Immunology of the Outer Membrane Proteins of Pasteurella Haemolytica A2, A7 and A9 in Sheep

Pneumonic pasteurellosis is a common respiratory disease of goats and sheep throughout the world, including Malaysia. In Malaysia, Pasteurella haemolytica A2 is most commonly isolated from cases of pneumonic pasteurellosis in sheep and goats followed by Pasteurella haemolytica A7 and A9. Vaccination has been used widely to control the disease with uncertain success rate. The reasons for vaccination failure in the field were due to incompatible strains, unsuitable antigen as vaccine component and improper vaccination programme. Therefore, the attentions have been focused on the concept of a novel vaccine, which includes subunit vaccine. The outer membrane proteins (OMPs) of Pasteurella haemolytica A2, A7 and A9 have been extracted using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SOS-PAGE). Each serotype gave two to three major polypeptide bands with some minor bands. Immunoblotting, carried out using homologous and heterologous antisera against the OMPs from all serotypes. The results showed that the 30 kDa band of Pasteurella haemolytica A7 could be recognised by all antisera, and was thus concluded as the major and common immunogen. The in vivo tests using the OMPs of the three serotypes revealed that sheep injected with the 100/lg OMP followed by a booster dose on day 21 showed highest antibody level on day 28 post-injection. Animals vaccinated with the OMP of Pasteurella haemolytica A7 showed good immune response upon challenge with significantly (p<0.05) less severe lung lesions regardless whether challenged with Pasteurella haemolytica serotype A2, A7 or A9. Those animals vaccinated with the OMP of Pasteurella haemolytica A2 failed to protect against challenge with live Pasteurella haemolytica A9 while those vaccinated with the OMP of Pasteurella haemolytica A9 failed to protect against challenge with live Pasteurella haemolytiva A2 and A7. It is concluded that the OMP of Pasteurella haemolytica A7 provides cross-protection to challenges uSing live Pasteurella haemolytica A2, A7 and A9. Thus, the OMP of Pasteurella haemolytica A7, particularly the 30 kDa, could be the best candidate for a subunit vaccine against pneumonic pasteurellosis in sheep

Identification of vibrio species isolated from marine fish using polymerase chain reaction

Vibrio species are found in marine and estuarine environments. Vibriosis can cause more than 50% mortality in fish culture facilities once an outbreak is in progress. The objectives of this study were to subculture and identify Vibrio spp. that were isolated previously from marine fish; to develop a technique for simultaneous identification of several Vibrio spp. (V. alginolyticus, V. parahaemolyticus, V. fluvialis and V. vulnificus) using polymerase chain reaction (PCR); and to compare the rapid identification kit and PCRtechniques commonly used for the identification of the Vibrio spp. In this study, 20 isolates from four Vibrio species, which consisted of five eachof V. alginolyticus, V. parahaemolyticus, V. fluvialis and V. vulnificus isolates were provided by National Fish Health Research Centre (NaFisH). The species of Vibrio were identified using an identification kit, API 20E system. These organisms were isolated from various marine fish such as Asian Seabass (Latescalcarifer), Grouper (Epinepheluscoioides), Silver Pomfret (Pampusargenteus) and Red Snapper (Lutjanuscampechanus). The isolates were previously stored at -80°C and subcultured onto TSA+. The pure cultures were then transferred to TSB+. These isolates were subjected to DNA extraction. Once the DNA is ready, PCR was used to optimise the products with the designated primers. All the PCR products were electrophoresed through 1% agarose gel for 1 h. The designated primers in this study were found suitable for the detection of V. alginolyticus,V. parahaemolyticus,V. fluvialis and V. vulnificus.Using the API 20E system, 15% (3/20) isolatesof Vibrio spp. were negative, indicating the the PCR technique is still required to confirm the result obtained by the use of the API 20E system

Role of heat stress in red tilapia streptococcosis

Streptococcus agalactiae is one of the causative agents associated with warm-water streptococcosis that produces massive mortality in aquaculture. The emergence of disease in tilapia farms usually occurs during high temperature seasons, which suggested higher susceptibility of tilapia to infection under this condition. Thus, the objectives of this study were to determine the pathogenesis of streptococcosis in heat-stressed tilapia using various routes of infection and the role heat stress in the development of streptococcal infection in tilapia. Red tilapias, including the control group without heat stress, were inoculated with 109 CFU/mL of S. agalactiae via intraperitoneal, immersion and immersion cut routes of inoculations and maintained at a water temperature of 34ºC for 24 hours. Samples of brain, eyes and kidneys were taken and subjected to bacterial isolation, PCR, histological examination and immunoperoxidase test. Diseased fish showed typical signs of bacterial septicaemia including skin and fin haemorrhage and exophthalmia. The fishes were more susceptible to intraperitoneal route of infection, followed by immersion cut and lastly immersion. The bacteria was isolated and detected by PCR from all organs of fishes with and without heat stress. The lesions were more clearly seen first in fishes with heat-stressed than those without. Fifty percent mortality occurred in the heat-stressed group infected via intraperitoneal route. However, no mortality was observed in the group without heat stress. Post-mortem revealed that the lesions were more severe in the heat-stressed group infected via the intraperitoneal route than those infected via the immersion cut and immersion routes. The lesions observed were haemorrhage, presence of inflammatory cells and bacteria in the brain, eyes and kidneys. There is significant (p0.05) difference was observed between routes of infection in most organs of fishes without heat stress. Immunoperoxidase test were positive in most organs. However, the intensity of the antigen-antibody reactions were greatest in the group infected via the intraperitoneal route followed by immersion cut and immersion groups. In conclusion, the severity of lesions observed in the brain, eye and kidneys are most marked in heat-stressed red tilapias infected with S. agalactiae via the intraperitoneal route, followed by the immersion cut and lastly the immersion route

