51.
Anthelmintic Activity Of Withania Coagulans Against Gastrointestinal Nematode Of Sheep In District Killa Saifullah, Baluchistan
by Yousaf Gul (2009-VA-145) | Dr. Muhammad Lateef | Dr. Saadullah Jan | Dr. Muhammad Imran Rashid | Dr. Wasim Shehzad.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Evaluation of anthelmintic activity of Withania coagulans was studied against GIT nematodes of sheep in district Killa Saifullah Baluchistan. Sheep of the district were screen out for the presence of GIT nematodes. Animal positive for GIT nematodes and having 150+ Egg per Gram (EPG) of feces was included in the drug trial. Animals were treated with extract(s) of locally available herbal plant (withania coagulans) and levamisole. Two types of plant formulations that is crude powder and crude methanole extract were prepared each with various dosages. The effect of both medicinal plant and levamisole was observed on different groups of animals and the results were analyzed with appropriate statistical tool. Eighty animals were randomly divided in to eight groups (10 animals in each group) i.e. A, B1, B2, B3, C1, C2, C3 and D. Animals in group A served as control untreated group. Animals in groups B1, B2 and B3 were treated with crude powder of Withania coagulans at the dose rate of 1, 2 and 3 g/kg body weight respectively. And Animals in groups C1, C2 and C3 were treated with crude methanol extract of Withania coagulans at 33.3, 66.6 and 100mg/kg equivalent dose rate of 1, 2 and 3 g/kg body weight respectively. Animals in group D were given Levamisole at the standard dose rate of 7.5 mg/ kg body weight. Data was analyzed by using SPSS version 20.0; comparative analysis was done by applying ANOVA. P value <0.05 was taken as significant. The analyzed data and the results revealed that Levamisole is still a better anthelmintic against ovine nematodes in district Killa Saifullah Balochistan. Efficacy of levamisole tested for 15 days in-vivo sheep was up to 92%. This efficacy was much higher than the various forms and dosages of medicinal plant. The efficacy of Levamisole was significantly higher (P<0.05) than all forms and dosages of medicinal plant. Group C3 treated with crude methanol extract of Withania coagulans at the dose rate of 10mg/kg equivalent to 3mg/kg showed highest efficacy of the plant that is up to 48%. The efficacy showed by the form of the medicinal plant used in group C3 against ovine GIT nematodes was significantly higher (P<0.05) than all other forms of the plant. Animals in group B1, B2, B3, C1 and C2 showed anthelmintic efficacy of 19.47%, 23.58%, 31.66%, 31.76% and 33.33% from day 0 to day 15th post-treatment. Gastrointenstinal nematodes of sheep have produced anthelmintic resistance against Levamisole at the dose rate of 7.5mg/kg. In previous studies Levamisole had showed efficacy of 99.99%, 99% and 98%. It is therefore recommended that further investigation on huge scale should be passed out concerning a great number of animals, quantities higher than those used in the present study, documentation of active principles, and calibration of dose and toxicity studies for drug development from the herbal plant.
Availability: Items available for loan: UVAS Library [Call number: 2688-T] (1).
52.
Clinico Epidemiology of tick Borne Hemoparasitic Diseases Using Single Round And Multiplex PCR Along With their Phylogenetic Analysis In Bovine
by Shahid Hussain Farooqi (2012-VA-447) | Dr. Muhammad Ijaz | Dr. Muhammad Hassan saleem | Dr. Muhammad Imran Rashid.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: CD Crupt Availability: No items available
53.
