1.
Identification And Genotyping Of Vp1 Genses Of Fmd Viruses
by Atia Bukhari | Prof. Dr. Irshad Hussain | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
print
Publisher: 2009Dissertation note: Within two decades after its first report in 1954 from Pakistan, Foot and mouth disease has become endemic in the country and poses a serious threat to large as well as small ruminant population. Foot and Mouth Disease (FMD) is prevailing in cattle and buffaloes and is caused by either 0, A, Asia-i serotype of the FMD virus in Pakistan. The present study was undertaken to study the mutation rate of FMD virus and also molecular typing of the strains prevalent in Pakistan was done.
A total of 60 samples from buffalo and cattle were collected from five districts of Punjab including Lahore, Faisalabad, Sialkot, Okara and Sheikhupura. Soon after extraction of their RNA, all of them were reverse transcribed and then subjected to amplification by using different sets of the primers including universal as well as serotype specific primers. Then their VPI portions were amplified by using VP1 specific primers. Among 60 samples, 48 were positive with universal primers. Other 12 samples were not amplified with these primers hence not processed.
Among 48 FMD positive samples, 24 were positive with serotype 0 specific primers, 16 with serotype Asia-i and remaining 8 were positive with serotype A specific primers. After their amplification, the amplicons were run on the gel. These amplicons were extracted by using DNA extraction kit. After their purification, they were sent to Macrogen® (Seopl, Korea) and Centre of Excellence for Molecplar Biology, Pakistan (CEMB) for sequencing. Each amplicon was sequenced thrice and the consensus sequence was established eliminating sequencing errors.
Sequence identity and multiple sequence alignment of molecular sequences (nucleotide and amino acids) were performed with Clustal W algorithm (Thompson et al., 1994). Neighbour joining trees were constructed by using MEGA version 4.0 (Kumar et al., 2004). Nucleotide distance matrices were computed by Kimura two parameter algorithm based on the total nucleotide substitutions and evolutionary trees for VP1 genes were constructed.
For FMDV serotype '0' phylogenetic analysis, 14 VPI sequences from various field isolates were compared with some previously published Pakistani FMD 0 type VP1 specific sequences available with GeneBank and some recently published VP1 sequences reported by countries bordering with Pakistan including India, Iran and Afghanistan Similarly, 12 VP 1 sequences of FMDV serotype Asia-I isolates of this study were compared with previously published sequences and their phylogenetic relationship was established. However, the sequencing results of serotype A were inconclusive and were not included for phylogenetic analysis. Three sequences of three locally available FMD vaccines were also studied and compared with the outbreak strains.
Polymerase chain reaction was optimized with respect to MgCI2, buffer pH, annealing temperature, primer concentration, template concentration, and Taq polymerase. A concentration of 2.5 mM of MgCl2 resulted in the best amplification of the target sequences (Figure 1). The buffer with pH 8.8 yielded the best results (Figure 2) Although, the suggested annealing temperatures for various primers (of various serotypes) ranged from 48 °C to 63 °C, however, a temperature of 56 °C was found to be the best with all sets of primers (Figure 3). The best intensity DNA bands were observed with 0.3 pM concentration of the primers (Figure 4). Moreover, the best cDNA template concentration giving optimum amplification was found to be 3.0 p1 per reaction (Figure 5). Lastly, a concentration of 0.5 U of Taq polymerase was not sufficient for amplification of cDNAs, however, 1.0 U of enzyme was found to yield better amplification (Figure 6).
VP 1 DNA sequences of six previously published Pakistani FMD serotype 0 strains were analyzed phylogenetically with VP 1 DNA sequences of 14 isolates of the study. Serotype 0 isolates of this study distributed themselves into two distinct clusters (Figure 19). First cluster comprised of Sheikhupura 1 and 2, Muridkey 1, Raiwind 1, Nankana 1, Gujranwala 1 and Gujrat I isolates (Figures 19 and 20), whereas the second cluster included Depalpur 1, Sahiwal 1, Okara I, Multan 1, Toba 1, Faisalabad I and Pattoki 1 isolates (Figures 19 and 21). The first cluster was found to be associated with previously published Pakistani isolates of 2006 mostly. However, it also showed association with Afghanistan's isolates of 2004 (Figure 20). The second cluster seemed to be mostly related to previously published Pakistani isolates of 2003 (Figure 21). The overall grouping of the 14 sequences, when compared with each other, depicted a three clustered phylogram (Figure 22). Serotype 0 isolates from Depalpur, Sahiwal, Okara, Multan, Pattoki, Toba Tek Singh and Faisalabad grouped together into a clan and had more than 85% sequence similarity with each other. The second cluster consisted of isolates of Sheikhupura, Nankana, Raiwind and Muridkey. These sequences had more than 86% similarity with each other. The third cluster consisted of only two isolates which were 100 % similar to each other. However the third cluster had only 74 % sequence similarity to cluster I and 73 % sequence similarity when compared with cluster 2.
When the phylogenetic relationships with previously reported isolates of Asia 1 was evaluated, FMD Asia I isolates of this study were found to be scattered into two distinct groups (Figure 16). Group one consisted of isolates of Lodhran, Toba and Hafizabad that were more closely related to Indian isolates sharing more than 98% identity with each other and more than 94 % sequence identity with isolates of Indian 2001 to 2004 (Table 5 and Figures 16 and 17). However, they shared more than 86% sequence similarity with Pakistani isolates of 2002-2005 (Table 5). Group two comprised of isolates of kasur, Lahore, Pakpattan, Okara, Faisalabad, Jhang, Rahim Yar Khan, Bahawalpur and multan alongwith vaccine A and B (Figure 16). The isolates of group 2 were found to be closely associated with previously published isolates of Pakistani and Afghani origin of year 2003 and 2004 (Figures 16 and 18). Collectively, they shared an overall 70% sequence identity with each other. However, isolates of Bahawalpur, Rahim Yar Khan and Multan shared more than 98% similarity with each other, a measurement of close relationship denoting a likely common origin as one clan or dade. Similarly, isolates of Pakpatan, Faisalabad, Okara, Kasur, and Lahore shared 88% sequence identity with each other and qualified as one clade.