Isolation and identification of pathogenic bacteria from red tilapia in cage-cultured system and its environment

Bacteria were isolated from the brain, eye and kidney of red tilapia, as well as water and debris samples. The weight and length of red tilapia were measured and the water quality as well. API test were done to identify the type of bacteria from the isolates. In Kenyir Lake, bacterial isolates that predominated in the fish were Micrococcus spp. and Aeromonas hydrophila at 13.64 %, in water samples it was Staphylococcus xylosus at 40% and in the debris samples, Pseudomonas aeruginosa and Enterobacter cloacae at 50%. In the Semantan River, the predominant bacteria in fish and debris samples were Aeromonas hydrophila at 23.53 % and 90 % respectively. In the water samples, Staphylococcus lentus and Staphylococcus xylosus were the predominant bacteria with 30 and 20%, respectively. The ammonia, sulphide, iron and nitrite-nitrogen levels in the Semantan River were over the acceptable limits and this may lead to high fish mortality. This study concluded that Aeromonas hydrophila and Staphylococcus spp. were the most predominant bacteria in red tilapia and poor water quality played a major role in red tilapia succumbing to infections by pathogenic bacteria

Efficacy of feed-based adjuvant vaccine against Streptococcus agalactiae in Oreochromis spp. in Malaysia.

This study was conducted to determine the systemic, mucosal immunity and protective capacity of the feed-based adjuvant vaccine (FAV) of Streptococcus agalactiae following oral vaccination against streptococcosis in tilapias. Two hundred and sixteen red tilapia fish were divided into three major groups. Each major group consisted eight tilapia kept in nine 2000 L glass aquaria. At day 0, all fish from the FAV group were fed with feed that had been incorporated with an adjuvant, while fish in the feed-based vaccine (FNV) group were fed with vaccine incorporated into the pellet without adjuvant. Fish in the control-unvaccinated group, FC, were fed with normal commercial pellet. Booster dose was performed on day 14 post immunization. Fish from each group were sacrificed on a weekly basis for the entire 7 weeks. Serum, body mucus and gut lavage fluid were evaluated for antibody responses by indirect ELISA, while histological examination was carried out on the gut following intraperitoneal challenge. The FAV group had a significantly higher protection (P < 0.05) following challenge with 3.4 × 109 CFU mL−1 of live S. agalactiae than FNV group. This level of protection may be due to high antibody responses, increase in size of gut-associated lymphoid tissue and high number of lymphocytes in the FAV grou

The effects of commercial flower honey and turmeric on dermal wound in rats

Twenty healthy rats, ten adults (2-month-old) and ten young (1-month-old), were used in this study. Four skin biopsies were created at the dorsum of each rat under general anesthesia. The wound was each treated with honey, turmeric powder, turmeric-honey paste and a blank (control). The wounds were photographed on day 0, 1, 3, 5, 7 and 9. Wound area reduction was measured on day 9 after which the rats were euthanized. The skin samples were taken for histology. The results showed that there was no significant difference in the healing between treatments in young and adult rats. However, honey was the best treatment with the highest healing scores, followed by control, turmeric and honey-turmeric paste. Honey-turmeric paste resulted in a severe wound infection thus delayed healing

Molecular and antigenicity characterisation of Vibrio sp. isolates from Asian seabass (Lates calcarifer)

Two species of vibrio (Vibrio fluvialis and Vibrio mimicus) isolated earlier from Lates calcarifer was sub-cultured into thiosulfate citrate bile sucrose (TCBS) agar. They were then grown into brain heart infusion broth (BHI) with the addition of 1% sodium chloride (NaCl). This culture was incubated with gentle shaking (50 rpm) for 24 h at 37ºC. Outer membrane protein (OMP) of both vibrio species was prepared from the culture in brain heart infusion broth. The bacteria were pelleted and subjected to sonication to break the bacteria cells from its membrane. The outer membrane of the vibrio species was then separated using sodium polyacrylamide gel electrophoresis (SDS-PAGE) in running buffer and subjected to 300 ma, 100 v for 1 h and 20 min. The gel containing polypeptides was then transferred into a nitrocellulose membrane for immunoblotting. Hyperimmune serum against the OMP of the vibrio spp. raised rabbits was used in this study. Immunodetection was done by subjecting the nitrocellulose membrane to the antiserum against the respective vibrio species. Horseradish peroxidase (HRP) was used as conjugate to facilitate binding of antigen antibody complex reaction at the nitrocellulose membrane. All photographed gels were scanned and analysed using gel analysis software gene tool. The results were compared to protein standard markers. This study shows that for vibrio fluvialis, the most antigenic OMPs were of molecular weights 50, 60a and 75 kda whereas the most antigenic proteins for the whole cells of this strain were of 33 and 75 kda. For vibrio mimicus the most antigenic OMPs were of molecular weight 40 and 80 kda while the most antigenic protein for the whole cells were of molecular weights 24 and 35 kda. These antigenic proteins can be good vaccine candidates against vibrio spp infections

The effects of oral vaccination of Streptococcus agalactiae on stimulating gut-associated lymphoid tissues (GALTs) in tilapia (Oreochromis spp.)

