51.
Study On The Status And Risk Factors Of Brucellosis In Small Ruminants Of District Bagh Azad Jammu And Kashmir
by Hafiz Muhammad Atique Ghafoor (2008-VA-160) | Dr. Aamir Ghafoor | Prof Dr. Khushi Muhammad | Dr. Aqeel Javeed.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Brucellosis is one of the most important zoonotic diseases of large and small ruminants with worldwide distribution. Brucellosis is declared as serious problem in 86 countries as it is widely distributed throughout the developing world (WHO, 1996). It mainly affects sexually mature animals and causes significant reproductive losses (Wadood et al. 2009). In Azad Jammu & Kashmir (AJK), research work on seroprevalence of brucellosis in small ruminants is very limited. In Bhimber Azad Jammu and Kashmir, 13.33% prevalence of brucellosis in goats has been reported (Din et al. 2013). In another study in Kashmir valley, overall 6.5% small ruminants were found positive by RBPT (STAT 2013). The data was based only on screening test. No confirmatory test like ELISA or PCR was performed to know the true picture of disease problem among RBPT positive ones. To find out seroprevalence of brucellosis in small ruminants, 400 animals ( n= 173 sheep, n=227 goat) were randomly selected and were screened for brucellosis in District Bagh Azad Jammu and Kashmir. A questionnaire was filled for each animal which carried entries including breed, specie, sex, lactation status, abortion history, housing, feeding, age and management etc. Sera samples from these animals were collected and examined by RBPT. Sera samples declared positive by RBPT were subjected to further analysis through ELISA. All lab analyses were done at University diagnostic Lab UVAS, Lahore. The data originated from this study was tested by using Chi square by using “SPSS version 20” and probability level <0.05 was considered significantly different. The results showed overall prevalence 30.75% through RBPT while 2.25 % by ELISA in sheep and goats of District Bagh Azad Jammu and Kashmir. The results showed that the prevalence through RBPT was 14.7 % in
Summary
33
sheep and 16 % in goats while through ELISA it was 1 % and 1.25 % in sheep and goats respectively.
The results revealed that overall prevalence was higher in goats than in sheep. Moreover the prevalence was higher in female then in male. The prevalence of brucellosis was higher in age group (1.6 Y to 3 Y) in both sheep and goats. The results showed that prevalence was higher in non-pregnant as compare to pregnant animals while prevalence was higher in dry as compare to lactating sheep and goats. Area wise results revealed that prevalence was higher in Bagh followed by Harighel, Arja and Mongbajri. Availability: Items available for loan: UVAS Library [Call number: 2616-T] (1).
52.
Molecular And Serological Characterization Of Soilborne Francisella Tularensis
by Javed Muhammad (2010-VA-65) | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad | Dr. Muhammad Wasim.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Tularemia, caused by Francisella tularensis (F. tularensis ), is a zoonotic disease transmitted through contact with infected animals and contaminated environment. The disease has been reported from many countries of the world but no study has been done in Pakistan. In the current project, a total of 2280 soil samples representing 456 villages of eight districts of Punjab province were collected from way-points having human-animal interaction, processed for genomic extraction and tested through real time PCR for presence or absence of F. tularensis. Association of risk factors was determined from data such as gender and age of animals, plough method, irrigation system, fertilizer type used, availability of veterinary services, level of farmer education, physical and chemical composition of the soil. Moreover, sero-prevalence against F. tularensis in cattle, buffaloes, sheep and goats was determined using ELISA. Seventy four soil samples (3.24 percent) were found positive for F. tularensis. Phylogenetic analysis showed 100 percent similarity index with F. tularensis sub specie holarctica reported from other regions like USA, Sweden, Spain, Turkey and Germany. Presence of F. tularensis in soil showed negative association with increase in number of human density (0.7159; 0.3834-0.2054). Prevalence of anti- F. tularensis ELISA antibodies were significantly higher (p<0.05) in large ruminants (cattle and buffalo) as compared to small ruminants (goat and sheep). Age and gender-wise analyses showed non-significant differences (p>0.05) between small and large ruminants. Whereas, rain-irrigation system (2.96: 1.35- 6.48), lack of veterinary services (4.77:1.26-18.03) and use of organic fertilizer (5.3: 11.38- 20.39) have positive association with prevalence of anti- F. tularensis ELISA antibodies in the serum. Sero-prevalence of Ft in the animals has significant association with quantity of clay in soil (p<0.05). A conventional PCR based test has also been optimized for
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detection of F. tularensis using tul4 gene specific primers. Specificity of primer showed F. tularensis detection in soil DNA in the presence of other cross-reactive organism. Sensitivity was determined in two fold dilutions with detection limit of up to 320 pg/μL. Utilizing pET28a vector, a construct was prepared containing transformed tul4 gene (450bp) showing 100 percent sequence homology to query gene sequence. For manufacturing diagnostic assays especially in developing countries where availability of BSL-3 facilities and positive control reagents is an issue, provision of tul4 gene based constructs in vector can act as positive control and biosafe to use.