Although, overall amino acid sequence similarity of our isolates was not strikingly different from that of the published isolates, however, amino acid substitutions with dissimilar properties were found with a scattered pattern of distribution. For example, 15th amino acid residue which is hydrophilic in the previously published isolates had a substitution with a hydrophobic amino acid residue in our three isolates namely Sheikhupura 2, Muridkey I and Raiwind I (Figure 25). Similarly, 14th amino acid residue which is hydrophobic in nature was found to be replaced with a hydrophilic one in our last five isolates. Amino acid residue number 13 (Figure 25) had a substitution with a hydrophobic residue in some of our isolates etc. etc. It is interesting to note that such substitutions with amino acids having dissimilar properties have also been found, albeit at lower rate, in previously published sequences by many researchers (Figure 25).
A comparison of the deduced amino acid sequences in the critical VP I region of FMD serotype Asia I revealed that most of this study isolates shared very high homology with sequences of Vaccine A. However, the sequences of isolates of Lodhran, Hafizabad and Toba did not match much with that of either vaccines, A or B (Figure 23). Sequences of Vaccine A had a "K" which seemed to be replaced by a "T" in the sequences of most of the isolates. Considering the properties of various amino acids, this change does not signify a major shift in the three dimensional picture of the protein as K is a lysine, a positively charged amino acid, whereas a T is threonine, a hydrophilic amino acid in nature. Next substitution in most of the isolates is a "P" for "A" in comparison to the vaccines. Again, it is not a significant change as both P and A share the same property, hydorphobicity. Similarly a K with an R can be substituted without much change in the overall shape of the protein molecule. Next amino acid substitution is a leucine instead of methionine. Again both are hydrophobic in nature; hence their impact on the overall picture is minute, if at all. However, glycine and arginine are two very different amino acids; the former is a hydrophobic amino acid whereas the latter is positively charged one. Such amino acid substitutions may have the potential to make a major impact in terms of the epitopic differences in the capsids of vaccinal and field viruses. A comparison of the deduced amino acids of FMD serotype 0 isolates also exhibited such changes with the vaccinal virus (Figure 24).
Of the three hyper immune sera raised against three different vaccines in rabbits, only one vaccine induced a measureable immune response yielding good precipitation line against various FMD virus antigens.
In summary, RT-PCR for diagnosis of serotypes A, 0 and Asia 1 of FMDV was optimized and could be used for prompt and precise diagnosis of FMD in the country. Although, RT-PCR data pertains to bovines in the current project, but PCR optimization parameters are equally applicable to FMDV infections in other FMD susceptible animal species such as sheep and goat. The combination of PCR and sequencing of the VP1 gene to detect and analyze FMDV in disease outbreaks is fast (less than 6 hours for PCR and about 24 hours for sequencing), and it can give an accurate immunologic characterization of the virus, thus providing a rational basis for choice of vaccine. In fact, the molecular epidemiology of field isolates is a powerful tool to monitor the circulation of viruses (Saiz et al., 1993).
Secondly, various isolates of serotypes 0 and Asia 1 were sequenced along with some vaccinal strains. Sequence similarity tree analysis indicated that most of our isolates were closely related to previously reported Pakistani isolates and to those of neighboring countries such as India, Afghanistan and Iran. Additionally, amino acid sequence similarity data of major immunogenic site that forms 13G-13H loop in FMDV serotypes revealed that serotype Asia 1 vaccinal strain and Asia 1 isolates of this study possessed high degree of similarity suggesting a likely host immune response against the vaccine that may afford some protection against most field isolates of serotype Asia 1 type. Lastly, of three vaccines tested, only one was found to afford protection against field isolates of FMDV suggesting more work on vaccine issue in the country.
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2.
Comparison Of Diagnostic Approaches For The Detection Of Bovine Viral Diarrhea Persistency In Dairy Herds
by Arfan Ahmad | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
print
Publisher: 2012Dissertation note: Bovine viral diarrhea is one of the most important diseases of cattle which are causing
continuous economic losses to the cattle industry primarily due to decreased reproductive
I performance. Without doubt, direct contact between BVDV persistently infected, and susceptible animals is the most important transmission route of virus. All control programs which are in use in many countries of the world, mainly depend upon the detection of PI animals, eliminating them and preventing their return into the herds. Therefore, in this study diagnostic suitability of ear notch biopsies and serum samples were compared for the detection of PI animals, as well as proficiency of various diagnostic approaches like VI, AC-ELISA, IHC and real time RT-PCR were evaluated using ear notch biopsies. A total of 468 samples were collected from 12 participating dairy cattle farms located at Prince Edward Island, Canada. The samples were divided into two groups on the basis of age, A " 6 months), and B (> 6 months).
PI calves remain immunotolerant to the infecting strain but if exposed to a heterogonous strain postnatally, they may develop low level of antibody. Accordingly, serum neutralization was applied for initial screening of samples for further testing. The samples of animals of group B, having SNT (:S 1 :64) were selected, while all samples of younger aged group A were processed without considering the serum neutralizing titres, because unlike older animals, P.1. animals below 6 months of age can have passive colostral antibodies in the course of persistency. Diagnostic suitability of ear notch biopsy and serum sample for confirmation of BVDV
A significant discrepancy was observed between ear notch biopsies (51198 positive) and
serum samples (71198 positive) during first round of testing by real time RT-PCR. However, on
follow up testing, 30 days post first round of testing, a complete agreement between ear notch
biopsies and serum samples was observed. On second round of testing, a total of 4 animals out of
197 (one positive animals died before re-sampling) were confirmed with PI, using both ear notch
biopsies and serum samples. The decrease in the positivity using RT-PCR on serum samples in
the second round of testing reflected the presence of 2 transiently infected animals. Ear notch
biopsy (EN) testing did not detect any transiently infected animal indicating the lack of
delectability of the virus in EN during transient infection under conditions of this study. After
follow up testing, 2 animals in each of group A and B were identified as PI. These findings have
led us to conclude, that either serum or ear notch biopsy can be used for the detection of
persistent infection. Of 468 collected and 197 tested samples, an overall 0.85% and 2.03%
prevalence of PI animals with BVDV was observed respectively. A complete agreement (P value=l) was observed when three diagnostic approaches (Real time RT- PCR, AC-ELISA, and IHC) were compared with standard of VI. A total of 197 ear notch biopsies (145 of group A and 52 of group B) were tested by the four diagnostic tests, four animals (2 from group A and 2 from group B) were found positive by all the tests applied. A complete agreement was observed between the first and the second round of testing. All four assays were found specific but real time RT-PCR was found to be more sensitive. Both, VI and IHC were found labour intensive, as diagnosis may take more than one week to be made. Further PI calves remain immunotolerant tothe infecting strain but if exposed to a heterogonous strain postnatally, they may develop low leved
ofantibody. Accordingly, serum neutralization was applied for initial screening of samples for further testing. The samples of animals of group B, having SNT (:S 1 :64) were selected, while all samples of younger aged group A were processed without considering the serum neutralizing titres, because
unlike older animals, P.1. animals below 6 months of age can have passive colostral antibodies in the course of persistency. Diagnostic suitability of ear notch biopsy and serum sample for confirmation of BVDV persistent animals were evaluated by real time RT-PCR. TaqMan probes and primers specific for BVDVI and BVDV2 were used. They were found specific and able to detect 10·s and 10-4 TCID50 units ofBVDVI and BVDV2, respectively.
