Tahir Yaqub

Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses.

The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked α2–6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic features

Virulent Newcastle disease virus (NDV) isolates from new sub-genotypes within genotype VII are rapidly spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) characterized by significant illness and mortality in poultry, suggesting the existence of a fifth panzootic. These viruses, which belong to the new sub-genotypes VIIh and VIIi, have epizootic characteristics and do not appear to have originated directly from other genotype VII NDV isolates that are currently circulating elsewhere, but are related to the present and past Indonesian NDV viruses isolated from wild birds since the 80s. Viruses from sub-genotype VIIh were isolated in Indonesia (2009-2010), Malaysia (2011), China (2011), and Cambodia (2011-2012) and are closely related to the Indonesian NDV isolated in 2007, APMV1/Chicken/Karangasem, Indonesia (Bali-01)/2007. Since 2011 and during 2012 highly related NDV isolates from sub-genotype VIIi have been isolated from poultry production facilities and occasionally from pet birds, throughout Indonesia, Pakistan and Israel. In Pakistan, the viruses of sub-genotype VIIi have replaced NDV isolates of genotype XIII, which were commonly isolated in 2009-2011, and they have become the predominant sub-genotype causing ND outbreaks since 2012. In a similar fashion, the numbers of viruses of sub-genotype VIIi isolated in Israel increased in 2012, and isolates from this sub-genotype are now found more frequently than viruses from the previously predominant sub-genotypes VIId and VIIb, from 2009 to 2012. All NDV isolates of sub-genotype VIIi are approximately 99% identical to each other and are more closely related to Indonesian viruses isolated from 1983 through 1990 than to those of genotype VII, still circulating in the region. Similarly, in addition to the Pakistani NDV isolates of the original genotype XIII (now called sub-genotype XIIIa), there is an additional sub-genotype (XIIIb) that was initially detected in India and Iran. This sub-genotype also appears to have as an ancestor a NDV strain from an Indian cockatoo isolated in 1982. These data suggest the existence of a new panzootic composed of viruses of subgenotype VIIi and support our previous findings of co-evolution of multiple virulent NDV genotypes in unknown reservoirs, e.g. as recorded with the virulent NDV identified in Dominican Republic in 2008. The co-evolution of at least three different sub-genotypes reported here and the apparent close relationship of some of those genotypes from ND viruses isolated from wild birds, suggests that identifying wild life reservoirs may help predict new panzootics.

Single or combined effects of mannan-oligosaccharides and probiotic supplements on the total oxidants, total antioxidants, enzymatic antioxidants, liver enzymes, and serum trace minerals in cyclic heat-stressed broilers

This study was intended to explore the oxidative status of broilers under cyclic heat stress (HS) as modulated by supplementation of mannan-oligosaccharides (MOS) and a probiotic mixture (PM). Two hundred fifty 1-d-old chicks were randomly divided into 5 groups. From d 22, birds were either kept in a thermoneutral zone (TN; 26.7°C for 24 h/d) or subjected to HS (35 ± 1.1°C and 75 ± 5% RH for 8 h/d from 1000 to 1800 h) to the conclusion of the study on d 42. Birds were fed either a corn-based diet (TN and HS groups) or the same diet supplemented with 0.5% MOS (HS-MOS group), 0.1% PM (HS-PM group), or their combination as a symbiotic (SYN; HS-SYN group). On d 42, birds were killed by cervical dislocation to collect serum for the determination of total oxidants, total antioxidants, paraoxonase, arylesterase, ceruloplasmin, aspartate aminotransferase, alanine aminotransferase, and trace minerals. Heat stress increased (P < 0.05) total oxidants and total antioxidants and decreased (P < 0.05) paraoxonase and arylesterase, with no change in ceruloplasmin, aspartate aminotransferase, and alanine aminotransferase activities. Dietary supplementation decreased (P < 0.05) total oxidants and total antioxidants, with no effect on the activities of other enzymes. Heat stress did not influence serum copper, zinc, and manganese concentrations of birds when compared with those in the TN group. However, MOS increased (P < 0.05) concentrations of all the trace minerals, whereas SYN increased (P < 0.05) concentrations of only zinc and copper. It was concluded that MOS or PM supplementation, alone or as a SYN, may reduce some of the detrimental effects of HS, whereas MOS alone or as a SYN may improve the absorption of trace minerals.

Infectivity and transmissibility of H9N2 avian influenza virus in chickens and wild terrestrial birds

Genetic changes in avian influenza viruses influence their infectivity, virulence and transmission. Recently we identified a novel genotype of H9N2 viruses in widespread circulation in poultry in Pakistan that contained polymerases (PB2, PB1 and PA) and non-structural (NS) gene segments identical to highly pathogenic H7N3 viruses. Here, we investigated the potential of these viruses to cause disease and assessed the transmission capability of the virus within and between poultry and wild terrestrial avian species. Groups of broilers, layers, jungle fowl, quail, sparrows or crows were infected with a representative strain (A/chicken/UDL-01/08) of this H9N2 virus and then mixed with naïve birds of the same breed or species, or different species to examine transmission. With the exception of crows, all directly inoculated and contact birds showed clinical signs, varying in severity with quail showing the most pronounced clinical signs. Virus shedding was detected in all infected birds, with quail showing the greatest levels of virus secretion, but only very low levels of virus were found in directly infected crow samples. Efficient virus intra-species transmission was observed within each group with the exception of crows in which no evidence of transmission was seen. Interspecies transmission was examined between chickens and sparrows and vice versa and efficient transmission was seen in either direction. These results highlight the ease of spread of this group of H9N2 viruses between domesticated poultry and sparrows and show that sparrows need to be considered as a high risk species for transmitting H9N2 viruses between premises.

Presence of virulent Newcastle disease virus in vaccinated chickens in farms in Pakistan.

