Reverse vaccinology-based identification of a novel surface lipoprotein that is an effective vaccine antigen against bovine infections caused by Pasteurella multocida.

Pasteurella multocida can infect a multitude of wild and domesticated animals, with infections in cattle resulting in hemorrhagic septicemia (HS) or contributing to bovine respiratory disease (BRD) complex. Current cattle vaccines against P. multocida consist of inactivated bacteria, which only offer limited and serogroup specific protection. Here, we describe a newly identified surface lipoprotein, PmSLP, that is present in nearly all annotated P. multocida strains isolated from cattle. Bovine associated variants span three of the four identified phylogenetic clusters, with PmSLP-1 and PmSLP-2 being restricted to BRD associated isolates and PmSLP-3 being restricted to isolates associated with HS. Recombinantly expressed, soluble PmSLP-1 (BRD-PmSLP) and PmSLP-3 (HS-PmSLP) vaccines were both able to provide full protection in a mouse sepsis model against the matched P. multocida strain, however no cross-protection and minimal serum IgG cross-reactivity was identified. Full protection against both challenge strains was achieved with a bivalent vaccine containing both BRD-PmSLP and HS-PmSLP, with serum IgG from immunized mice being highly reactive to both variants. Year-long stability studies with lyophilized antigen stored under various temperatures show no appreciable difference in biophysical properties or loss of efficacy in the mouse challenge model. PmSLP-1 and PmSLP-3 vaccines were each evaluated for immunogenicity in two independent cattle trials involving animals of different age ranges and breeds. In all four trials, vaccination with PmSLP resulted in an increase in antigen specific serum IgG over baseline. In a blinded cattle challenge study with a recently isolated HS strain, the matched HS-PmSLP vaccine showed strong efficacy (75-87.5% survival compared to 0% in the control group). Together, these data suggest that cattle vaccines composed of PmSLP antigens can be a practical and effective solution for preventing HS and BRD related P. multocida infections.

Immune response in dairy cattle against combined foot and mouth disease and haemorrhagic septicemia vaccine under field conditions.

BACKGROUND: Foot-and-mouth disease (FMD) and Haemorrhagic septicemia (HS) are two important diseases that are known to have caused significant economic losses to the cattle industry. Accordingly, vaccinations have been recognized as an efficient method to control and prevent both of the above-mentioned diseases. This study aimed to determine the immune response to FMD virus antigens and the recombinant outer membrane protein of HS (rOmpH) of Pasteurella multocida in cattle administered as a combination vaccine and compare antibody titers with the two vaccines given independently, under field conditions. Dairy cattle were divided into three groups. Each group was immunized with different vaccine types according to the vaccination program employed in this study. Antibody responses were determined by indirect ELISA, liquid phase blocking ELISA (LPB-ELISA) and viral neutralization test (VNT). Furthermore, the cellular immune responses were measured by lymphocyte proliferation assay (LPA). RESULTS: The overall antibody titers to HS and FMDV were above cut-off values for the combined FMD-HS vaccine in this study.The mean antibody titer against HS after the first immunization in the combined FMD-HS vaccine groups was higher than in the HS vaccine groups. However, no statistically significant differences (p > 0.05) were observed between groups. Likewise, the antibody titer to the FMDV serotypes O/TAI/189/87 and Asia 1/TAI/85 determined by LPB-ELISA in the combined vaccine were not statistically significantly different when compared to the FMD vaccine groups. However, the mean VNT antibody titer of combined vaccine against serotype O was significantly higher than the VN titer of FMD vaccine groups (p < 0.05). Moreover, the LPA results showed that all vaccinated groups displayed significantly higher than the negative control (p < 0.05). Nevertheless, no differences in the lymphocyte responses were observed in comparisons between the groups (p > 0.05). CONCLUSIONS: The combined FMD-HS vaccine formulated in this study could result in high both antibody and cellular immune responses without antigenic competition. Therefore, the combined FMD-HS vaccine can serve as an alternative vaccine against both HS and FMD in dairy cattle under field conditions.

Protective efficacy of phage PVN02 against haemorrhagic septicaemia in striped catfish Pangasianodon hypophthalmus via oral administration.