Availability: Items available for loan: UVAS Library [Call number: 1407,T] (1).
3.
Molecular And Serological Characterization Of Avian Influenza Viruses In Domestic And Wild Birds
by Mobeen Sarwar | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Avian Influenza virus (AIV) has been recognized one of the most important pathogen in poultry industry. AIVs play an important role in the pandemic spread that could cause high morbidity and mortality in human beings. A total of 1500 tracheal and cloacael swabs were collected from the seven live bird poultry markets of Lahore Pakistan for surveillance of AIV. The samples were processed for virus isolation in chicken embryos. The isolates were processed for HA test and AIV Antigen Rapid Test Kit to differentiate Newcastle disease virus and Avian influenza virus. Only four samples were positive for Avian influenza H9N2 subtype and 17 were positive for Newcastle disease virus. Four HA virus suspension of AIV showed high titers of anti AIV H9N2-HI antibody titer in chicken as well as rabbit serum, raised using Ottoman Pharma AIV-H9N2 (Oil based) vaccine.
The isolates of AIV H9N2 were confirmed using laboratory optimized RT-PCR, mRT-PCR and LAMP tests. All isolates were sequenced and analyzed to develop phylogenetic relationship. Two isolates A/Chicken/Pakistan/Micro-1/2009 and A/ chicken/ Pakistan/ Micro-2/ 2009 showed 99.1% nucleotide homology with each other and 95- 99% homology with the other Pakistani isolates, 95.1% homology with A/Chicken/Iran/B102/2005. The nucleotide sequence of "A/Duck/Pakistan/Micro-3/2009" showed 98.8% homology with "A/chicken/Pakistan/micro-4/2009", 98-98.7% homology with other Pakistani Isolates and 95.8% homology with "A/Chicken/Iran/B102/2005". The nucleotide sequence of "A/Chicken/Pakistan/Micro-4/2009" showed 99.6% homology with "A/duck/Pakistan/micro-3/2009", 95-96% homology with other Pakistani isolates and 94.3% homology with "A/Chicken/Iran/TH lBM863/2007".
Three out of four isolates had PARSSRGL cleavage sites and one isolate A/chicken/Pakistan/micro-1/2009 had PAKSSRGL cleavage site. The four isolates of the study contained a 226-Leu and 228-Gly at receptor binding sites. Substitution of Glutamine (Q) into Leucine (L) at 226 receptor binding site in HA glycoprotein increased the binding specificity for Sialic acid ? 2, 6 Galactose linkage of human receptor. The HA gene of the live poultry market isolates had 7 predicted glycosylation sites at 29-32, 105-108, 141-144, 298-301, 305-308, 492-495 and 551-554, positions. The NA gene of the live poultry market isolates had 8 predicted glycosylation sites at 44-46, 61-63, 69-71, 146-148, 200-203, 234-237 and 402-403, positions. Predicted glycosylation sites affected the structure and stability of NA protein.
It is concluded that continuous epidemiological and virological surveillance of live bird poultry markets may help scientists to develop an effective control and preventive measures for AI viruses.
Availability: Items available for loan: UVAS Library [Call number: 1536,T] (1).
4.
Epidemmiology Of Foot And Mouth Disease In Buffaloes Of Punjab Province
by Farhat Nazir Awan | Prof. Dr. Khushi Muhammad | Prof. Dr. Muhammad Akram Muneer.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2009Dissertation note: This study indicates that the ranking order of buffalo diseases, with respect to their incidence in descending order in Punjab province is Foot and Mouth Disease, Mastitis, Diarrhea, Haemorrhagic Septicemia, Sudden Death Syndrome and haemoglobinuria. Similarly the disease ranking order in cattle in descending order is FMD, Mastitis, Diarrhea, Hemorrhagic Septicemia, Haemoglobinuria and Sudden Death Syndrome. FMD is top most economic important disease both in buffaloes and in cattle in the province.
Morbidity rate in the adult cattle and buffalo was higher as compared to the younger stock. However, the mortality rate was higher in young stock as compared to the adult animals of both the species. Moreover, adult and young males of both the species were more susceptible to the disease as compared to females.
Cross-sectional survey revealed the economic loss of Rs. 41.32 million due to loss of milk, cost of dead animals and treatment cost of sick and complicated cases of FMD. The loss due to milk reduction was 57.3% of the total losses followed by mortality loss (26.4%), morbidity effect expenses (15.2%) and treatment charges in FMD complicated cases (1.0%). The findings of present study clearly indicate the association of age, feeding pattern, vaccination status and season as risk factors in the incidence of FMD in Punjab.
Data obtained from the EPI-Unit Lahore showed that 719 FMD outbreaks occurred in the district of Punjab during 2007-2008. The highest number of outbreaks (212) was recorded in Rahim-Yar-Khan followed by Bhakkar (118), T.T. Singh (81) and Faisalabad (72). Of the total 309 disease outbreaks in buffalo, 174 (56.3%) were recorded in adults, whereas this number in cattle was 169 (61%). The incidences of the outbreaks increased gradually following the post-monsoon period. The greatest number of outbreaks was observed during the winter season, from December to February. Data from FMD Research Center, Lahore revealed the involvement of only FMDV serotype "O" in all the outbreaks during 2007-2008.
