Pulmonary expression of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-8 in the acute phase of bovine pneumonic pasteurellosis.

Inflammatory cytokines are suspected to contribute to the pathogenesis of bovine pneumonic pasteurellosis (BPP) through neutrophil recruitment, leukocyte activation, and the induction of a broad array of soluble inflammatory mediators. An in vivo experimental model of BPP was used to characterize the pulmonary expression kinetics of tumor necrosis factor alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) genes and proteins during the acute phase of disease development. Cytokine expression in bronchoalveolar lavage (BAL) fluid, BAL cells, and pneumonic lung parenchyma was quantitated by northern blot analysis, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization at 2, 4, 8, 16, and 24 hours after endobronchial inoculation of Pasteurella (Mannheimia) haemolytica. Expression of TNFalpha, IL-1beta, and IL-8 was significantly increased in the airways and lung lesions of infected calves as compared with mock-infected controls. Although kinetic patterns varied, peak levels of cytokine mRNA occured within 8 hours postinfection (PI), and peak cytokine concentrations occurred within 16 hours PI. In all samples, IL-8 was expressed to the greatest extent and TNFalpha was least expressed. Expression of TNFalpha was restricted to alveolar macrophages. Alveolar and interstitial macrophages produced IL-1beta and IL-8 in the first 4 hours; bronchial and bronchiolar epithelial cells were also significant sources of IL-8 during this period. By 8 hours PI, neutrophils were the dominant source of both IL-1beta and IL-8. These findings demonstrate a spatial and temporal association between pulmonary expression of inflammatory cytokines and acute lung pathology, supporting the hypothesis that cytokines contribute to inflammatory lung injury in BPP.

Serotyping of Mannheimia (Pasteurella) haemolytica isolates from the upper Midwest United States.

Mannheimia (Pasteurella) haemolytica biotype A serotype1 (A1) is the primary bacterial agent responsible for the clinical signs and pathophysiologic events in bovine pneumonic pasteurellosis. The goal of this study was to determine the prevalence of other serotypes of M. haemolytica biotype A organisms obtained from the upper Midwest diagnostic laboratories. A total of 147 M. haemolytica isolates were collected from Minnesota, South Dakota, and Michigan. Isolates were tested against M. haemolytica antisera obtained from the National Animal Disease Center, Ames, Iowa. Results indicated that M. haemolytica serotype 1 represented approximately 60%, serotype 6 represented 26%, and serotype 2 represented 7% of the total examined isolates. In addition, 7% of the isolates were serotype 9, 11, or untypable. This finding suggests that M. haemolytica serotypes other than serotype 1 can be isolated from the lung lesions of diseased cattle and seem to be capable of causing the pathologic changes observed in the lung with pneumonic pasteurellosis.

Ehrlichia equi infection of horses from Minnesota and Wisconsin: detection of seroconversion and acute disease investigation.

Equine granulocytic ehrlichiosis (EGE) is caused by infection with Ehrlichia equi. EGE has been reported primarily in northern California, where E equi is transmitted by the tick Ixodes pacificus. Reports of EGE and the emergence of human granulocytic ehrlichia in Minnesota prompted a seroprevalence study of E equi in horses of Minnesota and Wisconsin. Tick (Ixodes scapularis) endemic areas of Minnesota and Wisconsin were compared to nonendemic regions of Minnesota. Indirect fluorescent antibody was used to detect the presence of serum antibodies to E equi. Serum samples from healthy horses, 375 samples from I scapularis endemic counties, and 366 samples from nonendemic counties were screened at a 1:40 dilution. Results demonstrated a seroprevalence of 17.6% in endemic areas versus 3.8% in nonendemic areas. Ehrlichial DNA from 2 samples was successfully amplified by polymerase chain reaction and 919 base pairs were sequenced. The DNA sequence of 1 Minnesota/Wisconsin strain differed from the GenBank strain (M73223) of E equi at positions 84 and 886 and from the MRK strain of E equi at position 84, and was identical to the human granulocytic ehrlichiosis (HGE) agent. The 2nd Minnesota/Wisconsin strain was identical to the 1st with the exception of a substitution of "A" at position 453 that is not present in E phagocytophila, E equi, or HGE agent strain sequences. Based on the results of this study, we concluded that E equi is present and causes infection in horses in Minnesota and Wisconsin. The occurrence of infection is higher in tick endemic regions.

Use of repetitive sequence-based polymerase chain reaction for molecular epidemiologic analysis of Streptococcus equi subspecies equi.