Haemorrhagic septicaemia caused by Aeromonas hydrophila in striped catfish (Pangasianodon hypophthalmus) is one of the most important aquatic diseases in the Mekong Delta, Vietnam. However, antibiotic-resistant A. hydrophila strains have become popular and resulted in inadequate control of the disease in striped catfish farms. This study investigates the protective efficacy of bacteriophage PVN02 against haemorrhagic septicaemia in striped catfish via oral administration. The phage-containing pellets were prepared by spraying the phage solution on food pellets at 20 ml/kg. The rate of phage desorption from the food pellets into the water was very low; the phage titres in the water were approximately log 1.0 PFU/ml or undetectable. The in vivo experiment evaluating the protective efficacy of PVN02 against haemorrhagic septicaemia in striped catfish was conducted using 21 groups of 1,260 fish in 50-L plastic tanks in triplicate. The catfish were fed twice daily with phage-sprayed pellets. Different densities of bacterial suspensions were added into the tanks for 24 hr. Without the existence of the phage, the highest mortality rate was 68.3 ± 2.9% at the highest density of bacterial suspension. In contrast, the mortality rate at the highest density of bacterial suspension was significantly reduced to 8.33 ± 2.9% or 16.67 ± 2.9% at the phage dose of log 6.2 ± 0.09 or log 4.2 ± 0.09 PFU/g. This study provides a very practical manner of applying phage therapy to prevent disease in large-scale striped catfish farms.

Potential DNA Vaccine for Haemorrhagic Septiceamia Disease.

Pasteurella multocida is the main cause of haemorrhagic septicaemia (HS) outbreak in livestock, such as cattle and buffaloes. Conventional vaccines such as alum-precipitated or oil-adjuvant broth bacterins were injected subcutaneously to provide protection against HS. However, the immunity developed is only for short term and needed to be administered frequently. In our previous study, a short gene fragment from Pasteurella multocida serotype B was obtained via shotgun cloning technique and later was cloned into bacterial expression system. pQE32-ABA392 was found to possess immunogenic activity towards HS when tested in vivo in rat model. In this study, the targeted gene fragment of ABA392 was sub-cloned into a DNA expression vector pVAX1 and named as pVAX1-ABA392. The new recombinant vaccine was stable and expressed on mammalian cell lines. Serum sample collected from a group of vaccinated rats for ELISA test shows that the antibody in immunized rats was present at high titer and can be tested as a vaccine candidate with challenge in further studies. This successful recombinant vaccine is immunogenic and potentially could be used as vaccine in future against HS.

Improving Village Animal Health Worker participation in national disease surveillance systems: A case study from Cambodia.

Para-veterinary systems have arisen in numerous developing countries to address the low capacity of national veterinary services in meeting livestock health demands of mainly smallholder farmers. In Cambodia, the village animal health worker (VAHW) system was established in the early 1990s, involving short training programmes to equip VAHWs to provide basic animal health services for smallholder farmers, particularly the vaccination of cattle for haemorrhagic septicaemia (HS). However, there are increasing expectations that VAHWs provide village level disease surveillance information to the national veterinary services, despite their low-level disease diagnostic skills. To identify opportunities to improve the disease reporting system in Cambodia, a closed-ended cross-sectional study of VAHWs (n = 80) from two provinces was conducted in 2015, examining their contact frequency with district and provincial animal health authorities. Ordinal logistic regression was used to determine factors associated with increased frequency of VAHW contact with the district animal health authorities responsible for national disease reporting. Positive associations between income generated from VAHW activities (p = .01) and the frequency of visiting farmers (p < .0001), with more frequent contacts with district animal health authorities, were identified. High levels of inappropriate use of antibiotics, with almost 90% of VAHW reporting they use antibiotics to treat FMD-affected animals, were reported. Although further research to increase the rates of reporting at the smallholder farmer and VAHW levels is required, increased emphasis on complying with the World Organisation for Animal Health's competency guidelines for veterinary paraprofessionals may be required if they are expected to contribute to national animal disease surveillance systems.

Intranasal inoculation of recombinant DNA vaccine ABA392 against haemorrhagic septicaemia disease.

