Dissertations / Theses on the topic 'Cattle – Vaccination'

4 pp.
Vaccinating for clostridial diseases is an important part of a ranch health program. These infections can have significant economic impacts on the ranch due to animal losses. There are several diseases caused by different organisms from the genus Clostridia, and most of these are preventable with a sound vaccination program. Many of these infections can progress very rapidly; animals that were healthy yesterday are simply found dead with no observed signs of sickness. In most cases treatment is difficult or impossible, therefore we rely on vaccination to prevent infection. The most common organisms included in a 7-way or 8-way clostridial vaccine are discussed below. By understanding how these diseases occur, how quickly they can progress, and which animals are at risk you will have a chance to improve your herd health and prevent the potential economic losses that come with a clostridial disease outbreak.

Conventional subunit vaccine regimes can be modified in order to stimulate strong immune responses resulting in memory cell formation. An adjuvant system that has gained increased attention in recent years is the use of particulate antigen delivery. Particulate systems have a number of advantages over conventional approaches since they are believed to be taken up preferentially by dendritic cells (DC) where they prolong the release of antigen resulting in enhanced T cell stimulation. Furthermore, they offer a versatile system that allows for targeted delivery of antigen and adjuvant to the same DC. A wide range of particle types have been used to enhance vaccine potency. Poly (Iactic-co-glycolic) acid (PLGA), in particular, has been used successfully by many groups. However, there are a great variety of means to synthesise and characterise the desired particles. This study, firstly, set out to develop and optimise a protocol to generate nanoparticles with defined properties. A number of parameters were evaluated including, particle size, protein loading, protein coating and surface charge. In addition to conventional methods, such as electron microscopy and dynamic light scattering, particles were characterised by novel flow cytometric methods. While particles have been shown to adjuvant candidate vaccine proteins, this property should be enhanced when the particle is targeted to dendritic cells by increasing specific uptake. Specific targeting has previously been performed through either targeting with natural receptor ligands or monoclonal antibodies. However, there is currently conflicting data in other studies as to whether this can be achieved. It was thus the second objective of this study to devise methods to implement this technology and to determine whether targeting can be achieved through either approach. It was 1 Abstract found that there was potential in targeting DC populations with monoclonal antibodies, while targeting with natural ligands yielded more mixed results. As a final component, the adjuvant properties of the particles rationally loaded with antigens and molecular adjuvant was tested in vivo in cattle using a viral challenge model. The experiment had a promising outcome with the vaccine particles inducing both T cell and antibody responses resulting in a degree of protection against virus challenge. Furthermore, it highlighted areas where the system, both the particles and the model, may be improved, which could form the basis of future work.

The study objective was to evaluate the effects of vaccination (respiratory and clostridial vaccination or no vaccination) and deworming (fenbendazole and levamisole or no deworming) of high risk stocker calves on-arrival on health and growth performance. Eighty sale barn origin calves were purchased three separate years (n = 240) from local order buyer. Steers (n = 61) and bulls (n = 179) were received over three days (d –3 to –1). On d 0 calves were stratified by arrival BW and FEC into 20 pens of 4 calves each, and treatment was applied to pens in 2 x 2 factorial. Vaccination increased the likelihood of BRD 1.7 times (P = 0.07) versus calves not vaccinated. Vaccination did not affect gain, but calves receiving dewormer had greater ADG than those not receiving dewormer. Calves that arrived uncastrated or with high fever (≥ 40.0 °C) gained less and were 1.7 and 4.3 times more likely (P < 0.10) to be treated for disease, respectively.

The persistent increase of bovine tuberculosis (bTB) over the past twenty years has put a substantial strain on both the British economy and the welfare of livestock. However, the development of an effective bTB vaccine has been continually hindered by the lack of knowledge on the immune response following Mycobacterium bovis (M. bovis) infection. In collaboration with the TB Research Group at the Veterinary Laboratories Agency (VLA, Surrey), this thesis is part of a much wider strategy managed by the Department of Environment, Food and Rural Agency (DEFRA) aimed at elucidating the immunopathogenesis of M. bovis and to develop more effective infection control measures. The specific focus of this thesis was to enable a stronger understanding of the bovine immune response over different periods of M. bovis infection and to apply this new knowledge in evaluating the efficacy of a novel BCG vaccination. Time Course Study: Knowledge of time dependent cytokine expression following M. bovis infection would aid vaccine development by revealing potential correlates of protection. Interferon gamma (IFN-γ), tumour necrosis factor alpha (TNF-α), interleukin (IL) 4 and 10 expression were analysed using quantitative (q) PCR in formalin fixed bovine lymph nodes following five, twelve and nineteen weeks of M. bovis infection. A strong pro-inflammatory/ T helper 1 (TH1) lymphocyte response was evident at five weeks post M. bovis infection, represented by IFN-γ and TNF-α expression (log2 copies of 6.5 and 2.15, respectively) in the absence of IL4. Between five and twelve weeks of infection, a significant increase was observed in IL10 (log2 copies from 5.97 to 8.27, p<0.01, Mann Whitney test), accompanied by an increase in both IFN-γ (log2 7.53) and TNF-α (log2 3.94). This data conformed to a recently described aspect of TH1 lymphocytes, a ‘self-limiting’ nature in which cells produced both IFN-γ and IL10 with the aim of controlling the heightened pro-inflammatory response. The role of IL10 as an immunosuppressive became evident when comparing cytokine expression between four different types of thoracic lymph node; the left bronchial (LB), cranial mediastinal (CRM), caudal mediastinal (CM) and cranial tracheobronchial (CRT) nodes. The LB and CRM lymph nodes produced significantly higher levels of IFN-γ expression (log2 copies between 8.2 and 10) as compared to the CM and CRT (log2 copies between 2.6 and 5.5, p<0.001, Mann Whitney test). Further analysis of the data as a profile of cytokine expression for each lymph node type revealed that IFN-γ was dominantly expressed within the LB and CRM nodes, whereas within the CM and CRT nodes, IL10 was the dominant cytokine. The former nodes also displayed a higher level of pathological damage (represented by mean percentage area coverage of granuloma, 33.6 and 20%, respectively) as compared to the CM (13%) and the CRT lymph node types (10.8 %). This suggests conflicting roles for IFN-γ and IL10 in the development of immune-associated pathology. Following nineteen weeks of infection, the expression levels of IFN-γ, TNF-α and IL10 reduced (log2 6.22, 3.02 and 7.03, respectively) implying a loss of the cellular response. The later stages of bovine tuberculosis have been shown within the literature to display characteristics of a humoral rather than cell mediated response. However, within this study at nineteen weeks post infection IL4 (an important cytokine in the development of the humoral response) remained undetectable. The results from this study therefore confirm the importance of the cell mediated immune profile in response to M. bovis infection as well as the integral role of IFN-γ in both protection and pathology. It also further demonstrates the involvement of IL10 in controlling the IFN-γ response and highlights this cytokine as being potentially important in future immunologybased vaccination studies. BCG Vaccination Study: The current vaccine used against human tuberculosis, BCG, has provided variable results on protection against infection in experimental bovine studies. The BCG bacterium has lost a comparatively large quantity of genomic DNA through attenuation since its primary production in 1921, of which the majority represented genes encoding antigenic proteins. MPB70 and MPB83 are differentially expressed between BCG sub-strains due to a single nucleotide polymorphism in the alternative sigma factor K (SigK). BCG Pasteur has been shown to produce low levels of these