Monitoring of the intra-dermal tuberculosis skin test performed by Belgian field practitioners.

The present study aimed to monitor skin test practices as performed by veterinarian field practitioners in Belgium. For this purpose, an anonymous postal questionnaire was elaborated and dispatched to veterinarians involved in bovine tuberculosis detection. The questionnaire included items focusing on the skin test performance. International experts in the field of bovine tuberculosis were asked to fill the questionnaire and a scoring scale was built as follows: 0 = 'ideal' answer, 1 = acceptable answer, whereas 2 = unacceptable answer. Furthermore, experts were asked to rank the questionnaire's items according to their possible impact on the risk of not detecting reactors. A global score was further calculated for each participant and a comparison of practices was carried out between the two regions of the country, i.e. Wallonia and Flanders. Significant differences were observed between both regions, a harmonization at the country level is thus essential. No veterinarian summed a null score, corresponding to the ideal skin test procedure, which suggests that skin-testing is far from being performed correctly. Field practitioners need to be sensitized to the importance of correctly performing the test. The authors recommend the questionnaire is suitable for application in other countries or regions.

The first International Standard anti-Brucella melitensis Serum.

The World Organisation for Animal Health (OIE) requested an International Standard anti-Brucella melitensis Serum (ISaBmS) to standardise diagnostic tests and reagents for sheep and goats. The agreed criteria were the highest dilution (in negative serum) of the standard which must give a positive result and the lowest dilution (in negative serum) which must simultaneously give a negative result. The two dilutions for each assay were, respectively: indirect enzyme-linked immunosorbent assay (iELISA) 1/64 and 1/750, competitive ELISA (cELISA) 1/8 and 1/300, fluorescent polarisation assay (FPA) 1/16 and 1/200, Rose Bengal test (RBT) 1/16 and 1/200. The OIE International Standard Serum (OIEISS) will remain the primary standard for the RBT; the ISaBmS is an additional standard. It was impossible to set criteria for the complement fixation test, therefore the OIEISS will remain the primary standard. The ISaBmS can be used to standardise iELISA, cELISA and FPA to diagnose sheep and goat brucellosis. This standard should facilitate harmonisation of tests used for brucellosis surveillance and international trade in these species.

Phenotypic and genotypic characterisation of Brucella strains isolated from cattle in the Gambia.

Thirty-five serum samples and six hygroma fluid samples were collected from sexually mature cattle in one herd with clinical signs of brucellosis (abortion and hygromas) in the Western Region of the Gambia in order to isolate and characterise Brucella species. Information on the sex, age, number of calvings, number of abortions, presence of hygromas, and presence of orchitis was also collected for each animal sampled. Twenty-six (74 per cent) of the serum samples were positive in the rose bengal test and 29 (83 per cent) were positive by indirect ELISA. Three isolates of Brucella, biotyped as Brucella abortus biovar 3, were cultured from six hygroma fluid samples. The multiple locus variable number tandem repeat analysis assay clustered the isolates as B abortus with the same profile for the three isolates, suggesting a common origin of contamination.

New assessment of bovine tuberculosis risk factors in Belgium based on nationwide molecular epidemiology.

This assessment aimed to elaborate a statistical nationwide model for analyzing the space-time dynamics of bovine tuberculosis in search of potential risk factors that could be used to better target surveillance measures. A database comprising Mycobacterium bovis molecular profiles from all isolates obtained from Belgian outbreaks during the 1995-to-2006 period (n = 415) allowed the identification of a predominant spoligotype (SB0162). Various databases compiling 49 parameters to be tested were queried using a multiple stepwise logistic regression to assess bovine tuberculosis risk factors. Two isolate datasets were analyzed: the first included all Mycobacterium bovis isolates, while the second included only data related to the SB0162 type strain. When all Mycobacterium bovis isolates were included in the model, several risk factors were identified: history of bovine tuberculosis in the herd (P < 0.001), proximity of an outbreak (P < 0.001), cattle density (P < 0.001), and annual amplitude of mean middle-infrared temperature (P < 0.001). The approach restricted to the predominant SB0162 type strain additionally highlighted the proportion of movements from an infected area during the current year as a main risk factor (P = 0.009). This study identified several risk factors for bovine tuberculosis in cattle, highlighted the usefulness of molecular typing in the study of bovine tuberculosis epidemiology, and suggests a difference of behavior for the predominant type strain. It also emphasizes the role of animals' movements in the transmission of the disease and supports the importance of controlling trade movements.

