Fasciola hepatica infection reduces Mycobacterium bovis burden and mycobacterial uptake and suppresses the pro-inflammatory response.

Bovine tuberculosis (BTB), caused by Mycobacterium bovis, has an annual incidence in cattle of 0.5% in the Republic of Ireland and 4.7% in the UK, despite long-standing eradication programmes being in place. Failure to achieve complete eradication is multifactorial, but the limitations of diagnostic tests are significant complicating factors. Previously, we have demonstrated that Fasciola hepatica infection, highly prevalent in these areas, induced reduced sensitivity of the standard diagnostic tests for BTB in animals co-infected with F. hepatica and M. bovis. This was accompanied by a reduced M. bovis-specific Th1 immune response. We hypothesized that these changes in co-infected animals would be accompanied by enhanced growth of M. bovis. However, we show here that mycobacterial burden in cattle is reduced in animals co-infected with F. hepatica. Furthermore, we demonstrate a lower mycobacterial recovery and uptake in blood monocyte-derived macrophages (MDM) from F. hepatica-infected cattle which is associated with suppression of pro-inflammatory cytokines and a switch to alternative activation of macrophages. However, the cell surface expression of TLR2 and CD14 in MDM from F. hepatica-infected cattle is increased. These findings reflecting the bystander effect of helminth-induced downregulation of pro-inflammatory responses provide insights to understand host-pathogen interactions in co-infection.

Evaluating the application of the dual path platform VetTB test for badgers (Meles meles) in the test and vaccinate or remove (TVR) wildlife research intervention project in Northern Ireland.

European badgers (Meles meles) are accepted as a wildlife reservoir host for Mycobacterium bovis, which causes bovine tuberculosis (bTB) in the British Isles. The objective of this study was to evaluate the use of Dual Path Platform (DPP) VetTB test (Chembio Diagnostic Systems Inc., Medford, NY, USA) within a Test and Vaccinate or Remove (TVR) wildlife research intervention project. Blood samples were collected from 456 individual badgers, trapped in 2015 and 2016, and tested in the field with DPP VetTB test using whole blood. Additionally, whole blood and serum samples were taken to the laboratory for further DPP VetTB testing and for gamma interferon (IFN-γ) testing. Swabs were taken from the oropharynx and trachea and submitted for bacteriological culture as were swabs from wounds, if present. Field DPP VetTB test positive badgers were euthanised and underwent post-mortem examination and bTB confirmatory testing. The results demonstrated that the test performed as well in the field using whole blood as DPP Vet TB tests in the laboratory using sera or whole blood, and as well as other established tests for M. bovis. Visual assessment of the DPP VetTB test using serum under laboratory conditions showed a high degree of consistency between raters. Using a relative gold standard (parallel interpretation of IFN-γ assay and oropharyngeal/tracheal sample/culture), sensitivity estimates for the DPP VetTB test using sera and whole blood were 0.5 (95%CI 0.34-0.66) and 0.42 (95%CI 0.24-0.66), respectively. Specificity estimates were 0.95 (95%CI 0.93-0.97) for sera and 0.89 (95%CI 0.86-0.92) for whole blood. Parallel interpretation of Band 1 (MPB83) and Band 2 (CFP-10/ESAT-6) of the DPP VetTB test was not superior to interpretation of Band 1 only. The results give confidence in the reliability and reproducibility of the DPP VetTB test for badgers under field conditions and therefore it is considered appropriate for use in a badger bTB control campaign.

Modelling the variation in skin-test tuberculin reactions, post-mortem lesion counts and case pathology in tuberculosis-exposed cattle: Effects of animal characteristics, histories and co-infection.

