1.
Microscopic Comparison Of Human Hair Amongst Three Male Generation Of Five Castes In Punjab Pakistan
by M. Farhan khan | Prof. Dr. Tahir yaqub | Dr. Muhammad tayyab | Dr. Sehrish.
Material type: ; Format:
print
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2009,T] (1).
2.
Production Of Laccase By Immobilized White Rot Fungi And Its Application For The Decolorization Of Textile Effluent Dyes
by Iqra Ghulam Rasool (2012-VA-579) | Ms. Faiza Masood | Dr. Muhammad Tayyab | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Textile wastewater effluent contains several types of dyes that are toxic, carcinogenic, and dangerous for environment (Nyanhongo et al. 2002). More than 10,000 different kinds of dyes and pigments are used in dyeing and textile industries. Approximately 8, 00, 000 tons colorant is produced annually and 10% of used dyes are enters the environment in the form of wastes. There are different types of textile dyes such as direct dyes, disperse dyes, reactive dyes, acid dyes, and basic dyes. Wastewater effluents discharge from textile industries contain more than 10-15% of these dyes (Kunamneni et al. 2007). Such wastewater effluents are being discharged into water stream without or after only partial treatments, causing water pollution and negatively affecting the aquatic life. The treatment of textile wastewater effluents are of major environment concerns (Nyanhongo et al. 2002).
White rot fungi (WRF) is a wide class of fungi and it is mostly comprised of basidiomycetes, ascomycetes and lignin-decomposing fungi (Wesenberg et al. 2003). WRF are the most abundant wood degraders, and are so named because they leave a bleached appearance of the wood fibers following their attack. WRF has the ability to degrade contaminants by virtue of the nonspecific nature of its extracellular ligninolytic enzyme system (Nyanhongo et al. 2002)
The white rot fungus is also known as lignin degraders because it degrades lignin effectively due to some enzymes present in it. The important enzymes involves in degradation of lignin are following: (i) lignin peroxidase: It oxidizes both phenolics and non pheolics compounds, (ii) manganese-dependent peroxidase, (iii) laccase: It oxidises phenolic compounds and produce phenoxy radicals and quinones; (iv) glucose oxidase and glyoxal oxidase used for H2O2 production, and (v) celloulobiose quinone oxidoreductase for quinone reduction (Kunamneni et al. 2007).
Laccase (oxidoreductase, EC 1.10.3.2) belongs to polyphenol oxidases group of enzymes. Copper atoms are present in the catalytic center of enzyme so it is also known as multicopper oxidases (Baldrain et al. 2006). The molecular mass of laccase is 50–100 kDa (Couto and Toca 2006). According to the mechanism of laccase, it carries out the reduction of oxygen to water along with the oxidation of its substrate. Laccases oxidize wide range of compounds such as polyphenols, methoxy substituted phenols, aromatic diamines, and other compounds (Baldrain et al. 2006).
The substrate specificity of laccase is very wide and broad. In ortho and para substituted mono and polyphenolics substrate, it carries out reduction by removing hydrogen atom from hydroxyl group. In aromatic amines, it removes one electron and produces free radicals. These radical are able of many other reactions such as depolymerization, repolymerization, demethylation, or quinone formation. During lignin degradation, oxidation of methoxyhydroquinones followed by auto-oxidation of the methoxysemiquinones. Furthermore, formation of superoxide anion radicals undergoes more chemical reactions. The activity of laccase may be increased by using different kind of activators, such as ABTS (2, 2-azinobis (3-ethylbenzthiazoline- sulfonic acid), 1-hydroxybenzotriazole, or compounds secreted by fungi (Abadulla et al. 2000). In the presence of ABTS, the decolorization efficiency increases up to 45% (Tong et al. 2007).
