Bovis-infected Cattle Research Articles

An injectable subunit vaccine containing Elongation Factor Tu and Heat Shock Protein 70 partially protects American bison from Mycoplasma bovis infection.

Mycoplasma bovis (M. bovis) is the etiologic agent of high mortality epizootics of chronic respiratory disease in American bison (Bison bison). Despite the severity of the disease, no efficacious commercial vaccines have been licensed for the prevention of M. bovis infection in bison. Elongation factor thermal unstable (EFTu) and Heat Shock Protein 70 (Hsp70, DnaK) are highly conserved, constitutively expressed proteins that have previously been shown to provide protection against M. bovis infection in cattle. To assess the suitability of EFTu and Hsp70 as vaccine antigens in bison, the immune response to and protection conferred by an injectable, adjuvanted subunit vaccine comprised of recombinantly expressed EFTu and Hsp70 was evaluated. Vaccinates developed robust antibody and cellular immune responses against both EFTu and Hsp70 antigens. To assess vaccine efficacy, unvaccinated control and vaccinated bison were experimentally challenged with bovine herpes virus-1 (BHV-1) 4 days prior to intranasal infection with M. bovis. Vaccinated bison displayed reductions in joint infection, lung bacterial loads, and lung lesions compared to unvaccinated controls. Together, these results showed that this subunit vaccine reduced clinical disease and bacterial dissemination from the lungs in M. bovis challenged bison and support the further development of protein subunit vaccines against M. bovis for use in bison.

Tuberculosis in wild animals in India.

India is renowned for its complex megadiverse ecosystems and abundant biodiversity. Bovine tuberculosis (bTB) often remains synonymous with Mycobacterium bovis infection in cattle. The domain of tuberculosis (TB) among wild animals, induced by members of the Mycobacterium tuberculosis complex organisms (MTBC), is often underexplored and underreported in India. Within this context, instances of wild animal tuberculosis (wTB) have manifested across both captive and free-roaming animals. The sources contributing to wTB in animals can be human, animal, or environmental factors, thus illuminating the complex transmission pathways. The diagnosis of wTB continues to pose a formidable challenge, a consequence of the expansive taxonomic diversity in both the host and the pathogen. Complications inherent in acquiring samples from wildlife, the absence of standardized diagnostic protocols, limited insights into infection prevalence, and resource constraints compound diagnosis. Amidst these, adopting the comprehensive One Health paradigm surfaces as an imperative, accentuating the interconnectedness bridging human, animal, and environmental health. Recognizing key stakeholders and fostering intersectoral collaboration to provide enhanced diagnostic techniques driven by skilled personnel and advanced infrastructure play pivotal roles in a comprehensive strategy. Additionally, leveraging vaccination efforts contributes to effective control. A national wTB surveillance program is a cornerstone, ensuring an integrated and holistic approach to disease management. Through this review, we delve into the current landscape of wTB in India, unveiling its multifaceted challenges, and further explore the multifarious strategies that the One Health approach proffers in this dynamic endeavor.

Confirmation of the invasive American mink (Neogale vison) as carrier of Mycobacterium bovis in southern Chile

Bovine tuberculosis (bTB) is a chronic infectious-contagious disease with worldwide distribution, caused by the zoonotic pathogen Mycobacterium bovis. It is believed that the existence of wild cycles may hamper the success of bTB control strategies worldwide, where wildlife species could be reservoirs of this bacterial agent across their native (e.g., European badgers, wild boars) or non-indigenous (e.g., brushtail possum in New Zealand) ranges. However, further studies are required to understand the potential risk posed by non-native wildlife in becoming carriers of M. bovis in other neglected latitudes, such as the Southern Cone of South America. In this study, we performed a specific M. bovis-RD4 real-time PCR (qPCR) assay to detect bacterial DNA in tissues from the invasive American mink (Neogale vison) in Los Ríos region, Chile. We detected M. bovis DNA in blood samples collected from 13 out of 186 (7 %) minks with known sex and age. We did not find any significant differences in bacterial DNA detection according to mink sex and age. We found that 92 % (12/13) of specimens were positive in lung, 39 % (5/13) in mediastinal lymph node, and 15 % (2/13) in mesenteric lymph node, which suggest that both respiratory and digestive pathways as possible routes of transmission between infected hosts and minks. Our study is the first report on M. bovis molecular detection in invasive minks in an area where the largest cattle population in the country is located. Furthermore, this area is characterized by a low within-herd prevalence of M. bovis infection in cattle, with a relatively low number of infected herds, and so far, no attempts at eradicating the disease have been successful.