Phylogenetic Analysis And Gis Mapping Of Boophilus Species Of Ticks Of Bovine And Buffalo Of District Peshawar
by Zulfiqar Ahmad (2015-VA-11) | Dr. Muhammad Imran Rashid | Dr. Muhammad Oneeb | Dr. Mamoona Chaudhry.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Livestock is playing a major role in the uplift of our national economy in terms of revenue from milk, meat and hides. In spite of this major contribution this sector is facing hurdles in its development. The infectious diseases and their vectors have occupied a mainstay in posing the uplift of this sector. Boophilus is an important biological vector for various protozoan and bacterial infections in animal as well as human. To date the identification of these ticks mainly rely on the morphological basis which have many variations among different identification keys. To make the identification more accurate at species level, the use of molecular tools is very necessary. Ticks were collected from the various areas of district Peshawar through random convenient sampling method. Briefly, 50 cattle and 50 buffaloes were sampled through forceps. Various ticks spp. were stored in 70% ethanol for further processing. Among the species Rhepicephalus microplus (Boophilus microplus) was the most prevalent specie (25.64103%) followed by Rhepicephalus annulatus (5.413105%) Rhipicephalus decloratus (5.128205%) Rhipicephalus distinctus (4.273504%), Rhipicephalus arnoldi (3.988604%), Rhipicephalus evertsi (5.698006%), while in Heamaphysalis species Heamaphysalis aciculifer highly prevalent (5.128205%) followed by Haemaphysalis parmata (4.843305%), Haemaphysalis excavatum (3.988604%) and Haemaphysalis houyi (3.988604%), so far Hyalomma species is concerned includes Hyalomma anatolicum (3.988604%), Hyalomma trancatum (4.843305%), Hyalomma detritium (5.982906%), Hyalomma egyptium (4.273504%), Hyalomma impeltatum (0.854701%) Hyalomma rufipes (1.709402%), Amblyomma pomposum (4.273504%), Dermacentor rhinocerinus (2.849003%), D. circumguttatus (3.703704%), and
Summary
44
Dermacentor marginatus (2.564103%) are highly prevalent in cattle. Among the buffalo, Rhipicephalus 173 (43.25 %) followed by Haemaphysalis 82 (20.5 %), Hyalomma 54 (13.5 %), Dermacentor 26 (6.5 %) and Amblyomma 9 (2.25 %). The species prevalent in Rhipicephalus are Rhipicephalus microplus 74 (42.78%), Rhipicephalus annulatus 15 (8.68%), Rhipicephalus decloratus 19 (10.99%), Rhipicephalus distinctus 14 (8.10%), Rhipicephalus arnoldi 16 (9.25%), Rhipicephalus evertsi 17 (9.84%) and Rhipicephalus kochi 18 (10.40%) followed by Haemaphysalis aciculifer 18 (21.96%), Haemaphysalis parmata 15 (18.30%), Haemaphysalis excavatum 23 (28.05%), and Haemaphysalis houyi 26 (31.70%), so far Hyalomma species is concerned, Hyalomma anatolicum 10 (18.52%), Hyalomma tranctum 7 (12.96%), Hyalomma detritium 9 (16.67%), Hyalomma egyptium 7 (12.96%), Hyalomma impeltatum 10 (18.52%), and Hyalomma rufipes 11 (20.37%) and Dermacentor rhinocerinus 9 (34.62%), followed by Dermacentor circumgutattus 8 (30.76%) and Dermacentor marginatus 9 (34.62%) and Amblyomma is concerned Amblyomma pomposum 9 (2.25%). DNA was extracted from the Rhipicephalus (Boophilus) ticks through phenol-chloroform method. The extracted product was then run by gel stained ethidium bromide. The gel was visualized and examined bands on UV illuminator. Different sequences were retrived from database and genus specific primer were designed for the amplification of ITS-2 gene of Rhipicephalus genus of hard ticks. A consensus sequence was retrieved, a set of primers were designed by using Bioedit softwere version 7.2.6. DNA was extracted from 100 ticks and then run by PCR. Specific primers were designed for ITS2 gene. Phylogenetic tree based on the DNA sequences amplified from extracted from all the comparison with ticks and determined Genus Rhipicephalus area that are ITS2 Rhipicephalus ITS2 ribosomal RNA gene sequence 18s, thus obtained from Genebank. Availability: Items available for loan: UVAS Library [Call number: 2848-T] (1).
54.
Molecular Characterization of Carbapenem Resistant Pseudomonas Aeruginosa Isolated from a Tertiary Case Hospital in Lahore
by Sarfraz Hussain (2015-VA-1326) | Dr. Muhammad Imran Najeeb | Prof. Dr. Aftab Ahmed Anjum | Dr. Muhammad Wasim.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Empty CD. Availability: Items available for loan: UVAS Library [Call number: 2834-T] (1).