Zoonotic tuberculosis and brucellosis in Africa: neglected zoonoses or minor public-health issues? The outcomes of a multi-disciplinary workshop.

Late in 2007, veterinary, medical and anthropological professionals from Europe and Africa met in a 2-day workshop in Pretoria, South Africa, to evaluate the burden, surveillance and control of zoonotic tuberculosis and brucellosis in sub-Saharan Africa. Keynote presentations reviewed the burden of these diseases on human and livestock health, the existing diagnostic tools, and the available control methods. These presentations were followed by group discussions and the formulation of recommendations. The presence of Mycobacterium bovis and Brucella spp. in livestock was considered to be a serious threat to public health, since livestock and animal products are the only source of such infections in human beings. The impact of these pathogens on human health appears to be relatively marginal, however, when compared with Mycobacterium tuberculosis infections and drug resistance, HIV and malaria. Appropriate diagnostic tools are needed to improve the detection of M. bovis and Brucella spp. in humans. In livestock, the 'test-and-slaughter' approach and the pasteurization of milk, which have been used successfully in industrialized countries, might not be the optimal control tools in Africa. Control strategies should fit the needs and perceptions of local communities. Improved intersectoral and international collaboration in surveillance, diagnosis and control, and in the education of medical and veterinary personnel, are advocated.

Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains.

An evaluation of a multiplex PCR assay (Bruce-ladder) was performed in seven laboratories using 625 Brucella strains from different animal and geographical origins. This robust test can differentiate in a single step all of the classical Brucella species, including those found in marine mammals and the S19, RB51, and Rev.1 vaccine strains.

Sanitary control in bovine embryo transfer. How far should we go? A review.

Embryo transfer is a globally executed technique which, when properly done, has both economic and sanitary advantages. International guidelines are available to prevent infection of the embryo with pathogens, both originating from the donor animals as from the environment. This manuscript describes the bacteria, viruses, protozoa, fungi and prions that are of major concern in the context of embryo transfer in cattle. In addition, the actual scientific knowledge on these pathogens is evaluated in terms of the current international and national guidelines and legislation.

Active membrane transport and receptor proteins from bacteria.

A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides.

IFN-gamma diagnostic tests in the context of bovine mycobacterial infections in Belgium.

In countries where cattle tuberculosis caused by Mycobacterium bovis (Mbov) and paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (Mptb) are present, testing strategies for the Mbov eradication have to discriminate between these two infections. Present indirect tests are based on the analysis of the specific cellular immune response (DTH, IFN-gamma) against crude mycobacterial antigens (avian and bovine PPD). In this study, we compared the evolution of the IFN-gamma responses of animals experimentally infected with Mbov, Mptb, or inoculated with Mycobacterium phlei. Mbov inoculation induced a strong IFN-gamma response that allows rapid classification of the status of the animals following interpretation criteria set up by us. Experimental inoculation with M. phlei induced sensitisation to mycobacterial antigens as detected by the IFN-gamma test but these reactions were of short duration, therefore, repeated testing allows us to define these animals as aspecific reactors. IFN-gamma response induced after oral inoculation of calves with Mptb was of low intensity and ratio of responses measured against avian versus bovine PPD did not allow a clear diagnostic at least for the six first month of infection.

Analysis of the antigen-specific IFN-gamma producing T-cell subsets in cattle experimentally infected with Mycobacterium bovis.