Studies of the factors (age, feeding pattern, stage of pregnancy and species) on the immune response of local trivalent FMD vaccine revealed that buffaloes of all age groups responded well to vaccination against disease. It was also observed that 7-9 months pregnant buffaloes elicited significantly lower antibody response to vaccine as compared to the control groups. Similarly, buffaloes on grazing have shown lower anti-FMD-CF GM titer as compared to buffaloes on manger feeding. Sheep and goat were found to be late and poor responder to vaccine as compared to cattle and buffalo.
Analysis of 300 serum samples from FMD affected buffaloes of 12 districts of the Punjab indicated the highest incidence of serotype "O" (62.3%) followed by Asia-1 (32.4%) and "A" (3.30%) in the population tested.
FMD virus was inactivated at 61 ºC within 15 minutes and at pH 4, 8, and 10 within 24 hours. However, ultraviolet radiation was unable to inactivate the virus even after 45 minutes. The disinfectants/chemicals evaluated in this study including sodium hydroxide, sodium carbonate, citric acid, acetic acid, formalin, sodium hypochlorite, virkon-s, aldekol and Gas-G were effective in inactivating the FMDV at recommended concentration levels of 2%, 4%, 0.20%, 4%, 0.15%, 3.0%, 1.0%, 0.50% and 0.1% after 60, 30, 60, 60, 30, 30, 30, 60 and 30 minutes, respectively, at 300C. Sodium hypochlorite and Gas-G were equally good in inactivating the virus at half (1.5% and 0.05%) of the recommended concentration.
Efficacy trial of local and imported oil based trivalent FMD vaccine in six villages, of the Faisalabad district clearly showed that 81.8% of FMD cases were prevented by the local inactivated vaccine in vaccinated animals whereas; this percentage was 70.6 in case where imported vaccines were employed. Moreover, efficacy of the local vaccine was higher than the imported vaccines.
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5.
Factors Affecting Biomass Producation Of Baby Hamster Kidney Cell Line In Roller Bottle Culture System For The Production of Foot and Mouth Diseas Virus
by Qaiser Akram | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Foot and Mouth Disease (FMD) is endemic in Pakistan and killed virus vaccines against prevalent serotypes are used for the control of disease. Baby Hamster Kidney (BHK-21) cells as monolayer culture are routinely used for the propagation of FMD viruses. Various nutritional factors: amount of growth medium and serum concentration as well as physical conditions: seeding density, rolling speed, growth time, and incubation temperature for the propagation of BHK-21 cells on roller culture bottles were optimized. The roller culture bottles having surface area of 480 cm2 were used for the propagation of cells. Feeding of cells with 100 ml of growth medium per bottle and supplementation of 5% serum supported the growth of the cells in optimum way. Seeding of ten million cells per bottle resulted into the development of complete monolayer with maximum cell density within 48 hours. The cultured cells remained confluent up to 60 hours while a rapid decline in the number of harvested cells was recorded after 72 hours of incubation. Growth rate of the cells was slower at 33° C that increases at 35° C, reaching to its maximum at 37° C while cells could not tolerate the temperature of 39° C. The bottles kept at rolling speed of 3 rpm yielded maximum amount of cells while higher or lower rotation speed negatively affected the cell proliferation.
Antibody response of buffalo calves to different levels of FMD virus immunogen in trivalent vaccine was investigated. The vaccine containing 106.2 units of immunogen/TCID50 of each of the three serotypes of FMD virus induced log2(1.3± 0.4) units of anti-FMD "O" Complement Fixing Cumulative Geometric Mean antibody (FMD"O" CFT-CGM) titer, log2(1.4±0.3) units of anti-FMD"A" CFT-CGM titer and log2(2.0±0.7) units of anti-FMD"Asia-1" CFT-CGM titer. The vaccine containing 2x106.2 units of immunogen of each of the three serotypes of FMD virus induced log2(2.2±0.2) units of anti- FMD"O" CFT-CGM titer, log2(2.1±0.25) units of anti- FMD"A" CFT-CGM titer and log2(3.4±0.8) units of anti-FMD"Asia-1" CFT-CGM titer. The vaccine containing 3x106.2 units of TCID50 of each of the three serotypes of FMD virus induced log2 (5.3 ± 2.0) units of anti-FMD"O" CFT-CGM titer, log2 (4.6±1.9) units of anti-FMD"A" CFT-CGM titer and log2 (5.0±2.2) units of anti- FMD"Asia-1" CFT-CGM titer. The vaccine containing 4 x 106.2 units of TCID50 of each of the three serotypes of FMD virus induced log2(5.5±1.5) units of anti-FMD"O" CFT-CGM titer, log2(4.4±1.9) units of anti-FMD"A" CFT-CGM titer and log2(5.2±1.9) units of anti-FMD"Asia-1" CFT-CGM titer. Moreover, buffalo calves (n=3) which were primed and boosted with 60 days interval using vaccine containing 2x106.2units of immunogen of each serotype of FMD virus, showed log25.0 and log26.3 units of anti FMD"O"-CFT-GMT antibody titer, log24.6 and log2 6.0 units of anti FMD"A"-CFT GMT antibody titer, log25.6 and log26.0 units of anti FMD"Asia-1"-CFT GMT antibody titer, on 30 and 120 days post boosting.
Each serotype of the virus grew well on BHK-21 cell line. The virus showed poor TCID50 (log 103.2±0.2) in BHK-21 cells having Glasgow Minimal Essential Medium (GMEM) without Fetal Calf Serum (FCS). Addition of FCS in the medium at the rate of one percent increased log 107.1±0.2 units of virus TCID50. Incubation temperature of 350 C and 370 C supported the multiplication and maintenance of BHK-21 cells and yielded log106.6±0.1 and log107.0±0.2units of virus TCID50, respectively. Each serotype of FMD virus showed log106.29±0.07 units of virus TCID50 in the stationary monolayer of BHK-21 cells in Roux flask (75cm2), log107.66± 0.02 units of virus TCID50 in roller bottles (490 cm2) and log108.34 ± 0.07 units of virus TCID50 on 0.2 g of micro-carriers suspending in 200 ml of the growth medium in stirring bottle. The infectivity titer/TCID50 of each of the virus serotypes was significantly higher in roller bottles than that achieved in Roux flasks (p<0.05) and was significantly higher in stirring bottle containing micro-carriers suspending in the growth medium than that of harvested in roller bottle (p<0.05). It is concluded that the infectivity titer of the virus is directly proportional to number of BHK-21 cells in the culture system.