Correctly identifying bovine tuberculosis (bTB) in cattle remains a significant problem in endemic countries. We hypothesized that animal characteristics (sex, age, breed), histories (herd effects, testing, movement) and potential exposure to other pathogens (co-infection; BVDV, liver fluke and Mycobacterium avium reactors) could significantly impact the immune responsiveness detected at skin testing and the variation in post-mortem pathology (confirmation) in bTB-exposed cattle. Three model suites were developed using a retrospective observational data set of 5,698 cattle culled during herd breakdowns in Northern Ireland. A linear regression model suggested that antemortem tuberculin reaction size (difference in purified protein derivative avium [PPDa] and bovine [PPDb] reactions) was significantly positively associated with post-mortem maximum lesion size and the number of lesions found. This indicated that reaction size could be considered a predictor of both the extent (number of lesions/tissues) and the pathological progression of infection (maximum lesion size). Tuberculin reaction size was related to age class, and younger animals (<2.85 years) displayed larger reaction sizes than older animals. Tuberculin reaction size was also associated with breed and animal movement and increased with the time between the penultimate and disclosing tests. A negative binomial random-effects model indicated a significant increase in lesion counts for animals with M. avium reactions (PPDb-PPDa < 0) relative to non-reactors (PPDb-PPDa = 0). Lesion counts were significantly increased in animals with previous positive severe interpretation skin-test results. Animals with increased movement histories, young animals and non-dairy breed animals also had significantly increased lesion counts. Animals from herds that had BVDV-positive cattle had significantly lower lesion counts than animals from herds without evidence of BVDV infection. Restricting the data set to only animals with a bTB visible lesion at slaughter (n = 2471), an ordinal regression model indicated that liver fluke-infected animals disclosed smaller lesions, relative to liver fluke-negative animals, and larger lesions were disclosed in animals with increased movement histories.

Bayesian latent class estimation of sensitivity and specificity parameters of diagnostic tests for bovine tuberculosis in chronically infected herds in Northern Ireland.

In the European Union, the recommended ante-mortem diagnostic methods for bovine tuberculosis (bTB) include the single intradermal cervical comparative tuberculin (SICCT) test and the interferon-gamma (IFN-γ) test as an ancillary test. The SICCT test has a moderate sensitivity (Se) and high specificity (Sp), while the IFN-γ test has good Se, but a lower Sp than the SICCT test. A retrospective Bayesian latent class analysis was conducted on 71,185 cattle from 806 herds chronically infected with bTB distributed across Northern Ireland (NI) to estimate the Se and Sp of the common ante-mortem tests and meat inspection. Analyses were also performed on data stratified by farming type and herd location to explore possible differences in test performance given the heterogeneity in the population. The mean estimates in chronically infected herds were: (1) 'standard' SICCT: Se 40.5-57.7%, Sp 96.3-99.7%; (2) 'severe' SICCT: Se 49.0%-60.6%, Sp 94.4-99.4%; (3) IFN-γ(bovine-avian) using a NI optical density (OD) cut-off difference of 0.05: IFN-γ(B-A)NI: Se 85.8-93.0%, Sp 75.6-96.2%; (4) IFN-γ(bovine-avian) using a standard 'commercial' OD cut-off difference of 0.1: IFN-γ(B-A)0.1: Se 83.1-92.1%, Sp 83.1-97.3%; and (5) meat inspection: Se 49.0-57.1% Se, Sp 99.1-100%. Se estimates were lower in cattle from dairy farms than from beef farms. There were no notable differences in estimates by location of herds. Certain population characteristics, such as production type, might influence the ability of bTB tests to disclose truly infected cases.

Experimental exposure of cattle to a precise aerosolised challenge of Mycobacterium bovis: a novel model to study bovine tuberculosis.

Non-aerosol models of bovine tuberculosis are limited in reproducibility and relevance to natural cases seen in farmed animals. Therefore, there is a need for aerosol models of infection in cattle that can reproduce bovine tuberculosis as seen in natural cases of the disease. This manuscript describes a cattle tuberculosis model based on the inhalation of a precisely defined dose of Mycobacterium bovis in aerosol form, and defines those sites of M. bovis deposition following aerosol inhalation. The dissemination of bacilli and the resultant pathological change following infection is also described. Cattle aged 4-5 months, were infected with approximately 10(4) colony forming units (CFU), using a Madison chamber that had been modified to deliver aerosols to calves. In Experiment 1, calves were examined for gross pathology at post mortem (PM) examination at 93 and 132 days post-infection (PI), respectively. In Experiment 2, pairs of calves were examined for gross pathology at PM examination at 1 day PI and 7 days PI, respectively. At PM examination, samples were taken for bacteriology. Retrospective counts showed that the calves inhaled between 3 x 10(4) and 8 x 10(4)CFU of M. bovis. In Experiment 1, pathology indicative of tuberculosis and detection of M. bovis by qualitative bacteriology was found throughout the lower respiratory tract (LRT). In Experiment 2, pathology was only observed in a single site of one calf at day 7 PI. Samples positive for M. bovis by bacteriology were predominantly in the LRT. The numbers of M. bovis CFU recovered and the distributions of positive sites were greater at day 7 PI than day 1 PI. This study describes an aerosol exposure method that can deliver a defined dose of M. bovis almost exclusively to the LRT. The distribution of M. bovis and lesions indicative of tuberculosis suggests this aerosol method replicates the primary mode of tuberculosis transmission in cattle.

