2.
Clinico-Epidemiological Study Of Multiple Drug Resistant Staphylococcus Aureus From Bovine Mastitis
by Muhammad Abdul Rauf Malik (2015-VA-832) | Dr. Muhammad Ijaz | Dr. Syed Saleem Ahmad | Prof. Dr. Aftab Ahmad Anjum.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Bovine mastitis is one of the most significant problems of livestock causing huge economic losses in dairy industry of Pakistan. Among other bacterial etiology of bovine mastitis, the Staphylococcus aureus is overwhelming to control and is well-known to cause subclinical and contagious mastitis. Methicillin resistant Staphylococcus aureus is our prevailing field issue. In view of the economic importance of Staphylococcus aureus mastitis, the current project was designed to study the Clinico-epidemiology of multiple drug resistant Staphylococcus aureus in bovine mastitis.
A total number of 900 milk samples (n=450 cattle, n=450 buffalo) were collected from Faisalabad district of Punjab, The collected samples were processed in laboratory of Microbiology and Medicine, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore. Primarily, screening of subclinical mastitis was done by Surf Field Mastitis Test (SFMT). Milk samples were spread out primarily on blood agar to rule out possibility of loss of later growth. Colonies with round and golden color characteristic were put to sub-culturing on Mannitol salt agar as differential and selective medium. The morphological and microscopic clarification was done under microscope using Gram’s staining technique. Various biochemical tests including coagulase and catalase were applied.
Prevalence of subclinical mastitis was found 55% (495/900), however significant differences were found among different tehsils of district Faisalabad. The prevalence of subclinical mastitis in cattle from district Faisalabad was found 54% (243/450) that upon comparison between different cities presented significant difference. While buffaloes presented 56% of subclinical mastitis. Comparisons of subclinical mastitis among different tehsils were
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found significant.
Among quarter based prevalence of subclinical mastitis and prevalence of blocked quarters the number of blocked quarters were found 5.58% (201/3600) with highest percentage of blocked quarter noted in case of front right followed by rear right , front left , and rear left with 6.89, 6.56, 4.67, and 4.22%, respectively. However, the quarter based prevalence was found 32% (1088/3399) from bovine. The association of bovine subclinical mastitis with different risk factors presented significant association with few exceptions. Age, parity and body health status of animal were found non- significant with prevalence of mastitis. The breed and open rearing system presented significant relation with chances of mastitis. Chi-square test was used to statistically correlate the risk factors and prevalence of subclinical mastitis. P-value less than 0.05 was considered as significant.
Molecular confirmation of S. aureus was done by using coag gene through PCR technique. The S. aureus which were isolated from milk samples were put to antibiotic (oxacillin) sensitivity test for estimation of prevalence of methicillin resistant S. aureus from the bovine mastitis. Molecular identification of mec-A gene in staphylococcus aureus was done through PCR. The PCR confirmed methicillin resistant S. aureus isolates from cattle (n=20) and buffalo (n=20) were tested for their in-vitro drug response. However, Ciprofloxcin, Moxifloxacine, Linezolid, and Trimethoprim + Sulphamethoxazole were found 100% effective against multiple drug resistant Staphylococcus aureus and Levofloxacin showed 90% efficacy in bovine. While Oxytetracycline, Tylosin, Gentamycin, Amikacin, Vancomycin, and Fusidic acid were also found sensitive moderately except cefoxitin which was responsible for 100% resistane in bovine. Gentamycin was found to much more effective in buffaloes rather than cattle.
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The study provided current status of Staphylococcus aureus infection with higher percentage in bovine mastitis. The prevalence of mecA gene revealed variation in methicillin resistant Staphylococcus aureus. In-vitro drug trial provided effective treatment possibilities against multiple drug resistance Staphylococcus aureus. Availability: Items available for loan: UVAS Library [Call number: 2815-T] (1).
3.
