Search

Paratuberculosis (PTB), also known as Johne's disease, is a chronic proliferative enteritis of ruminants caused byMycobacterium aviumsubsp.paratuberculosis(MAP). To date, PTB diagnosis, based on serology, fecal culture, and real-time polymerase chain reaction, has identified animals in advanced stages of infection. To detect MAP infection in animals earlier, the interferon-gamma (IFN-γ) test may be applied. This assay detects cytokines produced by T-lymphocytes of infected subjects after stimulation with purified protein derivatives (PPDs), extracted fromMycobacterium bovis(MB) and fromM. avium(MA). The study involved three bovine herds: one PTB-infected herd, one PTB-free herd, and one with an outbreak of bovine tuberculosis. The IFN-γ test was performed on 235 animals, using bovine PPD (PPDB), avian PPD (PPDA), and three experimental PPD Johnins (PPDJs) extracted from a synthetic liquid medium culture of MAP (PPDJ A, B, and C), to assess early MAP detection and avoid false reactions to MB. Furthermore, IFN-γ results were evaluated using 12 interpretative criteria (ICs), based on the differences and ratio between PPD optical density (OD) and IFN-γ basal OD values after lymphocytic stimulation. IC accuracy was expressed as area under the receiver operating characteristic curve. Through a longitudinal study, PPDJs proved to be specific and sensitive in the detection of MAP-infected animals. Among the evaluated ICs, six showed the best performance in terms of accuracy (p< 0.0001), highlighting PTB subclinical infections. In particular, the two best criteria reached sensitivity values of 100% [confidence interval (CI) 95%, 94.1–100%] with a specificity of 91.8% (CI 95%, 81.9–97.3%) and sensitivity levels of 80.6% (CI 95%, 69.1–89.2%) with a specificity of 100% (CI 95%, 94.1–100%). Thus, the IFN-γ assay proved to be a useful diagnostic tool to identify early subclinical MAP-infected animals, in order to manage infected cattle or those exposed to MAP and to monitor younger calves within a herd. Furthermore, the IFN-γ test can be considered an additional test to avoid the introduction of MAP-infected animals, especially in herds where disease has already been eradicated and preservation of the health status is required to maintain the PTB certification level.

BackgroundClostridium perfringensis the causative agent of several diseases and enteric infections in animals and humans. The virulence ofC. perfringensis largely attributable to the production of numerous toxins; of these, the alpha toxin (CPA) plays a crucial role in histotoxic infections (gas gangrene). CPA toxin consists of two domains, i.e., the phospholipase C active site, which lies in the N-terminal domain amino acid (aa residues 1–250), and the C-terminal region (aa residues 251–370), which is responsible for the interaction of the toxin with membrane phospholipids in the presence of calcium ions. All currently produced clostridial vaccines contain toxoids derived from culture supernatants that are inactivated, mostly using formalin. The CPA is an immunogenic antigen; recently, it has been shown that mice that were immunized with the C-terminal domain of the toxin produced inE. coliwere protected againstC. perfringensinfections and the anti-sera produced were able to inhibit the CPA activity. Monoclonal and polyclonal antibodies were produced only against full-length CPA and not against the truncated forms.ResultsIn the present study, we have reported for the first time; about the generation of a recombinant baculovirus capable of producing a deleted rCPA toxin (rBacCPA250–363H6) lacking the N-terminal domain and the 28 amino acids (aa) of the putative signal sequence. The insertion of theL21consensus sequence upstream of the translational start codon ATG, drastically increases the yield of recombinant protein in the baculovirus-based expression system. The protein was purified by Ni-NTA affinity chromatography and the lack of toxicity in vitro was confirmed in CaCo-2 cells. Polyclonal antibodies and eight hybridoma-secreting Monoclonal antibodies were generated and tested to assess specificity and reactivity. The anti-sera obtained against the fragment rBacCPA250–363H6 neutralized the phospholipase C activity of full-length PLC.ConclusionsTheL21leader sequence enhanced the expression of atoxic C-terminal recombinant CPA protein produced in insect cells. The monoclonal and polyclonal antibodies obtained were specific and highly reactive. The availability of these biologicals could contribute to the development of diagnostic assays and/or new recombinant protein vaccines.

