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.

Newcastle 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. To 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. The 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. Taken 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.

Background: Few serological tests are available for detecting antibodies againstMycoplasma capricolumsubsp.capripneumoniae, the causal agent of contagious caprine pleuropneumonia (CCPP). The complement fixation test, the test prescribed for international trade purposes, uses a crude antigen that cross-reacts with all the other mycoplasma species of the “mycoides cluster” frequently infecting goat herds. The lack of a more specific test has been a real obstacle to the evaluation of the prevalence and economic impact of CCPP worldwide. A new competitive ELISA kit for CCPP, based on a previous blocking ELISA, was formatted at CIRAD and used to evaluate the prevalence of CCPP in some regions of Kenya, Ethiopia, Mauritius, Tajikistan and Pakistan in an international collaborative study.Results: The strict specificity of the test was confirmed in CCPP-free goat herds exposed to other mycoplasma species of the “mycoides cluster”. Prevalence studies were performed across the enzootic range of the disease in Africa and Asia. Seroprevalence was estimated at 14.6% in the Afar region of Ethiopia, whereas all the herds presented for CCPP vaccination in Kenya tested positive (individual seroprevalence varied from 6 to 90% within each herd). In Mauritius, where CCPP emerged in 2009, nine of 62 herds tested positive. In Central Asia, where the disease was confirmed only recently, no positive animals were detected in the Wakhan District of Afghanistan or across the border in neighboring areas of Tajikistan, whereas seroprevalence varied between 2.7% and 44.2% in the other districts investigated and in northern Pakistan. The test was also used to monitor seroconversion in vaccinated animals.Conclusions: This newly formatted CCPP cELISA kit has retained the high specificity of the original kit. It can therefore be used to evaluate the prevalence of CCPP in countries or regions without vaccination programs. It could also be used to monitor the efficacy of vaccination campaigns as high-quality vaccines induce high rates of seroconversion.

Background: Approximately 170 million people are infected with Hepatitis C virus (HCV) worldwide. The prevalence of chronic HCV infections in Pakistan is about 5%, with most individuals being infected with HCV genotype 3a. Data on HCV genotypes distribution across various districts of the country are scarce. One example is district Mardan from where such data is available only from 17 individuals. Accordingly, the present study aimed at determining HCV genotypes distribution among 177 HCV RNA positive individuals from district Mardan. Findings: Serum samples (n = 215) from patients suspected of hepatitis C were collected and processed for Nested PCR based detection and subsequent genotyping. Gender-wise and age-wise differences in HCV prevalence and HCV genotypes distribution were determined by χ 2 test. Out of the total 215 serum samples, 177 were found to be positive for HCV RNA. The genotype 3a was the most predominant genotype among HCV RNA positive samples with a prevalence of 90.3%, followed by genotype 1a (5.6%), mixed genotypes (2.8%), genotype 3b (0.6%) and genotype 4 (0.6%). The HCV prevalence was higher in young individuals than old people and was indicative of reduced survival rate beyond 40 years. Conclusion: HCV genotype 3a is the most predominant genotype in district Mardan. The state of the art preventive and therapeutic strategies should be implemented to control the spread of HCV infections. Further temporal studies involving different geographical areas of Pakistan, are required to improve the control measures for HCV infection.