Laccases have been produced from different kind of sources such as some species of fungus like white rot fungi, different kinds of bacteria, and some insects (Heinzkill et al. 1998; Diamantidis et al. 2000; Dittmer and Kanost 2010). This enzyme is widely distributed in Ascomycetes, Deuteromycetes, and Basidiomycetes, WRF is the major source for the production of laccase enzyme because this fungi is involved in metabolism of lignin (Bourbonnais et al. 1995).
There are many applications of fugal laccases such as effluent decolorization discharged from industries, degradation of pulp released from paper and pulp industries, removal of phenolics compounds from alcohols, synthesis of organic compounds, biosensors, pharmaceutical sector (Yaver et al. 2001). This enzyme can also improve animal performance, increase nutrient digestibility when added to animal feed (Sharma et al. 2013). Fungal laccases have higher redox potential of +800mV as compared to plants or bacterial laccases that’s why there are several applications of laccase in biotechnology field especially in the decolorization of dyes. Enzymes can be produce in larger amount so that laccase based decolorization techniques are advantageous to bioremediation technologies (Devi et al. 2012).
Pleurotus is a species of WRF and few laccases have been isolated, purified and cloned from Pleurotus species. However, the physiological significance of laccase produced by the white rot fungi is not known. Literature reports that mycelia culture of Pleurotus florida produces at least two laccases (L1 and L2), one of which appears to be linked with the mycelia growth of the fungus (Das et al. 1997). The L1 isoenzyme is dominantly involved in the dye decolorization process.
Submerged fermentation (SmF) is a type of fermentation in which microorganism is grow in liquid broth and enzymes and other compounds are released in the broth. This technique used free liquid substrates such as nutrients etc. The substrates are utilized quite rapidly and constantly supplemented with nutrients. In fermentation broth, microorganisms are provided with appropriate nutrients and conditions such as high oxygen concentration for the production of microorganism in order to get desired products. In this technique, mycelium formation is takes place. Mycelium formation can lead to pellet formation which hinders the diffusion of oxygen and nutrients in the medium.
In recent times, wide variety of secondary metabolites has been produced commercially by fungal fermentation. Fungi are complex microorganism that is different morphologically and structurally at different phases of their life cycles form others. It is also differ in form between surface and submerged growth in fermentation media. Nature of liquid media also effect on the growth of fungi. Different culture conditions such as temperature, pH and mechanical forces are important for fungi growth but these parameters are different for different fungi (Kossen et al. 2000).
Enzymes act like catalyst and they speed up any chemical reaction without being used up in the reaction. The uses of enzymes are advantageous due to its several characteristics and features as compared to conventional chemical catalyst. However, there are some problems that can reduce the operational life time of any enzymes. These problems includes; non-reusability of enzyme, the instability of their structure, high cost of isolation, purification and characterization and their sensitivity to harsh condition of reaction.
These objectionable limitations of enzymes may be reduced by the use of immobilized enzymes. There are mainly four procedures present for immobilization of any cell (Kunamneni et al. 2007). These procedures are following: adsorption, gels entrapment or polymer entrapment, covalent coupling, and cross-linking to insoluble matrices (Brouers et al. 1989). For immobilization different kinds of matrices, such as agar, calcium alginate beads, polyacrylamide gel, etc have been used. In order to select suitable matrix and immobilization procedure, type of the cell, type of the substrate, medium conditions and products are major factors (Prasad et al. 2005).
During immobilization, enzyme is fixed to or within solid matrix in order to get heterogeneous immobilized enzyme system. Naturally enzymes are bounded to cellular membrane in living cells for most cases so in order to get the natural form of enzyme, immobilization of the cell is done. This immobilized system stabilized the structure and activity of the enzyme for longer period of time. When enzymes are immobilized, they are stronger and more challenging to harsh environment changes. Immobilization also allows easy recovery of enzyme and also it’s multiple re-use in processes. The Michaelis constant of immobilized enzymes increased and their activity usually lowered when compared to free enzyme. When immobilization procedure applied, different structural changes introduced to an enzyme which leads to these alterations. Immobilization helps to maintain the structure, stability and activity of enzyme for longer time without being de-activated (Kunamneni et al. 2007).