ABSTRACT One year after a virulent Newcastle disease virus (vNDV) outbreak in Pakistan, the causative strain was present in vaccinated chickens of multiple farms despite the existence of high-average NDV-specific antibody titers (>4.75 log2). The data suggest a possible role of vaccinated birds as reservoirs of vNDV.

Genetic diversity of Newcastle disease virus in Pakistan: a countrywide perspective

BackgroundNewcastle disease (ND) is one of the most deadly diseases of poultry around the globe. The disease is endemic in Pakistan and recurrent outbreaks are being reported regularly in wild captive, rural and commercial poultry flocks. Though, efforts have been made to characterize the causative agent in some of parts of the country, the genetic nature of strains circulating throughout Pakistan is currently lacking.Material and methodsTo ascertain the genetics of NDV, 452 blood samples were collected from 113 flocks, originating from all the provinces of Pakistan, showing high mortality (30–80%). The samples represented domesticated poultry (broiler, layer and rural) as well as wild captive birds (pigeons, turkeys, pheasants and peacock). Samples were screened with real-time PCR for both matrix and fusion genes (1792 bp), positive samples were subjected to amplification of full fusion gene and subsequent sequencing and phylogenetic analysis.ResultsThe deduced amino acid sequence of the fusion protein cleavage site indicated the presence of motif (112RK/RQRR↓F117) typical for velogenic strains of NDV. Phylogenetic analysis of hypervariable region of the fusion gene indicated that all the isolates belong to lineage 5 of NDV except isolates collected from Khyber Pakhtunkhwa (KPK) province. A higher resolution of the phylogenetic analysis of lineage 5 showed the distribution of Pakistani NDV strains to 5b. However, the isolates from KPK belonged to lineage 4c; the first report of such lineage from this province.ConclusionsTaken together, data indicated the prevalence of multiple lineages of NDV in different poultry population including wild captive birds. Such understanding is crucial to underpin the nature of circulating strains of NDV, their potential for interspecies transmission and disease diagnosis and control strategies.

Complete Genome Sequence of a Velogenic Neurotropic Avian Paramyxovirus 1 Isolated from Peacocks (Pavo cristatus) in a Wildlife Park in Pakistan

ABSTRACT Avian paramyxovirus serotype 1 (APMV-1) was isolated from an acute and highly contagious outbreak in peacocks (Pavo cristatus) in a wildlife park in Pakistan. A velogenic neurotropic form of APMV-1 caused a 100% case fatality rate and killed 190 peacocks within a week. Biological and serological characterizations showed features of a velogenic strain of APMV-1, and these results were further confirmed by sequence analysis of the cleavage site in the fusion protein. The complete genome of one of the isolates was sequenced, and phylogenetic analysis was conducted. The analysis showed that this isolate belonged to genotype VII, specifically, to subgenotype VIIa, and clustered closely with isolates characterized from Indonesia in the 1990s. Interestingly, the isolate showed significant differences from previously characterized APMV-1 isolates from commercial and rural chickens in Pakistan. The work presented here is the first complete genome sequence of any APMV-1 isolate from wild birds in the region and therefore highlights the need for increased awareness and surveillance in such bird species.

Prevalence of Neospora caninum Antibodies in Sheep and Goats in Pakistan

abstract:  The purpose of the present study was to obtain seroepidemiological information on the Neospora caninum infection status of sheep and goats in different areas of Punjab Province and Azad Kashmir (Pakistan). A cross-sectional study, with the use of a competitive ELISA, showed an overall 27.7% (35 of 128) (95% confidence interval [CI] ± 7.7%) and 8.6% (13 of 142) (95% CI ± 4.6%) seroprevalence of N. caninum antibodies in sheep and goats, respectively. The difference in seroprevalence between sheep and goat populations was statistically significant (P < 0.05). The highest prevalence (37.4% ± 13.2%) was recorded in the tailless breed of sheep.

Complete Genome Sequencing of a Velogenic Viscerotropic Avian Paramyxovirus 1 Isolated from Pheasants (Pucrasia macrolopha) in Lahore, Pakistan

ABSTRACT We report the complete genome sequence of avian paramyxovirus 1 (APMV-1) isolated from an acute and highly contagious outbreak in pheasants (Pucrasia macrolopha) in Lahore, Pakistan. Biological and serological characterization showed a velogenic strain of APMV-1, which was further confirmed by the sequence analysis of the cleavage site in the fusion protein. Complete genome sequencing and phylogenetic analysis indicated that this isolate belonged to genotype VII, specifically to subgenotype VIIa, and clustered closely with isolates characterized from Indonesia. Notably, the isolate showed significant differences from previously characterized APMV-1 from Pakistani commercial and rural chicken.

Seroprevalence of Neospora caninum in Dairy Buffaloes in Lahore District, Pakistan

Abstract Seroprevalence of Neospora caninum in dairy buffaloes (Bubalus bubalis) was assessed in the Lahore District of Punjab Province, Pakistan. The study revealed an overall prevalence of 54.7% for N. caninum antibodies determined through a competitive enzyme-linked immunosorbent assay performed on randomly collected serum samples. The highest prevalence was observed in buffaloes >3–5 yr of age (64.1%), followed by 57.9% for 5 to 6 yr olds, and 55.8% in 1-yr-old neonates, with high probability of infection under intensive dairy farming conditions. The pattern of prevalence was closely associated with the season as reflected by the highest prevalence (70.5%) in summer (May–August) and lowest (39.6%) in winter (November–January). Aborting buffaloes illustrated significantly higher (78.9%) exposure compared with non-aborting dams (59.8%). Prevalence in animals with canine contact was significantly higher (60.3%) than without contact (48.1%). This is the first reported prevalence of N. caninum in Pakistan.