Three 10 months old cattle were infected by the intratracheal route with 10(6)cfu of a field strain of Mycobacterium bovis. Blood samples were regularly collected for in vitro IFN-gamma production after antigenic stimulation. Peripheral blood cells of infected animals produced IFN-gamma in response to crude M. bovis antigens (live and heat-inactivated BCG and protein-purified derivative (PPD)) 3-4 weeks after infection. The ratio of the response to bovine PPD versus avian PPD indicated a specific sensitisation for M. bovis antigens. Three months post-infection (PI), animals were culled and M. bovis was cultured from tubercle lesions. At different time points, the frequency of specific M. bovis IFN-gamma producing CD4+, CD8+ and WC1+ T-cells in the peripheral blood was examined by flow cytometry. Two colour immunofluorescence staining of intracellular IFN-gamma and bovine cell surface molecules showed that both CD4+ and CD8+, but not WC1+, T-cells produced IFN-gamma following stimulation with PPD, live or killed BCG. In two animals analysed, the relative percentage of circulating IFN-gamma producing CD8+ cells decreased between week 5 and week 9 PI. The same evolution was not observed for IFN-gamma secreting CD4+ cells. Magnetic positive selection of T-cells from infected animals showed that CD4+ T-cells produced specific IFN-gamma only in the presence of antigen presenting cells (APCs). Positively selected CD8+ T-cells secreted IFN-gamma only in the presence of recombinant human IL-2 and APCs. In vitro depletion of the CD4+ T-cells, but not the depletion of CD8+ or WC1+ T-cells, resulted in abrogation of the specific IFN-gamma production showing the key role of this cell population for the specific IFN-gamma production.

Induction of immune response in BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of Brucella spp.

In this study, we evaluated the ability of DNA vaccines encoding the bacterioferritin (BFR) or P39 proteins of Brucella spp. to induce cellular and humoral immune responses and to protect BALB/c mice against a challenge with B. abortus 544. We constructed eukaryotic expression vectors called pCIBFR and pCIP39, encoding BFR or P39 antigens, respectively, and we verified that these proteins were produced after transfection of COS-7 cells. PCIBFR or pCIP39 was injected intramuscularly three times, at 3-week intervals. pCIP39 induced higher antibody responses than did the DNA vector encoding BFR. Both vectors elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the specific antigens or Brucella extract. In this report, we also demonstrate that animals immunized with these plasmids elicited a strong and long-lived memory immune response which persisted at least 3 months after the third vaccination. Furthermore, pCIBFR and pCIP39 induced a typical T-helper 1-dominated immune response in mice, as determined by cytokine or immunoglobulin G isotype analysis. The pCIP39 delivered by intramuscular injection (but not the pCIBFR or control vectors) induced a moderate protection in BALB/c mice challenged with B. abortus 544 compared to that observed in positive control mice vaccinated with S19.

Protection of BALB/c mice against Brucella abortus 544 challenge by vaccination with bacterioferritin or P39 recombinant proteins with CpG oligodeoxynucleotides as adjuvant.

The P39 and the bacterioferrin (BFR) antigens of Brucella melitensis 16M were previously identified as T dominant antigens able to induce both delayed-type hypersensivity in sensitized guinea pigs and in vitro gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells from infected cattle. Here, we analyzed the potential for these antigens to function as a subunitary vaccine against Brucella abortus infection in BALB/c mice, and we characterized the humoral and cellular immune responses induced. Mice were injected with each of the recombinant proteins alone or adjuvanted with either CpG oligodeoxynucleotides (CpG ODN) or non-CpG ODN. Mice immunized with the recombinant antigens with CpG ODN were the only group demonstrating both significant IFN-gamma production and T-cell proliferation in response to either Brucella extract or to the respective antigen. The same conclusion holds true for the antibody response, which was only demonstrated in mice immunized with recombinant antigens mixed with CpG ODN. The antibody titers (both immunoglobulin G1 [IgG1] and IgG2a) induced by P39 immunization were higher than the titers induced by BFR (only IgG2a). Using a B. abortus 544 challenge, the level of protection was analyzed and compared to the protection conferred by one immunization with the vaccine strain B19. Immunization with P39 and CpG ODN gave a level of protection comparable to the one conferred by B19 at 4 weeks postchallenge, and the mice were still significantly protected at 8 weeks postchallenge, although to a lesser extent than the B19-vaccinated group. Intriguingly, no protection was detected after BFR vaccination. All other groups did not demonstrate any protection.