Clostridium (C.) perfringens is the causative agent of several diseases and enteric infections in animals and humans. The pathogenicity of the bacterium is largely mediated by the production of a wide range of toxins. Individual C. perfringens strains produce only subsets of this toxin repertoire, which permits the classification in seven toxinotypes (A&ndash, G). In addition, a variety of minor toxins further characterizes the single strains. The aim of this work was to evaluate, using Polymerase Chain Reaction (PCR) assays, the diversity of 632 C. perfringens strains isolated in Italy over 15 years. The genotyped strains were analyzed to determine the presence of major and minor toxins (cpe, consensus, and atypical cpb2), their geographical origins, and the source of isolation (animal species or food). Our study shows that toxinotype A had the greatest representation (93%) and correlated mainly with consensus cpb2 in a variety of animal species, as well as with atypical cpb2 in the five food samples. Type D, associated with cpe and atypical cpb2 minor toxins, was identified in 3% of the cases, and type F was identified in 2.5%. Seven type C isolates (1.1%) were detected in cattle, whereas the only type B atypical cpb2 isolated in Italy was detected in a goat, and one type E cpe+atypical cpb2 was detected in a sheep. Type G was not detected.

The bovine leukaemia virus (BLV) envelope protein (Env) is synthesized as a polyprotein precursor (gp72) proteolytically cleaved into the mature surface (SU) and transmembrane (TM) glycoproteins. The amino-terminal region of SU contains conformational epitopes F, G and H, which require a glycosylated SU to be recognized by monoclonal antibodies (MAbs) and antibodies from BLV-infected cattle. The SU contains eight asparagine (N) residues that are putative N-glycosylation sites. The N129, N203, N230 and N251 appear involved in carbohydrate binding, play an essential role in the in vitro infection. To determine which sites were actually glycosylated, we generated mutated SU forms, where each N-glycosylation site was changed to alanine (A). Subsequently, these N to A mutations were inserted into the env gene to generate Env mutants. The increase of electrophoretic mobility of EnvA256 and EnvA271 derived SU showed that the asparagine residues N256 and N271 were also glycosylated. ELISA revealed that only the N129 oligosaccharide determined the antigenic conformation of SU. The syncytium formation induced by EnvA129 showed that fusogenic capacity was independent of amino-terminal SU glycan conformational structure. Finally, anti-BLV serum inhibited syncytia formation even with the EnvA129 mutant. The latter inhibition was higher than Env, suggesting that the oligosaccharides could be also involved in the glycan shield for viral escape.

Endotoxin contamination is a serious concern for manufacturers of biological products and vaccines in terms of not only quality but also safety parameters. We evaluated the endotoxin presence in different veterinary autogenous vaccines produced by the Pharmaceutical Unit at the Experimental Zooprophylactic Institute of Umbria and Marche "Togo Rosati" (IZSUM). According to the 3Rs principles (Replace, Reduce, Refine), which aim to progressively reduce animal use in the quality control process, we tested the vaccines obtained from gram-negative bacteria and adjuvants by the limulus amebocyte lysate (LAL) assay. The results revealed low endotoxin concentrations compared to available data in the literature and represent the first report of the application of the 3Rs principles to veterinary autogenous vaccines production in Italy.