55.
Development Of Novel Mtdna Metabarcodes For The Species Differentiation Of Class Amphibia
by Rehmatullah (2011-VA-365) | Dr. Muhammad Imran | Dr. M. Yasir Zahoor | Dr. Amjad Riaz.
Material type: Publisher: 2017Dissertation note: The Folmer COI mtDNA universal primers that are considered standard for DNA
barcoding of life contain so many mismatches against the target sequences of vertebrate origin
that they often end in failure to amplify many of vertebrate DNA extractions. This discrepancy
favors for the selection and designing of new metabarcode primers that can be used to identify all
individuals of vertebrates or at least all individuals represented in a class of Vertebrata such as
Class Amphibia. The current study embarks on such an endeavor. In this study development of
new mtDNA metabarcode (16SrRNA) that can be used as universal primers to amplify almost all
species of Class Amphibia for different forensic and molecular biodiversity analyses.
Tissue samples were collected from order Urodela of Class Amphibia (Toads , Bull frog
and skittering frogs sample were collected from Punjab, Pakistan). DNA was extracted from the
collected specimens through standard organic method, qualified and quantified and then PCRamplified
using novel universal primers selected from aligned mtDNA sequences originating
from order Urodela mitochondrial DNA genomes submitted to different online sequence
databases such as NCBI nucleotide database. The sensitivity of PCR also be assessed using a
range of DNA concentrations. The amplified products were sequenced on ABI Genetic Analyzer
following Sanger’s dideoxy method of sequencing. The correctness of obtained mtDNA
sequences were examined visually in Chromas Lite 2.1 software and then alignment of these
sequences were performed against highly similar DNA sequences in NCBI nucleotide databases
using BLAST in order to identify origin of unknown mtDNA sequences. With the help of
sequencing and phylogenetic studies specificity of the universal primer set confirmed and
Summary
67
presented as a novel metabarcode (16SrRNA) for species level identification of large number of
Amphibian species.
In summary, we present universal method for species classification of Amphibia using a targeted
parallel sequencing approach. Both sequencing and phylogenetic studies experiments confirm
specificity of universal primer set. Although promising results were obtained with current
settings, rapid improvement of bench top instruments will further develop method with less
hands-on, fewer sequencing errors and lower detection limit. So, in future, this barcode can be
used for species identification in various fields of study such as meat adulteration, illegal trade,
food mislabeling and molecular estimation of biodiversity. Availability: Items available for loan: UVAS Library [Call number: 2874-T] (1).
56.
Molecular Diagnosis Of Brucella Zoonosis As Blood Transfusion Hazard In Metropolitan Population Of Lahore And Okara
by Amna Azam (2011-Va-3560 | Dr. Wasim Shehzad | Dr. Iahtasham Khan | Dr. Muhammad Imran | Dr. Imran Rashid.
Material type: Publisher: 2017Dissertation note: Brucellosis and Coxiellosis are one of the most spreading zoonotic diseases. They both are facultative, intracellular, Gram negative and involved in bioterrorism and agro-terrorism attacks. Brucella abortus and Brucella melitensisare common among all specie of Brucella. Total two hundred Human Blood transfusion samples were collected from hospitals of Okara and Lahore. Blood was collected in vacutainers (without EDTA). After serum isolation serological test RBPT were performed of all samples. Eighty nine out of two hundred were positive to RBPT with seroprevalence of 44.5% (95% Confidence Interval [CI]: 37.61 – 51.4). DNA extraction was done. The concentration of DNA was analyzed through Nanodrop 2000. Then these samples were subjected to Genus specific Real-time PCR analysis. Forty one out of two hundred were positive to Genus specific Real-time PCR with seroprevalence of 20.5% (95% Confidence Interval [CI]: 14.9 – 26.09). Brucella genus positive samples were subjected to two species specific PCR Brucella abortus and Brucella melitensis respectively. Forty one out of forty one Brucella genuspositive samples were positive to Brucella melitensisand none of the sample was positive to Brucella abortus. These two hundred DNA samples were then subjected to Coxiella specific Real-time PCR analysis and 4 were found positive with seroprevalence of 2% (95% Confidence Interval [CI]: 0.06 – 3.94). Results were recorded in the form of Ctvalue. Results indicate that Real-time PCR is more efficient than RBPT and due to increasing seroprevalence in Blood transfusion samples its screening should be included in normal blood transfusion screening procedure through collaboration with Government to prevent transfusion transmitted infections (TTI’s). Availability: Items available for loan: UVAS Library [Call number: 2873-T] (1).
