Bovine CD4 T cell memory subsets and immunological markers in Mycobacterium bovis infection and vaccination

Mycobacterium bovis is the causative agent of tuberculosis in cattle. M. bovis is a member of the Mycobacterium tuberculosis complex (Mtbc) which also includes M. tuberculosis, M. caprae, M. microti, M. africanum, M. canetti, M. pinnipedii, M. suricattae, M. mungi, and M. bovis Bacillus Calmette Guerin (BCG) 1-3. Despite what the nomenclature may imply, members of this group are not host species specific. The host range of M. bovis appears to be the broadest of the complex, causing disease in a number of species including humans 4. Great strides have been made over the past century both toward the control of bovine tuberculosis (bTB) in cattle and also limiting the risk to human exposure (e.g., pasteurization of milk for dairy products); however, the disease still has great socioeconomic impact for livestock farmers. It is estimated that over 50 million cattle are currently infected worldwide, costing around $3 billion annually5. The WHO (World Health Organization), in conjunction with FAO (Food and Agriculture Organization of the United Nations) and OIE (Office International des ÃÂpizooties), classify bTB as a neglected zoonosis 6,7. A crucial component of the immune response to TB in humans, cattle and mice is the production of interferon (IFN)-γ by T helper 1 (Th1) CD4 T cells 8-12. Immune deficiencies affecting CD4 T cells [e.g., human immunodeficiency virus (HIV) infection] and IL-12/IFN-γ /STAT1 signaling pathways result in more severe disease upon TB infection in humans 13,14. In fact, IFN-γ release assays (IGRA) and delayed type hypersensitivity (i.e., skin test) responses are markers of infection in cattle and humans (reviewed respectively by Schiller et al. 15 and Walzl et al. 16). Diagnostic IGRAs are measures of ‘ex vivo’ immune responses relying on rapid production of IFN-γ in response to mycobacterial antigen stimulation in short-term (16–24 h) whole blood or peripheral blood mononuclear cell (PBMC) cultures. These ex vivo assays are generally considered a measure of T cell effector responses 12,17. Most protective bTB vaccines elicit ex vivo IFN-γ responses; however, not all vaccines inducing this response are protective 5,18. Cultured assays (i.e., culture of cells for 7-14 days before IFN-γ measurement) are assumed to quantify memory responses, primarily central memory T cells (Tcm) in humans 19-21. In light of the idea that cultured ELISPOT measures Tcm responses, several studies have shown its association with protection against malaria, suppression of viral recrudescence in hepatitis B virus carriers, low virus levels in HIV infection, and favorable outcomes in human TB 22-26. In cattle, responses measured by cultured IFN-γ ELISPOT following vaccination are the best-known positive correlate of protection in vaccine and challenge experiments 27-31. Increasing interest has risen in order to characterize and assess the role of polyfunctionality in both protective and detrimental immune responses in TB. Polyfunctional T cells simultaneously produce two or more cytokines with IFN-γ, IL-2, and tumor necrosis factor-α (TNF-α) being the most commonly measured Th-1 cytokines 32,33. Association between protection and vaccination-induced polyfunctional T cells has been mainly studied in small animal models 34,35. In humans, strong polyfunctional responses are detected in M. tuberculosis-infected individuals, high IL-2 production is associated with a positive clinical status (e.g., latent or treated disease), while a strong IFN-γ/TNF-α response is associated with a poor outcome (i.e., active TB) 36. Human polyfunctional responses to vaccination both prior to TB exposure and in previously exposed individuals (i.e., latent infection) are extremely variable. In cattle, T cell polyfunctionality has only been measured upon ex vivo recall stimulation 26,37.These studies found no association between polyfunctional responses measured before challenge and vaccine success. Instead, polyfunctional responses to infection were associated with increased pathology and poor disease outcome 26. Polyfunctional responses by long-term cultured cells for enrichment of Tcm responses have not been evaluated in spite of the fact that cultured IFN-γ ELISPOT is one of the most promising protection correlates in cattle 27-31. The research included in this thesis had the objective to characterize memory cells phenotype and aspects of their functionality in cattle, specifically in response to bTB infection and vaccination. The discrimination of cell phenotype involved in cytokine production under both cultured and ex vivo conditions may be necessary to identify specific correlates of vaccine efficacy, useful for vaccine candidates selection for costly efficacy trials in biosafety level 3 (BSL-3) facilities