antigenic proteins; however complementation of BCG Pasteur with a copy of sigK from BCG Russia resulted in up-regulating expression. It was therefore hypothesised that the recombinant BCG (sigK) Pasteur would prove more efficient in controlling M. bovis infection by inducing a stronger protective immune response post vaccination. IFN-γ, TNF-α, IL 4 and 10 expression were analysed using qPCR within the freshly dissected lymph nodes of five experimental cattle groups; BCG Pasteur vaccinated M. bovis challenged, BCG (sigK) Pasteur vaccinated challenged, non-vaccinated infected, non-vaccinated noninfected and BCG Pasteur vaccinated non-infected. Five weeks following infection, a strong IFN-γ mRNA response was detected in both the non-vaccinated and vaccinated cattle (mean log2 copies between 9.6 and 10.5 as compared to between 7.84 and 8.58 in the non-infected cattle). M. bovis infection also induced a significant reduction in IL10 mRNA levels in both vaccinated and non-vaccinated cattle (mean log2 14.4 in the infected groups compared to 15.5 in the non-infected cattle, p<0.005, Mann Whitney test) although there was little difference in TNF-α expression (mean log2 copies between 11.06 and 11.8 in all five groups). Interestingly, IL4 mRNA was detectable only within the two non-infected control groups (mean log2 12.4), further supporting the concept of a strong cell mediated response after five weeks of infection. Vaccination prior to challenge had an effect on IFN-γ mRNA levels only, as both the BCG Pasteur and BCG (sigK) Pasteur vaccinated groups displayed a smaller increase in IFN-γ mRNA following challenge (mean log2 10.3 and 9.6, respectively) as compared to the nonvaccinated group (mean log2 10.5). This reflected the role of vaccination in priming the immune response to enable more rapid elimination of the bacteria and subsequently inducing a lesser pro-inflammatory response. Interestingly, the BCG Pasteur vaccinated group appeared to control the immune response to a greater extent, as IFN-γ mRNA was significantly similar to that observed in the non-vaccinated non-infected group (mean log2 8.58, p>0.05, Mann Whitney test). In addition to the qPCR data, levels of IFN-γ and TNF-α protein (represented by the number of cells producing these proteins) were also analysed by immunohistochemistry. IFN-γ protein in the five experimental groups displayed the same pattern as that observed for IFN-γ mRNA expression (p<0.001, Spearmans correlation coefficient). However, analysis of TNF-α protein revealed significant differences between the five groups (p<0.005, Kruskal Wallis test) in contrast to that observed for the mRNA levels (p>0.05, Spearmans correlation coefficient) suggesting that posttranscriptional controls may play an important role in TNF-α translation. The difference in IFN-γ mRNA and protein expression between the two vaccination groups was also reflected within the pathological data. Although both BCGs reduced levels to below that of the non-vaccinated group (represented by mean percentage area coverage of granuloma, 59%), the BCG Pasteur group displayed less pathology (mean 6%) compared to the BCG (sigK) Pasteur cattle (mean 35%). It was suggested that the increased antigenic repertoire of the recombinant BCG (sigK) Pasteur did result in a stronger stimulation of the immune response post vaccination but that, as a consequence the bacterial threat was eliminated more rapidly.

Doctor of Philosophy
Department of Diagnostic Medicine/Pathobiology
Daniel U. Thomson
Escherichia coli O157:H7 is responsible for over 70,000 cases of human illness every year in the United States. Most cases occur in children under the age of five, the elderly, or other immune-compromised people. A small percentage of these cases will develop a life threatening complication, hemolytic uremic syndrome. Cattle are the reservoir host for E. coli O157:H7 and serve as the main source of contamination of meat products and other food sources. The beef cattle industry is diverse with producers caring for as few as one to as many as thousands of cattle. The first objective of this research was to examine three major production systems (conventional, organic, and natural) in the U.S. and the published performance effects of the various technologies used in each system. The second objective was to determine if a newly licensed E. coli O157:H7 SRP® (SRP) vaccine administered to cows pre-partum could achieve successful passive transfer in their offspring. The third objective was to determine if colostrum obtained from SRP vaccinated heifers could protect against an oral challenge with an E. coli K99+ strain. The fourth objective was to examine the shedding characteristics, health, and performance effects of calves born to SRP-vaccinated cows that also receive SRP vaccination themselves. The technologies used in conventional beef cattle production resulted in significant improvements in health and performance of beef cattle. Vaccinating cows pre-partum with SRP resulted in passive transfer in calves consuming their colostrum. Calves that achieved successful passive transfer shed less E. coli K99+ and had improved fecal consistency compared to placebo. When calves were vaccinated with SRP at branding, weaning, and arrival to the feedyard there was no difference in fecal E. coli O157:H7 shedding on arrival to the feedyard or at harvest. Vaccinating calves with SRP had no effects on performance or health outcomes. Vaccinating cattle with SRP may provide protection against other pathogenic E. coli strains and warrants further investigation. The timing of vaccination appears to be an important consideration in order to ensure maximum vaccine efficacy.

Shiga producing E. coli (STEC) O157 is a zoonotic pathogen. In humans STEC O157 causes bloody diarrhoea and potentially fatal renal failure. Cattle are the major reservoir, where bacteria are limited to the intestinal tract and do not cause clinical signs of disease. Previous studies indicate that shiga toxins produced by STEC O157, suppress STEC-specific cellular immune responses in vivo. This study aimed to initially examine the humoral immune response in cattle following natural challenge and the effects of a toxoid vaccination on this humoral STEC specific-immune response. We determined a statistically significant suppression in Tir specific IgA in STEC O157 positive cattle compared to O157 negative cattle but not in super shedding cattle. Following toxoid vaccination we determined a significant increase in flagellin specific IgG1 antibody levels in toxoid vaccinated animals despite lower numbers of positive faecal samples compared to placebo vaccinated controls. These results suggest that shiga toxins produced by STEC O157 are actively suppressing the STEC specific immune response in natural colonisation. To clarify this suppression further calves were orally challenged with STEC O157 (either a PT21/28 Stx2c+, PT32 Stx2c+ or PT21/28 Stx2a+Stx2c+ strain) and their STEC specific immune responses monitored. STEC specific systemic antibody responses were variable and weak in some cases. STEC specific local antibody responses were only significantly increased following challenge with the PT21/28 Stx2a+Stx2c+ challenge. Transcripts for genes associated with immune responses, and in particular B cell activation, at the terminal rectum were analysed by reverse transcriptase quantitative PCR. Suppression of IL2RA transcripts was observed in calves challenged with PT21/28 Stx2a+Stx2c+ compared to control calves but not with the other two STEC O157 strains tested. This study also aimed to determine the effects of cattle colonisation with STEC O157 on the immune response to a non-bacterial T-cell dependent antigen, ovalbumin (OVA). Cattle were orally challenged with either a PT21/28 Stx2c+, PT32 Stx2c+ or PT21/28 Stx2a+Stx2c+ strain or unchallenged. Calves were subcutaneously immunised with OVA five days post challenge, on two separate occasions with a two week interval. Lymphocytes from lymph nodes local to the immunisation site demonstrated significantly increased OVA-specific proliferation and OVA-specific activation of CD4+ and CD8+ cells in calves that were challenged with the PT21/28 Stx2c+ strain (but not with the other two challenge strains), compared to unchallenged controls. These results indicate that colonisation with STEC O157 can alter local adaptive immune responses to non-bacterial antigens in a strain dependent manner, unexpectedly enhancing the immune response rather than suppressing it. Circulating T cell responses were unaffected. In conclusion this study provides some further evidence of adaption of the host immune response by STEC O157, which is strain dependent, and variable. It seems unlikely from the data in this study that STEC O157 colonisation is having a major impact on the responses of cattle to other vaccines or infections in the field.