We evaluate the efficacy of recombinant DNA vaccine ABA392 against haemorrhagic septicaemia infection through intranasal administration route by targeting the mucosal immunity. The DNA vaccine was constructed and subjected to animal study using the Sprague Dawley (SD) rat. The study was divided into two major parts: (i) active and (ii) passive immunization studies, involving 30 animals for each part. Each group was then divided into five test groups: two test samples G1 and G2 with 50 and 100 µg ml-1 purified DNA vaccine; one positive control G5 with 106  CFU per ml formalin-killed PMB2; and two negative controls, G3 and G4 with normal saline and pVAX1 vector. Both studies were conducted for the determination of immunogenicity by total white blood cell count (TWBC), indirect ELISA and histopathological changes for the presence of the bronchus-associated lymphoid tissue (BALT). Our findings demonstrate that TWBC, IgA and IgG increased after each of the three vaccination regimes: groups G1, G2 and G5. Test samples G1 and G2 showed significant differences (P < 0·05) compared to the negative controls, G3 and G4, but no significant differences from the positive control G5. Groups G1, G2 and G5 showed more formation of BALT compared to the negative controls, G3 and G4. Our results show that intranasal inoculation of recombinant DNA vaccine ABA392 can provoke mucosal immunity which makes it a potential prophylactic against HS. SIGNIFICANCE AND IMPACT OF THE STUDY: New approach of combating haemorrhagic septicaemia disease among bovines by recombinant DNA vaccine is crucial to overcome the loss of edible products from the infected bovines. DNA vaccine can potentially serve as a better immunogen which would elicit both cellular and humoral immunity, and it is also stable for its molecular reproduction. This research report demonstrates an effective yet simple way of administering the DNA vaccine via the intranasal route in rats, to provoke the mucosal immunity through the development of immunoglobulins IgA, IgG and bronchus-associated lymphoid tissue which guard as the first-line defence at the host's mucosal lining.

Influence of amino acids and vitamins on the growth of gdhA derivative Pasteurella multocida B:2 for use as an animal vaccine.

Pasteurella multocida serotype B:2 is the causative agent of haemorrhagic septicaemia, a fatal disease in cattle and buffaloes. For use as a vaccine in the treatment of HS disease, an efficient cultivation of attenuated gdhA derivative P. multocida B:2 (mutant) for mass production of viable cells is required. In this study, the role of amino acids and vitamins on the growth of this particular bacterium was investigated. Initially, three basal media (Brain-heart infusion, Terrific broth, and defined medium YDB) were assessed in terms of growth performance of P. multocida B:2. YDB medium was selected and redesigned to take into account the effects of amino acids (glutamic acid, cysteine, glycine, methionine, lysine, tyrosine, and histidine) and vitamins (vitamin B1, nicotinic acid, riboflavin, pyridoxine, pantothenic acid, and biotin). High viable cell number was largely affected by the availability of micronutrient components and macronutrients. Histidine was essential for the growth whereby a traceable amount (20 mM) was found to greatly enhance the growth of gdhA derivative P. multocida B:2 mutant (6.6 × 109 cfu/mL) by about 19 times as compared to control culture (3.5 × 108 cfu/mL). In addition, amongst the vitamins added, riboflavin exhibited the highest impact on the viability of gdhA derivative P. multocida B:2 mutant (5.3 × 109 cfu/mL). Though the combined histidine and riboflavin in the culture eventually did not promote the stacking impact on cell growth and cell viability, nonetheless, they were still essential and important in either growth medium or production medium.

A novel approach for development, standardization, and safety testing of enriched alum-precipitated vaccine against hemorrhagic septicemia in different breeds of cattle.

Hemorrhagic septicemia is a fatal disease of cattle and buffalo all over the world including Pakistan and it causes heavy economic losses every year. The poor farmers cannot bear this loss in the form of less milk production and heavy expenditures on the animal treatment. An enriched alum-precipitated vaccine with reduced dose was prepared and standardized and safety testing of enriched vaccine was performed in Swiss albino mice as well as in natural host. In this experiment, a total of 36 cattle both male and female of different age groups ranging from 4 months to 4 years were used. All these animals belong to all major cattle breeds of Pakistan including Sahiwal, Red Sindhi (crossbred and purebred), Dhani (crossbred and purebred), Lohani (crossbred and purebred), and Cholistani, and exotic breeds including Holstein Friesian and Jersey. These animals were examined for current immune titer prior to vaccination. Animals were vaccinated subcutaneously with 2 mL and 4 mL dose of new vaccine and were observed for any untoward reaction for 48 h. All the animals were kept under close observation for the next 30 days and all were found safe. The experiment was designed to reduce the dose of the vaccine to 2 mL by using BHI as a growth medium, as well as to increase the number of doses prepared in the same infrastructure, hence reducing the cost of vaccine production. The study proved that vaccine with increased biomass in reduced dose is safe in local as well as in exotic breeds of cattle.