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6.
Biomass Production Of Pasteurella Multocida By Using Biofermentor For Preparation Of Montanoid Based Vaccine
by Noreen Sarwar | Prof. Dr. Khushi Muhammad | Dr. Atif Hanif | Prof. Dr. Muhammad.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Hemorrhagic septicemia is a contagious bacterial disease of large ruminants principally in cattle and buffalo with high morbidity and mortality. The disease is endemic in nature and outbreaks are common during hot, humid and wet season. The acute and fatal nature and brief duration of the disease limit the antimicrobial therapy. In Pakistan, the disease causes heavy economic losses to dairy industry. Vaccination therefore, is an option for controlling the disease. For a quality vaccine, biomass production of P. multocida along with well developed capsule (immunogen) is necessary. The problem associated with the production of a quality vaccine is poor biomass production of P. multocida when grown in ordinary or routine media.
Present study was designed to isolate P. multocida from sick animals and its molecular characterization in the laboratory and study factors (temperature, media composition, pH incubation time and agitation or shaking) affecting its immunogen production and "in process quality control" factors (biological titer, dry mass, adjuvant and storage time) that affect antibody response. Finally, biomass production of the organism using biofermentor and monitoring of the antibody response of buffaloes to inactivated Montanide ISA-70 based P. multocida vaccine.
Each of the field isolates showed grey, viscous, mucoid, translucent and non hemolytic colonies on blood agar. There was no growth on MacConkey's agar. It was Gram negative coccobacilli or thin rods and bipolar when stained with Leishman's stain. The isolates were positive for Catalase, Oxidase, Hydrogen sulphide and Indole production along with nitrate reduction while it was negative for urease production, citrate utilization and gelatin liquefaction. The bacteria fermented glucose, sucrose, mannitol, mannose, but failed to ferment arabinose, maltose, salicin, lactose, dulcito and inositol.
Polymerase chain reaction (PCR) was performed on isolated colonies by using P. multocida specific and HS causing serotype B specific primers. P. multocida specific PCR gave product of 465 bp while HS causing serotype B specific primers amplified a product of approximately 590 bp.
Growth of the bacteria in casein yeast sucrose broth was optimized under different conditions. CSY broth showed dense growth of P. multocida during incubation for 18 hours. A temperature in between 35°C and 40°C showed its optimum growth. Poor growth was observed below 30°C and no growth was detected at 50°C and above. No growth occurred at pH 0.5 and 10.0 but best growth was obtained at pH 7.0 and 8.0. There was positive correlation between shaking in terms of rpm and growth. There was optimum growth at 500 rpm for 24 hours.
Inactivated HS Vaccine was prepared from dense growth in biofermentor on the basis of dry mass and bacterial count. The effect of biomass, adjuvant, storage of the vaccine, priming alone or with boosting on its potency was also studied along with boosting effect of montanoid ISA 70 oil based vaccine. Dry mass 1.7 mg/dose produced protective antibody titer while bacterial count 10-14/ml was sufficient to produce the protective antibody titer. Montanoid ISA 70 based vaccine provided immunity to buffalo calves better than aluminium hydroxide gel and bacterins. Boosting with oil based vaccine can help to keep the animal immunized for whole year. For better results of vaccine, it can be stored at 4oC for six months.
It is concluded that the proposed study improved quality of the vaccine and reduced volume of the vaccine dose, cost of its production and frequency of vaccination.
Availability: Items available for loan: UVAS Library [Call number: 1581,T] (1).
7.
Production And Evaluation Of Peste Des Petits Ruminants Virus Vaccine
by Muhammad Aness | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2013Dissertation note: PPR is an acute highly contagious viral disease of small ruminants which is endemic in Pakistan. Present study was aimed to evaluate the freeze dried PPRV vaccine with variable biological titer to induce protective immune response in beetal goats. The comparative immune response of animals to adjuvant and non-adjuvant vaccine and duration of immunity was also studied. Effect of boosting on the humoral immune response of animals as well as shelf life of various vaccines was also evaluated. Each of the vaccines was inoculated in a group of five animals. Serum samples were collected at specified time intervals and antibody levels were detected through cELISA as PI values and neutralization test as MNA titer.
The virus was propagated on the Vero cells. It was estimated that infecting 2 x 107cells with 104.00 TCID50 virus concentrations added to a T-175 cell culture flask at the time of subculture yielded maximum virus titer in the cell culture harvest following three freeze thaw cycles of the contents. The freeze dried vaccine with a biological titer of 105.00 TCID50 per dose provoked maximum antibody titer followed by the ones with a titer of 104.00 or 103.00TCID50 which provoked nearly equivalent protective immune response while the animals inoculated with a vaccine having 102.00 TCID50virus concentrations developed minimum antibody titer. The oil adjuvant PPRV vaccines elicited significantly higher antibody titer in comparison to gel based vaccines but however minimum antibody titers were detectable in response to freeze dried vaccines. Although protective antibody level (? 10 neutralizing antibody units) was detectable in the animals vaccinated with either oil based, gel based or freeze dried vaccine containing biological titer of 104.00 TCID50 but however the extent and duration of immunity was found to be most superior in response to oil based vaccines. It can be concluded that a single shot of either gel or oil based vaccine can provide protection in the vaccinated animals for a minimum of one year duration. Goats receiving a booster dose of the vaccines had a significantly higher antibody tier in comparison to the ones who received single dose of the vaccines. The freeze dried and wet vaccine kept at 4 °C did not show any significant drop in the biological activity of the virus even after 12 months of storage. Immunogenicity of the both adjuvant and non-adjuvant vaccines, as measured through the immune response in the vaccinated animals, also remained unaffected after 12 months of storage at 4 °C.