OBJECTIVE: To determine whether repetitive sequence-based polymerase chain reaction (rep-PCR) could be used to differentiate Streptococcus equi isolates, to examine S equi isolates from throughout the world, and to determine whether a horse had > 1 subtype of S equi during an outbreak of disease. SAMPLE POPULATION: An initial group of 32 S equi isolates, 63 S equi isolates from various geographic areas, and 17 S equi isolates obtained during outbreaks of disease. PROCEDURE: An aliquot of S equi genomic DNA was amplified, using enterobacterial repetitive intergenic consensus primers. Gel electrophoresis was performed on 1.5% agarose gels, and a computed-assisted program was used to compare rep-PCR results. RESULTS: Use of these primers to analyze 100 ng of S equi genomic DNA resulted in patterns of 6 to 14 bands. The 32 initial isolates were separated into 7 rep-PCR subtypes. There were 30 rep-PCR subtypes found among 29 S equi isolates obtained from Minnesota, Michigan, Canada, and Australia and 34 S equi isolates obtained from Kentucky and other sources. Furthermore, the same clone was identified in several horses during an outbreak of disease. Infected horses on the same farm all had a single clone of S equi. CONCLUSION AND CLINICAL RELEVANCE: Analysis of these results suggests that rep-PCR is useful for delineating S equi into rep-PCR subtypes. Results revealed that isolates with the same geographic source or similar date of collection did not always have the same rep-PCR subtype. A single clone of S equi usually predominated during an outbreak of disease.

Use of bovine somatotropin at the time of surgery for left displacement of the abomasum in dairy cows. Minnesota Dairy Practitioners Study Group.

OBJECTIVE: To evaluate the effect of a single dose of recombinant bovine somatotropin (bST) on certain metabolic values, health, and milk production of dairy cows undergoing surgery for left displacement of the abomasum. DESIGN: Blinded clinical trial. ANIMALS: 413 cows with left displacement of the abomasum. PROCEDURE: A single 500-mg dose of bST was administered to dairy cows following surgery in field practice conditions for left displacement of the abomasum. A placebo of the same carrier without bST was administered to control cows in this blinded study. Metabolic and production responses in a short-term follow-up period were measured. RESULTS: Blood glucose concentrations in cows 3 to 5 days after surgery were statistically higher for treated cows than for control cows. A higher proportion of treated cows had improved urine ketone test results than did controls. Significant differences in other metabolic values, health, and milk production were not detected. CLINICAL IMPLICATIONS: Treatment of metabolically compromised cows with bST may have some positive effects, but further investigation is needed to confirm therapeutic value.

Effect of intravenous calcium administration on gentamicin-induced nephrotoxicosis in ponies.

OBJECTIVE: To determine whether supplemental i.v. calcium administration would attenuate or prevent gentamicin-induced acute renal failure, defined as an increase in serum creatinine concentration > or = 50% above baseline. ANIMALS: 10 healthy pony mares. PROCEDURE: Pony mares were randomly assigned to receive calcium at a dosage of 20 mg/kg of body weight or saline solution i.v., twice daily for 14 days. All pony mares received gentamicin at a dosage of 20 mg/kg i.v. every 8 hours for 14 days. Gentamicin pharmacokinetic, serum biochemical, and urinalysis data were measured every other day for the 14-day study period. Renal histologic examination was performed, and results were scored at the end of the 14-day period. RESULTS: 4 of 5 mares not receiving calcium supplementation developed acute renal failure. Only 1 of the 5 mares receiving calcium supplementation developed acute renal failure. Over the course of the study, pony mares receiving calcium supplementation had significantly fewer changes in urinalysis variables, and significantly less microscopic renal damage. CONCLUSION: Daily i.v. administration of calcium attenuated gentamicin-induced acute renal failure. CLINICAL RELEVANCE: Calcium supplementation may help diminish the risk of acute renal failure associated with aminoglycoside antibiotics.

Construction of an isogenic leukotoxin deletion mutant of Pasteurella haemolytica serotype 1: characterization and virulence.