The ABA392/pET30a protein of Pasteurella multocida provoked mucosal immunity against HS disease in a rat model.

Haemorrhagic septicaemia (HS) is a well-known high fatality septicaemic disease happening among bovines. The disease is caused by the Pasteurella multocida serotype B:2 bacteria. P. multocida B:2 has high mortality and morbidity rates and is spread through the intranasal and oral routes in bovines. In this study, our aim was to investigate the efficacy of the recombinant protein vaccine, ABA392/pET30a via intranasal inoculation by targeting the mucosal immunity. The constructed recombinant protein vaccine ABA392/pET30a was subjected to an animal study using Sprague Dawley rats. The study was divided into two parts: active and passive immunization studies. Both studies were carried out through the determination of immunogenicity (using Total White Blood Cell (TWBC) Count with Indirect ELISA) and histopathogenicity, analyzing (Bronchus Associated Lymphoid Tissue (BALT) formation) in lungs. As a result, the IgA and IgG development of both tested groups: group 1 (50µg/mL protein vaccine) and group 2 (100µg/mL protein vaccine) showed equivalent with the positive control group 4 (formalin-killed P. multocida B:2). However, there was a significant difference when compared with the negative control group 3 (normal saline). These results demonstrate that both the protein vaccine at the concentration 50µg/mL and 100µg/mL have the same efficacy as the commercially available positive control vaccine. From the studies, higher concentration of protein vaccine at 100µg/mL showed higher development of both IgA and IgG compared to 50µg/mL protein vaccine. Higher and rapid development of IgA compared to IgG showed that mucosal immunity has been induced through the intranasal administration of the protein vaccine. In addition, leucocytosis was observed at each dose of vaccination showed that the protein vaccine is capable to induce the immune responses of the host. Histopathogenicity studies of the vaccinated groups showed more BALT formation and no severe lesions after challenge compared to the negative control group. Besides, no inflammatory onsite or anaphylactic responses were observed after the intranasal inoculation which proved to be safer and provided longer lasting immunity. Therefore, recombinant protein vaccine ABA392/pET30a could be a potential candidate for intranasal administration which can provoke mucosal immunity against HS disease.

Intranasal immunization with a recombinant outer membrane protein H based Haemorrhagic septicemia vaccine in dairy calves.

Haemorrhagic septicemia (HS) is a contagious disease in cattle with high morbidity and mortality rates. HS vaccine in Thailand is an oil-adjuvant formulation, and is difficult to administer. The present study aimed to formulate and evaluate the protection in dairy calves conferred by immunization with an in-house intranasal HS vaccine. The intranasal vaccine was formulated in a total volume of 500 µl containing either 50 or 100 µg of the recombinant outer membrane protein H (rOmpH) of Pasteurella multocida strain M-1404 (serovar B:2), and 10 µg of Cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) as a mucosal adjuvant. Intranasal immunizations were conducted three times at three-week intervals. The antibodies post-immunization were detected by indirect ELISA and demonstrated efficient in vitro activity in suppressing a P. multocida strain from the complement-mediated killing assay. An intranasal vaccine induced both the serum IgG and secretory IgA levels that were significantly higher than the level conferred by the parenteral vaccine (P<0.05). Challenge exposure was conducted with a P. multocida strain M-1404 at day 72 of the experiments. The immunized calves had reduced clinical signs after challenge exposure that would normally result in disease proliferation. We conclude that intranasal vaccination of calves with rOmpH with CpG-ODN 2007 stimulated serum and secretory antibodies to rOmpH and whole cells of P. multocida strain M-1404 antigen. Moreover, it would result in protection in calves against artificial P. multocida infection.

Improved stability of live attenuated vaccine gdhA derivative Pasteurella multocida B:2 by freeze drying method for use as animal vaccine.

