Sentinel surveillance of selected veterinary and public health pathogens in camel population originating from Southern Punjab province, Pakistan.

An extended range of host susceptibility including camel has been evidenced for some of the important veterinary and public health pathogens, such as brucellosis, peste des petits ruminants (PPR) and bluetongue (BT). However, in disease endemic settings across many parts of the globe, most of the disease control interventions accounts for small and large ruminants, whereas unusual hosts and/or natural reservoirs, such as camels, remain neglected for disease control measures including routine vaccination. Such a policy drawback not only plays an important role in disease epizootiology particularly in settings where disease is endemic, but also serves an obstacle in disease control and subsequent eradication in future. With this background, using pre-validated ELISA and molecular assays [multiplex PCR, reverse transcriptase (RT)-PCR and real-time (rt)-PCR], we conducted a large-scale pathogen- and antibody-based surveillance for brucellosis, peste des petits ruminants and bluetongue in camel population (n = 992) originating from a wide geographical region in southern part of the Punjab province, Pakistan. Varying in each of the selected districts, the seroprevalence was found to be maximum for bluetongue [n = 697 (70.26%, 95% CI: 67.29-73.07)], followed by PPR [n = 193 (19.46%, 95% CI: 17.07-22.09)] and brucellosis [n = 66 (6.65%, 95% CI: 5.22-8.43)]. Odds of seroprevalence were more significantly associated with pregnancy status (non-pregnant, OR = 2.23, 95% CI: 1.86-5.63, p<0.01), farming system (mixed-animal, OR = 2.59, 95% CI: 1.56-4.29, p<0.01), breed (Desi, OR = 1.97, 95% CI: 1.28-4.03, p<0.01) and farmer education (illiterate, OR = 3.17, 95% CI: 1.45-6.93, p<0.01) for BTV, body condition (normal, OR = 3.54, 95% CI: 1.92-6.54, p<0.01) and breed (Desi, OR = 2.19, 95% CI: 1.09-4.40, p<0.01) for brucellosis, and feeding system for PPR (grazing, OR = 2.75, 95% CI: 1.79-4.22, p<0.01). Among the total herds included (n = 74), genome corresponding to BT virus (BTV) and brucellosis was detected in 14 (18.92%, 95 CI: 11.09-30.04) and 19 herds (25.68%, 95% CI: 16.54-37.38), respectively. None of the herds was detected with genome of PPR virus (PPRV). Among the positive herds, serotype 1, 8 and 11 were detected for BTV while all the herds were exclusively positive to B. abortus. Taken together, the study highlights the role of potential disease reservoirs in the persistence and transmission of selected diseases in their susceptible hosts and, therefore, urges necessary interventions (e.g., inclusion of camels for vaccine etc.) for the control of diseases from their endemic setting worldwide.

Evaluation of transmission potential and pathobiological characteristics of mallard originated Avian orthoavulavirus 1 (sub-genotype VII.2) in commercial broilers.

Newcastle disease (ND), caused by Avian orthoavulavirus 1 (AOAV-1), affects multiple avian species around the globe. Frequent disease outbreaks are not uncommon even in vaccinates despite routine vaccination and, in this regards, viruses of diverse genotypes originating from natural reservoirs (migratory waterfowls) play an important role in a disease endemic setting. Though genomic characterization of waterfowl originated viruses has been well-elucidated previously, there is a paucity of data on clinico-pathological assessment of mallard-originated sub-genotype VII.2 in commercial chickens. Hence, the current study was designed to evaluate its transmission potential, tissue tropism and micro- and macroscopic lesions in commercial broilers. Based on complete genome and complete F gene, phylogenetic analysis clustered the study isolate within genotype VII and sub-genotype VII.2 in close association with those reported previously from multiple avian species worldwide. The study strain was found to be velogenic on the basis of typical residue pattern in the F-protein cleavage site (112R-RQ-K-R↓F117), sever disease induction in chicken, tissue tropism and subsequent clinico-pathological characteristics. Giving a clear evidence of horizontal transmission, a 100% mortality was observed by 4th and 6th day post infection (dpi) in chickens challenged with the virus and those kept with the challenged birds (contact birds), respectively. The observed clinical signs, particularly the greenish diarrhea, and macroscopic lesions such as pinpoint hemorrhages in proventriculus and caecal tonsils were typical of the infection caused by an AOAV-1 in chickens. The virus exhibited a broad tissue tropism where genomic RNA corresponding to study virus was detected in all of the tissues collected from recently mortile and necropsied birds. The study concludes that mallard-originated Avian orthoavulavirus 1 is highly velogenic to commercial chicken and therefore ascertain continuous disease monitoring and surveillance of migratory/aquatic fowls to better elucidate infection epidemiology and subsequent potential impacts on commercial poultry.