Allelic replacement was used to generate two isogenic lktA deletion mutants of Pasteurella haemolytica serotype 1 that were incapable of synthesizing leukotoxin (Lkt). Southern blot data confirmed that lktA sequences were absent in the two P. haemolytica deletion mutants. Culture supernatants and whole cell lysates from the wild type P. haemolytica, D153 parent strain, but not the lktA deletion mutants, contained immunoreactive and bioactive leukotoxic protein. In addition, only the parent strain was haemolytic when grown on bovine and sheep blood agar plates. Virulence of the lktA deletion mutant, lktA 77, was compared with the parent in an experimentally infected calf model of pneumonic pasteurellosis. Results revealed significant reduction in virulence in the lktA mutant as measured by clinical and lung lesion scores. Notable differences in histological changes such as markedly reduced necrosis and lack of leukocyte degeneration occurred in calves infected with the lktA mutant in comparison with those infected with the parent wild-type strain. Thus, it appears that leukotoxin plays a important role in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.

Dairy calf pneumonia. The disease and its impact.

The author discusses the syndrome of respiratory, disease in dairy calves, reviewing the disease and the causative agents. Attention is given to the epidemiology of the disease with discussion of morbidity, mortality, proportionate mortality and risk factors associated with dairy calf pneumonia. The economic impact of dairy calf pneumonia is discussed in detail and management options for calf rearing are suggested.

Acquired B lymphocyte deficiency and chronic enterocolitis in a 3-year-old quarter horse.

This case report describes a 3-year-old American Quarter Horse with acquired immunodeficiency. Clinical signs included chronic diarrhea due to Salmonella typhimurium and bacterial pneumonia. Characterization of the immunodeficiency involved in vivo phytohemagglutinin (PHA) intradermal testing, in vitro lymphocyte proliferation in response to concanavalin A, immunofluorescence flow cytometry data on blood lymphocytes, serum protein electrophoresis and immunoglobulin (Ig) quantification. A diagnosis of B lymphocyte deficiency with resulting deficiencies in serum IgG, IgA and IgM and a concurrent decrease in T cell function was made based on these tests. Postmortem examination revealed no evidence of lymphosarcoma. This case represents a variation of young adult-onset B cell deficiency not previously described in the literature.

Evaluation of efficacy of three commercial vaccines against experimental bovine pneumonic pasteurellosis.

The objective of this study was to evaluate the efficacy of three commercial vaccines against experimental pneumonic pasteurellosis in cattle. The three vaccines were: (a) One Shot (SmithKline Beecham, West Chester, PA.), (b) Presponse (Langford Laboratories, Guelph, Ontario) and (c) Once PMH (BioCor, Omaha, NE.). Protective immunity was evaluated in terms of lower clinical and pneumonic lesion scores after endobronchial challenge with virulent P. haemolytica. The results indicate that One Shot elicited antibodies against leukotoxin (Lkt), capsular poly-saccharide (CP) and surface antigens (SA), while Presponse and Once PMH elicited antibodies against CP and SA. There was significant correlation between lung and serum antibody levels against Lkt (P < 0.0001), CP (P < or = 0.0001) and IROMPs (P < or = 0.035). Animals that received the One Shot had significantly (P < or = 0.05) lower mean pneumonic lesion score (36.6 +/- 10.97) as compared to the control group (48.6 +/- 25.92). A significant negative correlation (-0.41; P < or = 0.008) existed between serum antibody levels against Lkt and pneumonic lesion score. High serum antibodies against SA did not correlate with reduction in pneumonic lesion score. In addition, high antibody levels against CP did not correlate consistently with reduced pneumonic lesion scores. The results from this study demonstrates that commercial vaccines evaluated in this trial did not confer optimal protection in vaccinated calves, against experimental pneumonic pasteurellosis. However, One Shot vaccinates showed a better protective immunity compared to the other two vaccine groups.

Induction of nitric oxide production by bovine alveolar macrophages in response to Pasteurella haemolytica A1.

We assessed the kinetics of inducible nitric oxide synthase (iNOS) mRNA expression and production of nitric oxide (NO) in bovine alveolar macrophages (AMs) stimulated with purified lipopolysaccharide (LPS) from Pasteurella haemolytica strain 12296. The effect of LPS on iNOS gene expression was dose-dependent and was expressed maximally at 24 h after stimulation with 10 micrograms/ml of LPS. Production of NO measured as secreted nitrite in supernatants took place in a time and dose-dependent manner with peak production at 24 h after LPS stimulation. Recombinant bovine gamma interferon (rb gamma IFN) augmented the LPS-induced iNOS gene expression and production of NO. The ability of LPS to induce iNOS gene expression and NO production either alone or in combination with rb gamma IFN was significantly abrogated by polymyxin B. In addition, the iNOS inhibitor NG-monomethyl-Larginine (L-NMMA) significantly inhibited LPS and rb gamma IFN + LPS induced NO production. Our results also demonstrated that NO produced from an exogenous NO donor sodium nitroprusside (SNP), and NO generated from LPS-stimulated AMs (endogenous) caused cytotoxic injury to bovine pulmonary artery endothelial cells in a dose-dependent manner. The cytotoxic injury caused by NO generated from LPS stimulated AMs was inhibited by polymyxin B or L-NMMA. There was a markedly increased concentration of nitrite in the lung lavage fluids of calves following P. haemolytica infection. These findings support a role for NO in the pathogenesis of lung injury in bovine 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).