The efficacy of attenuated strain of gdhA derivative Pasteurella multocida B:2 mutant as a live vaccine to control haemorrhagic septicaemia (HS) disease in cattle and buffaloes has been demonstrated. In order to use P. multocida B:2 mutant as a commercial product, it is essential to optimise its formulation for high viability and stability of the live cells. The effectiveness of freeze-drying process using different protective agent formulations for improving cells viability was explored. Sugar and nitrogen compounds were used as protective agents in freeze-drying and the capability of these compounds in maintaining the viability of mutant P. multocida B:2 during subsequent storage was investigated. A complete loss in viability of freeze-dried mutant P. multocida B:2 was monthly observed until 6-12 months of storage at -30 °C, 4 °C and 27 °C when nitrogen compound or no protective agent was added. Trehalose and sucrose showed significantly high survival rate of 93-95% immediately after freeze-drying and the viability was retained during the subsequent storage at -30 °C and 4 °C. A smooth cell surface without any cell-wall damage was observed for the cells formulated with trehalose under scanning electron micrograph. This study presented a freeze-drying process generating a dried live attenuated vaccine formulation with high stability for commercial applications.

Host responses and bacterial colonization following inoculation of Pasteurella multocida P52 strain into unvaccinated and aluminum hydroxide gel hemorrhagic septicemia vaccinated mice.

Hemorrhagic septicemia is a highly infectious and contagious disease caused by Pasteurella multocida serogroup B:2 in tropical Asian and African countries. The acute inflammatory responses induced by Pasteurella multocida are the main cause of death in hemorrhagic septicemia. Therefore, present study was undertaken to examine the blood cytokine expression profiles (TNF-α, IL-1ß, and IL-6), bacterial colonization and histopathological changes of intraperitoneally and subcutaneously challenged vaccinated and unvaccinated mice with 102 CFU of P. multocida P52. The observations were made at 6, 12, 18, 24 h and 48 h intervals. Real-time PCR based blood cytokine profiles (TNF-α, IL-1ß, and IL-6) measurement revealed a significantly higher amount of pro-inflammatory cytokines expression in the unvaccinated challenged group of mice than the vaccinated challenged group. There was heavy bacterial load in all organs of mice viz. trachea, lung, spleen, within 6 h of challenge in both vaccinated and unvaccinated group of mice, but bacterial load increased in the unvaccinated challenged group of mice with respect to time whereas the load were constant in the vaccinated challenged group. Histopathological changes were mild in the vaccinated challenged group of mice in comparison to the unvaccinated challenged group. There was no significant difference in the bacterial load, histopathological changes and cytokines expression when challenged through different routes.

Interaction between Pasteurella multocida B:2 and its derivatives with bovine aortic endothelial cell (BAEC).

BACKGROUND: Pasteurella multocida B:2 causes bovine haemorrhagic septicaemia (HS), leading to rapid fatalities in cattle and buffaloes. An attenuated derivative of P. multocida B:2 GDH7, was previously constructed through mutation of the gdhA gene and proved to be an effective live attenuated vaccine for HS. Currently, only two potential live attenuated vaccine candidates for HS are being reported; P. multocida B:2 GDH7 and P. multocida B:2 JRMT12. This study primarily aims to investigate the potential of P. multocida B:2 GDH7 strain as a delivery vehicle for DNA vaccine for future multivalent applications. RESULTS: An investigation on the adherence, invasion and intracellular survival of bacterial strains within the bovine aortic endothelial cell line (BAEC) were carried out. The potential vaccine strain, P. multocida B:2 GDH7, was significantly better (p ≤ 0.05) at adhering to and invading BAEC compared to its parent strain and to P. multocida B:2 JRMT12 and survived intracellularly 7 h post treatment, with a steady decline over time. A dual reporter plasmid, pSRGM, which enabled tracking of bacterial movement from the extracellular environment into the intracellular compartment of the mammalian cells, was subsequently transformed into P. multocida B:2 GDH7. Intracellular trafficking of the vaccine strain, P. multocida B:2 GDH7 was subsequently visualized by tracking the reporter proteins via confocal laser scanning microscopy (CLSM). CONCLUSIONS: The ability of P. multocida B:2 GDH7 to model bactofection represents a possibility for this vaccine strain to be used as a delivery vehicle for DNA vaccine for future multivalent protection in cattle and buffaloes.

Improving Smallholder Farmer Biosecurity in the Mekong Region Through Change Management.

