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Home > Molecular Analysis of Staphylococcus Aureus Isolated from Infected Dairy Goats in Rawalpindi Pakistan

Molecular Analysis of Staphylococcus Aureus Isolated from Infected Dairy Goats in Rawalpindi Pakistan

Thesis Info

Access Option

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Author

Muhammad Asghar Qazi

Institute

Virtual University of Pakistan

Institute Type

Public

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2017

Thesis Completion Status

Completed

Subject

Software Engineering

Language

English

Link

http://vspace.vu.edu.pk/detail.aspx?id=140

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676720976496

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Staphylococcus aureus is the main cause of mastitis in dairy goats. This organism is recognized as major mastitis causing pathogen in dairy animals worldwide. In humans, it also caused various diseases such as food poisoning and Staphylococcal enterotoxins. This bacterium mostly infected the dairy herds due to poor sanitary conditions and untreated groundwater used to wash utensils during milking practice. The purpose of this study was to determine the prevalence of Staphylococcus aureus among infected dairy goats. Hundred milk samples were collected from infected dairy goats and grown on a blood agar. A total of 58/100 (58%) were positive for S.aureus that was further confirmed on the basis of biochemical tests namely catalase positive and coagulase positive. The positive isolates of S. aureus strains were analyzed using a molecular technique. DNA of positive S. aureus isolates was extracted using Macherey-Nagel kit method. The extracted isolates of S. aureus were run on agarose gel electrophoresis. Ethidium bromide dye was used to visualize DNA bands using UV illuminator. The amplification of genes encoding 16S rRNA of positive S. aureus isolates was performed by Polymerase chain reaction. Primers (Forward primer 5''-AGA GTT TGA TCC TGG CTC AG & Reverse primer 5''-AAG GAG GTG ATC CAG CCG CA) were designed using Primer 3 software. PCR clean up method was used in purification of sequences. These amplified gene products were further sequenced by using Sanger method. The sequenced results of these bacterial species were confirmed by comparing the sequences present in BLAST program at National Centre of Biotechnology Information. MEGA 7 software was applied for the construction of Phylogenetic tree. Some other bacterial species such as Staphylococcus epidermidis, Staphylococcus hominis, Enterobacter cloacae and Acinetobacter baumannii along with Staphylococcus aureus were also identified due to their genetic differences. The evolutionary history was inferred using the Neighbor-Joining method. The evolutionary distances were computed using the p-distance method and in the units of the number of base differences per site. The analysis involved 14 nucleotide sequences. There were a total of 371 positions in the final dataset. Antibiotics such as penicillin, erythromycin or tetracycline are used in the treatment of mastitis in goats. It is concluded that Staphylococcus aureus was more prevalent in the milk samples of infected goats and is an etiologic agent of mastitis in dairy goats. The consumption of raw milk may infect humans and lead to various infections.
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خراجِ محبت و عقیدت

منظوم خراج تحسین
ڈاکٹر شہزاد احمد ہیں فدائے مصطفی
از ازل تا بہ ابد ہیں یہ گدائے مصطفی

نعت کے شعبے میں ان کی اس قدر خدمات ہیں
عاشق سرکار ہیں! گویا نوائے نعت ہیں

ایک دن ہم نے سنی ان سے یہ پیاری سی نوید
ان سے وابستہ ہوئے ہیں حضرت شفقت فرید

دھیمی دھیمی سی مسلسل ان کی جو پرواز ہے
ان کے کاموں پر بھی اب کچھ کام کا آغاز ہے

’’ایم فل‘‘ ان پر ہوا ہے منفرداور کامیاب
کام یہ شفقت میاں نے کر دیا ہے لاجواب

ڈاکٹر شہزاد احمد کو مبارک ہو یہ کام
حضرت شفقت کو ہو اس کام پر میرا سلام

اوج پائے یہ مقالہ آپ کا شفقت فرید
ہے لب خاکی پہ اتنی سی دعا شفقت فرید
عزیزالدین خاکی

عصر حاضر میں وحدت الادیان کا تصور اور اسلامی نکتہ نظر

Religion is the cradle of human civilization and will always be a core influence in determining the trends, outlooks and progression of our society, which inevitably confronts questions and issues with religious undertones. As the world becomes more multi-religious and multi-ethnic, however, there is a seemingly diminished respect for the diverse existence of faiths and ethnicities, and more concerns rise towards this new threat to peace, which can be seen in the form of religious hatred, persecution, and conflicts often involving physical violence. This rising hostility, which has become a prolonged, unstable social factor, in all manifestations is an obstacle to peace. According to the different religious scholars of the semitic and nonsemitic religions it is recommended that religion plays a vital role to save the humanity. For this purpose many scholars recommend a religious alliance and unity for the whole universe as we can prevent the violence against humanity. In this paper the different views of the scholars in favour of religious alliance while the stance of Islamic Sharia has been discussed in detail as well.

