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Browsing PhD by Subject "Animal Husbandry"
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- ItemClinical and endocrinaological studies on postpartum ovarian activity in Lanka buffaloes ( bubalus bubalis)(University of Peradeniya, 1990-01) Mohan, VadivelLong calving interval has been recorded as the important cause of poor fertility in Lanka buffaloes. However, buffaloes raised in some locations show. short calving intervals and better fertility than animals in many other areas. The reasons for these differences could he genetic or environmental (ie. climatic, management, nutrition etc.) Further, the endocrinological changes associated with resumption of ovarian activity and the effect of suckling on these changes have not been studied in Lanka buffaloes. Therefore, four experiments were conducted to determine the following : 1. reason(s) Lor the differences in fertility in different locations; Zs endocrinological changes during the postpartum period and 3. effect of suckling on resumption of ovarian. activity and other fertility indices. First, sixteen buffalo cows from a “low fertility" area and a "high fertility" area (n=8 each) were brought to a different environment and maintained over a period of 3 years under a uniform system of management. Postpartum ovarian activity was monitored in these animals by observation for signs of oestrus, weekly rectal examination of internal genitalia and weekly measurement of plasma progesterone concentrations. Results showed that there were no differences (p>0.05) in calving interval, time taken for uterine involution, duration of postpartum anoestrus and in number of services required for conception between these two groups. Calvings were distributed throughout the year and the mean calving interval was similar to what was reported for this area. Second, the endocrinological changes associated with the early postpartum period was monitored in 14 Lanka buffalo cows. On days 7,14, 21 and 28 postpartum, sequential blood samples were collected at 15 min. intervals for 8 hour periods. Subsequently intravenous administration of two injections of 12.5 ug GnRH was done two hours apart, followed by a further 4 hours of blood sampling at the same frequency. Plasma LH concentrations were measured by a heterologus RIA (detection limit 0.25 ng/ml).The validity of this RIA for measuring biologically active LH was checked by assaying some of the samples with a specific bioimmunoassay developed during this study. The LH concentrations remained below the detection limit of the assay and LH pulses were not seen in these animals up to day 28 postpartum. The pituitary did not respond to low doses of exogenous GnRH by increased LH release during this period. Third, 18 pluriparous Lanka buffalo cows were allotted to 1 of 3 treatment groups to study the effect of suckling on pituitary and ovarian function. Calves were allowed to suckle continuously in group AS (ad lib. suckling) and for two periods of 20 minutes in group RS (restricted suckling). different environmental conditions. The pre GnRH mean LH concentrations increased in all three groups with the days postpartum. The mean LH for group RS was higher (p*%0.05) than that for other two groups. There was no differences (p>0.05) between groups AS and AS/S. The resumption of pulsatile LH secretion was delayed in group AS and AS/S when compared to the group RS. The absence of pulsatile LH release in animals belonging to group AS and AS/S was associated with long periods (more than 90 days) of anoestrus. Similarly the pituitary response to exogenous GnRH, which increased (p<0.05) with days postpartum in all three groups, was higher (p<0.05) in group RS. These results suggest that the long calving intervals commonly recorded in Lanka buffaloes are mainly due to’ long periods of postpartum anoestrus. The reproductive efficiency of Lanka buffaloes is influenced by environmental and managemental factors which may be responsible for the differences in the fertility of Lanka buffaloes in different locations. Further, suckling can delay the resumption of ovarian activity during the postpartum period in Lanka buffaloes. This acts by delaying the reappearance of pulsatile LH secretion postpartum. Thus restricted calf suckling could be a practical and effective method for improving the efficiency of reproduction in Lanka buffaloes.