Transboundary animal diseases including foot-and-mouth disease and haemorrhagic septicaemia remain a major constraint for improving smallholder large ruminant productivity in the Mekong region, producing negative impacts on rural livelihoods and compromising efforts to reduce poverty and food insecurity. The traditional husbandry practices of smallholders largely exclude preventive health measures, increasing risks of disease transmission. Although significant efforts have been made to understand the social aspects of change development in agricultural production, attention to improving the adoption of biosecurity has been limited. This study reviews smallholder biosecurity risk factors identified in the peer-reviewed literature and from field research observations conducted in Cambodia and Laos during 2006-2013, considering these in the context of a change management perspective aimed at improving adoption of biosecurity measures. Motivation for change, resistance to change, knowledge management, cultural dimensions, systems theory and leadership are discussed. Due to geographical, physical and resource variability, the implementation of biosecurity interventions suitable for smallholders is not a 'one size fits all'. Smallholders should be educated in biosecurity principles and empowered to make personal decisions rather than adopt prescribed pre-defined interventions. Biosecurity interventions should be aligned with smallholder farmer motivations, preferably offering clear short-term risk management benefits that elicit interest from smallholders. Linking biosecurity and disease control with improved livestock productivity provides opportunities for sustainable improvements in livelihoods. Participatory research and extension that improves farmer knowledge and practices offers a pathway to elicit sustainable broad-scale social change. However, examples of successes need to be communicated both at the 'evidence-based level' to influence regional policy development and at the village or commune level, with 'champion farmers' and 'cross-visits' used to lead local change. The adoption of applied change management principles to improving regional biosecurity may assist current efforts to control and eradicate transboundary diseases in the Mekong region.

Recombinant transferrin binding protein A (rTbpA) fragments of Pasteurella multocida serogroup B:2 provide variable protection following homologous challenge in mouse model.

Transferrin binding protein A (TbpA), an iron acquisition surface protein that also acts as virulence factor, is widely distributed among strains of Pasteurella multocida. In the present study, a total of seven clones of TbpA fragments (39D to F777; 39D to Q697; 188V to F777; 188V to Q697; 39D to P377; 188V to P377 and 39D to F187) belonging to P. multocida B:2 were constructed, over-expressed and purified as recombinant fusion proteins from Escherichia coli using affinity chromatography. Immunization of mice with rTbpA fragments resulted in a significant (p < 0.05) rise in antigen specific serum total IgG and subtypes (IgG1 and IgG2a) tires. All immunized mice challenged with 8 LD50 of P. multocida B:2 resulted in a variable protective efficacy up to 50%. The study indicated the potential possibilities to incorporate full length TbpA in subunit vaccine formulation composed of synergistic subunit antigens against haemorrhagic septicaemia (HS) in cattle and buffalo.

The Socioeconomic Impacts of Clinically Diagnosed Haemorrhagic Septicaemia on Smallholder Large Ruminant Farmers in Cambodia.

Haemorrhagic septicaemia (HS) is an acute fatal infectious disease of mainly cattle and buffalo and outbreaks occur commonly in Cambodia. Disease outbreak reports were examined to select five villages from three provinces for a retrospective investigation of HS epidemiology and socioeconomic impact on smallholders, with an aim of identifying potential benefits from improving disease prevention through biosecurity and vaccination. The Village Animal Health Worker (VAHW) or Chief in each village and 66 affected smallholders were surveyed. At the village level, 24% of all households were affected with an estimated mean village herd morbidity of 10.1% and mortality of 28.8%. Affected farmers reported HS disease morbidity and mortality at 42.7% and 63.6% respectively. Buffalo had a higher morbidity (OR = 2.3; P = 0.003) and mortality (OR = 6.9; P < 0.001) compared with cattle, and unvaccinated large ruminants a higher morbidity (OR = 2.9; P = 0.001). The financial impact varied depending on whether the animal survived, provision of treatment, draught replacement and lost secondary income. The mean cost per affected household was USD 952.50 based on ownership of five large ruminants. The impact per affected animal was USD 375.00, reducing the pre-disease value by 66.1%. A partial budget revealed an overwhelming incentive for farmers to practice biannual vaccination, with a net benefit of USD 951.58 per household based on an annual disease incidence rate of 1. Sensitivity analysis showed that a net benefit of USD 32.42 remained based on an outbreak every 20 years. This study indicates HS can cause a catastrophic financial shock to smallholders and remains a critical constraint to improving large ruminant productivity and profitability. Addressing HS disease control requires a focus on improving smallholder farmer knowledge of biosecurity and vaccination and should be priority to stakeholders interested in addressing regional food insecurity and poverty reduction.

Development of Pasteurella multocida-loaded microparticles for hemorrhagic septicemia vaccine.