Immobilization represents an attractive option to obtain enzymatic catalyst for dyes treatment. This technique provides different advantages: (i) it prevents enzyme leakage even under harsh conditions; (ii) it facilitates enzyme use in different types of reactors like packed bed, stirred tank and continuous bed; (iii) it causes stabilization of the enzyme tertiary structure, usually as a consequence of multipoint attachment of the enzyme to the support, providing enzyme rigidity. The stabilization provided by covalent bonding is usually counter balanced by partial enzyme deactivation. This negative effect can be mitigated by carefully optimizing the immobilization conditions in order to maximize the ratio between immobilized enzyme activity and activity of the primary enzyme solution (Pezzella et al. 2014).
Immobilization of laccase was extensively investigated with broad range of different techniques and substrates. Inactivation of enzyme occurs when oxidized products are absorbed on the surface of the immobilization matrix support (Kunamneni et al. 2007).
Textile industries discharged wastewater effluents comprised of toxic dyes are dangerous for aquatic life and have harmful impacts on the environment. There are different methods including physical and chemical methods which are use previously to decolorized dyes. These physical and chemical methods are quite costly, prolonged, ineffective and insecure (Shang and Chi 1996; Mechichi et al. 2006). In comparison to these methods, biological processes are quite suitable and helpful. Biological processes are less expensive, safe and take less time and effective. Biological processes used microorganisms to decolorize dyes. Laccase as an extracellular oxidative enzyme produced by white rot fungi are eco-friendly and cheap. In order to decolorize dye, three day old fermentation media is used and dyes is added in the broth. To get 70-75% decolorization in fungal culture, more than 48 hours are required. Pleurotus Species produced laccase efficiently and this laccase could decolorize malachite green dye upto 70% within 24 hours (Yan et al. 2009).
Laccases can degrade several dye structures such as phenol, polyphenols and diamines (Abadulla. et al. 2000) to degrade harmful compounds into less toxic compounds and may be helpful to reduce environmental pollution (Gianfreda et al. 1999). The specific features and mechanism of laccase helps to make it a versatile biocatalyst. Due to its versatility, it is suitable for several applications such as biopulping, biobleaching, and industrial wastewater treatment. Due to the severe environment legislation, the textile industry is trying to introduce new innovative technologies for the treatment of wastewater effluents discharged from textile industries. Laccase has potential to degrade dyes of various chemical structures so that development of techniques based on laccase seems an attractive and suitable solution in decolorizing dyes (Madhavi and Lele 2009). The decolorization and detoxification of the wastewater effluent would help to use it again and again in dying process in textile wet processing.
The major purpose of this research is to decolorize the textile effluents dyes discharged by industries after partial treatment and cause water pollution and have negative effect on aquatic life and ecosystem. It is necessary to established most effective and efficient method to produce sufficient amount of laccase enzyme through immobilized white rot fungus and then utilized it in the process of bioremediation.
Availability: Items available for loan: UVAS Library [Call number: 2208,T] (1).
3.
DNA Based Characterization Of Protease Gene From Geobacillussp.Sbs-4s
by Anam Shabbir (2012-VA-608) | Dr. Muhammad Tayyab | Ms. Huma Mujahid | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Proteases are hydrolytic enzymes responsible for the hydrolysis of proteins(Qadar et al.2004).These enzymes contribute major role in textile and leather industry,accounting 60% of the world wide enzyme market(Nascimento et al.2004).These enzymes are also being used in food ,pharmaceutical ,detergent, brewage sweet industry and as digestive additives in human and animal feed (Wilson, 2012).
Proteases are produced by microbes,animal and plants but microbial proteases are preferred due to ease in production and cheaper cost (Ningthoujam et al.2010).Microbes produce a variety of proteases according to their requirement that are specific in their function (Neurath 1999).Microbes might be involved in the production of intra or extracellular proteases.Extracellular proteases help the organism to absorb and utilize hydrolytic products from proteinious substrates in order to get energy by catabolism or to synthesize the biomolecules through anabolism reactions(Ningthoujamet al.2010).