- ItemImmunological response of buffalo cows to toxocara vitulorum - antigenic analysis(University of Peradeniya, 1987) Amerasinghe, Priyanie H.Immunological and haematological responses of pregnant and non-pregnant buffaloes to natural and experimental infections with Toxocara vitulorum were studied. In addition, analysis of some T. vitulorum antigen preparations was carried out. The immunogenicity of some of these antigens was looked into in the mouse model. Further, the immunological and haematological responses of pregnant and lactating bitches after experimental infection with T. canis were studied. The haematological values in general showed a wide range of variation among buffaloes. The WBC and RBC counts did not show any noticeable change referable to the infection or. parturition. However, the WBC and RBC counts appeared to show some relationship to the climate. Packed cell volumes and haemoglobin values also did not show a marked change in relation to parturition. Also, experimental infection of the naturally infected animals in the laboratory three months before parturition did not produce any striking differences in their haematological values when compared with those which had been naturally infected in a similar environment but were not experimentally infected. Parasitological examination of the pregnant and parturient animals was carried out for adult T. vitulorum infection. Few eggs were seen in three animals at early lactation. However, the results were not conclusive. Immunological responses to T. vitulorum antigens, in the parturient and non-pregnant cows were elucidated by means of gel diffusion precipitin (GPT) and Immunoelectrophoretic (IEP) analysis and the Enzyme-linked Immunosorbent Assay (ELISA). A strong GPT response to infective egg extract of T. vitulorum (TVE) was seen in most of the naturally infected buffalo cows. When these GPT titres were followed through lactation, in general, a rise was observed prior to parturition. This rise in titre was followed by a fall about the time of parturition. Also, ELISA titres rose three to four months prior to parturition and decreased during lactation. In general, the nature of the GPT reaction of individual animals indicated by the intensity of the reaction and the presence of a minimum number of bands, showed a direct relationship to the GPT titres. Sera with a strong precipitin reaction revealed more than a minimum of one band and relatively higher titres. In general, the precipitin reaction of the sera of Murrah buffaloes collected randomly were faint. The minimum number of bands could not be recognized. The limited number of sera collected from five beef cattle were negative for T. vitulorum precipitins. Similarly, fetal calf and fetal buffalo sera did not show any precipitin reaction. However, ELISA antibodies with adult TVE were detected in the sera of all these,animals except in those A obtained from buffaloes maintained free of T. vitulorum infection in Armidale, Australia. Similarly, the commercial preparation of fetal calf sera, and two samples of fetal buffalo sera did not show any antibodies measurable by ELISA. ‘Sephadex’ G-200 gel filtration was carried out in order to characterize the immunoglobin classes of anti-T. witulorum antibodies in the buffalo sera. The elution profiles among sera of the naturally infected buffaloes and those taken after experimental infection did not show a significant difference. In general, the sera of buffaloes separated into three peaks (1-3). The strongest GPT reaction to all the antigens was seen in the peak-2. Further, on separation by DEAE A-25 ion-exchange chromatography the two major isotypesof IgG could be distinguished as IgG The2 and IgG 1° latter was the major immunoglobulin responsible for the GPT and ELISA reactions. A preliminary analysis of antigens was carried out. The antigenicity of the excretions and secretions of T. vitulorum, infective larvae was elucidated by the in vitro larval precipitin technique. These larvae were immersed in naturally infected buffalo sera. Precipitates were noted at the natural orifices and they appeared to be more common at the oral rather than the excretory and anal orifices. Five antigen preparations were analysed. a) TVE - Extract of infective Eggs of Toxocara vitulorum b) TVE-u - Extract of infective eggs of Toxocara vitulorum harvested free of uterine tissues. c) TVAS - Excretory, secretory products of adult T. vitulorum d) TVAD - Extract of adult T. vitulorum e) TVPE - Perienteric fluid of adult T. vitulorum These were analysed first with sera from naturally infected buffaloes with Toxocara vitulorum. Strong precipitin reactions were seen with both TVE and TVE-u antigens. The adult dntigen preparations were reactive only with a very limited number of sera collected from five to six animals. These antigen preparations were fractionated by 'Sephadex' G-150 gel filtration and tested against (i) the sera of buffaloes Naturally infected with T. vitulorum and (ii) the anti- sera to The different preparations raised in the rabbits. The above antigens separated Into three to five peaks, The number of peaks varied with the antiren preparation. In the GPT reaction, in general, the resolution of the bands improved when the fractionated antigens were used.For example, in the TVE and TVE-u antigenicity determined by GPT and ELISA resided mostly in the second peak (TVE, ; TVE-u} – ELISA reaction with unfractionated adult antigens was not clear as it gave a high background colouration with the antigen control. However, fractionation removed most of the non- Specific colouration in the antigen controls. Different preparations of the antigens shared common antigen components. However, at least two stage specific (infective egg specific) antigen components were present in TVE, In general, rabbit anti-sera produced more bands in the GPT with the respective anti-sera. Also infective egg and adult worm extract (TCE; TCAD) of Toxocara canis showed a marked serological ceross-reaction with TVE and TVAD (when Tested with some sera of naturally infected buffaloes by means of the GPT). Immunofluorescence test Revealed some possible antigenicsites in the adult parasite. The cuticle, sensory region of the muscle layer, and the ovary appenred to be at least three of the major contributors towards the antigenic make up of the adult antigen preparation. Part of this study (periparturient immunosuppression in dogs) was carried out in the Department of Clinical Veterinary Medicine, University of Cambridge in collaboration with Dr. S. Lloyd. Owing to the seasonal nature of breeding in dogs this study was extended over 16-18 months. Therefore, I conducted only the initial half of the study i.e. examined some half of the dogs in the experiments presented. The remainder were completed by Dr. Lloyd. However, for completeness, the study is presented in its entirety, in this thesis with the permission of Dr. Lloyd. This study has been already published in the Journal of Small Animal Practice (Lloyd, Amerasinghe and Soulsby, 1983). Part of this study (periparturient immunosuppression in dogs) was carried out in the Department of Clinical Veterinary Medicine, University of Cambridge in collaboration with Dr. 8S. Lloyd. Owing to the seasonal nature of breeding in dogs this study was extended over 16-18 months. Therefore, I conducted only the initial half of the study i.e. examined some half of the dogs in the experiments presented. The remainder were completed by Dr. Lloyd. However, for completeness, the study is presented in its entirety, in this thesis with the permission of Dr. Lloyd. This study has been already published in the Journal of Small Animal Practice (Lloyd, Amerasinghe and Soulsby, 1983).