The Establishment of a Novel γ-Interferon In Vitro Release Assay for the Differentiation of Mycobacterial Bovis-Infected and BCG-Vaccinated Cattle.

BCG vaccination is increasingly reconsidered in the effective prevention of bovine tuberculosis (bTB). However, the primary challenge in BCG vaccination for cattle is the lack of a technique for differentiating between infected and vaccinated animals (DIVA). This study aimed to establish a novel DIVA diagnostic test based on an interferon-gamma in vitro release assay (IGRA). The plasmid encoding three differential antigens (Rv3872, CFP-10, and ESAT-6) absent in BCG genes but present in virulent M. bovis was previously constructed. Thus, a recombinant protein called RCE (Rv3872, CFP-10, and ESAT-6) was expressed, and an RCE-based DIVA IGRA (RCE-IGRA) was established. The RCE concentration was optimized at 4 μg/mL by evaluating 97 cattle (74 of which were bTB-positive, and 23 were negative) using a commercial IGRA bTB diagnostic kit. Further, 84 cattle were tested in parallel with the RCE-IGRA and commercial PPD-based IGRA (PPD-IGRA), and the results showed a high correlation with a kappa value of 0.83. The study included BCG-vaccinated calves (n = 6), bTB-positive cattle (n = 6), and bTB-negative non-vaccinated calves (n = 6). After 3 months post-vaccination, PPD-IGRA generated positive results in both vaccinated and infected calves. However, RCE-IGRA developed positive results in infected calves but negative results in vaccinated calves. In conclusion, this DIVA method has broad prospects in differentiating BCG vaccination from natural infection to prevent bTB.

Proteomic and Lipidomic Profiling of Calves Experimentally Co-Infected with Influenza D Virus and Mycoplasma bovis: Insights into the Host-Pathogen Interactions.

The role of Influenza D virus (IDV) in bovine respiratory disease remains unclear. An in vivo experiment resulted in increased clinical signs, lesions, and pathogen replication in calves co-infected with IDV and Mycoplasma bovis (M. bovis), compared to single-infected calves. The present study aimed to elucidate the host-pathogen interactions and profile the kinetics of lipid mediators in the airways of these calves. Bronchoalveolar lavage (BAL) samples collected at 2 days post-infection (dpi) were used for proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, lipidomic analyses were performed by LC-MS/MS on BAL samples collected at 2, 7 and 14 dpi. Whereas M. bovis induced the expression of proteins involved in fibrin formation, IDV co-infection counteracted this coagulation mechanism and downregulated other acute-phase response proteins, such as complement component 4 (C4) and plasminogen (PLG). The reduced inflammatory response against M. bovis likely resulted in increased M. bovis replication and delayed M. bovis clearance, which led to a significantly increased abundance of oxylipids in co-infected calves. The identified induced oxylipids mainly derived from arachidonic acid; were likely oxidized by COX-1, COX-2, and LOX-5; and peaked at 7 dpi. This paper presents the first characterization of BAL proteome and lipid mediator kinetics in response to IDV and M. bovis infection in cattle and raises hypotheses regarding how IDV acts as a co-pathogen in bovine respiratory disease.

Detection of Mycobacterium bovis in nasal swabs from communal goats (Capra hircus) in rural KwaZulu-Natal, South Africa.

