Anticytotoxin activity of bovine sera and body fluids against Pasteurella haemolytica A1 cytotoxin.

Toxin neutralizing activity of bovine sera and body fluids against Pasteurella haemolytica type A1 cytotoxin was evaluated by 51Cr release assay using bovine peripheral blood mononuclear leukocytes as the target cells. Sera collected from precolostral calves did not exert anticytotoxin activity at 10(-1) or higher dilutions, whereas randomly selected complement fixing antibody-negative sera neutralized on average over 90% of cytotoxin activity at the 10(-1) dilution and less than 50% of the toxin activity at 10(-2) or higher serum dilutions. Nasal secretions and lung washings of some of the cattle tested also contained cytotoxin neutralizing activity. The antibody nature of the cytotoxin neutralizing activity was demonstrated by its neutralization with bovine immunoglobulin G2 purified from pooled seropositive sera. Sera from a group of cattle which were vaccinated with a potassium thiocyanate extract of P. haemolytica, but which subsequently developed fibrinous pneumonia after aerosol challenge with bovine herpesvirus 1 and P. haemolytica, had significantly lower anticytotoxin activity than sera from another group of cattle which did not develop the disease after similar vaccination and challenge. Cattle which survived a natural outbreak of shipping fever had higher anticytotoxin activity than those having fibrinous pneumonia in the aforementioned experimental group, although there was no statistical difference between them and a randomly selected CF seronegative group. It is probable that this cytotoxin neutralizing antibody exerts a beneficial effect in protection of cattle against pneumonic pasteurellosis.     

Examination of the role of Mycoplasma bovis in bovine pneumonia and a mathematical model for its evaluation

The authors screened 34 large cattle herds for the presence of Mycoplasma bovis infection by examining slaughtered cattle for macroscopic lung lesions, by culturing M. bovis from lung lesions and at the same time by testing sera for the presence of antibodies against M. bovis. Among the 595 cattle examined, 33.9% had pneumonic lesions, mycoplasmas were isolated from 59.9% of pneumonic lung samples, and 10.9% of sera from those animals contained antibodies to M. bovis. In 25.2% of the cases M. bovis was isolated from lungs with no macroscopic lesions. The proportion of seropositive herds was 64.7%. The average seropositivity rate of individuals was 11.3% but in certain herds it exceeded 50%. A probability model was developed for examining the relationship among the occurrence of pneumonia, the isolation of M. bovis from the lungs and the presence of M. bovis specific antibodies in sera.     

Induction of immunity against pneumonic pasteurellosis following experimental infection in calves.

Immunity against pneumonic pasteurellosis was studied in calves after recovery from experimental respiratory disease with Pasteurella haemolytica. Nine calves were exposed to aerosols of parainfluenza-3 virus and Pasteurella haemolytica A1 six days apart to produce respiratory disease. After recovery from the disease, these nine principal and four control calves were challenged with aerosols of bovine herpesvirus 1 and P. haemolytica A1 four days apart. With this viral-bacterial challenge, the nine principal animals failed to develop clinical responses to this bacterial challenge and their lungs did not show the growth of P. haemolytica on cultures, whereas two of four control calves had elevated temperatures and developed necropurulent pneumonia with the isolation of P. haemolytica from the lungs. The principal calves had developed high levels of cytotoxin neutralizing antibodies in their sera following parainfluenza-3 virus-P. haemolytica infection. This demonstrated that immunity against pneumonic pasteurellosis can be achieved, with a suggestion that further search for an effective vaccine for P. haemolytica is warranted.     

Competitive enzyme-linked immunosorbent assay for the detection of the antibodies specific to akabane virus.

A competitive enzyme-linked immunosorbent assay (C-ELISA) using neutralizing monoclonal antibodies (MAbs) against Akabane virus (AKAV) was developed to detect antibodies to AKAV in cattle sera. The performance of the test using 7 different competitor MAbs was evaluated in sequential serum samples and sera from cattle infected with various bovine arboviruses. The dynamics of the antibody response expressed by percentage of inhibition (PI) in C-ELISA coincided with those of neutralizing antibody titers in sequential serum samples from 2 cattle experimentally infected with AKAV. The value of PI in C-ELISA for convalescent sera from cattle infected with arboviruses correlated with the neutralizing antibody titer to AKAV but was unaffected by the antibodies to other arboviruses. In the validation experiment of C-ELISA using 286 bovine sera previously examined for the AKAV antibody by serum neutralization (SN) test, the relative specificity of C-ELISA was more than 98%, whereas the relative sensitivities of individual MAbs ranged from 49% to 82.2%. Overall agreement between C-ELISA and the SN test varied from 72% to 90% depending on the MAb. These results suggest that the C-ELISA is acceptable as a rapid and specific method for detecting antibodies to AKAV and is a potential alternative to the SN test.     