Tuberculins purified protein derivatives (PPDs) are obtained by precipitation from heat treated mycobacteria. PPDs are used in diagnosis of mycobacterial infections in humans and animals. Bovine PPD (PPDB) is obtained from Mycobacterium bovis (Mycobacterium tuberculosis complex), while Avian PPD (PPDA) and Johnin PPD (PPDJ) are extracted, respectively, from Mycobacterium avium and M. avium subsp. paratuberculosis (M. avium complex). PPDB and PPDA are used for bovine tuberculosis diagnosis, while PPDJ is experimentally used in the immunodiagnosis of paratuberculosis. Although PPDs date back to the 19th Century, limited knowledge about their composition is currently available. The goal of our study was to evaluate Fourier Transform InfraRed (FTIR) spectroscopy as a tool to differentiate PPDB, PPDA, and three PPDJs. The results highlighted that the three PPDs have specific profiles, correlated with phylogenetic characteristics of mycobacteria used for their production. This analysis is eligible as a specific tool for different PPDs batches characterization and for the assessment of their composition. The entire PPD production may be efficiently controlled, since the N content of each preparation is related to IR spectra, with a reference spectrum for each PPD and a standardized analysis protocol.

Background Tuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB). Methods Proteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC–MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system. Results Three hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920. Conclusions This study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1172-1) contains supplementary material, which is available to authorized users.

The Brucella melitensis REV1 vaccine is the most widely employed vaccine for prophylaxis against brucellosis in sheep and goats. The objective of vaccination is disease control in herds or preventing infection in farms. In this study, we produced REV1 vaccine with a protocol, based on the use of liquid medium in a bioreactor, that resulted efficient, safe, relatively fast, and cost-effective. The live attenuated vaccine produced was tested in mice and sheep to investigate its immunogenicity and efficacy. Seventy-two female BALB/c mice were obtained and subdivided in 2 groups, one was stimulated with 1 × 106 colony-forming units (CFUs) of B. melitensis while the other with physiological solution alone and acting as control group. Furthermore, 25 sheep were subdivided into 5 groups: four were inoculated with a B. melitensis dose, ranging from 0.6 × 109 and 3.2 × 109 CFUs and the other was the control group. In addition, a serological diagnosis was performed for sheep by rapid serum agglutination and the complement-fixation test. Immunocompetent cells from both experiment were collected at different times post vaccination and immunostained to evaluate innate and adaptive-immune responses. In mice flow cytometry was used to detect macrophages, T lymphocytes, dendritic cells, memory cells, naive cells, natural killer cells, major histocompatibility complex type II, B lymphocytes, regulatory T lymphocytes, T helper lymphocytes, cytotoxic T lymphocytes and recently activated CD4+ and CD8+ lymphocytes. In sheep, macrophages, T helper cells, cytotoxic T lymphocytes, regulatory T lymphocytes, dendritic cells, memory cells and naive lymphocytes, by the same method, were analyzed. The results showed, both in mice and sheep, that the live, attenuated REV1 vaccine stimulated all immunocompetent cells tested, with a balanced innate and adaptive response. In the sheep experiment, the administered vaccine dose was very important because, at the lower doses, immunological tolerance tended to disappear, while, at the highest dose, the immunological tolerance remained active for a long period. In our experimental conditions, the optimal vaccine dose for sheep was 3.2 × 109 CFUs, although a good immune response was found using a dose of 1.6 × 109 CFUs. The vaccine produced in this study could be extensively employed in developing countries to control the brucellosis in sheep and goats.