Evaluation of three experimental subunit vaccines against pneumonic pasteurellosis in cattle.

The objective of this study was to evaluate the efficacy of three Pasteurella haemolytica A1 derived experimental subunit vaccines against pneumonic pasteurellosis in cattle. The three vaccines were: (a) culture supernatant (CS) containing leukotoxin (Lkt), lipopolysaccharide (LPS) and capsular polysaccharide (CP); (b) sodium salicylate extract (SSE) containing iron regulated outer membrane proteins (IROMPs), LPS and CP; (c) and a combination of the above two. Vaccine efficacy was defined in terms of reduction in clinical and pneumonic lesion scores after intrapulmonic challenge with live P. haemolytica. The results indicate that the CS vaccine elicited antibodies against both Lkt and CP, while the SSE vaccine elicited antibodies against IROMPs and CP. Animals inoculated with the combination vaccine showed increased levels of antibodies against IROMPs, Lkt and CP. There was significant correlation between lung and serum antibodies against Lkt, CP and IROMPs. Animals that received the combination vaccine had significantly lower mean pneumonic lung score as compared to SSE and control groups. The animals which received CS vaccine had mean pneumonic lung score significantly lower than that of control group. A strong negative correlation existed between serum antibody levels against Lkt, IROMPs, CP and pneumonic lung scores. The results from this study demonstrate the usefulness of CS vaccine alone or in combination with SSE vaccine in bringing about optimal protection in vaccinated calves, against experimental pneumonic pasteurellosis.

Increased tumor necrosis factor-alpha and interleukin-1 beta expression in the lungs of calves with experimental pneumonic pasteurellosis.

We used a well characterized pneumonic pasteurellosis model in calves to determine whether increased proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) expression and secretion were associated with pneumonic lesions. Bronchoalveolar lavage fluids, lavage cells consisting of alveolar macrophages and neutrophils with degenerative changes, and lung tissues were analyzed for the presence of TNF-alpha and IL-1 beta approximately 48 h following endobronchial inoculation of logarithmic phase Pasteurella haemolytica 12296 organisms. Levels of TNF-alpha and IL-1 beta mRNA were significantly increased in lavage cells of P. haemolytica-infected animals but not in cells from phosphate buffered saline (PBS) inoculated controls based on in situ hybridization analysis. Significantly increased levels of TNF-alpha, and IL-1 beta mRNA were also expressed within the pneumonic lesions from P. haemolytica-infected calves. In contrast, lung tissues from PBS-inoculated control calves had cytokine mRNAs expressed at extremely low levels. Increased levels of bioactive IL-1 and immunoreactive (not bioactive) TNF-alpha were found in lavage fluids from P. haemolytica-infected calves compared with lavage fluids from PBS-inoculated calves. These findings indicate that the proinflammatory cytokines TNF-alpha and IL-1, may be associated with pathogenesis of lung injury in bovine pneumonic pasteurellosis.

Aneurysm of the cranial mesenteric artery in a cow.

Exploratory laparotomy of an adult dairy cow, examined because of acute signs of persistent abdominal pain, revealed a firm pulsatile mass with associated fremitus just distal to the origin of the cranial mesenteric artery. The cow died acutely 2.5 days after surgery. A dilated, thin-walled, sacculated aneurysm, which had ruptured, was located along the proximal portion of the cranial mesenteric artery. It was postulated that the aneurysm developed secondary to structural defects in the arterial wall, but caused no clinical signs until enlargement and local tissue stretching or circulatory disturbances caused intestinal ischemia, resulting in abdominal pain. Aneurysms of visceral arteries in cattle should be considered as another differential diagnosis for signs of abdominal pain after more common causes such as severe bloat, mesenteric root volvulus, intussusception, cecal dilatation/volvulus, and uterine torsion have been excluded.

Purified Pasteurella haemolytica leukotoxin induces expression of inflammatory cytokines from bovine alveolar macrophages.