Foot-and-mouth Diseases (FMD) is an important livestock disease with economic implications on trade. In southern Africa, the epidemiology of FMD is complicated as a result of the role of African buffalo (Syncerus caffer) in the maintenance and transmission of the South African Territories (SAT) virus serotypes. The aim of this study was to evaluate the FMD vaccination of cattle at the wildlife/livestock interface. A structured questionnaire was administered to communal farmers through in-person interview using the local language (Shangaan) to evaluate their perceptions concerning the current FMD vaccination programme. Cross-sectional sampling by cluster at herd levels was used to estimate proportions of cattle with high titres to FMDV-structural proteins, which was assumed to indicate an immunological response to vaccine routinely administered bi-annually in the absence of recently recorded outbreaks. A prospective cohort study was employed to evaluate immune responses and the duration of antibody responses to an inactivated aqueous trivalent FMD vaccine (SAT 1, SAT 2 & SAT 3) with blood samples collected on fortnightly bases. One hundred and four farmers responded to the questionnaire with 73% (76/104) being cattle owners while the remainder being hired cattle handlers. The majority of the respondents (79%; 95%CI: 70%-86%) indicated high level of satisfaction with the current animal health programme. The education level of the farmers varied over levels of satisfaction with the median education level being standard 9 (IQR: 2-12) for non-satisfied respondents, standard 3 (IQR: 0-6) for the little satisfied and standard 7 (IQR: 2-11) for the very satisfied respondents (P=0.036). Non-satisfied respondents were more likely to treat sick animals themselves than seek veterinary assistance (P=0.002). The majority of respondents identified the African buffalo as a risk factor for FMD outbreak (92%, 95%CI: 85%-96%). Two hundred and eighty-six cattle were sampled within six months post-vaccination and relative to antibody titre of ≥1.6 Log10 (1:40 dilution), 20% (95%CI: 14%-26%) of cattle had serologically converted to SAT 1, 39% (95%CI: 32%-46%) to SAT 2 and 22% (95%CI: 17%-27%) to SAT 3. Overall, only 4%, 15% and 9% of cattle had antibody titre ≥2 log10 to SAT 1, SAT 2 and SAT 3 respectively over a median period of 189 days since the most recent vaccination. Within the longitudinal study, few cattle had evidence of pre-existing antibody responses to SAT viruses at the beginning of the study. However, 14 days post-vaccination, the proportion of seropositive cattle (≥2 log10 titre) to the three SAT type viruses varied between 39% - 77% with SAT 2 having the highest proportions. Antibody responses peaked up to 98%, 98% and 65% at 42 days post-vaccination for SAT 2, SAT 3 and SAT 1 respectively until starting to decline at 56 days-post-vaccination.
Dissertation (MSc)--University of Pretoria, 2014.
National Research Foundation, South Africa
Research Development Project
University of Pretoria
Production Animal Studies
MSc
Unrestricted

The aim of this study was to determine the efficacy of vaccination in preventing LSDV excretion in semen and negative effects on semen quality. Lumpy skin disease (LSD) is caused by a virus in the genus Capripoxvirus of the family Poxviridae. The virus has been reported to be excreted in the semen of experimental infected nonvaccinated bulls. Nevertheless, vaccination has been the most widely used method to reduce and prevent the spread of the disease. This work was done to determine the efficacy of lumpy skin disease vaccination in preventing the excretion of lumpy skin disease virus (LSDV) in semen of experimentally infected vaccinated bulls. It also determined further the effect of vaccination and experimental infection on semen quality. Six serologically negative bulls 11-16 months of age were vaccinated with an attenuated Neethling strain of LSD vaccine, and a repeated dose of vaccine was given twenty one days later. These bulls were then experimentally infected by intravenous injection with a virulent field strain of LSDV (V248/93). Six unvaccinated bulls were similarly infected to act as controls. All animals were observed for clinical signs, blood and semen was collected and evaluated twice a week until day 40 post vaccination and every two days until day 28 post-infection when the trial was terminated. Serology was done using the serum neutralization test and viraemia was determined by virus isolation. Semen was examined by polymerase chain reaction (PCR) for the presence of virus. Semen evaluation was done visually and microscopically. Two of the unvaccinated controls developed severe LSD, two showed mild symptoms and two were asymptomatic. No clinical abnormalities were detected following vaccination, and clinical signs were limited to mild lymph node enlargement in four bulls following challenge of the vaccinated bulls. There was a significant difference (P<0.05) in semen quality after experimental infection of the unvaccinated bulls. In the vaccinated bulls, semen quality showed no significant difference (P>0.05) following vaccination and challenge. Three of the vaccinated bulls were serologically positive at the time of experimental infection and four at the end of the trial. Five unvaccinated bulls were found to be viraemic during the course of the trial. No vaccinated bulls were found to be viraemic at any stage. Four unvaccinated bulls excreted the virus in their semen during the course of the trial. Viral nucleic acid was not detected in any semen samples following vaccination or challenge in vaccinated bulls. This study provides evidence that vaccination against LSD prevented the excretion of viral particles in semen. It also illustrated that LSD vaccination prevented any effect on semen quality after experimental infection with virulent virus.
Dissertation (MSc (Production Animal Studies))--University of Pretoria, 2006.
Production Animal Studies
unrestricted

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Este estudo avaliou os efeitos da administração oral de meloxicam nas respostas metabólicas, inflamatórias e de fase-aguda em bovinos de corte recebendo um desafio de lipopolissacarídeo (LPS) bacteriano (Exp. 1; d -1 a d 6) ou vacina contra patógenos respiratórios (Exp. 2; d 7 a d 21). Vinte e um novilhos (n=11) e novilhas (n=10) foram alocados em baias individuais no d -15 e receberam água, mistura de sais minerais e vitaminas, e feno de alfafa ad libitum até d 21. No d -1, os animais foram classificados por sexo e peso corporal (PC) e designado para um dos três tratamentos a seguir: 1) Administração via oral de Meloxicam (1mg/Kg PC, diariamente; Carlsbad Technologies Inc., Carlsbad, CA, EUA) do d -1 até d 6 (MEL8); 2) Administração via oral de Meloxicam (1mg/Kg PC; Carlsbad Technologies Inc., Carlsbad, CA, EUA) no d 0 e de monoidrato de lactose (1mg/Kg PC; Avantor Performance Materials, Center Valley, PA, EUA) no d -1 e do d 1 até d 6 (MEL1); ou 3) Administração via oral de monoidrato de lactose (1mg/Kg PC, diariamente; Avantor Performance Materials, Center Valley, PA, EUA) do d -1 até d 6 (CON). Para o Experimento 1, no d 0, os animais receberam infusão intravenosa de LPS (0,5 μg/Kg PC; Escherichia coli 0111:B4, Sigma-Aldrich, St. Louis, MO, EUA) simultaneamente à administração do tratamento. Temperatura retal (RTEMP) foi medida, e amostras de sangue coletadas nas horas −2, 0, 2, 4, 6, 8, 12, 16, 24, 36, 48, 60, 72, 96, 120, e 144 h relativa à administração de LPS. Não foram detectados efeitos de tratamentos (P ≥ 0,36) para RTEMP, concentração sérica de Fator de Necrose Tumoral Alfa (TNFα), concentrações plasmáticas de haptoglobina, cortisol, insulina e leptina, assim como para expressão de RNAm sanguíneo para TNFα e ciclooxigenase-2 (COX2), contudo todas as variáveis se elevaram (P < 0,01) dentre os tratamentos, após administração de LPS. No Experimento 2, os animais receberam os mesmos tratamentos para os quais foram designados no Exp. 1, dos dias d 7 até d 13 e foram vacinados contra patógenos respiratórios juntamente com a administração dos tratamentos no d 8. Amostras de sangue foram coletadas e RTEMP registradas nos mesmos momentos realizados no Exp. 1 com adição das horas 168, 240 e 336 h relativas ao momento da vacinação. Nenhum efeito de 27 tratamento foi detectado (P ≥ 0,26) para RTEMP, nem para nenhumas das mesmas concentrações plasmáticas e séricas avaliadas no Exp. 1, e concentrações séricas de anticorpos contra Mannheimia haemolytica ou títulos contra Vírus Sincicial Bovino, Herpesvirus-1, Diarreia Viral Bovina vírus-1, e Vírus Parainfluenza-3. Todas as variáveis aumentaram (P < 0,01) dentre os tratamentos após a vacinação, exceto para TNFα sérico e títulos contra Diarreia Viral Bovina vírus-1 (P ≥ 0,40). Coletivamente, este estudo não encontrou evidencias de que administração oral de meloxicam, nas doses e intervalos adotados, mitigaram reações metabólicas, inflamatórias e de fase-aguda desencadeadas pela infusão de LPS ou vacinação contra doenças respiratórias.