- ItemUse of liposome Incorporated complete lipopolysaccharide core types R1, R2 , R3 and R4 to control infections caused by escherichia coli in chickens(University of Peradeniya, 2008) Dissanayake, Dadigamuwage Ruchirangani AnuruddhikaInfections caused by Escherichia coli (E. coli) make an economically significant impact in the poultry industry and non-serotype specific vaccine appears to be the most logical method of controlling it. The core oligosaccharide region of bacterial lipopolysaccharide is well conserved and highly immunogenic. This study focused on developing a broadly cross protective complete lipopolysaccharide core vaccine to control E. coli infections in chickens. Five distinct lipopolysaccharide (LPS) core types namely R1-R4 and K12 have been identified in E. coli and the distribution of those oligosaccharide core types among avian pathogenic E. coli is important to determine the LPS core types to be incorporated in the vaccine. To identify putative avian pathogenic E. coli, serum resistance and the presence of three virulence genes namely temperature sensitive haemagglutination (tsh), increase serum survival (iss) and col V plasmid (eva C) were determined. Of the 143 clinical isolates examined 62% were R1, 22% were R3, 13% were R4 and 3% were R2. Fifty commensal isolates consisted of 58% with R1 core, 38% with R3 core, 4% with R4 core, and none with R2. None of the isolates were K12 core type. Distribution of core oligosaccharide types in clinical and commensal isolates were not statistically significant (P-0.51). Three genes, tsh, iss and eva C were found in E coli of all four core types. The genes zsh (P>0.001) and iss (P=0.03412) were significantly associated with R4 core oligosaccharide type and the isolates containing LPS with R4 core type were mainly confined to phylogenetic group D. The widely-spread R1 core type showed less ability to process virulence genes and 83% were in the phylogenetic group A. The E. coli with R4 core type were less common among commensals, possessed more virulence genes nd were related to phylogenetic groups pathogenic for poultry Results indicated the E. coli with R1, R2, R3 and R4 were important in causing infections in chickens. Four rough E colt strains possessing R1, R2, R3 and R4 core types were identified by polyacrylamide gel electrophoresis. A mixture of heat-killed bacterin prepared from those rough strains representing RI. R2, R3 and R4 showed significantly high levels of anti-LPS core antibody titres (P < 0.002) that protected the birds from heterologous challenge. Lipopolysaccharide extracted from the four selected strains by aqueous phenol method were incorporated into liposome consisted egg phosphatidylcholine and bovine brain phosphatidylserine and cholesterol to reduce toxicity Endotoxicity of liposome incorporated LPS and free LPS were measured by Limulus amoebocyte lysate assay. Liposome incorporated LPS were at least 1000 times less toxic than free LPS. Induction of nitric oxide production and expression of inflammatory genes by free LPS and liposome incorporated LPS when tested on chicken macrophage cell line (HD11) showed that liposome incorporated LPS produced significantly less amount of nitric oxide than free LPS Expression of the gene interleukin-lbeta (IL-18) and inducible nitric oxide synthase (INOS) were lower in cells treated with liposome incorporated LPS than that of cells treated with free LPS Chicks when immunized with 0.2µg. 1 µg and 5 µg of liposome encapsulated mixture of complete core types showed that the antigenic response increased with increasing dose and the birds received 5µg of liposome encapsulated LPS had significantly high (p < 0.001) anti- LPS core antibody titres than the chicks in all other groups and also protected the birds against lethal challenge with E. coli 078. The liposome encapsulated, mixture of complete LP'S core vaccine was non toxic and showed greater potential to protect chickens against heterologous challenge.