57.
Development Of Novel MTDNA Metbarcodes For Species Differentiation Of Class Pisces
by Hira (2010-VA-476) | Dr. Muhammad Imran | Dr. Saadat Ali | Dr. Amjad Riaz.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: The Folmer COI mtDNA universal primers that are considered standard for DNA barcoding of life contain so many mismatches against the target sequences of vertebrate origin that they often end in failure to amplify many of vertebrate DNA extractions. This discrepancy favors for the selection and designing of new metabarcode primers that can be used to identify all individuals of vertebrates or at least all individuals represented in a class of Vertebrata such as Class Pisces. The current study embarks on such an endeavor. In this study development of new mtDNA metabarcode (16SrRNA) that can be used as universal primers to amplify almost all species of Class Aves for different forensic and molecular biodiversity analyses.
Fins/tissue samples were collected from Class Pisces (one specimen from every order reported to be present in Pakistan). DNA was extracted from the collected specimens through standard organic method, qualified and quantified and then PCR-amplified using novel universal primers selected from aligned mtDNA sequences originating from all Pisces mitochondrial DNA genomes submitted to different online sequence databases such as NCBI nucleotide database. The sensitivity of PCR also be assessed using a range of DNA concentrations. The amplified products were sequenced on ABI Genetic Analyzer following Sanger’s dideoxy method of sequencing. The correctness of obtained mtDNA sequences were examined visually in Chromas Lite 2.1 software and then alignment of these sequences were performed against highly similar DNA sequences in NCBI nucleotide databases using BLAST in order to identify origin of unknown mtDNA sequences. With the help of sequencing and phylogenetic studies specificity of the universal primer set confirmed and presented as a novel metabarcode (16SrRNA) for species level identification of large number of Piscean species
In summary, we present universal method for species classification of Pisces using a targeted parallel sequencing approach. Both sequencing and phylogenetic studies experiments confirm specificity of universal primer set. Although promising results were obtained with current settings, rapid improvement of bench top instruments will further develop method with less hands-on, fewer sequencing errors and lower detection limit. So, in future, this barcode can be used for species identification in various fields of study such as meat adulteration, illegal trade, food mislabeling and molecular estimation of biodiversity.
Availability: Items available for loan: UVAS Library [Call number: 2899-T] (1).
58.
A Study Of Chicken Gut To Determine The Colonization Of Bacillus Licheniformis And Changes In The Histological Features
by Arooj Tahir (2015-VA-812) | Mr. Shahid Abbas | Dr. Muhammad Imran | Dr. Saif-ur-Rehman Kashif.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: The most important and commonly used source of animal protein in the whole world these days is poultry meat. With the increase in the world population the demand of poultry meat
is also increasing. Universally, for the treatment of infected chicken and for growth promotion,
antibiotic growth promoters are used. But the extensive and long term use of AGPs for growth
promotion and infection treatments has resulted in the survival of resistant bacterial strains that
poses a very drastic threat to both animal and human health. Because of these radical results,
some of the countries have restricted the use of AGP in poultry and are moved on towards the
use of probitics as growth promoters. The most commonly used strain for probiotics is from
genus Bacillus as they have the tendency to survive even in the harsh and industrial conditions.
Bacillus subtilis, Bacillus licheniformis and Bacillus pumilusare are the ones that are involved in
the spoilage of food and certain medical conditions.
In this study, we considered the effects of probiotics as an alternative of AGP in poultry.