Vaccination of fish by intraperitoneal (i.p.) injection and bath immersion against bacterial infections has been proven to be a commercial success. However, those routes of vaccination are not economical in practice due to several reasons such as high labour cost, highly time consuming, and causing stress to the fish. Meanwhile, oral vaccination is considered as the best route to vaccinate the fish due to less stress to the fish, ability to treat large batch at one time, and easy and practical to administer booster vaccination. In this study, effect of oral vaccination with various regimes in stimulating gut-associated lymphoid tissues (GALTs) against Streptococcus agalactiae infection was observed. In this vaccination experiments, four groups of fish with four replicates consisting of 15 tilapias each were used; four groups per treatment received antigen incorporated vaccine in different regimes. Group 1 was fed with vaccine once per week, Group 2 was fed three consecutive days per week, and Group 3 was fed five consecutive days per week, while Group 4 (control) was fed with standard commercial feed. Booster dose was administered at day-14 after the first administration, and humanely killed at day-28 post-booster vaccination. Ten fish from each group were collected for gut sampling and subjected for histological analysis using Olympus FIVE Image Analyzer. Aggregations of GALTs were observed in lamina propria of the gut. The sizes of GALTs were measured and the numbers of lymphoid cells were also counted. The diameter of GALTs showed no significant (p>0.05) difference between Groups 1 to Group 2 and Group 2 to Group 3, but a significant difference (p0.05) were found between Group 1 to Group 2 and Group 2 to Group 3; however, a significant difference (p<0.05) was observed between Groups 1 and Group 3. As a conclusion, the frequencies of administration play a role in stimulating the size of GALT which is correlated with the number of aggregated lymphoid cells in the gastrointestinal tract of tilapia

Determination of LD50 for Streptococcus agalactiae and Staphylococcus aureus infections in tilapia

One hundred and sixty fingerlings and 80 adult tilapias were experimentally infected with Streptococcus agalactiae and Stapylococcus aureus to determine their LDso. Four concentrations of Streptococcus agalactiae (109, 108,107, 106 CFU/mL) were used in this experimental infection. These tilapias were divided into 4 groups of 40 fingerlings and 20 adults per group. Groups 1, 2, 3 and 4 of the fingerlings were exposed to 109, 10,107, 106 CFU/mL of S. agalactiae by immersion in 2 L inoculum solution for 20 min. Similarly, the adult groups were exposed to the same concentrations of S. agalactiae but by intraperitoneal injection at the rate of 1 mL of the inoculum per gram. Similar procedures were repeated using exposure to Staphylococcus aureus alone or a combination of S. agalactiae and S. aureus. All test groups were observed for signs of infections. On Day 7 post-infection (pi), all fish that were still alive were humanely killed. The LDso of the adult tilapia that were exposed to S. agalactiae, S. aureus or mixed infection was 2.3884 x 107,2.8151 X 108 , and 4.2409 x io', respectively. For the fingerling groups, the LDso for S. agalactiae, S. aureus, and mixed infection was 2.9242 x 1020,2.8665 x 1017 , and 4.9748 x IO!', respectively. Experimental infection in adults could be established within 12 h post-injection to 6.3 x 109 CFU per mL and 9.7 x 109 CFU per mL of S. agalactiae and S. aureus, respectively. For fingerlings, infection could be established within 72 h following bath immersion to 6.3 x 109 CFU per mL and 9.7 x 109 CFU per mL of S. agalactiae and S. aureus, respectively

Carbon Dioxide (CO2) emission, energy consumption and economic growth: Evidence from selected Southeast Asia countries / Nur Hafizah Ismail … [et al.]

Southeast Asia countries have experienced rapid economic growth within the past decades with a significant increase in energy dependency and carbon dioxide (CO2) emissions. Continuous development in the urban area has stimulated a rise in energy consumption in many Southeast Asia countries, which resulted in an improvement of citizen's lifestyles and living standards due to increasing income and population. Understanding the relationship between economic growth, energy consumption, and carbon dioxide emissions help economies in formulating energy policies, enhancing energy security and developing a sustainable energy resource. Therefore, this study focuses on the economic growth, energy consumption and carbon dioxide emissions evolved in Southeast Asia by using Environment Kuznets Curve theory. This paper could be useful and beneficial for the Southeast Asia countries to form appropriate environmental policies to maintain the balance of energy demand and supply and to deal with environmental quality issues