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Acute phase proteins such as serum amyloid A, haptoglobin, and alpha 1-acid glycoprotein have been identified as markers of inflammation in cattle because they are produced by the liver in response to pro-inflammatory cytokines. This study was designed to assess whether they could be used to discriminate between acute and chronic inflammation. Their concentrations were measured in serum samples from 81 cattle in which inflammation was classified by thorough clinical examination, supported by postmortem findings, as being acute in severity in 31 and chronic in 50. The classical haematological markers of inflammation were also determined in blood from the animals. Serum amyloid A had a maximum (100 per cent) clinical sensitivity in discriminating between the acute and chronic cases, and haptoglobin had the highest clinical specificity of 76 per cent; counts of neutrophils and band neutrophils had sensitivities of 71 per cent and 42 per cent and specificities of 30 per cent and 72 per cent, respectively. It was concluded that serum amyloid A and haptoglobin may be used to discriminate between acute and chronic inflammatory conditions.

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Pasteurella haemolytica isolates from cattle and sheep, including representatives of different serotypes and untypable strains, were examined for leukotoxin (Lkt) production at the end of the log phase of growth in brain heart infusion broth. There were marked differences in leukotoxic activity in culture supernate samples, as measured by chemiluminescence-inhibition assays with bovine and ovine neutrophils, even between strains of the same serotype. There was also some variation in the amount and mol. wt of the Lkt protein produced by different strains, as judged by SDS-PAGE, immunoblotting and ELISA. Some strains produced normal amounts of Lkt protein which had only low leukotoxic activity. Most strains produced Lkt of 105 kDa whereas four strains produced a higher mol. wt form of c. 108 kDa, including two of the five serotype A2 strains examined. Thus, the P. haemolytica isolates showed considerable heterogeneity in terms of leukotoxin production, mol. wt and activity, even within a given serotype.

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Twenty-three isolates of Haemophilus somnus were typed by repetitive extragenic palindromic (REP) element-based PCR, enterobacterial repetitive intergenic consensus (ERIC)-based PCR, and PCR ribotyping. A total of 11 types were distinguished by REP-PCR, 13 types were distinguished by ERIC-PCR, and 5 types were distinguished by PCR ribotyping. PCR ribotyping produced a relatively simple pattern and a small number of distinct types, whereas REP- and ERIC-PCR both produced more complex banding patterns but increased the discrimination between strains. Clearly distinguishable profiles were obtained for respiratory and genital isolates of H. somnus by all three typing methods. The results suggest that a combination of all three primer sets provides a high-resolution fingerprinting method for epidemiological studies of H. somnus and for its differentiation from related species.

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Lipopolysaccharide binding protein (LBP) is a liver-derived acute phase protein which is implicated in modulating the host responses to lipopolysaccharide (LPS) from Gram-negative bacteria. LBP interacts with circulatory LPS to form complexes which bind to the CD14 receptor or cells of the monocytic lineage and neutrophils resulting in their activation. This causes the release of mediators and cytokines which are responsible for initiating the acute phase response. LBP-like activity has now been identified in bovine serum and in this study LBP has been purified from acute phase bovine serum using ion exchange chromatography. On sodium dodecyl sulphate polyacrylamide electrophoresis, bovine LBP demonstrated a single band with a molecular mass of 58 kDa. Bovine LBP enhanced the binding of LPS to human monocytes while enzymatic removal of the CD14 receptor abrogated this interaction. Furthermore, bovine LBP increased the sensitivity of monocytes to LPS by at least 100-fold. Depletion of LBP by means of antibodies to bovine LBP inhibited the serum mediated LPS binding to monocytes. Antibodies to rabbit LBP or recombinant human LBP did not cross-react with bovine LBP. Studies on the kinetics of LBP activity in calves during the acute phase response demonstrated a four-fold increase in the serum concentration 36 h after a single intratracheal inoculation of Pasteurella haemolytica A1. The findings of this study indicate that cattle possess a LPS detection mechanism comparable to that described in man and experimental animals in which LBP forms complexes in serum with circulatory LPS enhancing the signal to the immune system to mount a host response. The isolation of LBP will allow further investigations into LBP-mediated responses to LPS in cattle.

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Prevalences of Cryptosporidium parvum oocysts in faeces and of isotype-specific anti-C. parvum antibodies in serum of apparently healthy adult cattle on two farms were determined. On Farm 1 cryptosporidial diarrhoea had been recorded in more than 80% of calves born over the previous 5 years, whereas on Farm 2 cryptosporidiosis had never been reported. No differences were demonstrated in oocyst excretion or presence of antibodies between the two farms. C. parvum oocysts were detected in 62.4% of faecal smears collected from a total of 553 apparently healthy adult cattle. Sucrose flotation was performed on a proportion of the faecal samples. This proved a more sensitive technique, detecting oocysts in 92% of the samples tested, and highlighting the insensitivity of direct smears for detecting oocysts. More than 90% of the cattle had specific anti-C. parvum IgG, IgG1, IgG2 and IgM antibodies and 58% specific anti-C. parvum IgA antibodies. Results suggest that asymptomatic adults may play an important role in the epidemiology of cryptosporidiosis in calves.