Bacillus spp. can be used as probiotics, as they can efficiently colonize in the small intestine and
improve the growth enhancement in broilers. A selective strain of bacteria Bacillus licheniformis
was used as a probiotic for the broiler chicken. After 42 days, 15 birds from three different
groups were collected, 5 from each group, and slaughtered to collect cecum samples and
intestinal tissue samples. The samples were processed for DNA extraction, PCR and histological
methods to determine the probiotics colonization and growth differences between the normal
ones and the treated ones. There was a considerable increase in the height of the villi in the
treated ones as compared to the control ones which showed that the use probiotic helped increase
the surface area of the intestine for increased absorption. The extracted DNA from the cecum
sample was used for PCR amplification and sequencing, the results confirmed the presence of
Summary
64
Bacillus licheniformis. The results showed that the probiotic was efficiently colonized in chicken
gut and it improved the gut health and also helped chicken in gaining weight. Availability: Items available for loan: UVAS Library [Call number: 2893-T] (1).
59.
Consumer’s Awareness Of Food Safety From Shopping To Eating
by Khakshan Noor (2015-VA-1089) | Dr. Azmat Ullah Khan | Dr. Waqas Ahmed | Dr. Muhammad Imran Najeeb.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Considerable proportions of foodborne illness arises from malpractices in purchasing food stuff. Such as serving contaminated raw food, insufficient cooking reheating of left overs, and poor hygiene practices. There are no food safety regulations for domestic kitchens hence, houshold womens as primery food handlers have to ensure food safety hygiene practices at home. The present study was an attempt to assess food safety awareness of consumers from shopping to eating in Lahore to understand its status, as there is no previous study found in this field in Lahore . A sample of 1000 consumers from 10 towns of Lahore was collected. Simple Random Sampling was used to select shopping malls and bazars with in towns and convenient sampling for selection of consumers. A KAP survey study ws conducted using a closed-ended questionaire to assess status of food safety awareness among consumers. A variable sheet was generated on SPSS version 22.0. Descriptives statistics was used to calculate mean and percentages of responces and overall knowledge, attitude and practices . Chi-Square test was used to study significant association between knowledge, attitude, practices scores and education levels. And also to study association between knowledge and practices. Mean and standard deviation scores for overall knowledge of consumers was 37.61 ± 1.087 which showed negative food safety knowledge among majority of the consumers. And overall practices mean was 54.89 ± 1.052 which showed unhygieneic food handling practices among
Summary
67
majority of the consumers. Significant difference was observed between education and KAP scores and attitude and practices of consumers. Data obtained served as baseline knowledge and information for emphasis on continues improvement on the knowledge of consumers. Food handlers who is going to prepare food probably have poor knowledge and misunderstandings about food safety and are certainly engaged in culturally focused stereotypical food handling practices. Henceforth, consumers should commit and expose themselves to attaining the prerequisite knowledge and sources of information needed in reducing the probability of potential risks that will eventually lead to progressive food safety culture development in Pakistan. It is thus advised that there is a need for surveillance and interventions at domestic level with professionals assistance for consumers regarding food safety issues. Food safety educational programs through official and casual training and media must be advertised and repeated at specific intervals. Health and educational instituations can communicate and provide short training courses to consumers of all ages, especially brginning at school since education has a bigger impact on food safety KAP in current study. They need to be informed about the basic principles of food safety at home and as well as from purchase using “Five Keys to Safer Food” as Food Safety starts and ends with frequent behaviors of consumers. This study determines that there is a need for additional research to identify the possible risks that could pose to human health in regard to food safety from purchase to home. Availability: Items available for loan: UVAS Library [Call number: 2919-T] (1).
60.
Microbiological Evaluation Of Various Vegetables At Different Processing Stages
by Ammara (2015-VA-1088) | Dr. Zubair Farooq | Dr. Azmat Ullah Khan | Dr. Muhammad Imran Najeeb.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: The current research was planned to investigate the microbial load and identification of
bacteria in fresh vegetables from different locations such as local shops and superstores. Total
108 samples were collected 9 from each place. Samples collected carefully in sterilized bags and
processed in microbiological lab of UVAS Lahore.