Switzerland has been officially free of bovine tuberculosis (OTF) since 1960. Since 1980 the control of bovine tuberculosis (bTB) has been reduced to passive abattoir surveillance. Isolated cases of bTB, partly due to reactivation of human Mycobacterium bovis infections with subsequent transmission to cattle, have been noticed in the last years. In Europe, the overall prevalence of bTB is slightly increasing. Both OTF and non-OTF countries report increases in the proportion of bTB positive cattle herds. Current bTB eradication and control programs in Europe are facing a range of challenges. Whole herd depopulation is becoming a less attractive option for economic reasons and due to animal welfare concerns. Live animal trade is increasing both at national and international levels. Regarding these tendencies and taking into account the chronicity of bTB infection, pre-movement testing is becoming increasingly important as a central tool for eradication and for protection against re-introduction of bTB. Pre-movement testing, however specifically focuses on the infection status in individuals, requiring a high level of diagnostic accuracy to correctly diagnose infected animals. Current screening tests for bTB, however, have been designed to meet demands as herd tests. This illustrates that the modification of existing and/or the development of new diagnostics for bTB might be needed. The tuberculin skin test (TST), the primary screening test for bTB may in certain situations have low sensitivity. The interferon gamma (IFN-γ) assay is accepted to be more sensitive compared to TST. Reduced specificity, however, especially in areas of low bTB prevalence raises concerns. New antigen combinations including Rv3615c, OmpATb and others have been shown to complement ESAT-6 and CFP-10 in the whole blood IFN-γ assay and resulted in improved sensitivity (compared to ESAT-6 and CFP-10) and specificity (compared to tuberculins). Lesion detection after slaughter represents a cost-effective procedure for passive surveillance of bTB, especially in areas of low prevalence or in regions free of bTB; however, its sensitivity is very low. This illustrates that trade is linked with a certain risk to re-introduce bTB in OTF regions or countries and that there may be delays in detecting a re-introduction of bTB. In conclusion, regarding the fact that some parameters linked with bTB programs are changing, the development of improved diagnostic tests with a high reliability for use as individual animal tests will be important for future eradication of bTB, in line with international commitment to high standard animal health programs.

The study was carried out in cattle kept at 11 livestock experiment stations of Punjab by using single comparative cervical intradermal tuberculin (SCCIT) test. Sahiwal was the main breed kept at these farms. Sixty three percent of animals were between four and 10 years of age. Seventy-six percent of animals weighed between 300 and 400 kg and 66% produced 5-10l of milk/day. Animals other than cattle were present at about 64% of these farms. The positive SCCIT test was recorded in 7.6% of animals at the 11 farms. However, the prevalence of tuberculosis varied from 2.0% to 19.3% at these farms. Bivariate frequency analysis showed that the chances of a positive SCCIT test were higher in older animals, in cattle with higher number of calving and those produced up to 1800l of milk. However, the chances of positive SCCIT test decreases with further increase in milk production. Results of bivariate and/or multivariate logistic regression analysis after controlling for the farm showed a significant association of age of cattle, numbers of calving, total milk produced, per day milk, lactation length, presence of sheep at the farm and total numbers of animals at the farm with a positive SCCIT test. It can be concluded from the study that herd prevalence of tuberculosis was 100%, while animal prevalence was about 8% at these farms. The stronger risk factors identified by logistic analysis were the age of cattle, numbers of calving, total milk produced and lactation length, while the presence of sheep at the farm has protective effect.

The present study was undertaken to find out the association of certain risk factors with the positive SCCIT (single comparative cervical intradermal tuberculin) test in water buffaloes at Faisalabad and Okara, Pakistan. Seventy-six herds (697 buffaloes) at Faisalabad and 56 (395 buffaloes) at Okara were included in the study. The buffaloes were screened through SCCIT Test. Positive reaction to SCCIT test was recorded in 14% of herds and in 3% of buffaloes. The herds positive for this test were 18% when herds with less than 10 animals were excluded from the analysis and these were 19% when herds with less than 10 buffaloes were excluded. The results of logistic analysis (crude and adjusted) revealed the association of lactating status of buffaloes (OR = 1.8) and the presence of cattle at the farm (OR = 2–4) with positive SCCIT test. After controlling for the farm, the risk of a positive skin test was 1.5 times higher if we change the location of the animal. Similarly, the controlled analysis (for the farm, breed and other variables) revealed an increased risk (OR = 1.1) of a positive skin test with the increase in cattle at the farm. The breed controlled stratified analysis showed the association of a number of cattle at the farm with a positive skin test. It can be concluded from the study that the prevalence of tuberculosis on the basis of a positive skin test is higher at herd level and lower at animal level. Further the risk of a positive skin test is higher when cattle are present at the farm.

