Associations between exposure to viruses and bovine respiratory disease in Australian feedlot cattle.

Bovine respiratory disease (BRD) is the most important cause of clinical disease and death in feedlot cattle. Respiratory viral infections are key components in predisposing cattle to the development of this disease. To quantify the contribution of four viruses commonly associated with BRD, a case-control study was conducted nested within the National Bovine Respiratory Disease Initiative project population in Australian feedlot cattle. Effects of exposure to Bovine viral diarrhoea virus 1 (BVDV-1), Bovine herpesvirus 1 (BoHV-1), Bovine respiratory syncytial virus (BRSV) and Bovine parainfluenza virus 3 (BPIV-3), and to combinations of these viruses, were investigated. Based on weighted seroprevalences at induction (when animals were enrolled and initial samples collected), the percentages of the project population estimated to be seropositive were 24% for BoHV-1, 69% for BVDV-1, 89% for BRSV and 91% for BPIV-3. For each of the four viruses, seropositivity at induction was associated with reduced risk of BRD (OR: 0.6-0.9), and seroincrease from induction to second blood sampling (35-60 days after induction) was associated with increased risk of BRD (OR: 1.3-1.5). Compared to animals that were seropositive for all four viruses at induction, animals were at progressively increased risk with increasing number of viruses for which they were seronegative; those seronegative for all four viruses were at greatest risk (OR: 2.4). Animals that seroincreased for one or more viruses from induction to second blood sampling were at increased risk (OR: 1.4-2.1) of BRD compared to animals that did not seroincrease for any viruses. Collectively these results confirm that prior exposure to these viruses is protective while exposure at or after feedlot entry increases the risk of development of BRD in feedlots. However, the modest increases in risk associated with seroincrease for each virus separately, and the progressive increases in risk with multiple viral exposures highlights the importance of concurrent infections in the aetiology of the BRD complex. These findings indicate that, while efficacious vaccines could aid in the control of BRD, vaccination against one of these viruses would not have large effects on population BRD incidence but vaccination against multiple viruses would be expected to result in greater reductions in incidence. The findings also confirm the multifactorial nature of BRD development, and indicate that multifaceted approaches in addition to efficacious vaccines against viruses will be required for substantial reductions in BRD incidence.

Effects of exposure to Bovine viral diarrhoea virus 1 on risk of bovine respiratory disease in Australian feedlot cattle.

Viruses play a key role in the complex aetiology of bovine respiratory disease (BRD). Bovine viral diarrhoea virus 1 (BVDV-1) is widespread in Australia and has been shown to contribute to BRD occurrence. As part of a prospective longitudinal study on BRD, effects of exposure to BVDV-1 on risk of BRD in Australian feedlot cattle were investigated. A total of 35,160 animals were enrolled at induction (when animals were identified and characteristics recorded), held in feedlot pens with other cattle (cohorts) and monitored for occurrence of BRD over the first 50days following induction. Biological samples collected from all animals were tested to determine which animals were persistently infected (PI) with BVDV-1. Data obtained from the Australian National Livestock Identification System database were used to determine which groups of animals that were together at the farm of origin and at 28days prior to induction (and were enrolled in the study) contained a PI animal and hence to identify animals that had probably been exposed to a PI animal prior to induction. Multi-level Bayesian logistic regression models were fitted to estimate the effects of exposure to BVDV-1 on the risk of occurrence of BRD. Although only a total of 85 study animals (0.24%) were identified as being PI with BVDV-1, BVDV-1 was detected on quantitative polymerase chain reaction in 59% of cohorts. The PI animals were at moderately increased risk of BRD (OR 1.9; 95% credible interval 1.0-3.2). Exposure to BVDV-1 in the cohort was also associated with a moderately increased risk of BRD (OR 1.7; 95% credible interval 1.1-2.5) regardless of whether or not a PI animal was identified within the cohort. Additional analyses indicated that a single quantitative real-time PCR test is useful for distinguishing PI animals from transiently infected animals. The results of the study suggest that removal of PI animals and/or vaccination, both before feedlot entry, would reduce the impact of BVDV-1 on BRD risk in cattle in Australian feedlots. Economic assessment of these strategies under Australian conditions is required.

A Lucilia cuprina excretory-secretory protein inhibits the early phase of lymphocyte activation and subsequent proliferation.