Aromatic compound-dependent Brucella suis is attenuated in both cultured cells and mouse models.

The aroC gene of the facultative intracellular pathogen Brucella suis was cloned and sequenced. The cloned aroC gene complements Escherichia coli and Salmonella enterica serovar Typhimurium aroC mutants. A B. suis aroC mutant was found to be unable to grow in a defined medium without aromatic compounds. The mutant was highly attenuated in tissue culture (THP1 macrophages and HeLa cells) and murine virulence models.

Prevalence of paratuberculosis (Johne's disease) in the Belgian cattle population.

The national bovine paratuberculosis (PTB) seroprevalence (apparent prevalence) in the Belgian cattle population was determined by a serological survey that was conducted from December 1997 to March 1998. In a random sample of herds (N=556, 9.5%), all adult cattle of 24 months of age or older (N=13,317, 0.4%) were tested for the presence of antibodies using a commercially available absorbed ELISA test kit. The PTB median within-herd seroprevalence (proportion of detected animals within the seropositive herds) and the PTB individual-animal seroprevalence (proportion of detected animals) were, respectively, 2.9% (quartiles=1.6-5.6) and 0.87% (95% confidence interval (CI)=0.71-1.03). The PTB herd seroprevalence (proportion of detected herds) was 18% (95% CI=14-21). Assuming a test sensitivity and specificity of 45 and 99% [Sweeney et al., 1995. J. Vet. Diagn. Invest. 7 (4), 488; Sockett et al., 1992. J. Clin. Microbiol. 30 (5), 1134], respectively, the median true within-herd prevalence and the true individual-animal were estimated to be 7 and 2%, respectively. The true herd prevalence of Mycobacterium paratuberculosis infection was first estimated according to currently accepted methodology. This calculation revealed that the specificity of the used test has a dramatic effect on the estimation; assuming a test sensitivity of 45% and a true within-herd prevalence of 7%, the true herd prevalence estimation decreased from 36 to 0.8% if the test specificity decreased from 99. 9 to 99%, respectively. This sensitivity analysis showed that the practical limits of the accuracy of the used screening test jeopardize the estimation of the true herd prevalence within reasonable confidence limits, because the within-herd PTB true prevalence was low. For this reason we augmented the herd specificity for herds with larger adult herd size (>5). This was done by increasing the cut-off number of positive cattle required (>/=2) to classify a herd truly positive and including herds with one positive test result if there was historical evidence of PTB (previous diagnosis and/or clinical signs). This approach resulted in an estimated true herd prevalence of M. paratuberculosis infection of 6%. The true herd prevalence for dairy, mixed and beef herds was, respectively, 10, 11 and 3%.

First evidence of Johne's disease in farmed red deer (Cervus elaphus) in Belgium.

In a deer farm, chronic diarrhoea was seen in a 4-year-old hind. This animal died in poor condition on the farm and Johne's disease was suspected. Ziehl-Neelsen staining of the faeces of this hind were positive for the presence of clumps of small acid-fast bacilli, but faecal cultures remained negative. Direct and indirect tests were performed on 24 hinds and stags (yearlings, 2- and 4-year-old animals). The indirect tests performed were serology (Mycobacterium paratuberculosis antibody ELISA, HerdChek, Idexx), comparative cervical skin test (CCT) and lymphoproliferation test (LT) using Mycobacterium bovis purified protein derivative (PPD) and Mycobacterium avium PPD as antigens. Three positive serological results, three positive CCT and eight positive LT were observed in hinds and stags older than 2 years. No positive serological results were observed in the yearling group, whereas some sensitisation was observed in the CCT as well as in the LT for the same group of animals. The degree of concordance between these indirect tests was poor. The three seropositive animals were slaughtered and subjected to post-mortem examination. Histopathology was performed on mesenteric lymph nodes and on the terminal ileum. Visual changes in some mesenteric lymph nodes were observed, no gross lesion was seen in the intestine. Although Ziehl-Neelsen staining yielded no positive results, a catarrhal focal necrotic enteritis associated with a granulomatous lymphadenitis compatible with Johne's disease was evidenced. The mycobacterial cultures on organ samples from slaughtered animals were positive after 2 months for M. avium subspecies paratuberculosis and negative for M. bovis and M. avium. This is the first description of Johne's disease in a deer farm in Belgium.