To investigate the percentage reactor animals to SCCIT in sheep and goat the present study was carried out by using bovine and avian PPDs at 7 Livestock Experiment Stations of Punjab and villages around two cities. The overall percentage of reactor animals to SCCIT at farms in sheep and goat was 0.9% and 2.4%, while it was 0.4% and 0.0% around two cities, respectively. Hundred percent of goat and 86% of sheep farms under study had positive reactor animals. The reactor animals to SCCIT around two cities were 0% in goat and 0.4% in sheep. Goats (P0.05) and sheep (P0.001) kept with large ruminants at farms had higher percentage of reactor animals. Age showed significant (P0.01) association with tuberculosis in sheep and goat. In goats of more than 6 years of age, the reactor animals to SCCIT were 5, 8 and 11 times higher than goats of 4.1-6, 3-4 and3 years of age. The reactor animals were significantly (P0.05) higher in goats of30kg (3.5%) and40kg (3.5%) body weights, while these were less in goats weighing between 30 and 40kg (1.5%). Sheep and goat breeds showed significant (P0.001; P0.05) difference in percentage of reactor animals to SCCIT. Beetal (3.9%) and Teddy breeds (2.9%) had 3.3 and 2.4 times higher percentages of reactor animals to SCCIT as compared to Pak-Angora (1.2%) breed. However, no animal of Nachhi and Dera Din Pinah breeds showed positive reaction to PPDs.

Salmonellosis due to Salmonella enterica serovar Abortusovis (S. Abortusovis) is mainly characterized by abortion in sheep. Little is known about the immune response, which develops in the host as a result of infection. We evaluated the immune response of pregnant ewes vaccinated and successively exposed to full virulent S. Abortusovis. We found that vaccine constituted by inactivated S. Abortusovis induced both humoral and cellular-mediated immune response and that it provided protection against a challenge infection due to a fully virulent S. Abortusovis. Furthermore, we found an association between the lack of capability to produce IFN-gamma and abortion. This evidence suggests that protection against abortion can be associated to an IFN-gamma mediated mechanism. Our findings represent an interesting insight to better understand the interplay between host and S. Abortusovis and the effector mechanisms underpinning immune-based protection.

Bovine tuberculosis is one of the important diseases of dairy and wild animals. The disease is prevalent all over the world, though developed countries have tremendously reduced the prevalence through eradication campaigns. The prevalence of disease in Pakistan on the basis of tuberculin testing or culture isolation of the organism has been reported previously. It is, however, important to use the latest diagnostic tools, i.e. PCR to confirm the type of Mycobacterium infecting the animals in Pakistan. Therefore, the present study was carried out to assess the utility of direct PCR on milk samples and nasal swabs to confirm the type of Mycobacterium infecting the animals. This study was carried out on 215 cattle and buffaloes of more than 2 years of age present at two livestock farms. The tuberculin results showed 22.5 % prevalence at one farm and 25.9 % at the other with an overall prevalence of 24.7 %. The 92.5 % of milk samples and/or nasal swabs showed positive PCR for Mycobacterium genus, 86.8 % for Mycobacterium tuberculosis complex and 77.4 % for Mycobacterium bovis. The M. bovis by PCR was detected in 13.2 % of milk samples, 24.5 % of nasal swabs and 39.6 % of both milk samples + nasal swabs. The results suggested that there are 60 % higher chance for a nasal swab to yield a positive PCR for M. bovis than the milk sample. It can be concluded from the present study that tuberculin testing is a useful method in studying the prevalence of disease as the PCR for Mycobacterium genus was positive in 92.5 %, M. tuberculosis complex in 86.8 % and Mycobacterium bovis in 77.4 % cases.