Fresh vegetables are an important part of our daily food and especially fresh vegetables
with less processing like lettuce, carrot and beet root which are used as raw without cooking;
such type of vegetables became the cause of illness because of the microbial/cross
contamination. It is alarming situation for suitable agency to take some necessary action, make
guidelines to prevent potential food borne illnesses from the vegetables that contain pathogenic
bacteria, and find natural antimicrobials from plants that control spoilage and pathogenic
microorganisms in vegetables. Microbial quality of vegetables is not in good condition.
The vegetable samples were divided into two categories local shops and superstores.
Total six local shops and six superstores were selected for the collection of samples in triplicate
pattern. All the samples were evaluated for microbial parameters,Like TPC, TCC, detection of
salmonella ,E coli and staphylococcus aureus.
Data was analysed statistically by the two- way ANOVA (Analysis of Variance) for
microbial count with 5% probability. Means were compared by LSD (least significant design)
test (Steel et al.1997).
The aim of present study is to provide awareness and microbial trend data about different
fresh vegetables sold in North-West Lahore.Results of current study shows that the
microbiological quality of fresh vegetables collected from superstores is better than the local
Summary
62
shops. The result of present study shows that consumers should prefer to buy vegetables from
superstores as compared to local shops because the keeping quality and handling practices are
good in superstores than local shops. Availability: Items available for loan: UVAS Library [Call number: 1918-T] (1).
61.
Larvicidal And Adulticidal Effect Of Natural Herbs Against Mosquito Population
by Anam Shahwar (2015-VA-1347) | Dr. Nisar Ahmad | Dr. Muhammad Imran Rashid | Dr. Uzma Farid Durrani .
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Mosquitoes serve as vectors for a wide assortment of human and veterinary pathogens and cause pervasion of numerous infection. Many processes fall for the control of mosquito but the cross resistance didn’t let them successful. Plants are viewed as the natural industry of such chemicals which have promising medicinal and pesticidal properties. Ocimum basilicum regularly known as Basal and Calotropis procera which is famous as apple of Sodom has shown the insecticidal activities against mosquito. Ocimum basilicum and Calotropis procera has adulticidal and larvicidal activity against laboratory reared mosquito population. For the study plants of Calotropis procera and Ocimum basilicum has collected from Lahore city. Leaves stem and flower of Ocimum basilicum and leaves of Calotrops procera had ground after drying for their soxhelet extraction. Their methanol and aequeous extract was used against laboratory reared mosquito to check their efficacy. Serial dilutions of each plant was given to third and fourth instar larvae and three day old adult. Third and fourth instar larvae has shown complete mortality within one hour in 700ppm and 650 ppm of methanol extract of both plant whereas in water extract the concenteration were 450ppm and 500ppm. Mortality of larvae and adult mosquito has been recorded after every ten minutes for one hour and then after 24 hours. The lethal dose concentration from dose-probit model is 700ppm of and 650ppm (methanol extraction) and 450ppm and 500ppm (water extraction) of Ocimum basilicum and Calotropis procera. according The current study has checked the efficacy of indigenous plant extracts against mosquito for eco-friendly approach to control mosquito. Availability: Items available for loan: UVAS Library [Call number: 2949-T] (1).
62.
Bacterial Profiling And Development Of Molecular Diagnostic Assays For Detection Of Bacterial Pathogens Associated With Bovine Mastitis
by Aqeela Ashraf (2012-VA-388) | Dr. Muhammad Imran | Prof. Dr. Tahir Yaqub | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: The livestock sector plays a critical role in strengthening the economy of Pakistan. Control of livestock diseases is the primary objective of government livestock departments. Bovine mastitis is among the most significant diseases of livestock as reported by various field surveys in Pakistan. Despite considerable knowledge about mastitis and its etiology, this disease is still prevalent in many dairy herds; it remains most difficult to eradicate or control. It is an inflammation of mammary gland resulting in decreased milk production, veterinary care costs and culling losses.