Animal tuberculosis, caused by Mycobacterium bovis, presents a significant threat to both livestock industries and public health. Mycobacterium bovis tests rely on detecting antigen specific immune responses, which can be influenced by exposure to non-tuberculous mycobacteria, test technique, and duration and severity of infection. Despite advancements in direct M. bovis detection, mycobacterial culture remains the primary diagnostic standard. Recent efforts have explored culture-independent PCR-based methods for identifying mycobacterial DNA in respiratory samples. This study aimed to detect M. bovis in nasal swabs from goats (Capra hircus) cohabiting with M. bovis-infected cattle in KwaZulu-Natal, South Africa. Nasal swabs were collected from 137 communal goats exposed to M. bovis-positive cattle and 20 goats from a commercial dairy herd without M. bovis history. Swabs were divided into three aliquots for analysis. The first underwent GeneXpert® MTB/RIF Ultra assay (Ultra) screening. DNA from the second underwent mycobacterial genus-specific PCR and Sanger sequencing, while the third underwent mycobacterial culture followed by PCR and sequencing. Deep sequencing identified M. bovis DNA in selected Ultra-positive swabs, confirmed by region-of-difference (RD) PCR. Despite no other evidence of M. bovis infection, viable M. bovis was cultured from three communal goat swabs, confirmed by PCR and sequencing. Deep sequencing of DNA directly from swabs identified M. bovis in the same culture-positive swabs and eight additional communal goats. No M. bovis was found in commercial dairy goats, but various NTM species were detected. This highlights the risk of M. bovis exposure or infection in goats sharing pastures with infected cattle. Rapid Ultra screening shows promise for selecting goats for further M. bovis testing. These techniques may enhance M. bovis detection in paucibacillary samples and serve as valuable research tools.

Temporal dynamics of the early immune response following Mycobacterium bovis infection of cattle

Bovine tuberculosis is an infectious disease of global significance that remains endemic in many countries. Mycobacterium bovis infection in cattle is characterized by a cell-mediated immune response (CMI) that precedes humoral responses, however the timing and trajectories of CMI and antibody responses determined by newer generation assays remain undefined. Here we used defined-antigen interferon-gamma release assays (IGRA) and an eleven-antigen multiplex ELISA (Enferplex TB test) alongside traditional tuberculin-based IGRA and IDEXX M. bovis antibody tests to assess immune trajectories following experimental M. bovis infection of cattle. The results show CMI responses developed as early as two-weeks post-infection, with all infected cattle testing positive three weeks post-infection. Interestingly, 6 of 8 infected animals were serologically positive with the Enferplex TB assay as early as 4 weeks post-infection. As expected, application of the tuberculin skin test enhanced subsequent serological reactivity. Infrequent M. bovis faecal shedding was observed but was uncorrelated with observed immune trajectories. Together, the results show that early antibody responses to M. bovis infection are detectable in some individuals and highlight an urgent need to identify biomarkers that better predict infection outcomes, particularly for application in low-and-middle income countries where test-and-slaughter based control methods are largely unfeasible.

Antemortem detection of Mycobacterium bovis in nasal swabs from African rhinoceros

Mycobacterium bovis (M. bovis) infection has been identified in black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros populations in Kruger National Park, South Africa. However, it is unknown whether M. bovis infected rhinoceros, like humans and cattle, can shed mycobacteria in respiratory secretions. Limited studies have suggested that rhinoceros with subclinical M. bovis infection may present minimal risk for transmission. However, recent advances that have improved detection of Mycobacterium tuberculosis complex (MTBC) members in paucibacillary samples warranted further investigation of rhinoceros secretions. In this pilot study, nasal swab samples from 75 rhinoceros with defined infection status based on M. bovis antigen-specific interferon gamma release assay (IGRA) results were analysed by GeneXpert MTB/RIF Ultra, BACTEC MGIT and TiKa–MGIT culture. Following culture, speciation was done using targeted PCRs followed by Sanger sequencing for mycobacterial species identification, and a region of difference (RD) 4 PCR. Using these techniques, MTBC was detected in secretions from 14/64 IGRA positive rhinoceros, with viable M. bovis having been isolated in 11 cases, but not in any IGRA negative rhinoceros (n = 11). This finding suggests the possibility that MTBC/M. bovis-infected rhinoceros may be a source of infection for other susceptible animals sharing the environment.