Evaluation Considerations for Secondary Uses of Clinical Data: Principles for an Evidence-based Approach to Policy and Implementation of Secondary Analysis.

Objectives: To set the scientific context and then suggest principles for an evidence-based approach to secondary uses of clinical data, covering both evaluation of the secondary uses of data and evaluation of health systems and services based upon secondary uses of data. Method: Working Group review of selected literature and policy approaches. Results: We present important considerations in the evaluation of secondary uses of clinical data from the angles of governance and trust, theory, semantics, and policy. We make the case for a multi-level and multi-factorial approach to the evaluation of secondary uses of clinical data and describe a methodological framework for best practice. We emphasise the importance of evaluating the governance of secondary uses of health data in maintaining trust, which is essential for such uses. We also offer examples of the re-use of routine health data to demonstrate how it can support evaluation of clinical performance and optimize health IT system design. Conclusions: Great expectations are resting upon "Big Data" and innovative analytics. However, to build and maintain public trust, improve data reliability, and assure the validity of analytic inferences, there must be independent and transparent evaluation. A mature and evidence-based approach needs not merely data science, but must be guided by the broader concerns of applied health informatics.

Pathogenesis of bovine tuberculosis: the role of experimental models of infection.

In many countries, test-and-slaughter policies based on tuberculin skin testing have made a significant impact on the control of bovine tuberculosis (caused by infection with Mycobacterium bovis). However, in some countries these policies have not proved as effective and improved disease control strategies are required (including improved diagnostic tests and development of vaccines). The host pathogen interactions in bovine tuberculosis are very complex. While studies of the disease in naturally infected field cases of bovine tuberculosis have provided valuable information, detailed knowledge can also be gained through studies of disease models. A number of studies have developed M. bovis infection models employing a range of routes and challenge doses. An early objective was assessment of vaccine efficiency, and models of infection remain central to current work in this area. Development of the intra-nasal and intra-tracheal models have also advanced our understanding of the kinetics of the immune response. In many of these studies, understanding of pathogenesis has been improved by definition of the cells that respond to infection and those that are instrumental in modulation of host responses. Experimental models of infection have been adapted to study cattle to cattle transmission, modeling one of the fundamental routes of infection. This review provides a historical perspective on the types of experimental models used in over 100 years of research and outlines new opportunities to refine those methods for bovine and human tuberculosis and to contribute to improved diagnostics, advanced understanding of immunology and vaccine design.

Immune responses in bovine tuberculosis: towards new strategies for the diagnosis and control of disease.

In several countries, bovine tuberculosis (caused by infection with Mycobacterium bovis) is a major economic problem with the potential to be a significant public health risk. Where traditional test-and-slaughter policies based on skin testing with tuberculin have not been fully successful, new tools including additional diagnostic tests and improved vaccines are required urgently. This paper considers how recent developments in knowledge of immune responses and mycobacterial antigens can be used in the logical development of more efficient strategies for the identification of infected cattle.

Shedding of Mycobacterium bovis in the nasal mucus of cattle infected experimentally with tuberculosis by the intranasal and intratracheal routes.

Four groups of six calves were infected experimentally with either a low dose of approximately 10(4) colony-forming units (cfu) or a high dose of approximately 10(6) cfu of Mycobacterium bovis. Each dose was delivered by the intranasal and intratracheal routes. More severe disease was observed in the groups inoculated with the high dose. Visible lesions were identified in 21 of the 24 animals, all of which also gave positive skin tests and interferon-gamma (IFN-gamma) responses. Nasal shedding was detected in 15 of the 24 animals and the frequency of shedding was influenced by both the route and the dose of infection; no shedding was observed in the group infected intratracheally with the low dose. Two of the 15 confirmed shedders had no visible lesions at postmortem examination; both of these calves gave IFN-gamma responses but only one was skin test positive.