Proteases can be classified in different ways.On the basis of cutting preferences these can be divided in to two groups:endopeptidases and exopeptidases (Barret and Mcdonald 1985).Exopeptidases are involved in hydrolysis of the peptide bond near N or C terminal whereas endopeptidases are responsible for the hydrolysis of peptide bond, with the chain, distant from the peptide ends(Motyan et al .2013).On the basis of catalytic residues in active site the proteases can be divided into six groups including glutamate,serine, therionine cysteine,aspartate and metalloproteases(Li et al.2013).
Microorganisms occupy all possible environments including habitats that provides appropriate conditions for growth(Sharma et al.2009).Thermophiles have ability to grow at highertemperature whereas other microbes fail to survive.There has been increasing interest in thermophilic bacteria because of their thermostable enzyme(Obeidat et al.2012).Hyperthermophiles can survive in extremely hot environment. Hyperthermophiles occupy the most basal positions of the phylogenetic tree of life(Bouzas et al. 2006). About 70 species of hyperthermophilic bacteria and archea has been isolated from different terrestrial, marine and thermal areas in the world.Hyperthermophiles are very divergent in their phylogeny and physiological properties.Proteolytic enzymes from hyperthermophiles are catalytically active at high temperature and they can alsoretain their catalytic activity in the presence of detergent and other denaturing substances (Stetter et al.1993).
Geobacillusis widely distributed thermophiles isolated from geothermal areas (Chalopagorn et al.2014).On the basis of16SrRNA gene sequences, Geobacillus belongs to Bacillus genetic group 5. It is phenotypically and phylogeneticallyconsistent group of thermophilicbacilli (Rahman et al. 2007).Bacillus and Geobacillus species are the dominant workhorses in industrial biotechnology. These bacteria produce a variety of extracellular enzymes, such as amylases, xylanases, proteases, phytases, carbonic anhydrases, catalases, pectinases. Bacillus and Geobacillus species hasability to grow at acidic, alkaline, neutral pH and at elevated temperature has positioned them among the most important industrial enzyme producers(Satyanarayana et al. 2012).
Geobacillus are gram-positive, rod-shaped, aerobic,endospore-forming obligate thermophiles.The growth temperature for various Geobacillus species ranges from 37 to 75 °C and pH range of 6.0 to 8.5.The members of Geobacillusare homologus to each other and share homology 99% among them(Tayyab et al.2011). The genus Geobacillusthermophilicstrains, produce a variety of thermostable hydrolytic extracellular enzymes, such as proteases, amylases, and lipases used in various industrial applications (Wiegand et al. 2013)
GeobacillusSBS-4S was isolated from a hot spring located in Gilgit, Northern areas of Pakistan.Geobacillus SBS-4S strain is Gram positive, rod-shaped bacteria and occurs in chains. That could grow at a wide range of temperature (45 to 75˚C) and pH ranging 5.5 to 9.5.Geobacillus SBS-4S produced several extracellular enzymes including amylase, protease and lipase.The comparison of the strain SBS-4S with the already reported species of genus Geobacillus showed that SBS-4S is resistant to antibiotics such as streptomycine, spectinomycin and rifampicin(Tayyab et al.2011).
Availability: Items available for loan: UVAS Library [Call number: 2242-T] (1).
4.
Comparison Of Antifungal Activity Of Human Salivary Histatin Between Diabetic And Nondiabetic Individuals
by Farid-Ul-Haq (2013-VA-555) | Prof. Dr. Tahir Yaqub | Dr. Ali Raza Awan | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Histatins are antimicrobial proteins found in human saliva. These proteins have also been
observed to have the ability to aid in wound healing in various organisms. The genes HTN1 and
HTN3 have been studied to govern these proteins. Histatin proteins have a vast array of
antimicrobial properties. While a fungus, Candida albicans or C. albicans is a part of the human
normal gut flora, it is a threat to people who have a compromised immune system. An
overgrowth of the fungi belonging to the Candida family leads to candidiasis in humans, and oral
candidiasis has been reported to a large extent namely in diabetic patients. The antifungal
activity of histatin proteins laid the basis of the current research work.