Susceptibility of bovine antigen-presenting cells to infection by bovine herpesvirus 1 and in vitro presentation to T cells: two independent events.

The aim of the present study was to develop an in vitro system for presentation of bovine herpesvirus 1 (BHV-1) antigens to bovine T lymphocytes and to characterize the antigen-presenting cells (APC) which efficiently activate CD4(+) T cells. Two approaches were used to monitor the infection of APC by BHV-1 as follows: (i) detection of viral glycoproteins at the cell surface by immunofluorescence staining and (ii) detection of UL26 transcripts by reverse transcription-PCR. The monocytes were infected, while dendritic cells (DC) did not demonstrate any detectable viral expression. These data suggest that monocytes are one site of replication, while DC are not. The capacities of monocytes and DC to present BHV-1 viral antigens in vitro were compared. T lymphocytes (CD2(+) or CD4(+)) from BHV-1 immune cattle were stimulated in the presence of APC previously incubated with live or inactivated wild-type BHV-1. DC stimulated strong proliferation of Ag-specific T cells, while monocytes were poor stimulators of T-cell proliferation. When viral attachment to the surface of the APC was inhibited by virus pretreatment with soluble heparin, T-cell proliferation was dramatically decreased. Unexpectedly, incubation of DC and monocytes with the deletion mutant BHV-1 gD-/-, which displays impaired fusion capacity, resulted in strong activation of T lymphocytes by both APC types. Collectively, these results indicate that presentation of BHV-1 antigens to immune T cells is effective in the absence of productive infection and suggest that BHV-1 gD-/- mutant virus could be used to induce virus-specific immune responses in cattle.

Immune response of calves experimentally infected with non-cell-culture-passaged bovine respiratory syncytial virus.

The immune response of calves was studied following infection with non-cell-passaged Bovine respiratory syncytial virus (BRSV). Two groups of 6 specific pathogen free (SPF) calves were housed in separate isolation rooms. One group was inoculated intranasally with a non-cell-passaged BRSV strain and the control group was mock-infected. A BRSV specific antibody response was observed for all the BRSV infected calves. These antibodies were shown to have neutralizing activity. No lymphocyte proliferation response was detected in the mock-infected group whereas three animals in the infected group were positive three weeks after the infection. All BRSV-infected calves, except one, produced interferon-gamma (IFN-gamma) one week post-infection and IFN-gamma was observed in all six infected calves after three weeks. The control group showed no IFN-gamma synthesis. In spite of the limits of the BRSV infection model, humoral and cellular immune responses were actively developed by all the calves against this pathogen.

A Cryptosporidium parvum oocyst low molecular mass fraction evokes a CD4+ T-cell-dependent IFN-gamma response in bovine peripheral blood mononuclear cell cultures.

T-Cell antigens that induce the in-vitro interferon-gamma response during Cryptosporidium parvum infection of neonatal calves were identified. A total oocyst extract was separated into a high and a low Mr fraction by a microfiltration technique. Both the high and low Mr fractions evoked an in-vitro interferon-gamma response in naturally infected animals, although strong individual differences between the hosts were observed. Using a complement-mediated technique CD4+ T-cells or WC1+gammadelta T-cells were depleted, whereupon the remaining lymphocyte cultures were stimulated with the different antigen preparations. It was shown that the in-vitro interferon-gamma response of Cryptosporidium-infected calves is CD4+ T-cell-dependent.