Babesia ovis and Theileria ovis are among the important and main etiological agents causing ovine babesiosis and ovine theileriosis, causing severe economic losses among sheep and goats. The aim of the present study was to determine the prevalence and molecular diagnosis of B. ovis and T. ovis in Lohi sheep at Livestock Experiment Station Bahadurnagar, Okara, Pakistan. The prevalence of B. ovis and T. ovis was investigated in 200 Lohi sheep of mixed age and sex by PCR during 2011. The assay was employed using primers Bbo-F & Bbo-R, specific for a 549-bp fragment in B. ovis genomic DNA and primers TSsr 170F & TSsr 670R, specific for a 520-bp fragment in T. ovis genomic DNA. The animals were also screened for both haemoparasites through stained thin blood smears. Thirty two (16%), 48 (24%) and 26 (13%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites, respectively, through microscopy. Sixty eight (34%), 73 (37%) and 42 (21%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites, respectively, through PCR test. The results indicate the high sensitivity of PCR for surveying babesiosis and theileriosis and there is noteworthy prevalence of these diseases in sheep at an experimental station where environmental conditions are relatively controlled as compared to field conditions.

Hemorrhagic Septicemia (HS) caused by Pasteurella multocida is an economically significant disease of bovines that instigates high monetary losses due to unexpected mortality of animals in emerging countries like Pakistan. Similarly Black Quarter (BQ) caused by Clostridium chauvoei is also an acute infectious ailment of cattle and buffaloes in which severe inflammation of skeletal and cardiac muscles, severe systemic toxicity and a high mortality occurs. The only way to prevent losses due to both diseases is an effective and long lasting vaccination programme. Four HS+BQ combo vaccines adjuvanted with four different adjuvants were prepared. Two HS+BQ vaccines were prepared by addition of two oil adjuvants i.e Montanide ISA-50V-2 and Eolane-170 while one HS+BQ vaccine was prepared by addition of gel while fourth HS+BQ vaccine was prepared by addition of alum. The bacterial dry weight of P. multocida was adjusted to 2 mg/0.75 mL while the bacterial dry weight for Cl. chauvoei was set at 2.5 mg/0.75 mL. The dose of all vaccines was adjusted to 3 mL /animal. Four groups of rabbits having five rabbits in each group were selected at Veterinary Research Institute, Lahore. From four groups of rabbits one was inoculated with HS+BQ oil based vaccine containing Montanide ISA-50 V2 as adjuvant while other group was inoculated with HS+BQ oil based vaccine containing Eolane-170 oil as adjuvant. Third group was vaccinated with HS+BQ vaccine adjuvanted with gel while fourth group of rabbit was vaccinated with HS+BQ vaccin adjuvanted with alum. All rabbits were vaccinated at dose rate of 1mL / rabbit. Rabbits were again vaccinated with the same vaccines as booster dose after 27 days. Indirect HemAgglutination test was conducted after 141 days intervals on serum samples of all groups. The results showed that both HS+BQ combo oil based vaccines have higher antibody titers as compared to gel and alum based vaccines against HS disease.

Lumpy skin disease (LSD) of cattle, caused by the lumpy skin disease virus (LSDV), results in significant economic losses and restricted cross-border livestock trade. Although live attenuated vaccines are commercially available, the inactivated vaccines would be an appropriate substitute because they are devoid of any adverse reaction and cannot mutate to produce new viral strains. The objectives of this study were to develop an inactivated oil-adjuvanted vaccine using a local isolate of LSDV and to evaluate the immune response of this vaccine in rabbits. The LSDV was isolated from scab samples of suspected cows by inoculating embryonated chicken eggs via the chorioallantoic membrane route. One of the isolates was adapted to grow in Madin-Darby bovine kidney cells, followed by its inactivation with binary ethyleneimine. Montanide ISA50 V2 was added as an adjuvant. A total of 24 rabbits were divided into three groups of eight each; Group A received our inactivated vaccine while Group B was given a commercial live vaccine. Group C served as a non-vaccinated control. The mean antibody titers were statistically analyzed by Dunnett’s test. The D-value of 77.78 indicated that the vaccine produced protective antibodies.

Bacterium Pasteurella multocida type B:2 is the causative agent of Hemorrhagic Septicemia in bovines in Pakistan. This organism is also being used for oil adjuvanted (Montanide ISA 50 V2) vaccine for mass vaccination in cattle & buffaloes in Punjab, Pakistan. A study was conducted to study the impact of glycerol and dimethyl sulfoxide (DMSO) on persistence of P. multocida during its course of storage at 0, 4 and – 20 °C. Pure seed of P .multocida was grown in Brain Heart Infusion(BHI) broth at pH 7.4 and incubated at 37 °C for 18 hrs. in shaking incubator. The growth was observed for its purity microscopically as well as culturing on MacConkey’s & Nutrient agar slants. The pure culture was divided into three parts. One part was added with 5 % sterilized DMSO (final concentration) and second part was added with 10 % sterilized DMSO (final concentration) and 15 % glycerine (final concentration) while third part was kept as control without addition of DMSO and glycerine. All three parts were aliquoted (1 mL) in sterilized 1.5 mL eppendroff tubes and stored at three different temperatures i.e 0, 4 and – 20 °C. Eppendroff tubes from different temperatures were thawed in water bath (22 °C) and subjected to direct culturing on BHI broth, microscopic examination, purity testing on slants, culture turbidity / Optical Density (OD) values, dry biomass calculation and mice inoculation test for three months with 15 days intervals. The results indicated that pure P. multocida culture mixed with 10 % sterilized DMSO and 15 % sterilized glycerol stored at – 20 °C survived for three months and can be used for HS vaccine production without losing its survival.