Increased TNF-α/IFN-γ/IL-2 and Decreased TNF-α/IFN-γ Production by Central Memory T Cells Are Associated with Protective Responses against Bovine Tuberculosis Following BCG Vaccination

Central memory T cells (Tcm) and polyfunctional CD4 T cell responses contribute to vaccine-elicited protection with both human and bovine tuberculosis (TB); however, their combined role in protective immunity to TB is unclear. To address this question, we evaluated polyfunctional cytokine responses by CD4 T cell effector / memory populations from bacille Calmette Guerin (BCG) vaccinated and non-vaccinated calves prior to and after aerosol challenge with virulent Mycobacterium bovis. Polyfunctional cytokine expression patterns in the response by Tcm, effector memory, and effector T cell subsets were similar between BCG-vaccinated and M. bovis-infected calves; only differing in magnitude (i.e., infected > vaccinated). BCG vaccination, however, did alter the kinetics of the ensuing response to virulent M. bovis infection. Early after challenge (three weeks post-infection), non-vaccinates had greater antigen-specific IFN-γ/TNF-α and lesser IFN-γ/TNF-α/IL-2 responses by Tcm cells than did vaccinated animals. Importantly, these differences were also associated with mycobacterial burden upon necropsy. Polyfunctional responses to ESAT-6:CFP10 (antigens not synthesized by BCG strains) were detected in memory subsets, as well as in effector cells, as early as three weeks after challenge. These findings suggest that cell fate divergence may occur early after antigen priming in the response to bovine TB and that memory and effector T cells may expand concurrently during the initial phase of the immune response. In summary, robust IFN-γ/TNF-α response by Tcm cells is associated with greater mycobacterial burden while IFN-γ/TNF-α/IL-2 response by Tcm cells are indicative of a protective response to bovine TB

Virome characterization in commercial bovine serum batches : a potentially needed testing strategy for biological products

Bovine serum has been widely used as a universal supplement in culture media and other applications, including the manufacture of biological products and the production of synthetic meat. Currently, commercial bovine serum is tested for possible viral contaminants following regional guidelines. Regulatory agencies’ established tests focused on detecting selected animal origin viruses and are based on virus isolation, immunofluorescence, and hemadsorption assays. However, these tests may fail to detect new or emerging viruses in biological products. High-throughput sequencing is a powerful option since no prior knowledge of the viral targets is required. In the present study, we evaluate the virome of seven commercial batches of bovine serum from Mexico (one batch), New Zealand (two batches), and the United States (four batches) using a specific preparation and enrichment method for pooled samples and sequencing using an Illumina platform. A variety of circular replicase-encoding single-stranded (CRESS) DNA families (Genomoviridae, Circoviridae, and Smacoviridae) was identified. Additionally, CrAssphage, a recently discovered group of bacteriophage correlated with fecal contamination, was identified in 85% of the tested batches. Furthermore, sequences representing viral families with single-stranded DNA (Parvoviridae), double-stranded DNA (Polyomaviridae and Adenoviridae), single-stranded RNA (Flaviviridae, Picornaviridae, and Retroviridae), and double-stranded RNA (Reoviridae) were identified. These results support that high-throughput sequencing associated with viral enrichment is a robust tool and should be considered an additional layer of safety when testing pooled biologicals to detect viral contaminants overlooked by the current testing protocols

Immune responses to influenza D virus in calves previously infected with bovine viral diarrhea virus 2

