis. Lesions were found only in the right diaphragmatic lobe. By PIH 4, significant (P less than 0.01) increases were detected in lavage fluid total cell count, neutrophil count, total protein and albumin concentrations, and alkaline phosphatase (ALP) and lactic dehydrogenase (LD) activities. Myeloperoxidase and elastase activities did not increase. Neutrophil depletion ameliorated the lung lesions and prevented the increase in lavage fluid cell count, total protein, and albumin concentrations and ALP and LD activities. Treatment with the iron chelator, deferoxamine mesylatehydroxyethyl starch, attenuated the increase in total protein and albumin concentrations and ALP and LD activities at PID 4, but not PIH 6. Treatment with a neutrophil function inhibitor, pentoxifylline, prevented the increase in lavage fluid neutrophil numbers, but accentuated the increase in total protein and albumin concentrations, and ALP, LD, myeloperoxidase, and elastase activities.

The effects of stressful exercise on leukocytes in cattle with experimental pneumonic pasteurellosis.

Seven yearling bulls were treated with stressful exercise and intrabronchial Pasteurella haemolytica A1. Group 1 bulls (nos. 1-4) underwent treadmill exercise and, 24 days later, intrabronchial instillation of P. haemolytica A1. Group 2 bulls (nos. 5-7) underwent treadmill exercise, followed 30 min later by intrabronchial P. haemolytica A1. Blood lactic acid values were raised (p less than 0.05) by treadmill exercise only, but plasma cortisol was raised (p less than 0.05) by treadmill exercise and by P. haemolytica A1 infection. Neutrophils in bronchoalveolar lavage (BAL) differed from control values 24 h after treadmill exercise, and 1 h and 4 h after P. haemolytica A1 infection. Respiratory disease was more severe and the gross lung lesions were larger in group 2 bulls than in group 1 bulls. P. haemolytica A1 was recovered from the livers, spleens and mesenteric lymph nodes of group 2 but not group 1 bulls, suggesting that group 2 bulls had experienced bacteraemia. Decreased neutrophils in BAL fluid from group 2 bulls at 1 h and 4 h after infection suggests that exercise transiently inhibited neutrophil egress from the blood to the alveoli; BAL neutrophils peaked at 1 h and 4 h after infection in group 1 bulls but declined at 24 h. We conclude that group 2 bulls were made more susceptible to experimental pneumonic pasteurellosis by stressful exercise.