This study evaluated the effects of oral meloxicam administration on metabolic, inflammatory, and acute-phase responses of beef cattle receiving a lipopolysaccharide (LPS) challenge (Exp. 1; d −1 to 6) or vaccinated against respiratory pathogens (Exp. 2; d 7 to 21). Twenty-one Angus steers (n = 11) and heifers (n = 10) were housed in individual pens on d −15 and were offered free-choice water, mineral-vitamin mix, and hay until d 21. In Exp. 1, cattle were ranked on d −1 by sex and BW and assigned to 1) oral meloxicam administration (1 mg/kg BW daily) from day −1 to 6 (MEL8), 2) oral meloxicam administration (1 mg/kg BW) on d 0 and oral lactose monohydrate administration (1 mg/kg BW) on d −1 and from d 1 to 6 (MEL1), or 3) oral lactose monohydrate administration (1 mg/ kg BW daily) from d −1 to 6 (CON). On d 0, cattle received an intravenous LPS bolus (0.5 μg/kg BW) concurrently with treatment administration. Rectal temperature (RTEMP) was assessed, and blood samples were collected at −2, 0, 2, 4, 6, 8, 12, 16, 24, 36, 48, 60, 72, 96, 120, and 144 h relative to LPS administration. No treatment effects were detected (P ≥ 0.36) for RTEMP, concentrations of serum tumor necrosis factor α (TNFα), plasma haptoglobin, cortisol, insulin, and leptin, as well as blood mRNA expression of TNFα and cyclooxygenase-2, although all variables increased (P < 0.01) across treatments after LPS administration. In Exp. 2, cattle received the same treatments that they were assigned to in Exp. 1 from d 7 to d 13 and were vaccinated against respiratory pathogens concurrently with treatment administration on d 8. Blood samples were collected, and RTEMP was assessed as in Exp. 1 in addition to 168, 240, and 336 h relative to vaccination. No treatment effects were detected (P ≥ 0.26) for RTEMP, the same plasma and serum variables evaluated in Exp. 1, and serum concentrations of antibodies against Mannheimia haemolytica or serum titers against bovine respiratory syncytial virus, bovine herpesvirus-1, bovine viral diarrhea virus-1, and parainfluenza-3 virus. All variables increased (P < 0.01) across treatments after vaccination, except for serum TNFα and titers against bovine viral diarrhea virus-1 (P ≥ 0.40). Collectively, this study found no evidence that oral meloxicam administration, at the doses and intervals utilized herein, mitigated the metabolic, inflammatory, and acute-phase reactions elicited by LPS administration or vaccination against respiratory pathogens.

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The objective of this study was to evaluate the effect of supplementation with Matricaria chamomila CH12 through humoral immune response in vaccinated cattle with a single dose of antirabies vaccine. Twenty-eight female bovines, Pure Breed (PO), aged shape12 months old randomly divided into 2 groups (14 animals / group): control group (CG) received the mineral salt and rabies vaccine and group Testing (GT) received 4 grams of supplement with Matricaria chamomila CH12 added to mineral salt for voluntary intake. The individual titers of neutralizing antibodies were determined by the technique of neutralization based on the Rapid Fluorescent Focus Inhibition Test (RFFIT). The average of serological titers observed for both groups at 48 and at 90 days were compared by using unpaired t-test with Welch correction, and serological titers within each group, at 48 and at 90 days post-vaccination, by paired t test. Within each group, the proportion of animals immunized against rabies were compared by chi-square MacNemar. There was no statistical difference in antibody titers between groups. It was concluded that the use of Matricaria chamomila CH12 core added to the mineral salt does not alter the humoral immune response, and that a single dose was not sufficient to maintain the antirrábicos protective antibody titers 90 days post vaccination.
O objetivo desse estudo foi avaliar o efeito da suplementação com Matricaria Chamomila CH12 sobre a resposta imune humoral em bovinos vacinados com uma única dose de vacina antirrábica. Vinte e oito bovinos, fêmeas, Puro de Origem (PO), com idade de 12 meses foram divididos aleatoriamente em 2 grupos (14 bovinos/grupo): o grupo controle (GC) recebeu o sal mineral sem suplemento e vacina antirrábica e o grupo Teste (GT) recebeu 4 gramas de suplemento com Matricaria Chamomila CH12 adicionado ao sal mineral e por ingestão voluntária. Os títulos individuais de anticorpos neutralizantes foram determinados por meio da técnica de soroneutralização baseado no Rapid Fluorescent Focus Inhibition Test (RFFIT). Os títulos sorológicos médios observados para os dois grupos estudados aos 48 e aos 90 dias foram comparados pelo teste t não-pareado com correção de Welch, e os títulos sorológicos dentro de cada grupo, aos 48 e 90 dias pós-vacinação, pelo teste t-pareado. Dentro de cada grupo, a proporção de animais imunizados contra raiva foram comparados pelo teste de Qui-quadrado de MacNemar. Não houve diferença estatística nos títulos de anticorpos neutralizantes entre os grupos estudados. Conclui-se que o uso da Matricaria chamomila CH12 adicionado ao sal mineral não alterou a resposta imune humoral, e que uma única dose não foi suficiente para manter os títulos de anticorpos antirrábicos protetores pós 90 dias de vacinação.

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This study evaluated the effect of supplementation with a combination of probiotic microorganisms, added to a mineral mixture in different doses/day, in the production of serum antibodies against the rabies virus in primo vaccinated cattle. Forty-two male Nelore cattle, divided into three groups (n = 14): control group (CG) received 70 grams of mineral mixture/animal/day; probiotic groups 4 grams (G4P) and 8 grams (G8P) received 70 grams of mineral mixture/animal/day with 4 and 8 grams of added probiotics respectively. The individual anti-rabies antibody titers were determined using the technique of seroneutralization in cells based on the Rapid Fluorescent Focus Inhibition Test (RFFIT). The results obtained were compared using the unpaired t test with a 5% significance level. There were statistical differences in the mean serum concentrations between the CG, G4P and G8P groups, 30 and 60 days after the primo vaccination and after 60 days only the G8P group demonstrated minimum protective antibody titers in 100% of the animals. There is evidence of beneficial effects attributed to the association between probiotic microorganisms and the anti-rabies humoral immune response; in addition, an increase in the dose incorporated into the mineral mixture maintains high levels of serum antibodies in primo vaccinated cattle.