Bovine viral diarrhea virus (BVDV) induces immunosuppression and thymus depletion in calves. This study explores the impact of prior BVDV-2 exposure on the subsequent immune response to influenza D virus (IDV). Twenty 3-week-old calves were divided into four groups. Calves in G1 and G3 were mock-treated on day 0, while calves in G2 and G4 received BVDV. Calves in G1 (mock) and G2 (BVDV) were necropsied on day 13 post-infection. IDV was inoculated on day 21 in G3 calves (mock + IDV) and G4 (BVDV + IDV) and necropsy was conducted on day 42. Pre-exposed BVDV calves exhibited prolonged and increased IDV shedding in nasal secretions. An approximate 50% reduction in the thymus was observed in acutely infected BVDV calves (G2) compared to controls (G1). On day 42, thymus depletion was observed in two calves in G4, while three had normal weight. BVDV-2-exposed calves had impaired CD8 T cell proliferation after IDV recall stimulation, and the α/β T cell impairment was particularly evident in those with persistent thymic atrophy. Conversely, no difference in antibody levels against IDV was noted. BVDV-induced thymus depletion varied from transient to persistent. Persistent thymus atrophy was correlated with weaker T cell proliferation, suggesting correlation between persistent thymus atrophy and impaired T cell immune response to subsequent infections.Veterinary PathobiologyDean of Veterinary Medicin

Comparison of immune response induced by vaccination of calves Curraleiro and Nellore with Mycobacterium bovis-BCG.

Made available in DSpace on 2014-07-29T15:07:27Z (GMT). No. of bitstreams: 1 mayara MAGGIOLI.pdf: 1675170 bytes, checksum: 97b59ecd07956bdec51efc9f0ffbb42a (MD5) Previous issue date: 2010-03-05Curraleiro breed has adapted to the extensive breeding in the Brazilian savanna. These rustic animals seem to be more resistant to regional endemic diseases. Resistance to infection is directly correlated to the host immune response. In order to check if differences might exist among Curraleiro s immune response and the response of other exotic cattle breeds in the savannah region, Curraleiro and Nellore calves were assessed before and after immunization with BCG vaccine. Twelve young calves (around six months of age) were used. After quarantine, the animals were divided into four groups: Control (three calves from each breed) and Vaccinated (three calves from each breed). The mononuclear cells were obtained from peripheral blood at time zero (before vaccination), one, seven and, thirty days after the vaccination. The cells were submitted to phagocytosis test, NO production and analysis by flow cytometry of the following populations: NK, T, CD4 e CD8 as well as for their IFN-production. The intradermic test was realized in all calves forty-five days after vaccination. Macrophages from Curraleiro breed showed superior phagocytosis rates (7.650±3.661) than Nellore macrophages (2.970±1.282). Before BCG vaccination, Curraleiro calves had higher numbers of CD4+ cells (2049±402.3), CD8+ cells (1207±166), TWC1+ cells (1443/mL±384,6) in peripheral blood, than Nellore calves (CD4: 1240/mL ±337; CD8: 772.1/mL ±278.3; TWC1+: 745.5/mL ±67.22). The CD4 and CD8 IFN- positive cells were also higher in Curraleiro animals. Vaccination was able to generate specific immune response on both breeds. Thirty days after vaccination, Curraleiro animals presented greater levels of CD4IFN-positive (202.8±44.96) cells than Nellore ones (143.7±40.43). The findings of this study support the Curraleiro resistance profile in comparison to Nellore breed, due to a better capacity to induce specific immune response to M. bovis-BCG vaccine.A raça Curraleiro se desenvolveu e se adaptou à criação extensiva na região do cerrado brasileiro. Estes animais rústicos parecem ser mais resistentes às doenças endêmicas regionais. A resistência às infecções esta diretamente correlacionada a resposta imune do hospedeiro. Para verificar se existe alguma diferença na resposta imune de animais Curraleiros e outras raças exóticas criadas na região do cerrado, bezerros das raças Curraleiro e Nelore foram avaliados após a vacinação com Mycobacterium bovis- BCG. Foram utilizados animais com idade variando de 6 a 7 meses (n=12), provenientes do estado de Goiás. Depois da quarentena, os animais foram divididos em grupos: controles (3 de cada raça) e vacinados (3 de cada raça). Após obtenção das células mononucleares do sangue periférico nos dias zero (antes da vacinação), um, sete, quinze e trinta dias apos a vacinação, as mesmas foram submetidas a ensaios de fagocitose, produção de NO, quantificação por citometria de fluxo das populações celulares NK, T, CD4 e CD8 e de suas produções de IFN-. Quarenta e cinco dias após a vacinação, os animais foram submetidos ao teste intradérmico. Macrófagos provenientes da raça Curraleira apresentaram naturalmente maiores índices de fagocitose de leveduras não sensibilizadas (7,650±3,661) quando comparados aos dos Nelore (2,970±1,282). Além disso, antes da vacinação, bezerros Curraleiros tiveram no sangue periférico maior número de TCD4+(2049±402,3), TCD8+(1207±166), TWC1+(1443/mL±384,6) que os bezerros Nelores (CD4-1240/mL ±337; CD8-772,1/mL ±278,3; TWC1+-745,5/mL ±67,22). Em adição, as células CD4 e CD8 IFN- positivas foram superiores nos animais Curraleiros. A vacinação foi capaz de induzir resposta imune específica nas duas raças testadas. Trinta dias após a vacinação os Curraleiros apresentavam CD4IFN-positivas (202,8±44,96) superiores aos Nelores (143,7±40,43). Os resultados apresentados nesta dissertação favorecem o perfil de resistência da raça bovina Curraleira quando comparada a raça Nelore devido a melhor capacidade de induzir resposta imune específica à vacina M. bovis-BCG

