Comparative Studies On The Sensitivity Of Polymerase Chain Reaction (Pcr) And Microscopic Examination For The Detection of Trypanosoma Evansi in Horses
Material type: Book ; Format:
Publisher: 2005 Dissertation note: The polymerase chain reaction (PCR) was standardized and its efficacy was evaluated against microscopic examination i.e. Giemsa stained smear method ['or the diagnosis of Trypanosoma evansi infection (Surra) in horses. l3lood samples were collected from 100 suspected horses from different localities in Lahore.
Under aseptic precautions blood smears were prepared, after drying and fixing with methanol, slides were stained by Giemsa stain method of staining. By stained blood smear method 5 out of 100 horses were found positive For T. evansi infection. The polymerase chain reaction (PCR) was carried out on the blood of' the same suspected horses to evaluate its efficacy in the diagnosis of' T. evansi infection and to compare its diagnostic value against the microscopic examination method currently in use. For this purpose total genomic DNA was extracted from suspected blood samples. The PCR reaction was performed in a 50tl reaction mixture containing I X Taq BuFfer, 0.2 mM dNTP Mixture. I .5 mM MgCIl2 2.5 U/1i1 Taq Polymerase. 4uM of' each primer, 2 ul of DNA extracted and 31.5 p1 of DNase - free deionised water. The tubes containing the mixture were subjected to 30 cycles of amplification in a thermocycler. During each cycle the sample of' DNA was denatured at 93° C' For 30 seconds, annealed at 45° C For 30 seconds and extended at 720 C For I minute. Prior to the cycling and at the end of' cycling the mixture was subjected to incubation at 93° C for a period of 3 minutes and final extension at 72° C for a period of 5 minutes, respectively. PCR product was then characterized by 2.5% of agarose gel electrophoresis. To confirm the presence of DNA and to estimate its size it was compared with a DNA ladder and was photographed with a Polaroid camera. The polymerase chain reaction (PCR) revealed 16 positive cases out of 100 above mentioned suspected cases. These 16 positive cases diagnosed by polymerase chain reaction (PCR) also included animals, which were diagnosed by stained blood smear method.
It can be concluded that polymerase chain reaction (PCR) is a superior and sensitive (16%) in comparison with the microscopic examination i.e. Giemsa stained smear method (5%). Polymerase chain reaction (PCR) is more effective in cases where the parasitemia is low and this test could be used in other species of animals especially camels where the disease is more chronic and difficult to confirm by. other routine methods. PCR would not only ensure early diagnosis and treatment in individual animals but can detect animal reservoirs of infection and would help to eliminate threat to equine and camel herds which are grazed and housed together and where blood sucking mechanical fly vectors are ever present.
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Prevalence, Identification And Pathogenesis Of Clostridium Chauvoei In Cattle And Buffaloes In Punjab
Material type: Book ; Format:
; Literary form:
Publisher: 2012 Dissertation note: In the first phase of the project, the sampling of diseased animals presumably affected by Black quarter was carried out from six districts of Punjab belonging to three different zones. Around two hundred and fifty samples from each zone were collected and were subjected to bacterial culturing and isolation procedures followed by biochemical identification mechanism. The prevalence of Black quarter in Cattle and buffaloes were thus calculated for each district and zone. Highest prevalence of BQ in Zone II was observed (27.2%) for cattle while in case of Buffaloes highest prevalence (3.2%) was noted in Zone I. similarly higher Prevalence of BQ was noted in 1st quarter of year for Zone I followed by zone II and III while 2nd quarter of season was showing higher prevalence of BQ in zone II and III.
During 2nd phase of experiment tissue samples were inoculated in RCM and blood agar for the re-isolation of C. chauvoei, identified on the basis of colony characteristics and later on subjected to biochemical tests for the confirmation of the isolated organism. Then it was further confirmed through Polymerase chain Reaction for the identification of the causative agent i.e. C. Chauvoei on the basis of 16S rRNA gene sequence. Another set of primers corresponding to alpha toxin gene sequence of C. chauvoeui was also used which strengthened the belief that this strain of C. chauvoei possessed alpha toxin producing ability.