In animal health improvement, there is a paradigm shift from treatment of clinical illness to disease prevention. Recognition of disease is the foundation of disease control and prevention. California mastitis test and somatic cell counting are the most commonly used methods for diagnosis of bovine mastitis. These methods are unable to identify the causative agent. Detection of pathogen is critically important for better control of mastitis. Microbial culturing and biochemical tests are traditionally used methods for pathogen identification. But, these methods are very time consuming and can only detect viable bacteria from the sample and can lead to false negative results. The progress in molecular methods based on PCR has improved the veterinary diagnostics.
For the identification of bovine mastitis pathogens, an economical, rapid and sensitive molecular diagnostic assay was developed using multiplex PCR, detecting the pathogenic species-specific DNA. The target species areS. aureus,E. coli, S. uberis, S. agalactiae, S. dysagalactia, S. haemolyticus, S. epidermidis, S. chromogenes andM. bovis. Multiplex PCR assay was developed for the detection of these significantly important bacterial pathogen
causing bovine mastitis. Species specific primers were designed which have the ability to specifically amplify the particular gene in the target species. For this purpose various gene regions were selected for different bacterial species which included 16S rRNA, cpn60, phoA and rdr. Initially monoplex PCRs were optimized individually for each target species. For optimizing multiplex PCR assay, various combinations of individual PCRs with varying concentrations of primers and template DNA were used. The final protocol included all the nine sets of primer pairs, every set targeting a unique mastitic pathogen. Multiplex PCR assay was checked for its specificity and analytic sensitivity was calculated. Mastitic milk samples were collected aseptically from various farms. Initial screening was based on Surf field mastitis test and California mastitis test. Milk samples were cultured on nutrient agar, blood sheep agar and MacConkey’s agar. The bacterial isolates were identified and further sub-cultured in nutrient broth. All the isolates were identified on the basis of 16S rRNA sequencing analysis.
The developed multiplex PCR assay was used to detect the target bacterial pathogens from the collected milk samples. Limit of detection of developed assay was up to 50 pg for DNA isolated from pure cultures and 104 CFU/ml for spiked milk samples. The results obtained by 16S rRNA sequencing, bacterial culture based identification and multiplex PCR assay were compared. Sensitivity and specificity were calculated using latent class analysis, specificity was up to 88% and sensitivity was up to 98% for targeted mastitic pathogens. The developed multiplex PCR detected nine bacterial species in a single reaction. Multiplex PCR assay has also detected the bacterial pathogens in a few culture-negative mastitis milk samples. This is the first multiplex PCR assay which can efficiently detect nine important mastitic bacterial pathogens in a single reaction. The development of multiplex PCR assay is useful in early diagnosis and prevention & control of bovine mastitis.
Mycoplasma is often ignored as a major mastitis-causing pathogen due to lack of rapid and accurate diagnostic tools. In this study a LAMP assay was developed for the identification of M. bovis from clinical mastitic milk samples. LAMP primers were designed from three gene regions including uvrC, 16S rRNA and gyrB. Bacterial reference strains and mastitic milk samples positive for M. bovis were collected from Quality Milk Production Services, Cornell University, Ithaca, NY. Bacterial strains were further cultured on Hayflick medium containing 15% horse serum and incubated for up to 7 d at 37°C with CO2 enrichment. DNA was isolated from mastitic milk samples and bacterial culture using Qiagen DNeasy Blood and Tissue Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions with few modifications. PCR and LAMP assay was performed for all the samples obtained. Analytic sensitivity was calculated and the limit of detection was up to 50pg/reaction for LAMP assay which is higher as compared to PCR.
Sensitivity and specificity was calculated for each of the three tests. Cohen’s kappa values obtained were 0.940 for uvrC, 0.970 for gyrB and 0.807 for 16S rRNA. All three tests showed a high level of agreement between test results and the true mastitis status, indicated by the receiver operating characteristic (ROC) curve. A robust, sensitive and specific LAMP assay has been developed for the detection of M. bovis from mastitic milk. These novel molecular assays could be helpful for correct and timely identification of bovine mastitic pathogens, which is crucial for the control and treatment of the disease.Molecular diagnostic assayshave been developed in the current study based on multiplex PCR assay and loop-mediated isothermal amplification assay.
Availability: Items available for loan: UVAS Library [Call number: 2930-T] (1).