Prevalence, Molecular Diagnosis And Chemotherapy Of Degnala Disease In Large Ruminants Of Punjab.
by Mudassar Nazar (2005-VA-92) | Prof. Dr. Muhammad Sarwar Khan | Dr. Muhammad Ijaz | Prof. Dr. Aftab Ahmad Anjum.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: In Pakistan, Livestock is considered as a social security for poor villager as it can be a source of cash at the time of need. Degnala disease reduces the production of these animals directly. Along with other side issues related to Degnala disease, this study was done to diagnose the actual cause of Degnala disease by applying different latest scientific techniques. Prevalence along with risk factors was calculated in the rice growing areas of Punjab, Pakistan. Fungal isolation (n=40) was performed from the rice straw feedings of the Degnala disease affected animals through the technique of spot culture on SDA. Then these fungal isolates were identified through comparing their microscopic and macroscopic characters. Then toxigenic potential was checked for all these isolates through the application of TLC and HPLC. After that, from those isolates which were positive for mycotoxin production potential, most cytotoxic isolate was checked with the application of MTT assay. Then the most cytotoxic isolate was inoculated on non-contaminated rice straw and fed to the experimental animals to see a similarity of natural cases of Degnala disease. Finally treatment was conducted to see a proper combination of various drugs against this disease.
Toxigenic potential of different candidate fungi, isolated from rice straw feeding of Degnala disease affected bovines was analysed along with Species, age, gender and season wise prevalence. Out of 1536, 104 (6.77%) cases showed positive signs for this disease with a significant association (p<0.05) between rice straw feeding in buffaloes, winter season and bovines having an age of more than one year. Complete Blood Count showed marked increase in erythrocyte sedimentation rate and all white blood cells numbers, except lymphocytes in positive cases. There was a significant increase (p<0.05) in Alanine amino transferase, Aspartate amino transferase and Alkaline phosphatase noticed in Liver Function Test. At the same time, increased value of Creatinine was noticed in Renal Function Test. For isolation and screening of toxigenic fungi, rice straw samples (n=40) being fed to the positive cases were processed further, out of which there were 85 fungal isolates mainly of Aspergillus (57), Penicillium (10), Fusarium (04), Zygomycetes (03), Curvularia (01) and unidentified (10). All isolated fungi were subjected for mycotoxin production and only 11 showed mycotoxin producing capability (including Aspergillus, Penicillium and Fusarium isolates) analysed by Thin Layer Chromatography and quantified through High Performance Liquid Chromatography. It is concluded that all the fungi, contaminating rice straw feeding of Degnala affected animals are not toxigenic. This work will help in establishing major mycotoxin producing fungi leading to the probable cause of Degnala disease in bovine.
With the help of MTT assay on vero cell line, most cytotoxic fungus was identified. After an incubation with vero cells, OD values of all the candidate fungi were compared through one way ANOVA. Results of this analysis showed that Fusarium was at the highest ranking and then was the A. flavus with a significant value of 0.006 and 0.039.
Finally it was concluded through these systematic steps of converging the diagnosis that, out of all the 85 suspected fungi, Fusarium (isolate number S 8.1) was the most cytotoxic isolate obtained from the rice straw feedings of Degnala affected animals in our study.
For molecular diagnosis of the most cytotoxic isolate of Fusarium, PCR was conducted and the results showed that ultimately the final PCR product was successfully amplified against the mentioned primer of ITS conserved region for Fusarium genera and the DNA product was with a length of 570 base pairs.
Experimental feeding trials were conducted by inoculating Fusarium (the most cytotoxic isolate) and A. flavus (second most cytotoxic one after Fusarium) separately and in combination compared with the negative control group, all groups were of eight animals each. It was concluded that alone Fusarium was able to produce Degnala disease, while its combination with A. flavus was more lethal.
Ultimately the treatment trials proceeded with penta-sulphate, oxytetracycline and antiseptic topical application as therapeutic treatment were shown to be very effective against Degnala cases. While in all the affected animals feeding of affected rice straw was ceased. Only withdrawal of affected rice straw from the feedings of Degnala affected animals was not effective unless proper treatment as mentioned here was not conducted.
analysed The expected results of the study shall be helpful to make exact diagnosis and treatment of infected buffaloes and cattle that is further helpful for timely prophylaxis and control of the Degnala disease in the rice growing areas of Pakistan and South Asia.
Availability: Items available for loan: UVAS Library [Call number: 2960-T] (1).