Risk factors for visible lesions or positive laboratory tests in bovine tuberculosis reactor cattle in Northern Ireland.

An observational case-control study was conducted to investigate risk factors for confirmed bovine tuberculosis (bTB) infection in cattle reacting positively to the single intradermal comparative cervical test (SICCT) in Northern Ireland in the years 1998, 2002 and 2006. Macroscopic lesions were detected at slaughter (positive visible lesion (VL) status) in 43.0% of reactor cattle, whilst 45.3% of those sampled were confirmed as bTB positive due to the presence of lesions or positive histopathology/mycobacterial culture (positive bTB status). In 97.5% of the reactors, the VL status and bTB status were either both negative or both positive. Generalized linear mixed model analyses were conducted on data of 24,923 reactor cattle with the variables herd identifier, local veterinary office (DVO) and abattoir being used as random effects within all the models generated at univariable and multivariable level. The other variables within the dataset were used as fixed effects. Significant risk factors associated with VL status and bTB status at multivariable level (p<0.05) included age at death, breed, sex, test year, net increase in skin thickness at bovine tuberculin injection site, epidemiological status of skin test, total number of reactors at the disclosure test, mean herd size and prior response to the skin test. These risk factors are likely related to the time since infection, the strength of the challenge of infection and the susceptibility of the animal. These findings are important as the detection of visible lesions and the confirmation of bTB are an integral part of the overall bTB control programme in Northern Ireland and the veterinary meat inspection and hygiene programme. The visible lesion status and bTB status of an animal can affect the way in which bTB breakdowns are managed, since failure to detect visible lesions and recovery of Mycobacterium bovis can lead to a less stringent follow-up after other risk factors have been taken into account.

Development of a sensitive and specific time resolved fluorimetric immunoassay for the bovine acute phase protein haptoglobin (Hp).

Haptoglobin (Hp) is recognised as a major acute phase protein in the bovidae and its presence in serum is used as an indicator of inflammation. A mouse monoclonal antibody (1D9) specific for bovine Hp was labelled with a lanthanide (Eu) chelate and used to develop a competitive immunoassay. This competitive immunoassay allowed direct measurement of Hp in serum and was validated for intra- and interassay coefficients of variation (below 8%). Cross-reactivity with other serum proteins was measured (less than 0.1%) and limits of detection for Hp in serum were established for adult male (0.344 microgram/ml) and adult female cattle (1.589 micrograms/ml). The immunoassay was compared with an established haptoglobin-haemoglobin binding assay.

Immune responses in bovine tuberculosis.

Knowledge of the immune responses which develop in cattle following infection with Mycobacterium bovis is essential both to the understanding of disease pathogenesis and to the logical development of immune-dependent tools, such as diagnostic tests and vaccines, which can be used to combat the disease. Studies of field cases of bovine tuberculosis (TB) and of experimental bovine models of M. bovis infection have indicated that cell-mediated immune responses (CMI) predominate within a spectrum of immunity which exists. This paper reviews aspects of recent research and indicates how knowledge of T-cell antigenic targets in bovine TB along with increasing knowledge of T-cell subpopulations and their interactions with M. bovis -infected macrophages provides opportunities for the development of better methods for disease control.

IgG isotype antibody responses to epitopes of the Mycobacterium bovis protein MPB70 in immunised and in tuberculin skin test-reactor cattle.

Serological assays may have merit in identifying animals in advanced stages of bovine tuberculosis, but most tests have had sub-optimal sensitivities and specificities. The Mycobacterium bovis protein MPB70 has been identified as a B-cell target with diagnostic potential in measurement of pre- and post-skin-test antibody responses. One observation, which has potential practical application, has been that skin testing with tuberculin boosts IgG(1) anti-MPB70 antibody responses in cattle with tuberculous lesions. However, serological cross-reactivities with bacteria, such as Nocardia asteroides, have been described for this protein. With the aim of identifying candidate reagents for improved diagnostic tests, this study investigated IgG isotype antibody responses to MPB70 at the epitope level and, because of the previous findings, focused on IgG(1) responses following skin testing. Screening of a panel of overlapping synthetic peptides using sera from cattle immunised with MPB70 and cattle infected with M. bovis showed that two regions of the protein (residues 21-70 and 101-120) contain dominant B-cell epitopes. No individual epitope appeared to be selectively recognised by one isotype of IgG antibody. Investigation of IgG(1) responses showed that recognition of the epitope within residues 51-70 was boosted strongly by tuberculin injections in skin-test positive cattle and that this memory response was generally a feature of cattle which were found to have macroscopic, tuberculous lesions.