Bacteriological, serological, pathological and immunohistochemical studies of Mycoplasma bovis respiratory infection in veal calves and adult cattle at slaughter

Mycoplasma bovis is an important cause of calf pneumonia worldwide. In this study, we examined 140 cattle at slaughter comprising 70 veal calves and 70 beef cattle; 115 animals with pneumonic lesions and 25 without. Lung samples were submitted for bacteriological, histological, and M. bovis-immunohistochemical analyses. Serology for M. bovis was positive in 76% of beef cattle and 100% of veal calves. M. bovis was isolated only from veal calves in 16 out of 64 pneumonic cases. M. bovis was detected by immunohistochemistry in seven bacteriologically positive cases. M. bovis antigen was associated with bronchogenic necrosuppurative or fibrinonecrotizing lesions. Bacteriologically positive and immunohistochemical negative cases were associated with catarrhal bronchointerstitial pneumonia. Results suggest that M. bovis infection may develop into a severe necrosuppurative bronchopneumonia or fibrinonecrotizing pneumonia when associated with a high number of intralesional organisms or, conversely, into a mild catarrhal bronchointerstitial pneumonia when associated with a low number of organisms.     

Antibody response of horses to Rhodococcus equi antigens.

The antigens extracted from strains belonging to seven capsular serotypes of Rhodococcus equi, as well as from two wild strains isolated from pneumonic foals, were examined. Whole-cell antigens and soluble products present in broth culture supernatants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose, and stained with serum from hyperimmunized rabbits or foals. Foal sera used included sera from pneumonic animals with known titer to equi factors; from animals bled monthly on a farm with enzootic pneumonia, and from animals bled monthly on a farm with no history of R. equi pneumonia. The humoral response of foals to somatic antigen preparations was negligible, with few differences noted between sera from healthy, subclinically affected, and sick foals. The humoral response to R. equi broth culture supernatant products appeared more marked and was related to equi factor antibody titer. These findings suggest that the humoral response to R. equi whole-cell antigens is unimportant in protection against disease, which is consistent with the behavior of the organism as a facultative intracellular pathogen.     

Serum neutralization of cytotoxin from Pasteurella haemolytica, serotype 1 and resistance to experimental bovine pneumonic pasteurellosis

Sera from several groups of experimental calves were tested for cytotoxin neutralizing capacity. The relationship between this capability and resistance of the animals to an experimental challenge was examined. All undiluted bovine sera tested, other than fetal bovine serum, neutralized cytotoxin. Preadsorption of selected sera with formalin-killed P. haemolytica did not reduce their neutralizing capacity. Crude IgG fractions extracted from bovine sera retained neutralizing capacity as well. Cytotoxin neutralization titers were determined by serial dilution of sera from cattle which were previously unexposed, naturally exposed, or exposed by vaccination to the organism. Both live and killed vaccines were used. Prior exposure to live organisms resulted in the production of antibodies to both cell surface antigens and cytotoxin, whereas exposure to the killed vaccine resulted in the production of antibodies primarily to cell surface antigens. Resistance to experimental challenge with the organism correlated directly with serum cytotoxin neutralizing titers.     

An outbreak of acute pneumonia in young, single-suckled calves.

An outbreak of acute severe pneumonia which affected six to 14-week-old single-suckled calves, resulted in 45/77 requiring treatment. The examination of paired sera from all affected calves revealed that neither an adenovirus, non infectious bovine rhinotracheitis virus nor parainfluenza 3 virus was involved. The acute exudative interstitial pneumonia found at post mortem was typical of pneumonic pasteurellosis.     

The protective effect of vaccination against experimental pneumonia in cattle with Haemophilus somnus outer membrane antigens and interference by lipopolysaccharide.