Esse estudo avaliou o efeito da suplementação de uma associação de microrganismos probióticos, adicionados à mistura mineral em diferentes doses/dia, na produção de anticorpos séricos contra o vírus rábico em bovinos primovacinados. Quarenta e dois bovinos Nelore machos, divididos em três grupos (n=14): grupo controle (GC) recebeu 70 gramas de mistura mineral/animal/dia; grupos probióticos 4 gramas (G4P) e 8 gramas (G8P) receberam 70 gramas de mistura mineral/animal/dia adicionados respectivamente de 4 e 8 gramas de probióticos. Os títulos individuais de anticorpos antirrábicos foram determinados por meio da técnica de soroneutralização em células baseado no Rapid Fluorescent Focus Inhibition Test (RFFIT). Os resultados obtidos foram comparados pelo teste t não pareado, com 5% de nível de significância. Houve diferenças estatísticas significativas entre as médias de concentrações séricas entre os grupos GC e os G4P e G8P, após 30 e 60 dias da primovacinação e após 60 dias somente o G8P manteve 100% dos animais com títulos de anticorpos protetores mínimos. Existem evidências para se atribuir efeito benéfico à associação de microrganismos probióticos na resposta imune humoral antirrábica e ao aumento da sua dose incorporada na mistura mineral e sobre a manutenção de elevados níveis de concentração sérica de anticorpos nos bovinos primovacinados.

Weaning is defined as the physical separation of the cow-calf pair and the end of milk feeding. Natural weaning occurs between 7 and 14 months and is a gradual process. However, domestic weaning occurs between 6 and 8 months and occurs rapidly. Calves that are abruptly separated from their dam respond with increased vocalization and walking, and decreased eating and resting. The psychological stress the calf undergoes during weaning causes elevated glucocorticoid and catecholamine hormone concentrations that may predispose to increased morbidity and/or mortality from infectious diseases such as Bovine Respiratory Disease Complex. As an attempt to counter these changes, alternative weaning methods have been implemented and normally occur in two stages. Two-stage weaning begins with the cessation of milk feeding for approximately one week with the calf maintaining some contact with their dam and then permanent separation occurs. One of these methods uses a single fence to separate the cow-calf pair; this process allows the calf to see, hear and smell their dam, but does not allow the calf to suckle from its dam. Increases in cortisol, a glucocorticoid, have been linked to immunological alterations. Most notably, elevated cortisol concentrations decrease neutrophil function by down regulating the gene expression of CD62L and Fas. Cortisol also alters lymphocyte phenotype by decreasing ?δ T cells and increasing°? T cells in the circulation. Lastly, increases in cortisol can modify T cell cytokine production. The cytokines IL-12 and IFN? are secreted from T helper 1 cells while T helper 2 cells secrete IL-4 and IL-10; these T cells subsets also inhibit one another. During higher cortisol concentrations, these T cells are biased toward T helper 2 cytokine production. All these changes in immune function can lead to increased susceptibility to disease around the time of weaning. Therefore, two trials were conducted to test the hypotheses that abrupt weaning results in elevated concentrations of cortisol and subsequently alters immunological functions, and that fenceline weaning alleviates the increase in cortisol and alterations to immune function associated with weaning. In the fall of 2008, 12 Angus and Angus-X heifers (186°21 kgs; 174°16 days of age) were blocked by age and weight and randomly allotted into two groups, fenceline and abrupt. Blood samples were taken on day -7, 0, 7, 14, 21, and 42; fecal samples were taken on day -7, 0, and 3. All calves were weighed on day -7, 0, 7, 14, and 42. On day -1 all calves were separated from their dam and transported for 2 hours to another facility. On day 0 all calves were vaccinated with Brucella abortus (strain RB51). Serum was analyzed for IFN? and IL-4 as well as IgG1 and IgG2 specific antibodies to RB51. Fecal samples were analyzed for cortisol metabolites. Both IgG1 and IgG2 antibodies to RB51 increased from day 0 to day 14 (P<0.05), however no differences were detected between treatment groups. Fecal cortisol metabolites were higher on day 0 in abruptly weaned calves (P< 0.001) but did not differ between groups on day -7 or day 3. Fenceline calves had higher concentrations of IFN? in the serum on day -7 and day 0 as compared to the abruptly weaned calves (P<0.04). In the fall of 2009, forty-four Angus and Angus-X calves (19 heifers and 25 steers; 181°27 kgs; 148°17 days old) were blocked by age and gender and randomly allotted within block into two treatment groups, fenceline (FL) and abrupt (AB). Approximately half the fenceline calves were separated from their dams by a single fence at day -7 and the rest of the fenceline group at day -6; all calves were removed from their dam at day 0. Calves were vaccinated with Histophilus somni on day 1. Blood samples were taken at day -6, 1, 3, 8, 15, and 22. Fecal samples were taken on day -7, -6, 1 and 3. All calves were weighed on day -7, 0, 8, and 22. Serum samples were analyzed for IgG1 and IgG2 specific-H. somni antibodies, white blood cells were analyzed for lymphocyte phenotypes, and gene expression using 18S as the housekeeper gene. Fecal samples were analyzed for cortisol metabolites. Abruptly weaned calves had higher concentrations of cortisol metabolites in the feces than fenceline calves at day 1 (P<0.0001). No difference in average daily gain or H. somni specific antibodies between treatment groups was detected. There was a treatment*date interaction in lymphocyte and neutrophil populations (P<0.05); neutrophils from fenceline calves dropped from day -6 to day 1, but increased from day 1 to day 3, while abrupt calves decreased from day -6 to day 3. Lymphocytes from fenceline calves increased from day -6 to day 1, but decreased from day 1 to day 3, while lymphocytes from abrupt calves increased from day -6 to day 3. No difference in treatment groups was detected for lymphocyte phenotypes or gene expression; however, a date effect was detected. The CD4 and CD8 cell populations increased over time (P<0.0001) and WC1 and TcR1 decreased over time (P=0.0243 and P=0.0027 respectively) for both treatment groups. A decrease was detected over time for expression of GAPDH and CD62L (P<0.0001). The gene expression for the cytokines IFN?, IL-4 and IL-10 had no change over time. Results from the two studies suggest that fenceline weaning decreases the cortisol response associated with cow-calf separation, but does not have a significant effect on immunological parameters measured in this study.