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Outer-membrane protein (OMP) profiles of two serotype A1 isolates of Pasteurella haemolytica were compared by SDS-PAGE and Western blotting with bovine convalescent serum after growth (a) in vitro under iron-sufficient and -deficient conditions, (b) in vivo in the lungs of experimentally infected calves and (c) in vivo in diffusion chambers implanted into the peritoneal cavities of calves. Lung-grown bacteria differed from iron-sufficient in vitro-grown bacteria in having enhanced expression of the previously recognized 71, 77 and 100 kDa iron-regulated proteins, reduced expression of 18, 31, 39.5 and 50 kDa proteins, and expression of a 19 kDa protein. Differences were also apparent in the Western blot profiles of OMPs of in vitro- and lung-grown bacteria. These included the apparent lack of recognition of the 100 kDa protein in the lung-grown bacteria, but not in the in vitro-grown bacteria, and more intense staining of a 47 kDa protein in in vitro-grown bacteria, but not in lung-grown bacteria. The OMP profiles of the chamber-grown bacteria resembled those of the lung-grown bacteria in that expression of the 18, 19, 31 and 39.5 kDa proteins was similar. These similarities demonstrated that the chamber-grown bacteria had adapted to the in vivo environment, and that growth conditions within the chambers resembled, but not perfectly, those within the lungs. For example, expression of the three iron-regulated OMPs was very low in the chamber-grown bacteria compared to the lung-grown bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

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The concentrations of tumour necrosis factor-alpha (TNF alpha), serum amyloid A (SAA) and haptoglobin were determined in serum samples taken from four calves in the 10 hours after their intra-tracheal inoculation with Pasteurella haemolytica serotype A1. The concentration of haptoglobin did not increase but the concentration of SAA rose progressively from within two hours of inoculation. The concentration of TNF alpha reached a peak in all the animals two hours after inoculation but had returned to undetectable levels after a further four hours. TNF alpha is likely to be an important mediator of the acute phase response in cattle and SAA is a more rapid bovine acute phase protein than haptoglobin in its response to infection with P haemolytica.

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An intraperitoneal implant chamber was developed for the study of the in vivo growth of Pasteurella haemolytica in calves. The chamber had a volume of approximately 100 ml and featured an external sampling port which allowed multiple and sequential sampling of the chamber contents. A single polycarbonate diffusion membrane with a pore size of 0.22 micron allowed host peritoneal fluid to diffuse into the chamber and maintained the bacterial population free of white blood cells. Chambers were implanted into the peritoneal cavities of four five-month-old dairy-cross calves, demonstrated to be sero-negative by indirect haemagglutination assay. Three days later, four different P. haemolytica isolates, of serotypes A1 or A2, were inoculated into the chambers. In all cases, there was a slow decline in the viable bacterial numbers within the chambers. Western blot analysis of the antibody content of the chamber fluids revealed IgG antibodies to P. haemolytica OMPs in the fluid prior to inoculation and both 9 and 15 days after inoculation. Furthermore, there was no significant change in the IgG antibody content of the chamber fluid, either quantitatively or qualitatively, during the course of the experiment. Analysis of the bactericidal activity of pre-inoculation chamber fluid against the corresponding bacterial isolate suggested that an antibody-dependent complement-mediated process was not responsible for the decline in bacterial numbers. Overall, the chamber design was demonstrated to be extremely effective for in vivo studies of P. haemolytica in calves, allowing easy and regular sampling of the chamber contents and maintaining bacteria free of white blood cells. Although there was a slow decline in bacterial numbers over time, sufficient numbers of cells could be obtained for analysis of cell-surface antigens.

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The outer-membrane protein (OMP) and lipopolysaccharide (LPS) profiles of 29 isolates of Pasteurella haemolytica serotypes A1 (18 isolates) and A2 (11 isolates), obtained from pneumonic (13 isolates) or healthy (16 isolates) cattle, were compared by SDS-PAGE and Western blot analysis. Coomassie-blue-stained OMP profiles of serotype A1 and A2 isolates could be distinguished from each other by differences in both major and minor proteins. Whereas the OMP profiles of the serotype A1 isolates were extremely uniform in stained gels, there was variation in the mobilities of high-molecular-mass minor proteins and one of the major proteins of serotype A2 isolates. Differences in the OMP profiles of isolates within both the A1 and A2 serotypes were more clearly distinguished by Western blotting than by staining after SDS-PAGE. Thus, by Western blot analysis, four distinct OMP profiles were identified within the serotype A1 and A2 isolates, respectively. The profiles of the serotype A1 isolates were designated OMP types 1.1, 1.2, 1.3 and 1.4; those of the serotype A2 isolates were designated OMP types 2.1, 2.2, 2.3 and 2.4. Three distinct LPS profiles were recognized among the isolates which, by comparison with previously described LPS types, were identified as smooth LPS type 1 and rough LPS types 3 and 5. Isolates of serotype A1 consisted of LPS type 1 only. whereas isolates of serotype A2 consisted of LPS types 3 or 5. OMP and LPS analysis of P. haemolytica has applications in epidemiological and virulence studies.