Spatial association of Mycobacterium bovis infection in cattle and badgers at the pasture interface in an endemic area in France.

Despite control and surveillance programmes, Mycobacterium bovis, the main aetiologic agent of bovine tuberculosis (bTB), is still detected on cattle farms and in wildlife populations in France, especially in badgers in the French Côte-d'Or département. The aim of our study was to find out if infected badgers were trapped significantly closer to pastures of infected farms than non-infected badgers and, if so, to determine the most efficient distance around those pastures for badger trapping, particularly for surveillance purposes. We studied two subareas (southern and northern), chosen based on natural barriers to badger movements and according to the presence of pastures belonging to infected farms (POIFs) and infected or non-infected badgers. In each subarea, we computed the shortest distances D0 and D between badgers trapped a given year n between 2015 and 2019 (n=59 infected and n=1535 non-infected badgers for D0; n=53 infected and n=1476 non-infected badgers for D) and POIFs designated as infected between the year n - 4 and n+1 (respectively n=373 and n=388 POIFs). D0 was calculated without considering spoligotypes, while D was calculated considering the possible epidemiological link between infected badgers and POIFs by using bTB spoligotype information. Then, we computed the observed mean and median of the D0 and D distances and used a bootstrap analysis to test if infected badgers were found significantly closer to POIFs than non-infected badgers. We observed that infection of badgers was not independent of distance from POIF in both subareas but distances (D0 or D) were different between the northern and southern subarea. In the northern subarea, which displays a mosaic landscape (mean and median D distances were respectively 612m and 303m for infected badgers), infected badgers indeed were trapped closer to POIFs, considering D0 and D. In the southern subarea, predominantly forested, infected badgers were significantly closer to POIFs than non-infected badgers when considering D0 but not for D (mean and median D distances were respectively 7148m and 4831m for infected badgers). These results will help to determine the most efficient distance from POIFs to trap badgers to determine their infection status in countryside landscapes. They also highlight the need to better understand the epidemiological systems at play in more forested landscapes where badgers may behave differently or other susceptible sympatric wild species might play a more important role in the circulation of M. bovis, both phenomena contributing to badger infection at greater distances from POIFs.

Relationship between ambient temperature at sampling and the interferon gamma test result for bovine tuberculosis in cattle

Bovine tuberculosis (bTB) is a disease of significant economic and zoonotic importance, therefore, optimising tests for the identification of Mycobacterium bovis infected cattle is essential. The Interferon Gamma (IFN-γ) Release Assay (IGRA) can diagnose M. bovis infected cattle at an early stage, is easy to perform and can be used alongside skin tests for confirmatory purposes or to increase diagnostic sensitivity. It is known that IGRA performance is sensitive to environmental conditions under which samples are taken and transported. In this study, the association between the ambient temperature on the day of bleeding and the subsequent IGRA result for bTB was quantified using field samples from Northern Ireland (NI). Results of 106,434 IGRA results (2013–2018) were associated with temperature data extracted from weather stations near tested cattle herds. Model dependent variables were the levels of IFN-γ triggered by avian purified protein derivative (PPDa), M. bovis PPD (PPDb), their difference (PPD(b-a)) as well as the final binary outcome (positive or negative for M. bovis infection). IFN-γ levels after both PPDa and PPDb stimulation were lowest at the extremes of the temperature distribution for NI. The highest IGRA positive probability (above 6%) was found on days with moderate maximum temperatures (6–16 °C) or moderate minimum temperatures (4–7 °C). Adjustment for covariates did not lead to major changes in the model estimates. These data suggest that IGRA performance can be affected when samples are taken at high or low temperatures. Whilst it is difficult to exclude physiological factors, the data nonetheless supports the temperature control of samples from bleeding through to laboratory to help mitigate post-collection confounders.