Bovine CD4 T cell memory subsets and immunological markers in Mycobacterium bovis infection and vaccination

Mycobacterium bovis is the causative agent of tuberculosis in cattle. M. bovis is a member of the Mycobacterium tuberculosis complex (Mtbc) which also includes M. tuberculosis, M. caprae, M. microti, M. africanum, M. canetti, M. pinnipedii, M. suricattae, M. mungi, and M. bovis Bacillus Calmette Guerin (BCG) 1-3. Despite what the nomenclature may imply, members of this group are not host species specific. The host range of M. bovis appears to be the broadest of the complex, causing disease in a number of species including humans 4. Great strides have been made over the past century both toward the control of bovine tuberculosis (bTB) in cattle and also limiting the risk to human exposure (e.g., pasteurization of milk for dairy products); however, the disease still has great socioeconomic impact for livestock farmers. It is estimated that over 50 million cattle are currently infected worldwide, costing around $3 billion annually5. The WHO (World Health Organization), in conjunction with FAO (Food and Agriculture Organization of the United Nations) and OIE (Office International des à  pizooties), classify bTB as a neglected zoonosis 6,7. A crucial component of the immune response to TB in humans, cattle and mice is the production of interferon (IFN)-γ by T helper 1 (Th1) CD4 T cells 8-12. Immune deficiencies affecting CD4 T cells [e.g., human immunodeficiency virus (HIV) infection] and IL-12/IFN-γ /STAT1 signaling pathways result in more severe disease upon TB infection in humans 13,14. In fact, IFN-γ release assays (IGRA) and delayed type hypersensitivity (i.e., skin test) responses are markers of infection in cattle and humans (reviewed respectively by Schiller et al. 15 and Walzl et al. 16). Diagnostic IGRAs are measures of ‘ex vivo’ immune responses relying on rapid production of IFN-γ in response to mycobacterial antigen stimulation in short-term (16–24 h) whole blood or peripheral blood mononuclear cell (PBMC) cultures. These ex vivo assays are generally considered a measure of T cell effector responses 12,17. Most protective bTB vaccines elicit ex vivo IFN-γ responses; however, not all vaccines inducing this response are protective 5,18. Cultured assays (i.e., culture of cells for 7-14 days before IFN-γ measurement) are assumed to quantify memory responses, primarily central memory T cells (Tcm) in humans 19-21. In light of the idea that cultured ELISPOT measures Tcm responses, several studies have shown its association with protection against malaria, suppression of viral recrudescence in hepatitis B virus carriers, low virus levels in HIV infection, and favorable outcomes in human TB 22-26. In cattle, responses measured by cultured IFN-γ ELISPOT following vaccination are the best-known positive correlate of protection in vaccine and challenge experiments 27-31. Increasing interest has risen in order to characterize and assess the role of polyfunctionality in both protective and detrimental immune responses in TB. Polyfunctional T cells simultaneously produce two or more cytokines with IFN-γ, IL-2, and tumor necrosis factor-α (TNF-α) being the most commonly measured Th-1 cytokines 32,33. Association between protection and vaccination-induced polyfunctional T cells has been mainly studied in small animal models 34,35. In humans, strong polyfunctional responses are detected in M. tuberculosis-infected individuals, high IL-2 production is associated with a positive clinical status (e.g., latent or treated disease), while a strong IFN-γ/TNF-α response is associated with a poor outcome (i.e., active TB) 36. Human polyfunctional responses to vaccination both prior to TB exposure and in previously exposed individuals (i.e., latent infection) are extremely variable. In cattle, T cell polyfunctionality has only been measured upon ex vivo recall stimulation 26,37.These studies found no association between polyfunctional responses measured before challenge and vaccine success. Instead, polyfunctional responses to infection were associated with increased pathology and poor disease outcome 26. Polyfunctional responses by long-term cultured cells for enrichment of Tcm responses have not been evaluated in spite of the fact that cultured IFN-γ ELISPOT is one of the most promising protection correlates in cattle 27-31. The research included in this thesis had the objective to characterize memory cells phenotype and aspects of their functionality in cattle, specifically in response to bTB infection and vaccination. The discrimination of cell phenotype involved in cytokine production under both cultured and ex vivo conditions may be necessary to identify specific correlates of vaccine efficacy, useful for vaccine candidates selection for costly efficacy trials in biosafety level 3 (BSL-3) facilities.</p