Bovine leukaemia virus (BLV), an oncogenic C-type retrovirus, is the causative agent of enzootic bovine leucosis. Binding of BLV to its cellular receptor is mediated by the surface envelope glycoprotein subunit (SU). Previous studies have identified eight different epitopes (A through H) on the BLV SU. In this study, a new sequential epitope was identified using the monoclonal antibody 2G7 (MAb 2G7) on the C-terminal region of the BLV SU. To localise and refine the map of this epitope, a series of deleted forms in the C and N-terminal ends of the glycoprotein were made and synthesised in baculovirus and Escherichia coli expression systems. The synthetic proteins were analysed both in Western blot and MAb-capture ELISA assays. MAb 2G7 recognised a stretch of 11 amino acids, named epitope E′, corresponding to residues 189-SDWVPSVRSWA-199 (comprising the 33 amino acids signal peptide) overlapping with the E epitope of the SU. The data obtained by Enzyme-Linked Immunosorbent Assay (ELISA) revealed that the E′ epitope was hidden on whole BLV particles and that the variation in reactivity between epitope E′ and MAb 2G7 depends on the glycosylation state of SU. Similarly, the analysis of immunological data evidenced that the failure of interaction between the MAb anti-DD′ and its epitope was also due to a steric hindrance of the glycosylation. Finally, the ELISA assay analysis performed with the deleted and mutated forms of rSU evidenced that the conformational epitopes F, G and H lied into in the 34–173 amino-acids residues of N-terminal region of SU.

Mycobacterium bovis recognizes as hosts a wide spectrum of animal species. In particular epidemiological situations, high prevalence of infection is found also in pigs. In the present study, we evaluated the capability of the interferon-gamma (IFN-γ) assay to identify pigs infected with M. bovis. The results of the immune-diagnosis were correlated to the findings of the post mortem inspection and the bacterial culture of lymph nodes. Blood samples of 146 pigs, belonging to a local breed of Sicily reared in free or semi-free roaming conditions, were collected to assess the specificity and the sensibility of the IFN-γ assay. Thirty-one pigs, from M. bovis free herds, did not react to the IFN-γ assay, yielding a specificity of 100%. The IFN-γ assay identified 15 out of 19 animals positive to the bacterial culture and 22 out of 26 animals with tuberculous lesions, with a sensibility of 78.9-84.6%, respectively. Out of 26 reactors to the test, 15 pigs (57.7%) confirmed to be infected after the bacterial culture and 22 (84.6%) had tuberculous lesions. The IFN-γ assay was able to reveal 4 animals with no visible lesions (NVL). Together, these findings support the feasible use of the IFN-γ assay as an intra vitam tool for the surveillance and management of M. bovis infection in swine populations.

In this study, interferon-gamma (IFN-gamma) responses in whole blood cultures stimulated with tuberculins from different sources were compared with regard to their diagnostic reliability in cattle experimentally and naturally infected with Mycobacterium bovis. The IFN-gamma responses to different concentrations of purified protein derivatives (PPDs) from M bovis and Mycobacterium avium were quantified. Significant differences (P0.05) between sources and concentrations of PPDs used for stimulation were detected, indicating a need for standardisation of PPDs used in the IFN-gamma assay. Additionally, a tool named'relative potency 30' that allows rapid comparison of batches and sources of PPDs was defined.

Bovine leukaemia virus (BLV) causes disease in cattle and it is related to human T lymphotrofic viruses HTLV-1 and HTLV-2. The objective of this study was to express and purify deleted and stable forms of the gp51 envelope glycoprotein of BLV using a baculovirus system. Two forms of the gp51 were synthesised: one comprised the gp51 N-terminal 174 amino acids and a single 6xHis tag (Delta(175-268)gp51-His) and the second form contained the same amino acid sequence and a C-terminal Strep-tag II in addition to the 6xHis tag (Delta(175-268)gp51-STH). The two proteins were expressed and purified by immobilized metal-affinity chromatography (IMAC) or by Strep-Tactin column. The Strep-Tactin technology was more efficient than IMAC method and achieved a high pure recombinant deleted gp51. In addition the Delta(175-268)gp51-STH protein was further concentrated by IMAC. This purified antigen could be used for the isolation of immunoreactive molecules and to develop a competitive ELISA test.