The development of a protective immune response in sheep towards the presence of the larval stage of Lucilia cuprina has not been reported in the field. Upon investigation of the effects of larval excretory/secretory material on ovine T lymphocyte proliferation, we isolated a 56 kDa protein capable of inhibiting lymphocyte proliferation by at least 70%, compared with that in the presence of mitogen alone. This protein inhibited proliferation induced through cross-linking of the T-cell receptor as well as proliferation induced pharmacologically through the stimulation of the protein kinase C (PKC) pathway. The protein, named blowfly larval immunosuppressive protein (BLIP), was shown to bind directly to lymphocytes. Further investigation revealed that the BLIP prevented a proportion of lymphocytes from entering the first division following stimulation, by affecting the early events in lymphocyte activation. Subsequently, the BLIP reduced CD25 expression on T lymphocytes, reduced IL-2 mRNA expression, in addition to IFN-gamma, IL-4, IL-10 and IL-13 mRNA expression. Conversely, TNF-alpha and TGF-beta gene expression was up-regulated in response to the BLIP. These effects suggest suboptimal activation of T lymphocytes in the presence of the BLIP, and we propose that the BLIP presents an effective immune evasion tactic for the larvae of L. cuprina.

A study of autoantibodies to phosphatidyl-serine in Babesia bovis and Babesia bigemina infections in cattle.

Sera from cattle infected with Babesia bovis were found to contain antibodies to phosphatidyl-serine (PS), a negatively charged phospholipid normally found on the internal membrane of erythrocytes. In contrast, no autoantibodies were detected following Babesia bigemina infection indicating that the autoimmunity is not genus specific. During infection with Babesia bovis, PS translocates to the external membrane and it is suggested that this may result in PS behaving as an autoantigen owing to a transitional change. These autoantibodies may also play some role in the pathology of infection, especially the disturbed coagulation system associated with acute Babesia bovis infection.

The development of a recombinant Babesia vaccine.

Crude extracts of Babesia bovis parasites were shown to induce levels of protection in susceptible cattle equivalent to that resulting from natural infection. The crude material was systematically fractionated and tested in numerous sequential vaccination/challenge experiments in adult cattle. Antigens in protective fractions were then purified by affinity chromatography with monoclonal antibodies. Three highly protective (more than 95% reduction in parasitaemias) antigens were thus identified. None of these antigens was immunodominant; a number of immunodominant antigens were identified and all were immunosuppressive and/or non-protective. The three protective antigens were cloned and expressed as either beta-galactosidase or glutathione-S-transferase (GST) fusion proteins. Two of these, GST-12D3 and GST-11C5, when used in combination were almost as protective as has been previously shown for the commercially available live attenuated vaccine. A short fragment of a third antigen (21B4) has also been shown to be protective. In two of the antigens, repetitive segments have been shown to be non-protective while the third antigen (12D3) does not contain repetitive domains. Homologues of these antigens exist in other Babesia species and it is anticipated that these may be candidate antigens for protective vaccines against those species.

Babesia bovis: dextran sulphate as an adjuvant for and precipitant of protective immunogens.

Dextran sulphate, a chemical with some specificity for lipoproteins, was used to precipitate a fraction from a soluble extract of Babesia bovis-infected erythrocytes. The precipitate, in combination with dextran sulphate as an adjuvant, was used to vaccinate naive calves. The vaccinates and a group of control calves were challenged with virulent homologous B. bovis. The vaccinates showed delayed and decreased parasitaemias comparative to the controls. The antibody response to vaccination was primarily against the infected erythrocyte being of both IgG1 and IgG2 classes. We believe this is the first report of B. bovis antibody being detected in the IgG2 class. Lipase inhibition and chemical analysis suggested babesial lipid or lipoprotein was sufficiently immunogenic to produce serologically detectable antibody and presumably to elicit immunity.

Babesia bovis: immunity induced by vaccination with a lipid enriched fraction.

A chloroform extract from Babesia bovis-infected erythrocytes was used to vaccinate a group of five naive cattle. Following vaccination, the vaccinates, along with a group of control cattle, were challenged with a virulent heterologous strain of B. bovis. The vaccinates, comparative to the controls, showed delayed as well as decreased parasitaemias. The serological and initial biochemical studies suggested that the immune response was elicited by lipid of babesial origin.

Babesia bovis--effects of phospholipid translocation and adenosine tri-phosphate consumption.

The adenosine tri-phosphate concentration of Babesia bovis-infected erythrocytes was significantly (P less than 0.001) less than that of pre-infection erythrocytes. In addition, phosphatidyl serine was detected on the plasmatic surface of the infected erythrocyte. These two related findings could play important roles in the microvascular stasis characteristic of acute B. bovis infection.