An antigen capture ELISA test using monoclonal antibodies for the detection of Mycoplasma californicum in milk.

The use of monoclonal antibodies to detect Mycoplasma californicum was investigated in an antigen capture microtitre format. The finalized test was highly specific and no cross-reactions were detectable with any of the mastitis associated mycoplasma or bacterial antigens tested. Using a concentration step involving centrifugation, the sensitivity of the test could be improved from 10(5)-10(7) to 10(3)-10(5) colony forming units per ml with pure broth cultures, and from 10(7) to at least 10(6) colony forming units per ml in milk samples from two experimentally infected cows. The antigen detected was partially identified by immunoblotting, which demonstrated two polypeptides of 40 and 46 kD.

Production, preliminary characterisation and applications of monoclonal antibodies to porcine circovirus.

The preparation of monoclonal antibodies (mAbs) to porcine circovirus is described. Preliminary characterisation was carried out on nine mAbs obtained from two fusions and included isotyping, virus neutralisation assays and indirect immunofluorescence staining patterns obtained following immunostaining of both a porcine circovirus (PCV)-persistently infected pig kidney (PK/15/W) and Vero (Vero-PCV) cell line. Significant differences in the staining patterns were observed in both cell lines which appeared to be dependent on the subculture status of the Vero-PCV cultures. The development of a mAb-based antigen capture enzyme linked immunosorbent assay (ELISA) as an aid to virus purification is also described. The use of mAbs for the detection of PCV antigen in cryostat sections from a pig experimentally infected with the virus leading to identification of the sites of replication of PCV is also reported.

The role of WC1(+) gamma delta T-cells in the delayed-type hypersensitivity (DTH) skin-test reaction of Mycobacterium bovis-infected cattle.

Delayed-type hypersensitivity (DTH) skin-testing with mycobacterial antigens is often used as a means of identifying Mycobacterium bovis-infected cattle. Better understanding of the cellular basis underlying the DTH reaction is required if diagnostic methods are to be improved upon. Previous studies have shown that gamma delta T-cells, particularly those bearing the WC1 molecule, are present at an early stage of developing DTH responses and that such cells may modulate the developing immune response immediately following M. bovis-infection. However, their role, if any, in the DTH response remains unclear. In the present study we have used an in vivo model to deplete WC1(+) gamma delta T-cells, from cattle with established M. bovis-infection, prior to skin-testing. Results indicate that, although WC1(+) gamma delta T-cells do infiltrate the skin-test site in normal calves, they do not appear to be essential for the development of DTH skin swelling, as indicated by effective development of skin responses in calves depleted of circulating WC1(+) gamma delta T-cells.

Identification of Mycobacterium bovis antigens by analysis of bovine T-cell responses after infection with a virulent strain.

Purification and characterization of individual antigenic proteins are essential for the understanding of the pathogenic mechanisms of mycobacteria and the immune response against them. In the present study, we used anion-exchange chromatography to fractionate cell extracts and culture supernatant proteins from Mycobacterium bovis to identify T-cell-stimulating antigens. These fractions were incubated with peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle in lymphoproliferation assays. This procedure does not denature proteins and permits the testing of mixtures of potential antigens that could be later identified. We characterized protein fractions with high stimulation indices from both culture supernatants and cell extracts. Proteins were identified by two-dimensional gel electrophoresis followed by N-terminal sequencing or MALDI-TOF. Culture supernatant fractions containing low molecular weight proteins such as ESAT6 and CFP10 and other proteins (85B, MPB70), and the novel antigens TPX and TRB-B were associated with a high stimulation index. These results reinforce the concept that some low molecular weight proteins such as ESAT6 and CFP10 play an important role in immune responses. Also, Rv3747 and L7/L12 were identified in high stimulation index cell extract fractions. These data show that protein fractions with high lymphoproliferative activity for bovine PBMC can be characterized and antigens which have been already described and new protein antigens can also be identified in these fractions.