It is recommended that similar studies may be done in other parts of Pakistan to have spatial distribution of F. tularensis all over Pakistan. In future studies, other sources of transmission like water, ticks and rodents may be considered with soil for complete analysis. Transportation of whole genome of F. tularensis has been prohibited by Russian government, ATCC and CDC, WHO is working on designing a complete protocol for transportation of this bacteria or genome to other countries. Under such situation, conventional PCR optimization can be done for diagnosis of F. tularensis and pET28+tul4 constructs, developed in this study, can be used as a PCR positive control reagents. Availability: Items available for loan: UVAS Library [Call number: 2640-T] (1).
53.
Amelioration Of Pathological Effects Of Newcastle Disease Affected Broiler Chicks By Feeding Propolis
by Muhammad Adeel Manzoor (2009-VA-121) | Prof. Dr. Zafar Iqbal Chaudhry | Dr. Raheela Akhtar | Prof. Dr. Khushi Muhammad.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Poultry industry is playing an important economic role in Pakistan. Unfortunately many diseases are a continuous threat for this industry. One of the most alarming disease is Newcastle disease (ND). It is endemic in most of the parts of world and it causes huge economic losses. According to researches, flavones from propolis were observed to have anti-viral effect. Scientific researchers also reported that propolis has immuno- modulatory properties so it is helpful in reducing pathological effects of ND. It is reported that supplementation of propolis enhances the immune status and reduce the inflammatory reactions. So this research project was design to observe the effect of propolis on lymphoid organs, growth performance and antibody response in ND challenged birds. The propolis sample was collected from Apis mellifera bees held in HBRI Rawalpindi. Analysis by GC-MS shown that the main components of ethanol extract of propolis (EEP) are flavonoid, esters and aromatic phenylcarboxylic acid. For this research a total of 90 broiler chicks were purchased. The birds were divided into six experimental groups i.e. A, B, C, D, E and F. Fifteen (15) birds were kept in every group. Group A birds, were negative control birds (without infection and without supplementation), in group B were positive control for ND Infection (experimentally infected without any supplementation). The birds in group C were positive control for propolis and were given 500mg propolis per kilo gram (kg) of diet and were not given ND infection. Group D, E and F were supplemented with different concentrations of propolis i.e. 250, 500 and 750 mg/kg in diet respectively. Supplementation of propolis was provided 1st day to the end of the experimental study. Infection was given on day 14. On day 1, 13 (pre-exposure), 17, 19 and 21 (post-exposure) blood was collected from 4 randomly selected birds of each group to check the antibodies titre against ND by HI test. On day 17, 19 and 21, lymphoid
Summary
65
organs were collected for histopathology. Group F shows protective antibody titer (GMT of Lag2 of HI = 3.25) as compared to positive control group B (GMT of Lag2 of HI = 0.00) antibody titre. So propolis feeding groups show significant differences from diseased birds in other groups. The average weight gain was highest for group F which was 1097.50g at the end of experiment which is significantly higher than positive control group whose weight was 727.50g. Mortality rate in positive control group was 100% while in group F, mortality rate was 26.67%. This difference shows that propolis decreased the mortality rate significantly. Gross pathological lesions were also significantly different with respect to proventricular hemorrhages, button ulcers in intestine, tracheal lesions and hemorrhages in brain. Group F shows lowest gross pathological lesions as compared to other NDV infected groups. A significant difference was observed in the weight of lymphoid organs of birds, treated with propolis as compared to birds without propolis treatment. Weight of thymus, bursa and spleen in group F was 4.02g, 0.78g and 0.93g respectively which is significantly different from positive control group having 4.28g, 0.60g and 1.99g weights respectively. Histopathology of bursa, spleen, thymus, cecal tonsils, trachea and lungs shown significant differences from group B to other groups. Group B, being positive control group, has shown maximum lesions of histopathology. From this study it was concluded that ND caused immune suppression and damage of vital organs in broiler while propolis have immuno modulatory and antiviral effects. Propolis protects the bird from ND virus by interfering with its multiplication process. It also promotes growth performance of broiler birds and help the birds to survive against lethal ND infection. Availability: Items available for loan: UVAS Library [Call number: 2667-T] (1).
54.