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The optimal conditions for the detection of outer membrane proteins (OMPs) and lipopolysaccharide (LPS) of Pasteurella haemolytica by immunoblotting were evaluated. The variables examined included the equilibration time of the gels before transfer, composition of the transfer buffer, type of blotting membrane, blocking agent, effect of the zwitterionic detergent Empigen BB on protein renaturation, and the development reagent. The composition of the transfer buffer and time of gel equilibration significantly affected the efficiency of transfer of both OMPs and LPS. However, the optimal conditions for the transfer of OMPs were not the same as those for LPS. Thus, optimal transfer of OMPs occurred in Tris-glycine buffer, with prior equilibration of the gels to allow for expansion, whereas optimal transfer of LPS was achieved in Tris-glycine-methanol buffer with no equilibration of the gels. In Tris-glycine-methanol buffer, gel equilibration resulted in a significantly reduced transfer of both OMPs and LPS, probably due to the removal of SDS from these components. The use of Zeta-Probe blotting membrane which, unlike nitrocellulose, does not require methanol for optimal protein binding, did not result in improved binding of OMPs or LPS in the absence of methanol and, even after prolonged blocking (> 2 h), gave higher background staining than did nitrocellulose. Effective blocking of nitrocellulose was achieved with 3% (w/v) gelatin, 2.5% (w/v) skimmed milk or 0.3% (v/v) Tween 20, whereas increased background staining occurred with 1% (w/v) bovine serum albumin or 1% (w/v) ovalbumin. The incorporation of Empigen BB in the primary antibody buffer did not improve antibody recognition of proteins as a result of their renaturation. For the horseradish-peroxidase enzyme development system, the substrate 3,3'-diaminobenzidine tetrahydrochloride was more sensitive, and developed more quickly, than 4-chloro-1-naphthol, but faded more rapidly after drying of the membrane. 4-chloro-1-naphthol was more suitable for identifying OMPs because less background staining occurred, whereas 3,3'-diaminobenzidine tetrahydrochloride was more suitable for the detection of LPS due to its greater sensitivity.

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Bovine serum amyloid-A (b-SAA) was purified from a pool of acute phase serum using hydrophobic interaction chromatography and gel filtration. Serum was applied at a low salt concentration to a phenyl-sepharose column and SAA was eluted with a gradient of 0 to 6 M guanidine-HCl. Fractions containing SAA were pooled, concentrated and further purified by gel filtration on Superose-12. The concentration of SAA in bovine serum was quantified by an indirect ELISA using rabbit anti-human SAA and horseradish peroxidase conjugated donkey anti-rabbit IgG. Dilutions of an acute phase bovine serum sample were used as working standards. The SAA concentration of this standard was determined by comparison with purified b-SAA on SDS-polyacrylamide gel electrophoresis followed by densitometry at 590 nm. The assay detection limit was 3 micrograms ml-1; the intra-assay coefficient of variation was 4 per cent and interassay coefficients of variation were 5.5 per cent and 7.2 per cent at 66 and 178 micrograms ml-1 SAA, respectively. In calves experimentally infected with Pasteurella haemolytica type A1 the ELISA was able to detect a 10-fold increase of SAA within 24 hours of inoculation.