Viability of Veterinary-Relevant Viruses in Decomposing Tissues over a 90-Day Period Using an In-Vitro System

Depopulation is frequently employed during outbreaks of high-impact animal diseases. Security breaches in sites managing mortality may jeopardize pathogen control efforts as infected carcasses can serve as an infection source. This study evaluated the viability and nucleic acid detection of veterinary-relevant viruses or their surrogates in decomposing tissues. The used viruses were: Senecavirus A1 (SVA), feline calicivirus (FCV), bovine viral diarrhea virus (BVDV), porcine epidemic diarrhea virus (PEDV), bovine alphaherpesvirus 1 (BoHV-1), and swinepox virus (SwPV). Viruses were spiked in three decomposing tissues (swine bone marrow and spleen, and bovine bone marrow) and maintained for 90 days. Samples were kept under two temperature conditions resembling the average soil temperature in central Oklahoma, US, during the winter and summer (5.5 °C and 29.4 °C). At 5.5 °C, SVA and FCV remained viable over the 90 days of the study, followed by BVDV (75 days), BoHV-1 and SwPV (60 days), and PEDV (10 days). At 29.4 °C, SVA remained viable for 45 days, followed by BVDV and BoHV-1 (14 days). SwPV was viable for 10 days, whereas FCV and PEDV were viable for 5 days. Overall, viral nucleic acid detection was not significantly altered during the study. These findings support decision-making and risk management in sites overseeing animal mortality

Resposta imune celular de bezerros Curraleiro Pé-duro e Nelore após vacinação com Mycobacterium bovis-BCG