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8.
Epitope Mapping Of Fusion And Hemagglutinin Genes Of Ndv Isolates From Pigeon And Peacock, And Efficacy Of Lasota Vaccine In Broiler Against The Isolates
by Sameera Akhtar (89-ag-433) | Prof. Dr. Mohammad Akram Muneer | Prof. Dr. Khushi Muhammad.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Poultry industry in Pakistan has experienced huge economic losses in the recent past by NDV. Simultaneous appearance of disease outbreaks and an increased genetic dissimilarity between the isolates recovered from commercial, backyard poultry and captive wild-birds raised the concerns whether the commonly used vaccine strains and vaccination schedules were good enough to provide protection against the circulating diverged strains reported by various researchers (Munir et al., 2012a; Shabbir et al., 2012a; Shabbir et al., 2013; Siddique et al., 2013; Rehmani et al., 2015). The fact that wild birds are known to act as reservoirs of viruses of low virulence that may emerge as vNDVs with the mutation in F0 cleavage site especially for chickens (Alexander et al., 2012) and that phylogenetically related vNDVs of class II causing outbreaks in chickens have previously been isolated from wild birds (Miller et al., 2010, Dortmans et al., 2011), viruses (in this study) were isolated from disease outbreaks in pigeons and peacock and the evolutionary trends of the isolates were studied. Deduced amino acid profiling of F and HN genes of the isolates along with their pathogenic potential and virulence for the broilers vaccinated with currently used live LaSota and killed ND vaccines were also studied.
Both the isolates had the genome size of 15,192 nt similar to NDV genotypes reported previously. The complete genome and residue analysis of F and HN genes of the isolates revealed a low divergence to APMVs which was classified as genotype VI/lineage 4 and VII/lineage 5, respectively for pigeon and peacock isolates. The deduced amino acid residue analysis of cleavage site of the study isolate suggested that both the isolates carried a motif characteristic for velogenic/mesogenic ND viruses. The presence of polybasic F-protein
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cleavage site, mean death time (48 – 61 hrs), severe clinical, macroscopic and microscopic lesions, all highly suggestive of the virulent nature of under-study isolates.
The sites for glycosylation and cysteine residues are thought to be conserved for F and HN protein. However, in comparison to each other and to representing genotype particularly the vaccine strain, we found differences in residues composition for a given glycosylation site and variations in both number and site of cysteine residues. Further, comparison of functional domains of F and HN protein to other genotypes and vaccine strain revealed several substitutions that were more often in F protein than HN protein. The substitutions particularly in fusion peptide, hydrophobic regions and transmembrane region of F protein and neutralizing epitopes of HN protein could results in altered proteins that may result in increased or decreased capacity of the these protein to bind to the host cells. However, it may be noted that percent divergence for fusion protein in both isolates when compared with the vaccine strain was found higher for nucleotides than the deduced amino acids indicating synonymous nucleotide substitutions (homologous recombination) for amino acid residues. Though there exists low frequency of such recombination in negative sense RNA viruses especially in non-segmented, such homologous recombination in all the coding and some non-coding region of NDV particularly the vaccine and circulating lineages is supposed activity and neutralizing escape variants (Hu et al., 2010; Wang et al., 2015). They play an important role in generating genetic diversity and evolution to NDV (Chare et al., 2003; Wang et al., 2015). Taken together, variation in nucleotide and subsequent substitutions/alternations in amino acid profile such as observed in this study is consistent with previously described theories of evolution of RNA viruses particularly the NDVs (Yu et al., 2001; Umali et al., 2013).
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F and HN are glycoproteins. Two oligomers of HN are connected through disulphide bonds to form a dimer and two such dimers get associated noncovalently to form a tetramer which is a functional protein. F protein, on the other hand, is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. The role of cysteine residues in forming intermolecular as well as intramolecular bonds is important for proper functioning of HN protein. Similarly other amino acid residues also play important role in the proper folding of HN and F proteins to maintain their integrities and biological activities. Generally speaking, amino acid substitutions with residues having similar properties do not result in major changes in the overall three dimensional shape of a protein molecule. For example if a hydrophobic amino acid is substituted with another hydrophobic amino acid in a primary sequence of a protein, the overall three dimensional shape of the protein remains more or less conserved. However, if a hydrophobic amino acid residue is replaced with a hydrophilic amino acid residue, this kind of substitution results in a major change in the overall three dimensional structures or shape of a protein molecule impacting the epitopes. Although, overall amino acid sequence similarity of our isolates was not strikingly different from that of the published isolates, however, amino acid substitutions with dissimilar properties were found with a scattered pattern of distribution. For example, 36th amino acid residue (isoleucine) of HN gene of peacock isolate (Figure 4.13) in our study is hydrophobic but it is polar (threonine) in the previously published NDV isolates. On the other hand, 50th aa in our isolate (peacock) is polar but it is hydrophobic in previously reported isolates. Similar substitutions could be appreciated at various regions of the genes. It is interesting to note that such substitutions with amino acids having dissimilar properties have also been found, albeit at lower rate, in previously published sequences (Figure 4.13). The
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implications of these changes in the critical regions of F and HN genes could be serious as these changes may mean escape of the virus from the established immunity. Since the peacock flock had the history of vaccination with lentogenic strain (LaSota), identification of cleavage motif similar to velogenic strain raises concerns for type of vaccine used to protect the flock and need for post-vaccine evaluation. Substitution and subsequent mutations at fusion peptide, HR regions and transmembrane domain could affect the fusion activity of NDV (Umali et al., 2014) and alteration in antigenic epitopes particularly those that are involved in virus attachment could result in escape variants and subsequent vaccine failure (Cho et al., 2007; Wang et al., 2015).