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Lipopolysaccharide (LPS) from 40 isolates of Pasteurella haemolytica, comprising 23 serotype A1, seven serotype A2, one serotype T4, one serotype T10 and eight untypable isolates, obtained from diseased and healthy cattle or sheep, was characterized by SDS-PAGE and Western blotting. Ten different SDS-PAGE LPS profiles, five smooth and five rough, were identified among the biotype A and untypable isolates and designated LPS types 1-10. LPS types 1 and 2 were smooth, had similar O-antigen banding-patterns but differed in the low-molecular-mass or core-oligosaccharide regions; type 3 LPS was rough but had a core-oligosaccharide region similar to that of LPS type 1. No similarities were observed between these LPS types and types 6, 7 and 9, which were smooth, and types 4, 5, 8 and 10, which were rough. Most serotype A1 isolates (19/23) were of LPS type 1, whereas two isolates each had LPS of types 2 and 3. The majority (5/7) of serotype A2 isolates possessed type 3 LPS, whereas the remaining two isolates each had LPS of types 4 and 5. There was much greater heterogeneity within the untypable group of isolates, which comprised LPS of types 1 and 9 (two isolates each), and 6, 7, 8 or 10 (one isolate each). Western blotting analysis demonstrated that LPS types 1 and 2 had immunologically identical O-antigen side-chains but differed in their core-oligosaccharide regions, whereas the core-oligosaccharide region of rough LPS type 3 was immunologically very similar to that of LPS type 1. The other LPS types were immunologically unrelated to these three LPS types. The majority (20/23) of serotype A1 isolates originated from cattle and possessed LPS types 1 or 2, different from most (5/7) of the serotype A2 isolates which originated from sheep and possessed LPS of types 3 or 4. However, two of the three ovine serotype A1 isolates had the same type 3 LPS as occurred in most of the ovine serotype A2 isolates, suggesting a possible correlation between LPS type and host specificity. This study has demonstrated that LPS diversity within different serotypes of P. haemolytica is greater than was previously thought and that certain LPS types might be host-specific.

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Growth characteristics, as well as outer-membrane protein (OMP) and lipopolysaccharide (LPS) profiles in SDS-polyacrylamide gels, of two serotype A1 isolates of Pasteurella haemolytica were examined under different in vitro growth conditions. The two isolates were chosen as representatives of disease (S/C 82/1) and non-disease (W/D 83/4) isolates, respectively. The growth rates and final cell densities of both isolates increased as the degree of aeration increased. In particular, the final cell densities varied significantly according to the degree of aeration. Under anaerobic conditions, however, both the growth rate and final cell density were significantly reduced. There was reduced expression of a 40.5 kDa protein under anaerobic conditions in both isolates, whereas in S/C 82/1 expression of the 71, 77 and 100 kDa iron-regulated proteins increased as aeration decreased. There were also differences in low-molecular-mass components of LPS between cells grown anaerobically and those grown aerobically. Growth in the presence of 5% CO2 did not significantly alter the growth rate and had little, if any, affect on OMPs or LPS. Differences in the expression of certain proteins occurred as growth progressed from the exponential to the stationary phase. Growth in the presence of the iron chelators 2,2'-dipyridyl, ethylenediamine-dihydroxyphenylacetic acid (EDDA), desferrioxamine mesylate (desferal), ovotransferrin (conalbumin) and bovine transferrin was inhibited within a very narrow concentration range. In the presence of 2,2'-dipyridyl, EDDA or desferal, 71 and 100 kDa iron-regulated OMPs increased in both isolates whereas a 77 kDa protein increased in isolate S/C 82/1 only. In the presence of ovotransferrin or bovine transferrin there was, in both isolates, increased expression of the 71 kDa protein, a slight increase in expression of the 100 kDa protein but no expression of the 77 kDa protein; there was also increased production of the 40.5 kDa protein, and synthesis of two additional proteins of 23 and 26 kDa. Other differences occurred after growth in foetal and newborn calf sera. In foetal calf serum there was enhanced expression of the 71 but not of the 100 kDa protein. In newborn calf serum there was no enhanced expression of the 71, 77 or 100 kDa proteins, but expression of novel proteins of 97 and 98 kDa as well as a high-molecular-mass protein occurred. There was also slight quantitative differences in the LPS profiles of cells grown in foetal or newborn calf sera compared to those of cells grown in other media.(ABSTRACT TRUNCATED AT 400 WORDS)

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Five dairy herds with a high incidence of clinical mastitis were investigated. Two of these herds had a high incidence of mastitis caused by Staphylococcus aureus yet had a relatively low rolling mean cell count. The mean cell counts of samples from clinical cases submitted by the dairymen from these two farms were significantly lower than the counts of similar samples submitted from the other farms. It is suggested that a possible explanation of the low cell count on these two farms was the proficiency of the dairy-men in detecting clinical mastitis.

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Distribution of Pasteurella haemolytica in the respiratory tracts of calves with no apparent clinical signs of illness and those infected experimentally with Dictyocaulus viviparus was determined so as to define carrier sites for this organism. The calves had been positive by nasopharyngeal swab for either P haemolytica A2 or A1 for at least two months or for over a month, respectively, before slaughter. P haemolytica A1 was acquired following horizontal spread from other infected calves. It was observed post mortem that P haemolytica A1 or A2 resided in the tonsils and retropharyngeal lymph nodes of calves of both groups. In addition to these sites, P haemolytica A1 was also isolated from the right cranial lung lobe of one of the calves from the D viviparus infected group although there was no evidence of pasteurella associated pneumonia. It was concluded that tonsil and retropharyngeal lymph nodes appear to be the most important carrier sites for P haemolytica when compared to other tissues of the bovine respiratory tract.

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