Evaluation of the accuracy of the IDvet serological test for Mycoplasma bovis infection in cattle using latent class analysis of paired serum ELISA and quantitative real-time PCR on tonsillar swabs sampled at slaughter.

Mycoplasma bovis (Mbovis) was first detected in cattle in New Zealand (NZ) in July 2017. To prevent further spread, NZ launched a world-first National Eradication Programme in May 2018. Existing diagnostic tests for Mbovis have been applied in countries where Mbovis is endemic, for detecting infection following outbreaks of clinical disease. Diagnostic test evaluation (DTE) under NZ conditions was thus required to inform the Programme. We used Bayesian Latent Class Analysis on paired serum ELISA (ID Screen Mycoplasma bovis Indirect from IDvet) and tonsillar swabs (qPCR) for DTE in the absence of a gold standard. Tested samples were collected at slaughter between June 2018 and November 2019, from infected herds depopulated by the Programme. A first set of models evaluated the detection of active infection, i.e. the presence of Mbovis in the host. At a modified serology positivity threshold of SP%> = 90, estimates of animal-level ELISA sensitivity was 72.8% (95% credible interval 68.5%-77.4%), respectively 97.7% (95% credible interval 97.3%-98.1%) for specificity, while the qPCR sensitivity was 45.2% (95% credible interval 41.0%-49.8%), respectively 99.6% (95% credible interval 99.4%-99.8%) for specificity. In a second set of models, prior information about ELISA specificity was obtained from the National Beef Cattle Surveillance Programme, a population theoretically free-or very low prevalence-of Mbovis. These analyses aimed to evaluate the accuracy of the ELISA test targeting prior exposure to Mbovis, rather than active infection. The specificity of the ELISA for detecting exposure to Mbovis was 99.9% (95% credible interval 99.7%-100.0%), hence near perfect at the threshold SP%=90. This specificity estimate, considerably higher than in the first set of models, was equivalent to the manufacturer's estimate. The corresponding ELISA sensitivity estimate was 66.0% (95% credible interval 62.7%-70.7%). These results confirm that the IDvet ELISA test is an appropriate tool for determining exposure and infection status of herds, both to delimit and confirm the absence of Mbovis.

Diagnostic Accuracy of an Indirect Enzyme Linked Immunosorbent Assay (iELISA) for Screening of Babesia bovis in Cattle from West Africa.

The epidemiology of corresponding tick-borne diseases has changed as a result of the recent introduction of Rhipicephalus (Boophilus) microplus to West Africa. The current study aimed to assess the diagnostic performance of an indirect ELISA for the detection of Babesia bovis infection in cattle. In a cross-section study, using a Bayesian Latent Class Model and iELISA diagnostic test for cattle babesiosis due to Babesia bovis, accuracy has been assessed with RT-PCR as an imperfect reference test. A total of 766 cattle were tested. The optimal diagnostic performances were obtained with 5% percentage of positivity. Sensitivity and specificity were, respectively, 0.94 [Cr. I.: 0.85-0.99] and 0.89 [Cr. I.: 0.87-0.92]. Additional diagnostic characteristics revealed that the Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were 96.6% [Cr. I.: 92.7-100%] and 82.2% [Cr. I.: 72-93%]. Overall, this test well discriminates an infected status from an uninfected status considering the area under the ROC curve (AUC) which was 0.78 [Cr. I: 0.72-0.85] and a Diagnostic Odds Ratio (DOR) of 127.8 [Cr. I.: 10.43-1562.27]. The AUC was significantly higher than 0.5 (p < 10-5). In consequence, this serologic assay could be suitable in moderate to high prevalence assessments.