A semi-purified outer membrane anionic antigen (AA) fraction was isolated from Haemophilus somnus by a modified procedure of anion exchange chromatography to yield a protein fraction free of lipopolysaccharides (LPS). The AA fraction (1 mg) was administered with or without the homologous lipopolysaccharide (10 micrograms/kg body weight) as vaccines to groups of cattle twice, three weeks apart. A control group which did not receive any antigen was included in the trial. Six weeks after the first vaccination, the animals were challenged intratracheally with a virulent pneumonic strain of H. somnus (70986) and observed for clinical signs of respiratory disease. The cattle were euthanized six days later and the lungs were evaluated for the severity of lesions macroscopically as well as histopathologically. Vaccination with AA alone provided the best protection against pneumonia as indicated by significantly lower clinical scores, less extensive gross lung lesions and mild histopathological lesions with immune cell infiltration. However, when AA was combined with LPS in the vaccination, this protective effect was negated and the animals showed more detrimental histopathological lesions than the controls.     

Efficacy of sulbactam-ampicillin in an induced Pasteurella haemolytica pneumonia model in calves

Therapeutic efficacy of sulbactam, a beta-lactamase inhibitor, in combination with ampicillin was evaluated in an ampicillin-resistant Pasteurella haemolytica pneumonia model in cattle, using an IV agar emboli method of infection. Groups of cattle given vehicle (group 1, n = 19) or ampicillin (group 2, n = 8) had 74% and 50% mortality, respectively, whereas group 3 (n = 11) given sulbactam-ampicillin had no mortality. Morbidities were 100% in groups 1 and 2 and 27% in group 3. Retrospectively, mortalities and morbidities were significantly (P less than 0.001) lower for group 3 given sulbactam-ampicillin when compared with those in groups 1 and 2 given vehicle or ampicillin, respectively. Evidence of embolic pneumonic pasteurellosis was observed histologically.     

Range of viral neutralizing activity and molecular specificity of antibodies induced in cattle by inactivated bovine viral diarrhea virus vaccines

The range of neutralizing activity to bovine viral diarrhea (BVD) virus and viral protein specificity of antibodies induced by 3 inactivated vaccines were evaluated by use of samples of sera obtained from 13 cattle 14 days after vaccination. Viral neutralizing antibodies wee detected in all cattle to each of 10 noncytopathic and 10 cytopathic isolates of BVD virus. A viral-induced polypeptide (53,000 to 56,000 daltons) was detected by radioimmunoprecipitation with serum from all vaccinates. Other viral-induced polypeptides of 115,000, 80,000, 48,000, and 25,000 daltons were precipitated with sera from some vaccinates. Precipitation of those polypeptides was related to the vaccine used. When multiple viral polypeptides were precipitated, the 53,000- to 56,000-dalton polypeptide appeared immunodominant.     

An outbreak of Pasteurella multocida pneumonia in lambs during a field trial of a vaccine against Pasteurella haemolytica

A vaccine against pneumonic pasteurellosis was evaluated for efficacy at two dilutions in lambs transported by sea from New Zealand to Saudi Arabia. The experimental vaccine was a killed Pasteurella haemolytica serotype A1 and A2 preparation. There was no evidence of either dilution of the vaccine leading to a lower pneumonia death or lesion rate than for the control group. However, bacteriological examinations to establish the causality of the pneumonia cases showed Pasteurella multocida to be the dominant organism, while P. haemolytica types A1 and/or A2 occurred at only a very low incidence.     

Naturally occurring Mycoplasma bovis-associated pneumonia and polyarthritis in feedlot beef calves.