Submitted by Franciele Moreira ([email protected]) on 2018-02-05T17:42:10Z No. of bitstreams: 2 Artigo - Mayara Fernanda Maggioli - 2013.pdf: 535310 bytes, checksum: f435100d6019169f8177b50438580397 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2018-02-15T13:22:52Z (GMT) No. of bitstreams: 2 Artigo - Mayara Fernanda Maggioli - 2013.pdf: 535310 bytes, checksum: f435100d6019169f8177b50438580397 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2018-02-15T13:22:52Z (GMT). No. of bitstreams: 2 Artigo - Mayara Fernanda Maggioli - 2013.pdf: 535310 bytes, checksum: f435100d6019169f8177b50438580397 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-12O presente estudo teve como objetivo avaliar níveis sanguíneos de células CD4, CD8 e γδ no sangue periférico de bezerros Curraleiro Pé-Duro, bem como a produção específica de IFN-γ por essas células em resposta à vacinação com BCG, através de citometria de fluxo. A resposta imune específica contra BCG também foi avaliada por teste tuberculínico, realizado antes e 45 dias após a vacinação. Para fins de comparação, os mesmos parâmetros foram investigados em bezerros da raça Nelore, uma raça bovina exótica com resistência demonstrado anteriormente. Naturalmente, animais da raça Curraleiro Pé-Duro apresentaram maiores níveis de CD4, CD8 e linfócitos γδ. Em resposta a vacina, Curraleiro Pé-duro mostrou maior capacidade de responder especificamente ao BCG, gerando perfil de resistência (Th1), evidenciado pelo maior número de células CD4+ específicas produtoras de IFN-γ e maior reação cutânea a por tuberculina. Os maiores níveis basais de linfócitos, maior produção de IFN-γ e reação cutânea à prova tuberculínica provavelmente desempenham um papel positivo na proteção/resistência ao Mycobacterium tuberculosis.The present study aimed to assess the CD4, CD8 and gd blood levels for Curraleiro Pé- -duro, as well as the specific IFN-g response after BCG vaccination using flow cytometry. The specific immune response against BCG was also evaluated by tuberculin skin test, performed before and 45 days after the vaccination. For comparison purposes, the same parameters were investigated on Nellore calves, an exotic bovine with resistance previously demonstrated. Naturally, Curraleiro Pé-duro animals had greater levels of CD4, CD8 and γδ lymphocytes (p<0.05). In response to vaccine, Curraleiro Pé-duro showed greater ability to respond specifically to BCG, generating resistance profile (Th1), evidenced by greater number of antigen specific CD4+ cells producing IFN-γ (p<0.05) and also higher tuberculin skin test reaction (p<0.05). Additionally, vaccinated Curraleiro Pé-duro calves had higher CD4 cells numbers than both Nellore control (p<0.05) and vaccinated groups (p<0.05). Curraleiro Pé-duro calves’ higher basal lymphocytes blood level and stronger response in both IFN-γ and tuberculin skin test parameters probably play a positive role on protection/ resistance to Mycobacterium bovis

Evaluation of Ultraviolet Type C Radiation in Inactivating Relevant Veterinary Viruses on Experimentally Contaminated Surfaces

Many swine farms employ UVC treatment in employees’ personal belongings and small tools entering farms as part of the biosecurity protocol to decrease the risk of pathogen introduction into the operation. However, the UVC efficacy in some veterinary viruses is not fully evaluated. This study evaluated the efficacy of ultraviolet type C (UVC) radiation in inactivating seven relevant veterinary viruses: Swine Poxvirus (SwPV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Epidemic Diarrhea Virus (PEDV), Swine Influenza Virus (SIV), Bovine Viral Diarrhea Virus (BVDV), Porcine Parvovirus (PPV), and Senecavirus A (SVA). The experimentally contaminated materials included polystyrene and filter paper. The samples were exposed to UVC for 5 min (total dose of 360 mJ/cm2). The UVC treatment caused a decrease over 4 log10 in SwPV titer on the polystyrene surface, whereas it consistently reduced about 5 log10 in PPV and SVA samples. No viable virus was recovered from PRRSV, PEDV, SIV, and BVDV samples. In filter paper, conversely, the efficacy was reduced. This study provides essential information on the inactivation effectiveness of a specific dose of UVC on important veterinary viruses, further supporting the rational application and strategic guidance for UVC radiation use to disinfect materials