Differential detection of IgM and IgG antibodies to chimeric antigens in bovine tuberculosis

Recent studies have suggested the potential of innovative serologic tests for accurate and rapid detection of bovine tuberculosis (bTB). Dual Path Platform (DPP) technology has been used to develop rapid animal-side antibody tests for Mycobacterium bovis infection in a range of livestock and wildlife host species. The present study evaluated diagnostic performance of DPP BovidTB IgM/IgG assay designed for differential detection of bovine IgM and IgG antibodies against two chimeric antigens, DID38 and TBf2, respectively, using 662 well-characterized serum samples from M. bovis-infected and bTB-free cattle collected in the United States, Great Britain, France, and South Africa. Test sensitivity and specificity ranged from 71% to 100% and from 95% to 100%, respectively, depending on the country, with overall accuracy of 83%. No significant risk of cross-reactivity with serum samples from cattle infected with most relevant species of mycobacteria other than M. bovis was found. The DPP BovidTB IgM/IgG assay may be suitable for use in multi-test algorithms to improve current strategies for bTB surveillance.

Bovine Mastitis in Egypt: Bacterial Etiology and Evaluation of Diagnostic Biomarkers

In current study a total of 153 tuberculous lesion samples collected from cattle after PM examination (69 from El-Menufia, 47 from El-Sharkia and 37 from El-Qaliobia governorates) were subjected to bacteriological examination. Eighty-three samples showed the AF bacilli in direct smears stained by ZN stain at a percentage of 54.2% while by the culture method, M. bovis was isolated from 100 (65.4%) samples. Sixty-five isolates, confirmed to be M. bovis by conventional PCR targeting Mpb70, were tested for the sensitivity to anti-tuberculous drugs and 25 isolates showed resistance to at least one tested drug. The overall resistance to EMB was 3.1% and resistance to STR, INH and RIF were 20, 30.8 and 32.3% respectively. MDR was detected in 14 (21.5%) isolates. For DNA sequencing, PCR targeting rpoB gene, conferring rifampicin resistance, was performed on selected 6 isolates and identified three mutations designated as H526Y (4/6), S531L (1/6) and D516V (1/6) and the sequences were submitted to the NCBI GenBank with the accession numbers (OM448574, OM448575, OM448576, OM448577, OM448578 and OM448579). The obtained results are epidemiologically significant and augment the necessity of further investigation on ante-mortem identification of M. bovis infection in cattle to minimize public risk.

Bacteriological and Molecular studies on Mycobacterium Bovis in Cattle, with Special Reference to its Antimicrobial Resistance

In current study a total of 153 tuberculous lesion samples collected from cattle after PM examination (69 from El-Menufia, 47 from El-Sharkia and 37 from El-Qaliobia governorates) were subjected to bacteriological examination. Eighty-three samples showed the AF bacilli in direct smears stained by ZN stain at a percentage of 54.2% while by the culture method, M. bovis was isolated from 100 (65.4%) samples. Sixty-five isolates, confirmed to be M. bovis by conventional PCR targeting Mpb70, were tested for the sensitivity to anti-tuberculous drugs and 25 isolates showed resistance to at least one tested drug. The overall resistance to EMB was 3.1% and resistance to STR, INH and RIF were 20, 30.8 and 32.3% respectively. MDR was detected in 14 (21.5%) isolates. For DNA sequencing, PCR targeting rpoB gene, conferring rifampicin resistance, was performed on selected 6 isolates and identified three mutations designated as H526Y (4/6), S531L (1/6) and D516V (1/6) and the sequences were submitted to the NCBI GenBank with the accession numbers (OM448574, OM448575, OM448576, OM448577, OM448578 and OM448579). The obtained results are epidemiologically significant and augment the necessity of further investigation on ante-mortem identification of M. bovis infection in cattle to minimize public risk.