Epidemiology Of Influenza Virus H5n1 In Islamabad Capital Territory
by Zahida Fatima (2005-VA-246) | Prof. Dr. Muhammad Athar Khan | Dr. Khalid Naeem | Prof. Dr. Mansur Ud Din Ahmad | Prof. Dr. Khushi Muhammad.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: The poultry sector in Pakistan is the second largest industry that contributes to the national gross domestic products (GDP) and remains a major source of nutrition (protein and energy) for human population in Pakistan. Highly Pathogenic Avian Influenza (HPAI) outbreaks due to H5N1 virus in poultry have been recorded in over 62 countries, indicating the contagious nature of the disease and its potential to infect various avian species. These HPAI outbreaks in poultry have lead to killing/culling of around 120 million birds in various countries. During 2009, the Avian Influenza continues to occur in poultry in China, Hong Kong, India, Egypt, Nepal, Bangladesh and Canada . In Pakistan, an HPAI outbreak due to H7N3 virus was first observed in 1994-95 and those due to H9N2 virus in broiler and layer chickens were recorded between late 1990’s and early 2000. During the period between 2006 and 2008, poultry heavily suffered due to multiple outbreaks caused by H5N1 virus.
The country experienced several and severe HPAI subtype H5N1 outbreaks during 2006-2008 in commercial poultry farms mostly, causing mass economic losses. In Pakistan all the four poultry production system exists being identified by FAO. The present study was conducted in peri-urban areas of ICT Islamabad, capital of Pakistan. The objectives of the present study were to investigate the outbreaks due to HPAIV H5N1 in 2006-2007 in ICT and identify the pattern and trends of these outbreaks. For this purpose descriptive epidemiological study was conducted and data was collected on a predesigned questionnaire regarding farm demography, culling, morbidity and mortality. The result statistical analysis showed a significantly (P< 0.05) higher morbidity, mortality, case fatality and culling rate in layers farms than breeders and broilers respectively. Layers and breeders of old ages were mostly affected with having higher mortality and culling in comparison to younger age layer and breeder commercial farms. The mean morbidity and mortality rates ranged 57–95% and 5-43% correspondingly.
After the HPAIV H5N1 first reported outbreak in Pakistan in 2006 culling strategy was adopted after devastating outbreaks regularly reported from throughout the country. The reasons behind these emerging epidemics were unknown and several hypotheses were given birth after these outbreaks. Knowledge regarding potential risk factors responsible for HPAIV H5N1 epidemics in commercial poultry farms in Pakistan was lacking. Therefore we conducted a longitudinal cross sectional survey (1:1 matched case control study) to identify potential risk factors at farm level responsible for 2006-2007 HPAIV H5N1 infection in poultry in ICT. Information on farm characteristics, biosecurity practices and farm management were collected. Logistic regression model on data was used to unveil the potentially associated risk factors with cases (farms confirmed HPAI H5N1 Positive). Several candidate variables were studied and investigated for association. The results multivariable logistic regression showed that farm location such as in urban area (P<0.05: OR=18.50), wild birds entry (P<0.05: OR= 12.66) and farms situated in highly dense poultry populated area (P<0.05:OR=4.50) were found significantly associated with outbreaks of HPAIV H5N1 infection in commercial poultry farms during 2006-2007 epidemics in the study area.
Live bird markets (LBMs) are essential for poultry marketing in developing countries like Pakistan. One year active disease surveillance for influenza viruses in avian species in LBMs in ICT area was conducted in 2011. LBMs in Pakistan are typically urban that brings together many avian species produced by different suppliers. Which make LBMs in Pakistan a potential source of HPAIV viruses as well as other emerging poultry pathogens i.e. new castle disease virus,infectious bronchitis etc. The results of the present surveillance data showed that seroconversion against H5N1 and H9N2 is present in LBMs bird species which were isolated from different samples like serum, cloacal, nasal samples and organ samples.This indicates the continuous threat of AIV viruses circulating in the live bird markets set up of Pakistan.
Findings of these studies will help to tailor control and prevention measure against devastating outbreaks in future regarding the local circumstances of commercial poultry farms as well as in LBMs. These studies also succeeded to unveil the true reasons behind these devastating outbreaks and their higher impact on poultry industry. Such type of surveillance programs will be useful in future to investigate several emerging diseases and outbreaks in Pakistan and other developing countries. Availability: Items available for loan: UVAS Library [Call number: 2700-T] (1).
55.
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
Discussion
74
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).
Discussion
75
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
Discussion
76
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
Discussion
77
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
Discussion
78
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
Discussion
79
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
Discussion
80
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
Discussion
81
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).
56.
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.
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