Production and preliminary characterization of monoclonal antibodies to chicken anemia agent.

Mice were immunized with partially purified preparations of the Cux-1 isolate of chicken anemia agent (CAA), and their splenocytes were fused with NSO myeloma cells. Three patterns of staining of CAA-infected cells were recognized when the resulting hybridomas were screened by indirect immunofluorescence (IIF). Hybridomas representative of each staining pattern were cloned, and the monoclonal antibodies (MAbs) were characterized. Type 1 staining was indistinguishable from that produced by polyclonal chicken antisera to CAA. Type 2 staining was confined to large nuclear inclusions. Type 3 staining was predominantly nuclear and granular, and differed from type 1 in being more intense and occurring in a higher proportion of nuclei. Three MAbs producing type 1 staining were predominantly Cux-1-specific by IIF; they also reacted to lower titers with the Gifu-1 isolate but not at all with three other CAA isolates. These MAbs had very slight neutralizing activity against Cux-1. Another MAb giving type 1 staining reacted with all CAA isolates tested to high titers in IIF and neutralization tests. MAbs with type 2 and type 3 staining reacted by IIF with all CAA isolates tested but possessed no neutralizing activity. The availability of MABs to CAA should facilitate development of diagnostic tests for the virus.

Serum haptoglobin: an objective indicator of experimentally-induced Salmonella infection in calves.

Experimental models of Salmonella -induced gastroenteritis have previously relied on crude subjective clinical markers of infection to assess disease severity. The aim of this study was to investigate the possibility that changes in serum levels of the acute phase protein, haptoglobin, may be used as an objective, quantitative measurement of infection. Eight 3- to 4-week-old animals were challenged with a mixture of three Salmonella serotypes containing 6 x 10(10)bacteria and compared with five animals given a placebo preparation. Animals were monitored and characteristic clinical symptoms of infection; diarrhoeal scores, morbidity scores and rectal temperature, were recorded. Serum samples, from both animal groups, taken prior to challenge and again on days 1, 3, and 5 post-challenge, were analysed for haptoglobin levels using a direct serum binding assay. Prior to challenge, all 13 animals had normal levels of haptoglobin in their serum. By day 3 post-challenge six of eight animals challenged with Salmonella had abnormal serum haptoglobin levels (median level = 212 microg ml(-1)), while haptoglobin levels remained normal in placebo-challenged animals (median level = 0 microg ml(-1)). The change in haptoglobin levels during the 5-day observation period was statistically significant in the Salmonella -challenged animals (P = 0.0003, H = 16.477). Serum haptoglobin levels showed a statistical correlation with clinical measures of disease severity; diarrhoeal scores (P = 0.0015, H =8. 988), morbidity scores (P = 0.0004, H = 15.711) and rectal temperature (P = 0.0001, Z = 4.304). Thus, serum haptoglobin levels closely reflect the clinical symptoms of infection and are therefore a useful marker of infection severity in salmonellosis in calves.

Effects of dietary vitamin E and selenium on in vitro cellular immune responses in cattle.

Four groups of calves depleted of alpha-tocopherol and selenium (Se) were supplemented with alpha-tocopherol or Se or alpha-tocopherol and Se or received no supplement. In vitro lymphocyte proliferative responses were measured in fetal calf serum (FCS), in autologous serum and in pooled sera from each group. In FCS, the responses to pokeweed mitogen were significantly enhanced for calves supplemented with alpha-tocopherol. In autologous serum, the mean responses to keyhole limpet haemocyanin (KLH) were greatest for calves supplemented with Se alone. In pooled sera from each group, lymphocytes from calves supplemented with Se alone showed enhanced responses to KLH in the presence of serum from calves supplemented with alpha-tocopherol. The calves depleted of alpha-tocopherol had increased circulating percentages of BoCD2 lymphocytes, apparently due to changes in the BoCD4 subpopulation. The percentages of B cells were greatest in calves supplemented with alpha-tocopherol and Se. The results indicate that alpha-tocopherol and Se have interactive effects on lymphocyte responses to antigen and suggest that micronutrient status is important when interpreting the results of in vitro assays of lymphocyte function.