High-resolution transcriptomics of bovine purified protein derivative-stimulated peripheral blood from cattle infected with Mycobacterium bovis across an experimental time course

ObjectivesImproved bovine tuberculosis (bTB) diagnostics with higher sensitivity and specificity are urgently required. A better understanding of the peripheral blood transcriptional response of Mycobacterium bovis-infected animals after bovine purified protein derivative (PPD-b) stimulation of whole blood—an important component of current bTB diagnostics—will provide new information for development of better diagnostics. MethodsRNA sequencing (RNA-seq) was used to study the peripheral blood transcriptome after stimulation with PPD-b across four time points (−1 wk pre-infection, and +1 wk, +2 wk, and +10 wk post-infection) from a 14-week M. bovis infection time course experiment with ten age-matched Holstein-Friesian cattle. ResultsIn vitro PPD-b stimulation of peripheral blood from M. bovis-infected and non-infected cattle elicited a strong transcriptional response. Comparison of PPD-b stimulated, and unstimulated samples revealed higher expression of genes encoding cytokine receptors, transcription factors, and interferon-inducible proteins. Lower expression was seen for genes encoding proteins involved in antimicrobial activity, C-type lectin receptors, inhibition of signal transduction, and genes encoding metal ion transporters. ConclusionsA transcriptional signature associated with the peripheral blood response to PPD-b stimulation consisting of 170 genes was identified exclusively in the post-infection time points. Therefore, this represents a panel of potential biomarkers of M. bovis infection.

Parameter estimates to support future risk assessment of Mycobacterium bovis in raw milk cheese

Zoonotic tuberculosis, caused by Mycobacterium bovis, is mainly linked to the consumption of raw milk from infected cows. In many countries, cases are rare, due to pasteurisation of milk and national programmes to control M. bovis infection in cattle. Speciality cheeses, which are often produced using raw milk, present challenges to risk managers in countries where M. bovis is endemic or (re-) emerging. A key concern is the potential risk of zoonotic transmission of M. bovis via the consumption of dairy products produced using raw milk originating from herds infected with M. bovis (bovine tuberculosis, bTB). The aim of this study was to determine parameter estimates to support the future risk assessment of M. bovis in raw milk cheese. In this study, the hazard was identified as viable M. bovis organisms in raw milk cheese. Parameters of interest in this study related to exposure assessment (the estimated extent of human exposure to viable M. bovis organisms) and hazard characterisation (the risk posed to human health following exposure to viable M. bovis organisms). The pathway for exposure assessment was visualised using a conceptual framework, which describes the steps through which M. bovis may be transferred from an infected animal(s) through manufacturing to the final cheese product. Estimation of most parameters for exposure assessment and hazard characterisation was undertaken using systematic literature reviews. Estimates could be derived for many parameters, but not all. In particular, the number of M. bovis organisms excreted in the milk and present in the faeces of infected cattle are unknown. There is zero-tolerance for M. bovis in foods of animal origin destined for human consumption in European legislation. This work has highlighted important gaps in knowledge, and areas for further research. For each of the parameters for which estimates are available, we outline the types/sources of uncertainty as reflected in relevant published papers. In any future application of these parameter estimates, care will be needed to reflect the uncertainties associated with these elements of exposure assessment.

Morphometric analysis of schistosome eggs recovered from human urines in communities along the shoreline of Oyan River Dam in Ogun State, Nigeria.

There are growing concerns that communities characterized with surface water, where both humans and livestock interact for agricultural, domestic, cultural and recreational purposes, are likely to support hybridization between schistosome species infecting humans and livestock. This study therefore investigated the morphometrics of schistosome eggs recovered from human urine samples in four schistosomiasis endemic communities (Imala-Odo, Abule-Titun, Apojula and Ibaro-Oyan) along the banks of Oyan River Dam in Ogun State, Nigeria. Recovered eggs were counted, photographed, and measured with IC Measure™ for total length, maximum width and a ratio of egg shape. A total of 1984 Schistosoma eggs were analysed. Two major egg morphotypes were identified: the first represented 67.8% of the eggs, with the typical round to oval shape and mean length and width of 166 μm, 66.8 μm, respectively; the second represented 32.2% of the eggs and are more elongated, with a mean length of 198 μm, and width of 71.3 μm. Our results revealed significant variations in sizes of the schistosome eggs recovered (length: t = -35.374, degrees of freedom (df) = 1982, P = 0.000; weight: t = -10.431, df = 1982, P = 0.000), with the atypical shaped eggs appearing more elongated than expected. These eggs might represent individuals with some degree of contribution from Schistosoma bovis or possibly other Schistosoma species known to be present in Nigeria. Hence, this observation calls for further molecular studies to establish the genetic information about the miracidia from both atypical and typical eggs. It is also important to establish the presence of bona fide S. bovis infection in cattle and vector snails in the presumptive areas of hybridization.