Master of Science

A Leucose Enzoótica dos Bovinos (LEB) é uma enfermidade neoplásica, infectocontagiosa, pluri-sintomática, de evolução crônica, que compromete os órgãos linfopoiéticos e está associada ao desenvolvimento de linfocitose persistente (LP) e linfossarcoma. Acarreta diminuição na produção, quer por seus efeitos danosos diretos, quer pelos indiretos. No entanto, seu efeito na função e na quantidade das diferentes subpopulações de linfócitos, assim como seu papel no estabelecimento de outras doenças oportunistas, ainda não está claro. O presente estudo avaliou a resposta imunitária de bovinos da raça Holandês Preto e Branco naturalmente infectados pelo vírus da LEB (VLB), após desafio antigênico fornecido por vacinação contra o vírus da febre aftosa. Para tal, foram coletadas amostras sanguíneas antes do desafio e, após o desafio, semanalmente, por sete semanas, de dez vacas soropositivas, sem LP; de dez vacas soropositivas, manifestando LP; e de dez vacas soronegativas. Foram avaliadas as alterações quantitativas das diferentes subpopulações de leucócitos circulantes; a função dos linfócitos B, por meio da quantificação de diferentes isotipos de imunoglobulinas (Ig) séricas; os índices de proliferação linfocitária; os índices de morte celular por apoptose ou por necrose; e as concentrações séricas de interleucina-10 (IL-10), IL-12, inteferon- (IFN-γ) e fator de necrose tumoral-α (TNF-α). Foi verificada a normalidade da distribuição dos resultados obtidos, utilizando-se do teste de Anderson-Darling, e sua homoscedasticidade, utilizando-se do teste F (para dados que apresentaram distribuição normal) ou do teste de Lavene (para dados que não apresentaram distribuição normal). Para a avaliação das diferenças entre as médias dos resultados obtidos, de acordo, respectivamente, com a ocorrência ou não de homoscedasticidade, foram feitos, para dados com distribuição normal, os testes de análise de variância (One-way ANOVA), seguida do teste de Tukey-Kramer ou o teste t; e, para dados que não apresentaram distribuição normal, o teste de Mann-Whitney ou o teste de Kruskal-Wallis. Para todos os resultados, foram consideradas significantes as análises que apresentaram p≤0,05. Verificou-se que não houve diferença nas concentrações séricas de IgG1, de IgM e de IgA, tanto entre os tempos de coleta, quanto, a cada tempo, entre animais pertencentes aos diferentes grupos. As concentrações séricas de IgG2 aumentaram, após a vacinação, em todos os animais (p<0,05). Todavia, 17 dias após o desafio antigênico, as concentrações séricas de IgG2, em animais manifestando LP foram, a cada tempo de coleta, menores (p<0,01) que aquelas verificadas nos animais pertencentes aos demais grupos, indicando que animais com LP apresentam resposta humoral menos intensa e menos duradoura. Observou-se que ocorreu um aumento no índice de proliferação de linfócitos sanguíneos (p<0,01), 24 dias após a vacinação contra o vírus da febre aftosa, independente da presença de infecção pelo VLB. A partir deste momento, ocorre um aumento na porcentagem de linfócitos γσ circulantes (p<0,05) e posterior diminuição nas concentrações séricas de IgG2 (p<0,05), indicando regulação desta resposta humoral por linfócitos γσ. Em bovinos com LP, o aumento na porcentagem de linfócitos circulantes foi maior (p<0,05), ocasionando diminuição mais intensa e mais precoce nas concentrações séricas de IgG2. Constatou-se que a LP ocorre em decorrência de menor índice de apoptose, posto que as porcentagens de leucócitos sofrendo processo de apoptose foram menores (p≤0,001) entre as células obtidas de animais manifestando LP, do naquelas coletadas dos animais pertencentes aos demais grupos. Verificou-se que as concentrações séricas das citocinas de perfil Th1, IL-12 e IFN-γ, são maiores em amostras sangüíneas de animais infectados pelo VLB, alinfocitóticos (p<0,01), ao passo que as concentrações séricas das citocinas de perfil Th2, IL-10 e TNF-α, são maiores em amostras sangüíneas de animais infectados manifestando LP (p<0,01), indicando que alterações no perfil sérico de citocinas podem ser causa ou consequência da LP. Em resposta ao desafio vacinal, ocorre uma elevação nas concentrações séricas de IL-10 (p<0,01), de TNF-α (p=0,005) e de IFN-γ (p<0,01), três dias após o desafio, e de IL-12 (p<0,001), dez dias após o desafio. A elevação na concentração sérica de IL-10 perdura até 31 dias após o desafio e pode ser responsável pelo maior índice de proliferação de linfócitos γσ verificado a partir de 31 dias após a vacinação. Foi observado que a maioria dos linfócitos B circulantes, em bovinos, consiste de linfócitos B1 e, em animais infectados pelo VLB, a LP ocorre em decorrência de um aumento na porcentagem de linfócitos B1a (p<0,05). Além disso, em animais infectados pelo VLB, apresentando LP, as relações entre linfócitos T auxiliares e citotóxicos são menores (p<0,01) e a porcentagem de linfócitos γσ é maior (p<0,01), indicando atividade viral nas células infectadas. Assim, os resultados permitem-nos concluir que animais infectados pelo VLB, manifestando LP, apresentam alterações na resposta imunitária frente vacinação contra o vírus da febre aftosa.
Enzootic Bovine Leukosis (EBL) is an infectious, multi-symptomatic, chronic, neoplastic disease, which undermines the lymphopoietic organs and is associated with the development of persistent lymphocytosis (PL) and lymphosarcoma. Infected animals present a decrease of production, either by its direct or its indirect harmful effects. However, its effect on the function and quantity of different lymphocyte subpopulations, as well as its role in the establishment of other opportunistic diseases, are unclear. This study evaluated the immune response of Holstein dairy cattle naturally infected with Bovine Leukosis Virus BLV, after antigen challenge provided by vaccination against foot and mouth disease (FMD) virus. To this end, blood samples were collected before challenge and after challenge, weekly, for seven weeks, from ten seropositive cows without PL, from ten seropositive cows expressing PL, and from ten seronegative cows. We evaluated the quantitative changes of different subpopulations of leukocytes; the function of B lymphocytes, through the quantification of different isotypes of immunoglobulins (Ig) serum concentration; the rate of lymphocyte proliferation; the rate of cell death by apoptosis or necrosis; and the serum concentrations of interleukin-10 (IL-10), IL-12, inteferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). It was verified the normality of distribution of the results using the Anderson-Darling test, and their homoscedasticity, using the F test (for data with normal distribution) or the Levene test (for data without normal distribution). For the evaluation of differences between the average results, according respectively to the presence or absence of homoscedasticity, we used, for data with normal distribution, One-way ANOVA test, followed by the Tukey-Kramer test or the t test, and, for data without normal distribution, the Mann-Whitney test or the Kruskal-Wallis test. Results with p≤0.05 were considered statistically significant. There were no differences related to serum IgG1, IgM, and IgA concentrations, both among sampling time and, every time, among animals belonging to different groups. IgG2 serum concentrations increased after vaccination in all animals (p<0.05). However, in animals expressing PL, each collection time, 17 days after antigen challenge, IgG2 serum concentration was lower (p<0.01) than those observed in animals belonging to other groups, indicating that animals with PL present less intense and less enduring humoral response. It was observed that there was an increase in the rate of lymphocyte proliferation (p<0.01) 24 days after vaccination against FMD virus, irrespective of the presence of infection by BLV. From this moment, there was an increase in the percentage of γσ-lymphocytes (p<0.05) and a subsequent decrease in serum IgG2 (p<0.05), indicating regulation of this humoral response by γσ-lymphocytes. In cattle with PL, the increase in the percentage of γσ-lymphocytes was higher (p<0.05), leading to more intense and earlier decrease in IgG2 serum concentration. It was found that PL is due to a lower rate of apoptosis, since the percentage of leukocytes undergoing apoptosis was lower (p≤0.001) among cells obtained from animals expressing PL, when compared to those collected from animals from the other groups. It was found that serum concentrations of Th1 cytokines, specifically IL-12 and