In this study, Pasteurella multocida-loaded alginate microparticles (MPs) for subcutaneous vaccination was developed by emulsification-cross-linking technique. Formulation parameter was varied as a ratio of polymer and bacterin. Optical microscopy revealed spherical particles with uniformly distribution. A mean particle size of approximately 6 µm has been successfully constructed using simple mixer and ultrasonic probe. The zeta potential of the MPs showed negatively charge of approximately -23 mV determined by Zeta Pals® analyzer. The entrapment efficiency and the in vitro bacterin released profile could be controlled by varying the amount of alginate. The high entrapment efficiency up to 69% was achieved with low concentration of alginate. The MPs possessed a slow bacterin release profile, up to 30 days. In vivo safety and potency tests were proved that the alginate MPs were safe and induced protective immunity in mice. In addition, after storage for 6 months at either 4 °C or room temperature, the protective immunity in mice was maintained.

Impacts on rural livelihoods in Cambodia following adoption of best practice health and husbandry interventions by smallholder cattle farmers.

To better understand how smallholder farmers whom own the majority of Cambodian cattle can contribute to efforts to address food security needs in the Mekong region, a five-year research project investigating methods to improve cattle health and husbandry practices was conducted. Cattle production in Cambodia is constrained by transboundary animal diseases (TADs) including foot-and-mouth disease (FMD) and haemorrhagic septicaemia (HS) plus poor nutrition, reproduction and marketing knowledge. The project worked in six villages in Kandal, Takeo and Kampong Cham province during 2007-12. Farmers from three 'high intervention' (HI) villages incrementally received a participatory extension programme that included FMD and HS vaccination, forage development and husbandry training. Evaluation of project impacts on livelihoods was facilitated by comparison with three 'low intervention' (LI) villages where farmers received vaccinations only. Results of knowledge, attitude and practice (KAP) and socio-economic surveys conducted in 2012 of 120 participating farmers identified that farmer knowledge in the HI project sites exceeded LI sites on the topics of biosecurity, internal parasites, nutrition and reproduction. HI farmers adopted biosecurity practices including a willingness to vaccinate for FMD and HS at their own cost, separate sick from healthy cattle, grow and feed forages and displayed awareness of the benefits of building fattening pens. HI farmers that grew forages observed time savings exceeding two hours per day each for men, women and children, enabling expansion of farm enterprises, secondary employment and children's schooling. Logistic regression analysis revealed that farmers in the HI group significantly increased annual household income (P < 0.001), with 53% reporting an increase of 100% or more. We conclude that improving smallholder KAP of cattle health and production can lead to improved livelihoods. This strategy should be of interest to policymakers, donors, researchers and extension workers interested in addressing TAD control, food insecurity and rural poverty in Southeast Asia.

Efficacy of intranasal vaccination of field buffaloes against haemorrhagic septicaemia with a live gdhA derivative Pasteurella multocida B:2.

The efficacy of an intranasal haemorrhagic septicaemia vaccine containing live gdhA derivative Pasteurella multocida B:2 was tested in buffaloes in Sabah. Sixty buffaloes, kept grazing in the field with minimal human intervention were devided into three groups of 20 buffaloes per group. Buffaloes of group 1 were exposed intranasal to 5 ml vaccine containing 10(6) CFU/ml of live gdhA derivative P multocida B:2. Buffaloes of group 2 were not exposed to the vaccine but exposed to PBS and were allowed to commingle and graze in the same field as the buffaloes of group 1 while buffaloes of group 3 were similarly exposed to PBS and were grazing separately. Booster was on group 1, two weeks later. Twelve months after the first vaccination, three buffaloes from each group were brought into the experimental house and challenged subcutaneously with 10(9) CFU/ml of live wild-type P multocida B:2. All challenged buffaloes of groups 1 and 2 survived with only mild, transient signs while all control unvaccinated buffaloes developed severe signs of haemorrhagic septicaemia and were euthanased between 28 hours and 38 hours postchallenge with signs and lesions typical of haemorrhagic septicaemia. These data showed that the gdhA mutant strain, given intranasally as two doses two weeks apart, successfully induced systemic immunity in exposed buffaloes and also led to spread of vaccine strain to the in-contact animals, where it acted as an effective live vaccine to protect both exposed buffaloes and in-contact buffaloes against challenge with the virulent parent strain.