Mycoplasma bovis is perceived as an emerging cause of mortality in feedlot beef cattle. This study examined the lesions and infectious agents in naturally occurring M. bovis-associated bronchopneumonia and arthritis and the relationship of this condition with bovine viral diarrhea virus (BVDV) infection. Standardized pathologic, immunohistochemical, and microbiologic investigations were conducted on 99 calves that died or were euthanized within 60 days after arrival in 72 feedlots. Cranioventral bronchopneumonia with multiple foci of caseous necrosis was identified in 54 of 99 calves, including 30 with concurrent fibrinosuppurative bronchopneumonia typical of pneumonic pasteurellosis. Mycoplasma bovis was consistently identified in these lesions by culture and immunohistochemistry, but also commonly in healthy lungs and those with pneumonia of other causes. Focal lesions of coagulation necrosis, typical of pneumonic pasteurellosis, were often infected with both Mannheimia haemolytica and M. bovis. Arthritis was present in 25 of 54 (46%) calves with M. bovis pneumonia, and all calves with arthritis had pneumonia. BVDV infection was more common in calves with lesions of bacterial pneumonia than in those dying of other causes, but BVDV infection was not more common in calves with caseonecrotic bronchopneumonia than those with fibrinosuppurative bronchopneumonia. Retrospective analysis identified cases of M. bovis pneumonia in the early 1980s that had milder lesions than the current cases. The findings suggest that, in at least some calves, M. bovis induces caseonecrotic bronchopneumonia within the lesions of pneumonic pasteurellosis.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of infectious bovine rhinotracheitis infection, with results of a preliminary survey

An indirect enzyme linked immunosorbent assay (ELISA) procedure was evaluated against the serum neutralisation test (SNT) for the detection of antibodies to infectious bovine rhinotracheitis virus (bovine herpesvirus type l), using 2028 sera from 166 dairy and 172 beef cattle herds. The results showed the ELISA to give high levels of agreement with the SNT in classifying positive and negative sera (98% and 97% respectively). Such disagreements as did occur involved weakly reactive sera with SNT titres of % or less. A number of sera (n=123) with trace neutralising activity of doubtful diagnostic significance were found to give marginal reactivity with ELISA. ELISA absorbance values were found to be highly correlated with SNT titres (r=0.909) on an overall basis, though agreements were lower with individual sera. The ELISA procedure was quicker, cheaper, and detected more reactors than the SNT. It also allowed results to be obtained with a number of sera which were unsuitable for testing by SNT because of their cytotoxic nature. Analysis of ELISA results showed reactors to be present in 57% of tested sera, representing 81% of cattle herds. Reactor rates for sera and herds in the South Island, (37% and 58%), were significantly lower than for those in the North Island (64% and 88%). Antibody prevalence was also found to be significantly lower in districts having a low annual rainfall (<850 mm), and to be lower in beef cattle than in dairy cattle. A surprising exception to the latter occurred in low rainfall districts, where dairy cattle showed significantly lower reactor rates than local beef animals.     

Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of infectious bovine rhinotracheitis viral antibody in cattle.

An enzyme-linked immunosorbent assay was developed to detect bovine serum antibody to infectious bovine rhinotracheitis virus. The specificity of this assay in 304 bovine sera, collected from an infectious bovine rhinotracheitis virus-free herd, was 100%; in sera from 62 cattle inoculated with an intranasal vaccine, its diagnostic sensitivity was 27.4% at one month and 100% at six months, postvaccination. In 303 bovine sera with standard serum neutralizing antibody titers of greater than or equal to 1:2 it showed 100% sensitivity; and in 463 random diagnostic samples, comparative tests indicated that enzyme-linked immunosorbent assay detected more seropositive animals (61.6%) than the standard serum neutralizing test (49.9%). The enzyme-linked immunosorbent assay method was considered to be technically superior as a routine diagnostic test for the detection of infectious bovine rhinotracheitis viral antibody in bovine sera.     

Atypical Interstitial Pneumonia in Cattle

Atypical interstitial pneumonia is described as two clinical syndromes in young cattle. One syndrome occurs in animals which have clinical evidence of pneumonic pasteurellosis, responds initially to treatment for one to two days and then develops acute signs of atypical interstitial pneumonia. The second syndrome involves acute respiratory distress in young calves due to atypical interstitial pneumonia with antecedents of enzootic pneumonia. The postmortem lesions are described along with discussion of the possible pathogenesis of the condition and treatment.     

A field evaluation of the efficacy of two vaccines against ovine pneumonic pasteurellosis

Two vaccines against pneumonic pasteurellosis were evaluated for efficacy in lambs transported by sea from New Zealand to Saudi Arabia. One vaccine contained whole cell antigens of Pasteurella haemolytica A2 grown under iron restricted conditions. The other contained Pasteurella haemolytica A1 cell surface and leucotoxin antigens. There was no clear evidence of either vaccine leading to a lower pneumonia death or lesion rate than for the control group.     