Cardiorespiratory patterns during alarms in infants using apnea/bradycardia monitors.

Evaluating the significance of alarms at home in infants monitored for apnea/bradycardia depends on subjective parental observations. Retrospective analysis of 165 event recordings made during alarms in 90 monitored infants indicated that alarms were due to prolonged (greater than 15 s) apnea (6%), bradycardia (14%), shallow breathing (19%), mechanical malfunction (55%), or other causes (6%). Also, 68 infants had pneumograms. Of the 37 infants with an abnormal pneumogram, 14% had an abnormal event recording. Of the 31 infants with a normal pneumogram, 16% had an abnormal event recording. All monitors were discontinued without complication after a negative event recording. It may be concluded that (1) event recordings can document cardiorespiratory patterns during alarms, (2) the majority of alarms occurring at home are not significant, and (3) pneumograms do not appear to indicate which infants are at risk for a future significant alarm.

Babesia bovis: studies of parameters influencing microvascular stasis of infected erythrocytes.

Bovine erythrocytes infected with Babesia bovis were analysed for parameter changes known to influence rigidity and deformability of erythrocytes. Marked increases in malonyldialdehyde were detected indicating that lipid peroxidation occurs during infection. Consequently, increases in membrane lipid, methaemoglobin and membrane-bound haemoglobin were detected. Conversely, decreases in the antioxidant vitamin E and decreases in sialic acid were also detected. The cumulative effect of these changes would be to increase erythrocyte rigidity and decrease deformability thus contributing to the microvascular stasis characteristic of acute B bovis infection.

An enzyme linked immunosorbent assay to diagnose Babesia bovis infection in cattle.

The optimum gel filtration fraction from lysate of Babesia bovis infected erythrocytes was determined for use as an antigen in an ELISA to diagnose B. bovis infection in cattle. Of four enzyme labels tested, horseradish peroxidase was the most suitable. The assay is both sensitive and specific in detecting antibody for 2-4 years after a single infection. False positive reactions were obtained only with some sera from some Anaplasma marginale infected cattle.

Babesia bovis--analysis and vaccination trial with the cryoprecipitable immune complex.

Plasma samples from cattle recovering from acute Babesia bovis infection contain cryoprecipitable immune complexes (IC). Production of bovine and rabbit antisera to IC and subsequent serological assays indicated IC contained antigens of both babesial and erythrocytic origin. Vaccination of naive cattle with IC produced low titred antibody to B. bovis but the vaccinates did not survive challenge with a heterologous strain of B. bovis.

Babesia bovis: vaccination studies with three groups of high molecular weight antigens from lysate of infected erythrocytes.

A void volume fraction and fractions of mean sizes 800 kdalton and 300 kdalton were isolated by gel filtration from lysate of bovine erythrocytes infected with Babesia bovis. All fractions had good serological activity, as assayed by ELISA and IFA. Groups of four splenectomized calves were vaccinated with each fraction and then challenged, together with groups of four control calves, with a homologous strain of B. bovis. The group vaccinated with the 300 kdalton fraction showed some protection, as indicated by delayed and significantly lower parasitaemias and by a 75% survival in the group. In contrast, all animals in the relevant control group died from infection. No evident protection was obtained with the other two fractions.

Babesia bovis: vaccination trial with a dominant immunodiffusion antigen in splenectomised calves.

The dominant immunodiffusion antigen of Babesia bovis was prepared from the lysate of infected erythrocytes by cation exchange chromatography, gel filtration and preparative native acrylamide electrophoresis. It was seemingly free of other babesial antigens and tested as a vaccine. In vaccinated calves, compared to controls, there was a delay in parasitaemia and at times a statistically significant difference in parasite numbers. However, the vaccinates showed little difference in pathophysiological parameters or survival rates from the controls. It was concluded that serodominance cannot necessarily be correlated with protection.

Babesia bovis: vaccination of cattle against heterologous challenge with fractions of lysate from infected erythrocytes.

The distilled water lysate of erythrocytes infected with Babesia bovis was separated by gel filtration on Sephadex G200. The void volume fraction or a pool of retained fractions which had immunodiffusion activity were injected into two groups of five cattle. These were challenged 2 weeks after the final vaccination with a heterologous strain of B. bovis. A control group of five cattle was similarly challenged. Two of the five control animals died from the challenge, whereas none of the vaccinated animals died. There were significant differences in parasitaemia and pathophysiological parameters between the vaccinated groups and the control group.