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

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

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

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

Effect of route of infection on development of streptococcosis in Red tilapia

The effect of route of infection on the development of streptococcosis in the red tilapia was assessed using histopathological, PCR, microbiological and immunohistochemical methods. Three hundred healthy, adult Red tilapias (Oreochromis spp.) were obtained from a commercial hatchery. Forty-eight fish were randomly taken from the same age and weight (150 g) per group. Fish were clinically examined and screened one week prior to the experiment. The experiment was divided into three groups with duplicates. Group 1 was exposed to Streptococcus agalactiae intraperitoneally (IP), Group 2 was exposed to S. agalactiae through an immersion broth, and Group 3 was exposed through a skin cut immersion. The fish were necropsied at the end of 24 hours exposure, and the brain, eye and kidney samples were taken for microbiological, histopathological examinations and indirect immunoperoxidase test. Statistical analysis (ANOVA) from the histopathological lesions revealed that there was significant (p<0.05) difference between the route of infection in the brain but not in the eye and kidney, while IP route showed severe lesions at 8 hours post-inoculation. In conclusion, S. agalactiae was pathogenic to Red tilapia causing septicaemia and severe pathological changes through different routes of infection

In vitro antigenicity and cross-reaction of the outer membrane proteins of Pasteurella haemolytica A2, A7 and A9

The outer membrane proteins of Pasteurella haemolytica A2, A7 and A9 were subjected to SDS-PAGE and immunoblotting. The molecular weights of the polypeptide bands ranged between 33 to 97 kDa. The major polypeptide bands for P. haemolytica A2 were 33.4, 39.2 and 45 kDa while the minor polypeptide bands were 50, 58.7, 66.2, 84.7 and 97.4 kDa. Analysis of the outer membrane proteins of P. haemolytica A7 revealed two major protein bands of 33.4 and 45 kDa and three minor polypeptide of 40, 50 and 66.2 kDa. There were three major (33.4, 37.5 and 45 kDa) and one minor protein band (50 kDa) in the outer membrane proteins of P. haemolytica A9. There was one major protein band from each of the P. haemolytica A2, A7 and A9, which was unique to the respective serotype and appeared to represent the respective serotype. These were the 39.2 kDa band for P. haemolytica A2, the 40 kDa band for P. haemolytica A7 and the 37.5 kDa band for P. haemolytica A9. Following homologous immunoblot, all the serotypes showed pronounced antigenicity at the 30 kDa band. Heterologous immunoblot using the antiserum of P. haemolytica A2 did not reveal any antigenic band of P. haemolytica A9 but revealed antigenic bands at 30 and 31 kDa of P. haemolytica A7. Heterologous immunoblot using the antiserum of P. haemolytica A7 revealed antigenic band at 30 kDa of all the three serotypes while the antiserum of P. haemolytica A9 failed to reveal any common antigenic band between all three serotypes. Thus, the 30 kDa band of P. haemolytica A7 may be a suitable candidate for a sub-unit Vaccine against pneumonic pasteurellosis of sheep and goats

Male Reproductive System

The overall reproductive process consists of both the human sex organs which include the male and female reproductive system. The ability to produce offsprings that have similar characteristic as their parents is the goal of reproduction. The sexual type of reproduction takes place in human and both male and female reproductive system is required. Male reproductive system is mainly concerned with production of semen (whitish viscous fluid emitted from the male reproductive tract that contains sperm and fluids) and transferring it into the female reproductive tract. In this review, we will discuss the latest findings in the research pertaining the male reproductive system and its contribution towards the research in advancement of reproductive physiology

Male reproductive system

The overall reproductive process consists of both the human sex organs which include the male and female reproductive system. The ability to produce off springs that have similar characteristic as their parents is the goal of reproduction. The sexual type of reproduction takes place in human and both male and female reproductive system is required. Male reproductive system is mainly concerned with production of semen (whitish viscous fluid emitted from the male reproductive tract that contains sperm and fluids) and transferring it into the female reproductive tract. In this review, we will discuss the latest findings in the research pertaining the male reproductive system and its contribution towards the research in advancement of reproductive physiology