During third phase of project blood samples collected were subjected to hematological estimation for buffaloes and cattle having confirmed as BQ This study revealed significant effect on RBC's count and white blood cells count (P<0.05), while Differential leukocyte count were also showing significant different as compared to Non-infected (P< 0.05). Serum samples were tested for the change in levels of different enzymes. It was found that blood-glucose level and ALT levels were not significantly higher (P>0.05) when compared with control values, Values of AST, CPK and LDH were found significantly higher (P< 0.05) in all infected animals.
Histopathology of affected muscle tissues of both cattle and buffaloes was done to study microscopic changes in the muscle fibers and surrounding tissues. Lesions were somehow disappointing as compared to the magnitude of gross lesions. There were segmental degeneration, Zenker necrosis, discrete edema, occasional neutrophils and emphysema in affected muscle.
Finally, alpha toxin (hemolysin) in culture supernatant of RCM broth was titrated against 2% washed RBC's of cattle, buffalo, sheep, goat, chicken, rabbit and mice to study the hemolytic activity of the toxin. It was found that highest percentage of hemolysis was observed in mice followed by cattle, sheep, buffalo, chicken and rabbit respectively at 25°C. Higher the dilution of toxin, lower the extent of hemolysis. At 37°C variable results were obtained. It showed the biological activity of alpha toxin is also temperature dependant.
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Clinico-Pathological And Pathomorphological Studies On Co-Infection Of Avian Influenza (H9n2) With Escherichia Coli In Broiler Chicken
Material type: Book ; Literary form:
Publisher: 2016 Dissertation note: E.coli is an important pathogen of domestic poultry and is prevalent in commercial poultry. LPAIV H9N2 infections are emerging respiratory problems in poultry industry, causing huge economic losses especially in the presence of other co-infecting pathogens such as E.coli. Co-infections do occur but are not easily detected, and the impact of co-infections on pathobiology is unknown. The mixed infections may provide increased virulence, posing a substantial risk to poultry and public health. Moreover, mixed infections of low pathogenic avian influenza with bacteria can also lead to devastating pandemics and a major threat to poultry health, worldwide in future. Co-infections do occur but are not easily detected, and the impact of co-infections on pathobiology is unknown. The mixed infections may provide increased virulence, posing a substantial risk to poultry and public health. Moreover, mixed infections of low pathogenic avian influenza with bacteria can also lead to devastating pandemics and a major threat to poultry health, worldwide in future. The aim of the present study was to investigate the infection of LPAIV A/chicken/Pakistan/10RS3039-284-48/2010 (H9N2) in chickens challenged with E.coli (O78:K80).
This study had three objectives. First, it is designed to develop co-infection experimental models LPAIV (H9N2) + bacteria (E.coli) in the avian model. Second, it aims to study the hematological and biochemical alterations during co-infection in avian model. Finally to study the pathological and histological alterations during co-infection in avian model, this study will help researchers and veterinarians in implementation of necessary control measures. E.coli stockculture was prepared by inoculating MacConkey’s agar with a loop full of reference E.coli strain culture and incubating at 37°C for 24 h. The estimated colony count was confirmed by plating 0.1 ml of a 104 and a 105 dilution of the final culture onto separate MacConkey’s agar plates. Avian influenza A virus, A/chicken/Pakistan/10RS3039-284-48/2010 (H9N2) was obtained from Poultry Research Institute (PRI) Rawalpindi Pakistan.
Viral stocks were prepared and titrated in 9-day-old to 10-day-old chicken embryonated eggs the median embryo infectious dose (EID50) was computed using previously reported approaches The viral stocks were diluted in medium containing antimicrobials to give a final titre of 106 EID50/ ml The study were ran on 80 broiler chicks (3week old), procured from local hatchery. All fowl were held serologically innocent and free from flu virus by haemagglutination inhibition (HI).