Environmental Concerns of Poultry Manure Applied into Agriculture Fields of Khyber Pakhtunkhwa: Special Emphasis on Antibiotics and Heavy Metals

Heavy metals and antibiotics are growing environmental contaminants leading to public health concern.Poultry manure is enriched with antibiotics and heavy metals that are commonly used as organic fertilizer for agricultural crops. Antibiotics (Suphonamide, lincomycine, erythromycin, oxytetracycline and tylosine) and heavy metals (Pb, Cr, Cd, As Ni, Co and Cu) are used as growth promoters and therapeutic agents in poultry feed. Some of these antibiotics and all heavy metals are not biodegradable in the body tissues of chicken, get deposited in meat as parent compounds and ultimately excreted via poultry droppings into the environment.In the first study an attempt have been done to present a better understanding of emerging contaminants in environment and their associated health effects. In this part we investigated the use of antibiotics in poultry sector and their release into the environment which develop antibiotics resistance bacteria (ARB) along with antibiotics resistance genes (ARGs). The development of ARGs and ARB might lead to huge physical and economic losses, as these bacteria cannot be treated with commonly used antibiotics, proving fatal for thousands of people every year in the world. These antibiotics after entering into food chains seriously affect human immune system, growth and metabolism of the body. Therefore, remediating the ARB, ARGs and antibiotics from the agriculture soil by environmental friendly technique is important to protect the future human health risks. Manure composting and biochar application might be the possible ways to reduce the risk and spread of ARGs in environment. In the second study, heavy metals were quantified in poultry manure, soil and food plants of agriculture fields from eleven districts of Khyber Pakhtunkhwa province. The concentrations of selected metals (Cd, Cr, Ni, Co, Cu, Ba, Pb) in most of the soils (ranged 0.036-0.3±0.086, 1.05-54.1±17.3, 1.8-35.2±9.1, 0.47-16.9±4.1, 0.056-32.5±9.8, 48.5-200.8±45, 0.26-15.7±4.57 mg kg-1, respectively) and manure (ranged 0.1-0.3±0.06, 6.75-52.4±11.9, 9.18 23.6±4.5, 2.86-16.7±4.4, 15.0-82.6±19.7, 24.7-86.9±20.9, 2.67-11.1±2.4 mg kg-1, respectively) samples were observed below the maximum allowable viii limits set by United States, Environmental Protection Agency (USEPA). The concentrations of Cu (0.81 to 247 mg kg-1) and Cr (0.18 to 9.81 mg kg-1) in most of the food plants were significantly (Cu-P<0.05) (Cr-P<0.01) higher and crossed their respective permissible limits (Cu-20 mg kg-1 and Cr-0.5 mg kg-1) set by SEPA, China. The mean concentrations of Pb (ranged 0.41-11.4 mg kg1) in all the food plants were significantly (P<0.01) higher and crossed the limit (0.3 mg kg-1) of FAO and WHO (2001). The values of Cd in radish (0.45 mg kg-1), clover (0.31 mg kg-1), lettuce (0.34 mg kg-1) and argula (0.28 mg kg1) were also above the allowable limits (0.2 mg kg-1) of SEPA. Metals like Pb, Cr, Cd, Cr and Cu were shown higher (>1) bioaccumulation values in the selected plants like radish, garlic, barley and wheat.Among selected heavy metals, Co, Ni and Ba were shown lower BCF values and assumed to have no serious health risk.The health risk index (HRI) values were found >1 in barley (for Cu and Cd), wheat and radish (for Cu, Cd and Pb) and pea and onion (for Cd), which were considered, that may cause potential health risks to the local community. Therefore, heavy metal concentrations in poultry manure must be reduced before its application into cultivated fields through safe and environmentally friendly technology such as biochar and composting. The third study determined the health risk of total As (TAs) and its species in selected food plants grown on poultry manure-based agriculture soils of Khyber Pakhtunkhwa, Pakistan. The concentrations of total arsenic (TAs), arsenobetaine (AsB), monomethyarsenate (MMA), dimethylarsenate (DMA), arsenite (As3+) and arsenate (As5+) were investigated in soil, poultry manure, food plants and poultry feed. The TAs concentration in soil of the study area ranged from 4.56 to 9.98 mg kg−1, showing the high variability in the selected sites. Inorganic arsenic (iAs) was found highest in Charsadda (9.9 mg kg−1) and lowest in district Swat (4.02 mg kg−1) The mean TAs concentrations in the poultry manure and feed ranged from 0.64 to 1.60 mg kg−1 and 0.06 mg kg−1 to 0.13 mg kg−1, respectively. The mean TAs concentrations in the edible parts of the food plants was in range from0.096 to 1.25 mg kg−1, which were found above the safe permissible limits (0.1 mg kg−1) of FAO/WHO (2011) in 83% offood plants, except in pea (0.096 mg kg−1) and mint (0.093 mg kg−1). The ix maximum concentration was observed for wheat (1.25 mg kg−1), cucumber (0.67 mg kg−1) and mallow (0.63 mg kg−1). Furthermore, human health risk was assessed through health risk index (HRI), hazard quotient (HQ), dialy intake rate (ADI) and life time cancer (LTCR). The ADI of As in the different food plants was ranged from 1.42×10−6 mg kg−1 d−1 to 6.62 ×10−5 mg kg−1 d−1 in the adults and 4.07×10−6 mg kg−1 d−1 to 1.89 ×10−4 mg kg−1 d−1 in the children with highest value for Malva neglecta (leafy plants) and lowest for Allium sativum. The total ADI for children (6.33×10-4 mg kg−1day−1) was higher than the USEPA oral reference dose (3 × 10-4 mg kg−1day−1) posing non cancer risk. The HQ values for TAs were less than 1 for the ingestion of the tested food plants, revealing less non-carcinogenic risk in the area. The results revealed that HI value for children (2.1×10+0) were three times higher than the value for adults (7.87×10−1), posing non cancer health risks (HI>1) in the children. The long term potential cancer risk (LTCR) was found slightly higher for children (1.53 × 10-4) than the permissible limits of USEPA, while minimal cancer risk was observed for adults via consumption of selected food plants.This research highlights a need to reduce the level of As in the poultry feed and further suggests to treat poultry manure before their application to the crop field. A fourth study was carried out on the development of ARG and ARB due to manure application in agriculture field. Three different samples of poultry manure (M), soil amended manure (S+M) and soil without manure (S) were collected for assessment of ARGs in the agriculture field area of Khyber Pakhtunkhwa, Pakistan. Total number of 285 ARGs was observed by means of HT-qPCR (High Throghput quantitative PCR), which targeted most common classes of resistance genes. Poultry manure addition significantly (P<0.05) raised the abundance copies of ARGs in manure based soil and have changed composition of bacterial community. The detected resistance genes in manure amended soil gave resistance to antibiotics such as multidrug, aminoglycoside, MLSB, beta-lactmase, tetracycline, Vancomycine, tansposase and sulphoniamide. Total 130 unique ARGs were developed, 76 ARGs were shared by all three groups, 47 x ARGs were transferred from manure to soil and 49 ARGs were shared by manure and manure-based soil. Gene sequencing of 16S rRNA was carried out for the samples, which concluded that Proteobacteria, Bacteroidetes Actinobacteria, Fermicutes, Acidobacteria and Chloroflexi were the common phyla of microbial community, having about 87% of total microbes of 16S rRNA gene sequences. Manure has increased Bacteroidetes, Actinobacteria and Fermicutes in the soil. Hence manure has elevated the ARGs and antibiotic resistance bacteria (ARB) by horizontal gene transfer from manure based soil to indigenous soil microbiota. This study will help us to know the dissemination and fate of ARGs in this ecosystem to efficiently use poultry manure in crop land and highlight antibiotics and ARGs risks managements in the agriculture area of Pakistan.