Effective surveillance of bovine tuberculosis (BTB) in developing countries where reliable data on disease prevalence is scarce or absent is a precondition for considering potential control options. We conducted a slaughterhouse survey to assess for the first time the burden of BTB in Southern Chad. Altogether, 954 slaughter animals were consecutively sampled and tested using the single intra-dermal comparative cervical tuberculin (SICCT) test, a recently developed fluorescence polarization assay (FPA) and routine abattoir meat inspection after slaughter. Gross visible lesions were detected in 11.3% (CI: 9.4-13.5%) of the animals examined and they were mostly located in the lymph nodes and the lung. Significantly more Mbororo zebus (15.0%) were affected by lesions than Arab zebus (9.9%; OR=2.20, CI: 1.41-3.41%; p0.001). Of all animals tested, 7.7% (CI: 6.2-9.6%) reacted positively to SICCT if OIE guidelines were applied. However, receiver operating characteristic (ROC) analysis using Mycobacterium tuberculosis complex (MTBC) infected animals as the positive population and lesion negative animals as the negative population, revealed a better SICCT performance if the cut-off value was decreased to2mm. SICCT reactor prevalence rose to 15.5% (CI: 13.3-18.0%) and FPA did not perform better than SICCT, when this setting adapted cut-off was applied.

The study was conducted to ascertain the epidemiology, together with effects of bovine tuberculosis, on certain haematological parameters and serum proteins at two Livestock Experiment Stations in Pakistan. The results on prevalence of tuberculosis in buffaloes on the basis of comparative intradermal tuberculin test revealed it to be from as high as 8.48% (14/165) to as low as 2.45% (4/163) on the basis of positive reaction to bovine PPD. However, a doubtful reaction was observed in 8.58% (14/163) of buffaloes at farm 2 with low prevalence. It was also observed that the reaction to bovine or avian PPD was much stronger in buffaloes compared with indigenous cattle. The result on certain epidemiological factors studies revealed higher positive reactors of greater than 6 years of age and the majority were between 6-8 years old (P = 0.03). Results also revealed that a significantly higher number of animals had a body mass of less than 550 kg (P = 0.005), had less than three parturitions (P = 0.001) and milk production of 5-7 litres. The number of animals present at the farm, number of species, the nearby village, etc., played a significant role in the prevalence of the disease, rather than the condition of the farm. The results on haematological studies revealed a considerable decrease in RBC, while the decrease in ESR in a significantly (P = 0.05) greater number of buffaloes showed a positive or doubtful reaction to mammalian PPD. Total leukocyte count and lymphocyte percentages were higher in a considerably greater number of buffaloes. However, while changes in monocyte and eosinophil count were less significant, combined monocyte count in both positive and doubtful reactors was higher in a considerable number of buffaloes. While the results on serum proteins revealed an increase in serum total proteins and globulins in a significantly greater number of buffaloes, they had a positive reaction to mammalian PPD. A significant, positive relationship of serum total proteins with globulins in positive (r = 0.918; P = 0.0001) and doubtful reactors (r = 0.691; P = 0.0015).

orrigendum to ‘‘Bovine tuberculosis in Europe from the perspective f an officially tuberculosis free country: Trade, surveillance and iagnostics’’ [Vet. Microbiol. 151 (2011) 152–159] ene Schiller *, W. Ray Waters , H. Martin Vordermeier , Thomas Jemmi , ichael Welsh , Nicolas Keck , Adam Whelan , Eamonn Gormley , aria Laura Boschiroli , Jean Louis Moyen , Carmen Vela , Monica Cagiola , yce M. Buddle , Mitchell Palmer , Tyler Thacker , Bruno Oesch l

In this trial we assessed the effect of soluble alginates on murine cells. Mouse peritoneal monocytes were stimulated in vitro with a solution of alginate. The production of TNF-alpha and nitric oxide (NO), the expression of surface molecules CD80 and CD86, and the ability of monocytes to phagocyte bacteria were assessed, in order to evaluate the effect of alginate on cell functionality. We showed that mouse peritoneal monocytes stimulated with alginate produce NO and TNF-alpha. In addition, alginate is able also to increase their phagocytic activity and to a lesser extent also to increase the expression of CD80. Even with different degrees, it implies that alginates per se act directly on immune response, being able to effectively stimulate proinflammatory activity. These findings corroborate the idea that alginates can represent interesting adjuvants to use to increase the efficacy of antigenic stimulation.