Chicken were infected under experimental conditions with E.coli (O78:K80) and low pathogenic avian influenza (LPAI) strain (A/chicken/Pakistan/UDL-01/08) (H9N2) alone or in combination. The experimental groups were identified as follows: negative control, E.coli, AI, and E.coli plus AI. Infected birds showed clinical signs of differing severity, with the most prominent disease signs appearing in birds of the E.coli plus AI group. Moreover, birds in E.coli plus AI group showed significant decrease in weight, enhanced macroscopic and microscopic pathological lesions. Specifically, the survival rate was 60%, 90%, and 100% in birds inoculated with E.coli + AI, E.coli and control negative or AI virus alone, respectively. Hematological studies revealed anemia, thrombocytopenia and leukopenia especially in co-infected birds. Biochemical studies revealed a significant decrease in total protein, glucose and albumin concentration with significant increase of activities of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. Prominent increase in creatinine, urea and uric acid were significantly detected in the infected chicken. The results showed that experimental co-infection of E.coli and H9N2 increased the severity of clinical signs, mortality rate and gross lesions and suggest than E.coli infection can induce higher economic losses and mortality if H9N2 LPAIV is also present. The HI titer against LPAIV infection in the co-infected group was significantly higher than the HI titer of AI group, which may indicate that E.coli could promote the propagation of H9N2 LPAIV or stimulate the immune response. The present study revealed that co-infection E.coli and H9N2 LPAIV caused more serious synergistic pathogenic effects and indicates the role of both pathogens as complicating factors in poultry infections.
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Immunomodulatory Effects Of Feeding Allium Sativum Against Infectious Bursal Disease (IBD) Vaccinated Broiler Birds On IBD Vaccine.
Material type: Book ; Literary form:
Publisher: 2016 Dissertation note: A number of feed additives including antibiotics have been extensively used in
poultry diets for the purpose of weight gain to improve feed efficiency and growth rate.
However, use of antibiotics has restricted due to the bacterial resistance and the issue of
residues which make the chicken meat harmful for human consumption. So the medicinal
plants are gaining interest as alternative feed strategies now a day because of their low cost,
easy availability and presence of no residues. Garlic was used for the medicinal purposes and
as a health supplement by the ancient Egyptians. It is a natural feed additive and is
antimicrobial, immune stimulator, antiviral, antifungal, anti-parasitic, antithrombotic,
antioxidant, anti-cancerous, and vasodilator activities. Previous studies indicate that it has
beneficial effects on the immune system and is a best immune stimulator then the other
herbal plants and medicines.
Therefore, the present study was designed to estimate the immunomodulatory effect
of garlic to commercially available IBD vaccine in enhancing the immune system. Total
N=99 day old broiler chicks were purchased and kept in the experimental shed of CVAS
Jhang. Birds were divided into three groups A, B, C and group B and C were further divided
into three subgroups (B1, B2, B3 and C1, C2, C3). Group A was treated as control group and
was administered with commercially available IBD and ND vaccine and routine diet while
group B was administered with garlic at the rate of 4%, 5%, and 7% along with vaccine to see
the impact of different levels of garlic (Allium sativum) on the immune system and to see the
toxic effect (if any) of high dose of garlic. Group C was only administered with garlic in fee
at the same rate as to group B. At the end of study birds were slaughtered to check the effects
of garlic administration. Positive effect of garlic has been reported by many studies. Garlic is
a medicinal herb used for the prevention and treatment of many diseases, because of having
antiviral, antibacterial and antifungal activities. Also act as a good growth promoting agent
and have beneficial effects on the immune system. Results of the study indicate that
administration of garlic powder in different doses alone and combined with commercial IBD
vaccine have good effects on the growth, blood parameters and the immune system of the
Availability: Items available for loan: UVAS Library [ Call number: 2561-T] (1).