In this study, the antifungal activity of saliva from a total of 64 healthy and diabetic
human samples against Candida albicans has been evaluated. The samples of both healthy and
diabetic human samples belong from different age ranges: 15-25, 25-35, 35-45 and 45-55 years
in order to change in antifungal activity with respect to age of an individual. Antifungal activity
was observed through both agar well and agar disk diffusion methods, with agar disk diffusion
methods showing positive results. According to the outcomes of this study at least 120μL of
healthy saliva sample is required to create a zone of inhibition. Saliva from diabetic individuals
showed no antifungal results.
This occurrence led to the next part of this study involving amplification of HTN3 gene.
The nucleotide sequences of both healthy and diabetic individuals were compared together and
showed that the absence of antifungal activity in diabetic individuals might have reasons other
than a genetic one, according to this study. The results observed from the present study indicate
that healthy human saliva possesses antifungal activity against Candida albicans. In accordance
Summary
39
to these results, the naturally occurring antimicrobial activity of histatin proteins present in
human saliva can have immense use in the field of medicine. Availability: Items available for loan: UVAS Library [Call number: 2341-T] (1).
5.
Genetic Evolution And Development Of Recombinant Vaccine Against Newcastle Disease For Chicken In Pakistan
by Abdul Wajid (2009-VA-705) | Dr. Muhammad Wasim | Prof. Dr. Tahir Yaqub | Dr. Muhammad Tayyab.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2017Dissertation note: Newcastle disease (ND) is one of the most contagious diseases of poultry worldwide. The
disease is endemic in Pakistan and recurrent outbreaks have been reported in commercial poultry
flocks, domestic pet and migratory birds since 1963 an inception of commercial poultry farming
in the country. Disease surveillance is necessary to determine the incidence of the disease as well
as to identify the etiological agent of the disease status in the region. The analysis of the field
data provides a clue for the higher authorities to take steps for the remedy of the devastating
outbreak. A virulent form of Newcastle disease virus caused an outbreak in the northern region
of Pakistan during the mid of 2011. The virus was identified as a virulent viscerotropic vvNDV
and characterize, belonging to the sub genotype VIIi. However, the virus of this genotype is still
circulating in the field though the intensity of the strain to succumb the chickens to cause
mortality does not exist. The particular thing in this genotype was its susceptibility to other avian
species like pheasants, peafowls, ducks turkeys, peacocks, sparrows and parakeets. As this
genotype is circulating since 2011 2016 and still spill over in these avian species. Thus for the
last five years (2011-16), 3500 healthy, diseased and dead chickens, pheasants, peacocks,
turkeys, peafowls, ducks, sparrows, exotic parakeets, rosy-faced parrots, pigeons, and partridges
from 750 different locations s were monitored. Samples were collected from the Northern region
of the country Punjab, Khyber Pakhtoonkhawa, Azad Kashmir, including Gilgit,Baltitssan and
from Southern region, Karachi, Hyderabad , Mirpursakro and other small cities where the poultry
farms are located. The samples were collected by the local veterinarians, poultry Assistants and
Animal health practitioners who assist during the surveillance program. Samples were also
collected from the farmers who brought their birds for inspection in the lab with the details of the
141
farm. Mostly sampling was done where there was reports of NDV outbreak, tissues were
collected usually the trachea, spleen and brain, moreover, the pharyngeal and cloacal swabs not
only from the infected birds but also from the healthy birds were collected to assess the virus
shedding in the flock. Blood samples were also collected (1% of the birds at farm), for serum
collection to assess the immune status of the flock using Haemagglutination Inhibition (HI) test
and Enzyme linked immunosorbant assay (ELISA). The Survey Form meet the international