Novel polyprotein antigens designed for improved serodiagnosis of bovine tuberculosis

Recent studies have demonstrated potential for serologic assays to improve surveillance and control programs for bovine tuberculosis. Due to the animal-to-animal variation of the individual antibody repertoires observed in bovine tuberculosis, it has been suggested that serodiagnostic sensitivity can be maximized by use of multi-antigen cocktails or genetically engineered polyproteins expressing immunodominant B-cell epitopes. In the present study, we designed three novel multiepitope polyproteins named BID109, TB1f, and TB2f, with each construct representing a unique combination of four full-length peptides of Mycobacterium bovis predominantly recognized in bovine tuberculosis. Functional performance of the fusion antigens was evaluated using multi-antigen print immunoassay (MAPIA) and Dual Path Platform (DPP) technology with panels of monoclonal and polyclonal antibodies generated against individual proteins included in the fusion constructs as well as with serum samples from M. bovis-infected and non-infected cattle, American bison, and domestic pigs. It was shown that epitopes of each individual protein were expressed in the fusion antigens and accessible for efficient binding by the respective antibodies. The three fusion antigens demonstrated stronger immunoreactivity in MAPIA than that of single protein antigens. Evaluation of the fusion antigens in DPP assay using serum samples from 125 M. bovis-infected and 57 non-infected cattle showed the best accuracy (∼84 %) for TB2f antigen composed of MPB70, MPB83, CFP10, and Rv2650c proteins. Thus, the study results suggest a potential for the multiepitope polyproteins to improve diagnostic sensitivity of serologic assays for bovine tuberculosis.

The Risk of False-Positive Serological Results for Paratuberculosis in Mycobacterium bovis-Infected Cattle.

Both bovine tuberculosis (BTB) and paratuberculosis (paraTB) continue to cause significant economic losses in cattle breeding; in addition, their etiological agents have zoonotic potential. Although the diagnostics of both diseases are still being improved, problems still remain, such as the potential for cross-reactivity to the antigens used in tests. The aim of the present study was to confirm whether animals known to harbor Mycobacterium bovis antibodies are at increased risk of yielding positive results in paraTB serotesting and, additionally, to verify the accuracy of three commonly used methods for confirming M. bovis infection: ELISA, the tuberculin skin test (TST), and the presence of gross lesions. Material was collected from 98 dairy cattle suspected of BTB due to TST-positive results. During postmortem examination, gross lesions were assessed visually. Blood, lymph nodes, and TB-suspected organs were collected. Serum was obtained from the collected blood and tested serologically for TB and paraTB. The tissues underwent standard microbiological testing for M. tuberculosis complex. Among the 98 TST-positive individuals, tuberculous gross lesions were detected in 57 (58.1%), MTBC were isolated in 83 (84.7%), and the ELISA test was positive for 21 (21.4%). None of the lesions characteristic for paraTB were detected. The chance of obtaining a positive TB result by ELISA was seven times higher using the ELISA-paraTB method; hence, there is a significant risk of obtaining false-positive serological results for paraTB in M. bovis-infected cattle. However, the hypothesis that infection of M. bovis or prior TST performance may have boosted the host immune response and therefore increased the sensitivity of the paraTB-ELISA cannot be excluded.