Phylogenetic analysis of complete genome and hypervariable region (F gene, 374 bp) of pigeon-originated APMVs revealed evolutionary relationship to lineage 4/genotype VI (sub-genotype VIbii), which is predominantly represented by the pigeon isolates (PPMV-1). The NDVs belonging to same genotype have been reported previously in Sindh province (genotype VI, Khan et al., 2010) and Khyber PakhtunKhwa province (genotype VIc, Shabbir et al., 2013) indicating both are in circulation in the environment. However, this is the first report detailing complete genetic and clinicopathological characterization of pigeon-originated NDVs (PPMV-1, VIbii) from Pakistan. The isolate represents currently circulating PPMV-1 since it had cleavage motif (112RRQKR↓F117) different than the viruses isolated prior to 1980s (112GRQKR↓F117). Given the significant genetic variability in PPMV-1 belonging to VIb, the viruses were divided into two major sub-groups (VIbi and VIbii). The isolates belonging to VIbii have been observed as predominant strains in the latter period of pigeon-originated panzootic while strains of VIbi are known to be diminished in the late 1980s (Aldous et al., 2003; Awu et al., 2015) imitating selective pressure from vaccine usage (Aldous et al., 2004). Though it is difficult to speculate
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about the origin of the study virus of genotype VIbii, the clustering and genetic relatedness to isolate from Russia than recently reported PPMVs-1 from China [(VIa and VIb), (Awu et al., 2015; Wang et al, 2015)] indicates possible introduction through migratory birds from North to South Pole.
The peacock isolate clustered to lineage 5/genotype VII (sub-genotype VIIi) and was found to be closely related to isolate previously reported from chickens in Pakistan. Lineage 5/genotype VII is thought to originate from the Far East with the first isolation from Taiwan in 80s (Yang et al., 1999). Since then, the presence of this genotype has been indicated from various parts of the globe (Aldous et al., 2003). Varying at sub-lineage level, a number of vNDVs of class II have been reported from many Asian countries including those that shared borders with Pakistan. Genetically related NDVs to sub-genotype VIIa have been previously reported from wild birds while sub-genotype VIc, VIIb and novel VIIi were reported from backyard and commercial poultry. Interestingly, novel sub-genotype VIIi was reported previously from backyard and commercial poultry (Munir et al., 2012a; Siddique et al., 2013; Rehmani et al., 2015). Nevertheless, it is the first time that this genotype has been identified from peacock, a wild bird, indicating that this genotype has the potential for its transmission between the two species (peacocks to poultry).
We observed sudden deaths in challenged birds and in one of the vaccinate group. This was not unexpected since death with no apparent clinical indications are considered the most noteworthy evidence of velogenic NDVs. Similar observations have been reported by Samuel et al (2013) in immunogically naive birds challenged with virulent isolates of African origin. Though severity
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of observed clinical symptoms was relatively less for pigeon isolate than that of the peacock, it resulted in almost comparable morbidity, mortality and shedding even in vaccinates. All the birds in Ch group died on day 7 p.i. The nervous symptoms were observed in non-vaccinates and vaccinates however it varied in duration (days p.i): symptoms were evident on day 6 p.i in non-vaccinates while it were observed on day 9 p.i in vaccinates. In general, even with the characteristic F protein cleavage site for velogenic strains (112GRQKRF117 or 112RRKKRF117 or 112RRQKRF117), most of the pigeon-originated NDVs do not result in significant disease in poultry and differences in pathogenicity index vary from moderate to no virulence for chicken (Collins et al., 1994; Dortmans et al., 2010). In an effort to characterize pigeon-originated PPMVs, Awu et al. (2015) suggested a low rate of viral replication and an increased antibody level to the low pathogenicity of pigeon upon challenge to chicken. However, differences in pathogenicity and efficiency of viral RNA replication have been described previously demonstrating varying level of proneness to different host species (Dortmans et al., 2010). Some PPMV-1 have been reported to be highly pathogenic for chicken after passage either in chicken or chicken embryo indicating their potential to cause ND outbreaks (Dortmans et al., 2011) similar to what has been observed in this study. Furthermore, variants of genotype VIb originating from pigeon have been shown to produce neurological symptoms (Ujvari et al., 2003).
While comparing pre- and post-challenge antibody titers, we found varying but an increased immune response indicating that challenge virus have replicated. The immune response generated by pigeon isolate was found greater than peacock isolate indicating its efficient replication than peacock’s isolate (Fig.15). Furthermore, nervous symptoms were evident one day earlier in birds challenged with pigeon isolate than that of the peacock. The potential reason
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could be the fact that both group of vaccinates received killed vaccine containing genotype VII that may have hindered replication of challenged virus of genotype VII to some extent than genotype VI. Viral shedding together with increase in antibody titer suggests that the commonly practiced vaccine types and schedule (LaSota and killed vaccine of genotype VII) are not able to provide protection or provide only partial protection (if at all) from ND. Genetic and antigenic differences as observed in the functional domains and neutralization epitopes of F and HN protein of study isolates and vaccine strain could be attributed for increased virulence and escape from vaccine. The deduced amino acid residues (F and HN protein) for pigeon isolates were 11.8% and 12.1% while it were 11.6 and 13.5 for peacock isolate, respectively. It is believed that genome-heterologous vaccines may prevent the disease but are unable to prevent infection and subsequent shedding of challenged viruses compared to genome-homologous vaccination (Yu et al., 2001; Hu et al., 2009; Miller et al., 2009). For example, in an immunization and subsequent challenge experiment, Samuel et al. (2013) have attributed variations in pre- and post-challenge immune response of immunized birds, viral replication and shedding to the genetic distance between vaccine and challenge strains. Although, they did not find disease upon challenge but infection, shedding and increased titer were noted for strains that were much divergent to vaccine strains and had subsequent break-through replication. Contrary to this, Susta et al., (2014) revealed that genomic variation in vaccine and challenge strains did not affect virus shedding in the presence of protective immune response. Comparing classic vaccine (LaSota) and adopted vaccine containing F and HN protein of challenged virus (genotype VII virus NL/93), Dortmans et al. (2012) revealed that classic vaccines are well able to protect from disease and results in reduced shedding even with suboptimal vaccine dose of classic vaccine against virulent strains of NDV. Further, they reported that it may be poor flock immunity due to inadequate vaccine
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practices than genetic and subsequent antigenic evolution for potential outbreaks and spread of vNDVs. Beside potential compromise in procedures used in vaccine storage and administration, the expected genetic distance between vaccine strains and study isolate seems to be well-explained by Wang et al. (2015) through cross-HI assay. While evaluating the antigenic diversity of different strains through cross-HI assay, they reported a lower R-value (0.13-0.18) for interaction of PPMVs to LaSota than between PPMVs (VIa and VIb, 0.7) indicating an obvious antigenic difference with vaccine strain. This is consistent with our results where we observed an increased antibody response in vaccinated birds challenged with genotype VI than birds challenged with genotype VII indicating lack of or partial cross-reactivity of genotype II and VII to genotype VI.