standard was filled for each farm for recording the information required to find the diagnostic
clue as well as the molecular characterization of the isolates. Pool of five pharyngeal swabs were
processed after the passage into 9-day old chicken embryonated eggs and confirming the positive
HA test and then confirmed by real time PCR (RT-PCR). In addition, sera were tested against
NDV by HI and ELISA tests. The targeted samples were sequenced by complete fusion gene and
whole genome using 22 pairs of overlapping primers. The observations indicated that the
commercial broiler industry is highly susceptible to virulent NDV and confirmed by data
available in the laboratory in the survey form. Contrary to that a little is known regarding the
maintenance and enzootic trends of vNDV infection level in domestic and wild birds. Poor
strategy of the use of vaccines and vaccination as well as the existence of virulent form of NDV
in the domestic and pet birds indicate a possibility of the root cause of the ND eruption in the
developing countries. A continuous isolation of virulent viruses of the panzootic Newcastle
disease virus of sub-genotype VIIi since (2011-2016 from commercial chickens and from various
other avian species in the country provide evidence for the existence of epidemiological links
intermingling of the strain among them. Therefore, to avoid the huge economical losses in the
commercial poultry the second largest industry in Pakistan, their close proximity should be
strictly avoided. The mass vaccination of the poultry flocks is in practice in all commercial
142
poultry farms in Pakistan. However, the use and availability of a reliable and standard vaccine, as
well as the correct usage of vaccine dose of the live attenuated LaSota vaccine are the key factors
to improve their efficacy in the field. Minor outbreaks have been occurring in the field even
though a severe outbreak was occurred in 2011-12 collapsed the poultry industry with other pet
and wild birds. To minimize the continuity of these minor outbreaks in the field for long time
there is a need for more effective vaccine to control the particular genotype of the ND virus. In
the present study, DNA vaccine was developed using the SFR-55 NDV strain antigens, in the
form of fusion (F) and hemagglutinin-neuroaminidase (HN), namely pcDNA3.1-F and
pcDNA3.1-HN. In vitro expression of both genes construct was assessed by reversetranscriptase-
PCR (RT-PCR) and western blotting. In the trial an inactivated oil-based emulsion
vaccine was prepared using the field strain SFR-55 and compare with the commercial vaccine
LaSota strain commonly used by the poultry industry. Birds were divided into six groups, the
first two groups were immunized with pcDNA3.1-F and pcDNA3.1-HN alone respectively and
third group with was vaccinated with both antigens pcDNA3.1-F+HN. The other two groups
were immunized with inactivated (wvSFR-55) and LaSota vaccines as described above, the last
group was injected with empty vector as control. The birds were immunized twice at 14 and 21
days of age intramuscularly (DNA vaccine), subcutaneous and eye-drop by inactivated and
LaSota vaccines respectively. The birds were challenged with live virulent NDV strain using a
dose of 10,000 ELD50/0.1ml per chicken. Results indicate that Inactivated and LaSota vaccines
provided high protection (>80%), as compared to pcDNA3.1-F, pcDNA3.1-HN, pcDNA3.1-
F+HN gave 70%, 75% and 20% respectively. There was 100% mortality in control chickens.
The administration of two vectors expressing F and HN antigens induced high immune response,
and provide protection than when used separately. However, the groups immunized with
143
pcDNA3.1-F, pcDNA3.1-F+HN and inactivated vaccine resulted in lower amount of virulent
virus shed after challenge when compared to the group immunized with standard LaSota. In
summary, the co-administration of both NDV glycoprotein antigens increased protection than
use separately. DNA-based vaccine can be used safely to reduce mortality and most importantly
lower the risk of virus transmission due to decreased level of virulent virus shedding. Availability: Items available for loan: UVAS Library [Call number: 2910-T] (1).
6.
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).