We obtained biologically active purified leukotoxin (Lkt) from Pasteurella haemolytica serotypel, strain 12296 using preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three species of Lkt of molecular masses 95, 100, and 104 kDa were obtained. Purity of all three species of Lkt was confirmed by analytical SDS-PAGE and Western blot (immunoblot) analysis. Results from the chromogenic Limulus amebocyte lysate assay and silver staining of SDS-PAGE patterns indicated that the preparations were free of contaminating lipopolysaccharide. We then studied the kinetics of TNF alpha and IL-1 beta mRNA expression in bovine alveolar macrophages stimulated with the purified 104 kDa Lkt. Subcytolytic concentrations of Lkt induced TNF alpha and IL-1 beta gene expression and peak induction was observed at a concentration of 1 leukotoxin unit/ml. Both TNF alpha and IL-1 beta mRNA expression were detectable at 1 h after stimulation with 1 leukotoxin unit/ml. The expression peaked at 2 h, steadily declining up to 6 h, and was undetectable by 10 h. Secreted TNF alpha measured by bioassay peaked at 4-6 h and accumulated at a lesser concentration after 6 h. By contrast, secreted IL-1 peaked at 6 h and decreased significantly by 10 h. The ability of purified Lkt to induce TNF alpha and IL-1 beta gene expression and secretion of bioactive proteins was suppressed by Ca2+ chelating agents, 5 mM EDTA and 5 mM EGTA, but not polymyxin B. Heat-inactivation of the purified Lkt that had lost its cytocidal property completely abrogated induction of TNF alpha and IL-1 beta gene expression and secretion in bovine AMs.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of inflammatory cytokines in bovine alveolar macrophages following stimulation with Pasteurella haemolytica lipopolysaccharide.

Bovine tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) cDNAs were generated by reverse transcription and then by PCR amplification from lipopolysaccharide (LPS)-stimulated alveolar macrophage RNA. The amplified cDNAs were cloned into pPow and expressed in Escherichia coli DH5 alpha. The expressed proteins were confirmed as TNF-alpha and IL-1 beta by Western blot (immunoblot) analysis and bioassays. We then used the cloned genes as probes in Northern (RNA) blots and investigated the kinetics of TNF-alpha and IL-1 beta mRNA expression in bovine alveolar macrophages stimulated with purified LPS from Pasteurella haemolytica 12296. The effect of LPS on TNF-alpha and IL-1 beta gene expression was dose dependent, and induction was observed at a concentration of 0.01 microgram/ml. Both TNF-alpha and IL-1 beta mRNA expression were detectable within 0.5 h after stimulation with 1 microgram of LPS per ml, peaked at 1 to 2 h, steadily declined up to 16 h, and were undetectable by 24 h. Secreted TNF-alpha measured by bioassay peaked at 4 h and accumulated at a lesser concentration in conditioned medium throughout the 24 h. By contrast, secreted IL-1 beta was induced at 8 h and reached a maximal concentration at 24 h after stimulation. The ability of LPS to induce TNF-alpha and IL-1 beta gene expression and secretion of bioactive proteins were suppressed by polymyxin B. Our findings support a role for LPS from P. haemolytica in the induction of inflammatory cytokines in bovine pneumonic pasteurellosis.

Interaction of bovine respiratory syncytial virus with bovine alveolar macrophages in vivo: effects of virus infection upon selected cell functions.

The effect of bovine respiratory syncytial virus (BRSV) upon alveolar macrophage (AM) function was investigated using an in vivo calf inoculation model. Alveolar macrophages were collected sequentially from live calves at multiple time points during the 14 day period following viral inoculation. Alveolar macrophages from bronchoalveolar lavage fluids were purified by density gradient centrifugation (> 95% AM) prior to in vitro evaluation of cell functions. There were significant but variable and inconsistent differences in the functions of AM from the BRSV inoculated calves compared to the control calves. Fc-receptor mediated phagocytosis was either increased or unchanged by BRSV inoculation. Nonopsonized phagocytosis was decreased during the early postinoculation period and later increased. There was a variable effect on AM phagosome lysosome fusion with increased fusion activity on postinoculation days 2 through 5, 7 and 12 but reduced activity on days 6 and 10. The AM respiratory burst, as measured by nitroblue tetrazolium dye reduction, was essentially unaffected with a reduction in activity on day 10 only. In this model, BRSV inoculation of calves primarily resulted in an alteration of the membrane associated phagocytic functions of the alveolar macrophages (p < 0.05).