IFN-γ, were higher in blood samples from nonlymphocytotic infected animals (p<0.01), whereas serum concentrations of Th2 cytokines, particularly IL-10 and TNF-α, were higher in blood samples from infected animals expressing LP (p<0.01), indicating that changes in serum cytokines profile may be a cause or a consequence of PL. IL-10 (p<0.01), TNF-α (p=0.005), and IFN-γ (p<0.01) serum concentrations increased three days after the challenge, and IL-12 serum concentration increased (p<0.001), ten days after the challenge. The increase in IL-10 serum concentration lasts until 31 days after the challenge and may account for the higher rate of γσ-lymphocyte proliferation found from 31 days after vaccination. It was observed that the majority of circulating B lymphocytes in cattle consists of B1 lymphocytes and that, in BLV-infected animals, PL occurs due to an increase in the percentage of B1a lymphocytes (p<0.05). Moreover, in lymphocytotic BLV-infected animals, the rate between helper and cytotoxic Tlymphocytes are smaller (p<0.01) and the percentage of γσ-lymphocytes is greater (p<0.01), indicating viral activity in infected cells. Thus, results allow us to conclude that lymphocytotic BLV-infected animals show changes in the immune response after vaccination against FMD virus.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Vaccines with antigenic markers also known as differential vaccines have been largely used for control and prevention of bovine herpesvirus 1 (BoHV-1) infection. With this purpose, a Brazilian BoHV-1 isolate (SV56/90) was submitted to deletion of the glycoprotein E (gE) gene for a potential use in vaccines. BoHV-1 gE gene deletion was performed by homologous recombination, being the gE gene replaced by the green fluorescent protein (GFP) gene for selection. Upon co-transfection of MDBK cells with genomic viral DNA plus the GFP-bearing gE-deletion plasmid, three fluorescent recombinant clones were obtained (and nominated as BoHV-1ΔgE). The recombinant viruses formed smaller plaques in MDBK cells yet with similar kinetics and grew to similar titers to those of the parental virus, showing that gE deletion had no deleterious effect on the replication efficiency in vitro. Thirteen calves inoculated intramuscularly (IM) with the recombinant BoHV-1ΔgE developed virus neutralizing (VN) antibodies at day 42pi (titers from 2 to 16), demonstrating his ability to replicate and to induce a serological response in vivo. Furthermore, the serological response induced by the recombinant virus could be differentiated from that induced by wild-type BoHV-1 by the use of an anti-gE antibody ELISA kit. Experiments to determine the safety, immunogenicity and protection were performed with the BoHV-1ΔgE candidate vaccine strain. In the safety test, five three months-old calves were inoculated with approximately 10-100 times the usual vaccine dose (108.5TCID50 per animal). The inoculated animals remained healthy and did not shed virus, confirmed by the absence of virus in nasal secretions and lack of seroconversion by sentinel calves kept in contact. In addition, the recombinant virus was not shed upon dexamethasone administration (at day 42pi) showing the inability of reactivation and/or shedding after attempts of reactivation of latent infection. In the immunogenicity test, calves (8 to 10 months-old) were vaccinated once IM (group I, n=8) or subcutaneously (group II, n=9) with live BoHV-1ΔgE or twice (30 days apart) with inactivated virus plus aluminum hydroxide (group IV, n=13) or MontanideTM Gel 1 (Seppic - group V, n=14). As controls, three animals (group III) were vaccinated once IM with the parental virus. All calves vaccinated with live virus developed VN titers of 2 to 8 (group I, GMT: 2; group II, GMT: 1.65; group III, GMT: 1.65) at day 42pv. Animals of groups IV and V developed VN titers of 2 to 16 (GMT: 2.45) and 2 to 128 (GMT: 3.9), respectively. All calves vaccinated with the BoHV-1ΔgE remained negative in the gE ELISA. In a vaccination-challenge experiment, six calves (three to four-months-old) were vaccinated with live virus (107.3TCID50/animal) and four calves were kept as controls. Forty-seven days after vaccination, the calves were challenged with a heterologous BoHV-1 strain (107.5TCID50/animal) by the intranasal route. Vaccinated animals developed only mild and transient nasal signs comparing with the control calves. Virus shedding by vaccinated animals was also significantly reduced compared to controls. These results demonstrate that the recombinant BoHV-1ΔgE is safe/attenuated, immunogenic for calves both in a live or inactivated, adjuvanted vaccine formulation. Moreover, it induces a humoral response that can be distinguished from that induced by the wild type virus. Thus, the recombinant BoHV-1ΔgE presents suitable properties to be used in vaccine formulations.
Vacinas com marcadores antigênicos também denominadas vacinas diferenciais tem sido amplamente utilizadas no controle e prevenção da infecção pelo herpesvírus bovino tipo 1 (BoHV-1). Com este objetivo, uma amostra brasileira de BoHV-1 (SV56/90) foi submetida à deleção do gene da glicoproteína E (gE) para potencial uso em vacinas. A deleção do gene da gE foi realizada por recombinação homóloga, sendo o gene da gE substituído por um marcador para seleção (green fluorescent protein, GFP). Após co-transfecção de plasmídio de deleção e DNA genômico viral em células MDBK, três recombinantes expressando a GFP foram obtidos (e denominados BoHV-1ΔgE). Os vírus recombinantes produziram placas menores em células MDBK, porém com cinética e em títulos semelhantes ao vírus parental, demonstrando que a deleção da gE não afetou negativamente a sua capacidade replicativa in vitro. Treze bezerros inoculados pela via intramuscular (IM) com o recombinante BoHV-1ΔgE desenvolveram anticorpos neutralizantes (títulos entre 2 e 16), demonstrando a sua capacidade replicativa e imunogênica in vivo. Além disso, a resposta sorológica induzida pelo recombinante pode ser diferenciada daquela induzida pelo vírus parental pelo uso de um teste ELISA específico para anticorpos anti-gE. Posteriormente, o vírus recombinante foi submetido a testes de segurança/atenuação, de imunogenicidade e de proteção frente a desafio in vivo. No teste de segurança, cinco bezerros de três meses de idade foram inoculados pela via IM com o recombinante em uma dose aproximadamente 10-100 vezes a dose contida em vacinas comerciais (108,5TCID50 por animal). Os animais inoculados permaneceram saudáveis e não excretaram o vírus, confirmado pela ausência de vírus em secreções nasais e pela ausência de soroconversão em bezerros sentinelas mantidos em contato. O vírus recombinante também não foi excretado após administração de dexametasona (dia 42pi), demonstrando a incapacidade de reativar e/ou ser excretado após tentativa de reativação de infecção latente. No teste de imunogenicidade, bezerros com 8 a 10 meses de idade foram vacinados uma vez pela via IM (grupo I, n=8) ou subcutânea (SC, grupo II, n=9) com o recombinante BoHV-ΔgE viável, ou duas vezes (30 dias de intervalo) com o vírus inativado, conjugado com hidróxido de alumínio (grupo IV, n= 13) ou com MontanideTM Gel 1 (Seppic - grupo V, n=14). Como controle, três animais (grupo III) foram vacinados com o vírus parental (uma dose pela via IM). Todos os bezerros vacinados com o vírus viável desenvolveram anticorpos neutralizantes em títulos de 2 a 8 (grupo I, GMT: 2; grupo II, GMT: 1,65; grupo III, GMT: 1,65) no dia 42pv. Os demais animais desenvolveram títulos de 2 a 16 (grupo IV, GMT: 2,45) e de 2 a 128 (grupo V, GMT: 3,9). Todos os animais vacinados com o BoHV-1ΔgE permaneceram negativos no teste ELISA anti-gE. No teste de vacinação com desafio, seis bezerros (3-4 meses de idade) foram vacinados com o recombinante BoHV-1ΔgE viável na dose de 107,3TCID50/animal e quatro foram mantidos como controle. No dia 47pv, os animais foram submetidos a desafio com uma cepa heteróloga do BoHV-1 (dose de 107,5TCID50/animal) pela via intranasal. Os animais vacinados desenvolveram sinais clínicos mais brandos e por período mais curto e, excretaram vírus em menores títulos e por menos tempo do que os controles. Esses resultados demonstram que o vírus recombinante BoHV-1ΔgE é seguro/atenuado e imunogênico para bezerros, tanto na forma replicativa quanto na forma inativada. Além disso, induz resposta sorológica capaz de ser diferenciada daquela induzida pelo vírus parental. Sendo assim, o BoHV-1ΔgE apresenta propriedades adequadas para ser usado em formulações vacinais.