Given the antigenic similarity (serotyping) among all APMVs, lentogenic strains (LaSota, B1) are being used as live vaccine to protect birds from virulent NDVs (vNDVs). These strains cluster phylogenetically closely to the viruses isolated approximately 60-70 years ago, and have a high genetic gap in relation to viruses isolated in the fields (Miller et al., 2007; Munir et al., 2012a). These classic vaccines are known to prevent disease but not infection, replication and shedding of the virus even in vaccinates that may be reduced compared to immunologically naive or non-vaccinates. This reduced shedding depends but not limited to species affected and its immune status, concentration and virulence of challenged isolate, vaccine type and its dose as well as the time between vaccine and challenge (Miller et al., 2009). Relating to differences in virus shedding upon challenge to vaccinates, differentiation in antigenicity have been reported at the level of classes and genotypes by cross HI assays (Miller et al., 2009; Li et al., 2010; Gu et al., 2011). Since the genetic distance or dissimilarities exist between classic and prevailing
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strains of vNDVs, spread as well as control of disease is always a matter of concern. The situation becomes further worse particularly in settings (e.g., Pakistan) where there lacks routine vaccination to backyard poultry and wild-birds, lack of or infrequent serological monitoring of the poultry flocks, uneven vaccination schedules and breach in bio-security practices.
Currently, lentogenic (LaSota) strains are being used to vaccinate commercial poultry flocks. Killed vaccine has also been added in the schedule following frequent ND outbreaks in the recent years. However, vaccination schedule widely varies particularly in broiler flocks. Together, in a life-time of approximately 40 days, 3 time administration of LaSota and one time intramuscular injection of indigenous prepared killed vaccine (type and genome characteristic are unknown but most probably genotype VII) is being practised in broilers. The backyard poultry remains unvaccinated in Pakistan; however, occasionally LaSota or Muketesawr strains are being applied depending upon the owner awareness and access of government institutes to birds. The backyard poultry, characterized by small flock with an absolute lack of biosecurity measures and poor management, often intermingles with wild birds including pigeons and could serve as potential hot-spot for virus spread and disease transmission. Beside concerns that whether these vaccine strains are able to elicit a protective immune response against the diverged circulating variants, the excessive use of vaccination could be problematic than benefits. It is interesting to note that classic vaccines are supposed to give better protection against the vNDVs isolated in 1930-70s than the variants isolated in the recent years (Czegledi et al., 2006; Munir et al., 2012a).
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CONCLUSIONS
Two isolates originated from pigeon and peacock were genotypically and pathobiologically characterized. Both the isolates had the potential to cause disease and subsequent shedding even in vaccinates following the commonly practiced vaccine schedule. The result presented may be useful in revising the vaccine schedule being practiced currently in Pakistan. Furthermore, it ascertains the need to establish and maintain the active surveillance for appropriate diagnostics and control of NDVs that could be spilled out in the environment by wild birds. Availability: Items available for loan: UVAS Library [Call number: 2740-T] (1).
9.
Spatial Ecology And Distribution Of Soil Borne Burkholderia Mallei In Punjab, Pakistan
by Muhammad Asad Ali (2002-VA-73) | Prof. Dr. Khushi Muhammad | Prof. Dr. Aftab Ahmad Anjum | Prof. Dr. Mansur-Ud-Din Ahmad.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Burkholderia mallei is a causative agent of glanders, the disease of equines. The disease is characterized by pulmonary, nasal and cutaneous forms. B. mallei is excreted through nasal discharge, lacerated skin/wounds and expiration. Diseased animals shed bacteria through the discharges contaminating soil, water, fodder and other susceptible animals in its vicinity. The present study was designed to map and investigate the association of different physical factors and soil chemistry analytes with persistence of B. mallei genome in soil of 10% percent villages (n=456) from eight selected districts of Punjab province, Pakistan.
Eleven (0.48%) out of 2, 280 soil samples were positive for B. mallei genome in varied locations of Punjab. Higher prevalence (2.37%) for genome was detected in Sheikhupura district followed by Chakwal district (2.10%). None of the samples from Gujranwala, Sahiwal, DG Khan, Attock, Faisalabad and Sargodha districts were found positive for B. mallei genome. The genome of B. mallei was distributed in 25% study districts of Punjab, Pakistan. In Chakwal district, the genome of B. mallei was strongly associated with moisture (p=0.008) in all positive samples ranging from 0.80 to 39.20%, Phosphorous (p=0.050) ranging from 1.74 to 21.75 mg/Kg. While, this association in Sheikhupura district soil samples was with Sodium (p=0.018) and moisture (0.026) ranging from 1.90 to 133.59 mg/Kg and 0.80 to 39.20%, respectively. The odds of detecting DNA of B. mallei were recorded higher (1.4, 6.8, 5.0, 2.8 and 10.6 ) when soil sample sites were < 500 meters away from vehicular traffic roads, < one kilometer from animal markets, < 100 meters from canal, animal density < 1,000 animals and human population < 300 houses/village. While the odds of detecting DNA of B. mallei were 0.1, 0.3, 0.4, 0.2 and 0.5 when soil sample sites were > 500 meters from vehicular traffic roads, > one kilometer from animal markets, > 100 meters from canal, animal density > 1000 animals and human population > 300 houses/village, respectively. Soil-borne B. mallei DNA is more likely to be detected in areas closer to roads with vehicular traffic along the interstate routes in Punjab and soil containing low level of moisture.
It was concluded that soil of two districts out of eight selected was positive for B. mallei genome in Punjab province. Odds of less distance from main road to animal farm and high animal density at farm were positively associated with B. mallei DNA persistence in soil. Moisture, sodium and phosphorus were positively associated with persistence of B. mallei DNA in soil.
Availability: Items available for loan: UVAS Library [Call number: 2900-T] (1).