Kinetics of neutralization of two strains of Myxovirus Parainfluenza 3 in the presence of complement and anti-gammaglobulin-serum.

Using the kinetics of neutralization, it was established that two strains of Myxovirus Parainfluenza 3 of cattle and sheep origin which are immunologicaly identical have a different sensitivity to early neutralizing antibodies of reconvalescent calf sera. Complement-dependent neutralizing antibodies, supplied by adding 5% guinea pig serum to the calf serum, appear earlier than the usual neutralizing antibodies. Goat anti-cattle gammaglobulin serum decreases the neutralizing activity of early reconvalescent serum and release some of the neutralizing virus from the virus-antibody complex. It is believed that the lability of the virus-antibody complex depends not only on the activity of the antibody, but also on some properties of the strains. The addition of complement stabilizes this complex. The previous addition of adenovirus antigen to bivalent cattle serum against Myxovirus Parainfluenza 3 and cattle adenovirus, decreases the complement-dependent neutralizing antibody of the serum against Myxovirus Parainfluenza 3. It is suggested that this is the possible mechanism for the mutual activation of mixed virus infections.     

Mannheimia haemolytica and the pathogenesis of enzootic bronchopneumonia

Mannheimia (M.) haemolytica (formerly Pasteurella [P.] haemolytica) is the primary aetiological agent of pneumonic pasteurellosis--one of the most important respiratory diseases in cattle and sheep. While bovine pneumonic pasteurellosis is regarded to be mainly caused by M. haemolytica serotype A1, and in Germany during the last years also by serotype A6, sheep can be infected by all serotypes although there is an increased prevalence of serotypes A2 and A5-7. The obligate pathogenicity of M. haemolytica is proven by isolation of pure cultures from pneumonic lungs as well as by infection studies. Knowledge about the virulence mechanisms of M. haemolytica and their molecular basis are fragmentary, most probably due to the complex gene regulation of virulence associated factors in lung tissues. This review summarizes the current literature covering virulence factors to substantiate a model of pathogenesis. After serotype A1 strains have colonized the bovine upper respiratory tract they replace other serotypes by mechanisms unknown to date. After fulminant proliferation in the upper respiratory tract the microorganisms colonize the lower respiratory tract, finally entering alveolar spaces. An inflammatory cascade is initiated by M. haemolytica LPS and Leukotoxin, causing activation of the complement system and release of cytokines. Pathognomonic for bovine pneumonic pasteurellosis is the strong influx of neutrophiles accompanied by accumulation of fibrin, finally causing necrosis of alveolar spaces. Depending on lesion size this fibronecrotizing pneumonia can result in death of the animals. In addition, possible protective antigens are discussed. There is still a great effort in the development of efficacious vaccines against pneumonic pasteurellosis in cattle and sheep caused by various M. haemolytica serotypes worldwide. The scarce knowledge concerning presence and distribution of virulence associated factors in M. haemolytica strains and their role in pathogenesis made it difficult to determine a suitable vaccine candidate in the past. In addition, there is lack of knowledge concerning the variability of virulence factors in individual isolates. Genome sequence analysis of M. haemolytica, enabling proteomics and transciptomics, hopefully will give new insight into the pathogenesis of pneumonic pasteurellosis.     

Morphological study of bacterial pneumonia of feedlot cattle: Determination of age of lesions

Lungs from 48 feedlot cattle that had died from bacterial pneumonia were examined grossly and microscopically. Criteria based on microscopic lesions were adopted to age these pneumonias. In 38 cases, pneumonic lesions were of relatively uniform age throughout the affected tissue. In eight other cases, the presence of older lesions confined to one or two lobes suggested a previous episode of pneumonia. The aging criteria adopted were in agreement with the duration of the observed clinical signs in 26 cases. In 13 other cases, the pneumonia was estimated to be of longer duration than suggested by the history, whereas in the remaining nine cases, it was estimated to be more recent. Areas of tan discoloration of the parenchyma surrounded by white or yellow borders were considered the best areas to examine microscopically since they offered the best chances of revealing necrosis and fibrosis, the main lesions used to age the pneumonia.