Foot-and-mouth disease (FMD) is a viral disease endemic in Africa, the Middle East, South America, Asia and parts of Eastern Europe. It is a major constraint to international trade in livestock and livestock products in many African countries. In southern Africa, African buffaloes (Syncerus caffer) are reservoirs of the South African Territories (SAT) 1, SAT 2 and SAT 3 FMD viruses, and cattle raised in the vicinity of wildlife conservation areas are at constant risk of becoming infected with FMD viruses. In Mozambique, control of FMD is fundamentally based on vaccination of cattle in zones around protected areas. However, the vaccination protocol recommended by the vaccine producer (two primary vaccinations followed by four- to six-monthly boosters) is logistically impractical and financially not suitable for most countries. As a result, the double primary vaccination is usually not implemented. A commercially available bivalent FMD vaccine, containing the SAT 1 and SAT 2 serotypes, was assessed for its ability to induce and sustain immunity in cattle for at least 6 months following a single primary inoculation. The study was conducted with cattle reared in Limpopo National Park (LNP), Mozambique, and adjacent areas. One hundred and seventy five seronegative cattle aged between 6 and 18 months were vaccinated and 42 others from the same areas were left unvaccinated, as controls. A group of 39 vaccinated cattle were revaccinated 4 months after initial vaccination and 63 others were revaccinated 6 months after initial vaccination. The vaccinated and unvaccinated cattle were bled at predefined intervals (at vaccination, and at 1, 4, 5, 6, 8, 10 and 12 months post vaccination) and sera were tested with a liquid phase blocking ELISA to measure the antibody level against FMD virus. A high proportion (73%) of vaccinated cattle had seroconverted (log10 titre ≥1.6 for any SAT serotype) at one month after vaccination with a single primary dose and there was no significant difference between the proportions of animals that seroconverted to SAT 1 compared to SAT 2. A higher proportion of animals within LNP (82%) than outside LNP (50%) had seroconverted at one month after vaccination (P = 0.001). Overall, however, only relatively few animals (27% for SAT 1, 35% for SAT 2 and 7% for SAT 3) had protective antibody titres (log10 titre ≥2). At 4 months after vaccination, a very low proportion (8.3%) of vaccinated cattle had antibody titres ≥1.6 to any of the SAT serotypes, and there was no significant difference between the proportions of animals with antibodies to SAT 1 (2.1%) compared to SAT 2 (7.3%) (P = 0.17). No cattle had a protective titre (≥2) to SAT 1 at 4 months and only 4.2% to SAT 2. The revaccination at 4 months after initial vaccination elicited antibody titres ≥1.6 in 22% of vaccinated animals at one month after revaccination; this rose two months later to 90% and remained high (91%) at 10 months post first vaccination before dropping to 65% at 12 months. However, only 15% of cattle had protective titres (≥2) to any of the SAT serotypes at 12 months. For cattle revaccinated at 6 months after first vaccination the percentage of cattle that had a titre ≥1.6 two months after revaccination was also high (80%), remained high (89%) at 10 months post first vaccination and dropped to 54% at 12 months after first vaccination. Only 11% of cattle had protective titres (≥2) at 12 months. The research findings indicate that, although the vaccine is able to induce production of antibodies against SAT 1 and 2 in a significant percentage of cattle within one month after a single primary vaccination, these antibodies are short lived and have largely disappeared by 4 months post vaccination. This suggests that a protocol of a single primary vaccination is inadequate in naïve animals, even if revaccination takes place four months later. Revaccination improved the immune response for a longer period, resulting in detectible titres in the majority of animals for 6-8 months post revaccination. This can be used in disease control programmes to ensure some protection of cattle particularly when it is applied immediately before identified high risk periods of occurrence of FMD outbreaks. However, it seems unlikely that six-monthly revaccination is sufficient to maintain adequate levels of protective immunity. The study highlighted several difficulties associated with the vaccination of livestock under field conditions and the conduction of field trials. These included difficulties with cold chain maintenance, poor infrastructure for animal handling, and loss of follow-up due to loss of animal identification or poor owner/herder compliance. Copyright
Dissertation (MSc)--University of Pretoria, 2012.
Production Animal Studies
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Corridor disease is an economically important tick-borne disease of cattle in southern Africa. The disease is caused by Theileria parva and transmitted by the vectors, Rhipicephalus appendiculatus and R. zambeziensis. There is currently no vaccine to protect cattle against T. parva that is permitted in South Africa. To develop recombinant anti-tick vaccines against Corridor disease, comprehensive databases of genes expressed in the tick’s salivary glands are required. Therefore, in Chapters 2 and 3, mRNA from the salivary glands of R. appendiculatus and R. zambeziensis was sequenced and assembled using next generation sequencing technologies. Respectively, 12 761 and 13 584 non-redundant protein sequences were predicted from the sialotranscriptomes of R. appendiculatus and R. zambeziensis and uploaded to public sequence domains. This greatly expanded the number of sequences available for the two vectors, which will be invaluable resources for the selection of vaccine candidates in future. Further, in Chapter 3, differential gene expression analysis in R. zambeziensis revealed dynamic expression of secretory protein transcripts during feeding, suggestive of stringent transcriptional regulation of these proteins. Knowledge of these intricate expression profiles will further assist vaccine development in future. In Chapter 4, comparative sialotranscriptomic analyses were performed between R. appendiculatus and R. zambeziensis. The ticks have previously shown varying vector competence for T. parva and this chapter presents the search for correlates of this variance. Phylogenetic analyses were performed using these and other publically available tick transcriptomes, which indicated that R. appendiculatus and R. zambeziensis are closely related but distinct species. However, significant expression differences were observed between the two ticks, specifically of genes involved in tick immunity or pathogen transmission, signifying potential bioinformatic signatures of vector competence. Furthermore, nearly four thousand putative long non-coding RNAs (lncRNAs) were predicted in each of the two ticks. A large number of these showed differential expression and suggested a potential transcriptional regulatory function of lncRNA in tick blood feeding. LncRNAs are completely unexplored in ticks. Finally, in Chapter 5, concluding remarks are given on the potential impact the R. appendiculatus and R. zambeziensis sialotranscriptomes may have on future vaccine developments and some future research endeavours are discussed.
Life and Consumer Sciences
Ph. D. (Life Sciences)