Circulation of multiple subtypes (A, G and CRFs 02_AG) of human immunodeficiency virus type 1 (HIV-1) in selected districts of Punjab province, Pakistan.

Owing to consistent genetic mutation and recombination, various escape mutants and/or drug-resistant mutants of human immunodeficiency virus (HIV-1) are now emerging worldwide. Therefore, an understanding of the genetic characteristics of prevailing strains, particularly with regard to drug-resistance-associated substitutions, is essential for devising and implementing treatments and disease control interventions in endemic settings such as Pakistan. We processed a total of 130 plasma samples originating from HIV-treatment centers in selected districts of Punjab province, Pakistan. The samples were first screened using an HIV-1 Ag/Ab Combo test followed by amplification of the pol gene (1084 bp) from samples that were positive either for the antigen or for both the antigen and antibodies simultaneously. Screening revealed that a total of 45 samples were positive (34.62%; 95% CI: 26.99-43.13) for either antigen or both antigen and antibodies (n = 18, 40%; 95% CI: 27.02-54.55) or for antibodies alone (n = 27, 60%; 95% CI: 45.45-72.98). A largest number of positive samples was from the district of Lahore (n = 19/43, 44.18%; 95% CI: 30.44-58.9) followed by Faisalabad (n= 12/36, 33.33%; 95% CI: 20.21-49.66), Gujranwala (n = 05/23, 21.7%; 95% CI: 9.66-41.9) and Sargodha (n = 09/28, 32.1%; 95% CI: 17.93-50.66). The probability of occurrence of HIV infection was significantly associated with individuals having a history of injecting drug use (68.08%; OR = 11.15; 95% CI: 53.84-79.61, p = 0.0001). Phylogenetic analysis based on the pol gene showed that the sequences from this study clustered into three distinct clades representing recombinant form 02_AG (n = 14, 77.0%; 95% CI: 54.79-91.00), and subtypes A (n = 2, 11.1%; 95% CI: 3.1-32.8) and G (n = 2, 11.1%; 95% CI: 3.1-32.8). Although we screened 18 samples for drug-resistance-associated mutations, except for an accessory mutation (M46K) in the protease (PR) region in one subject, we found a lack of drug-resistance-associated substitutions in the PR region. On the other hand, we found two subjects (2/18) carrying a resistance-associated mutation (V106I) conferring a low level of resistance against non-nucleoside reverse transcriptase inhibitors. The present study shows that multiple subtypes of HIV-1 are present in the affected population. Continuous disease surveillance coupled with evaluation of drug resistance at higher resolution should be done in future studies.

Seroprevalence of Bluetongue virus in small and large ruminants in Punjab province, Pakistan.

Bluetongue (BT) is a vector-borne disease of immense economic importance for small and large ruminants. Despite frequent disease reports from neighboring countries, a little is known about current disease status and prevalent serotypes in Pakistan. We screened a total of 1312 healthy animals for group-specific antibodies and serotype-specific genome for BT virus through competitive ELISA and real-time PCR, respectively. An overall prevalence of group-specific VP7 antibodies [28.81% (n = 378/1312, 95% CI = 26.4-31.4)] was observed. The prevalence was higher in goats [40.75% (n = 194/476, 95% CI = 36.4-45.3)] followed by buffalo [29.34% (n = 81/276, 95% CI = 24.3-34.9)], sheep [18.40% (n = 60/326, 95% CI = 14.5-22.9)] and cattle [17.94% (n = 42/234, 95% CI = 13.56-23.4)]. The odds of seropositivity were more in buffalo of Nili breed (OR = 2.06, 95% CI = 1.19-3.58) as well as those found with a presence of vector (OR = 2.04, 95% CI = 1.16-3.59). Buffalo and cattle with history of abortion [(OR = 3.95, 95% CI = 1.33-11.69) and (OR = 5.89, 95% CI = 1.80-19.27) respectively] were much likely to be infected with the disease. Serotype 8 was detected in all animal species while, serotypes 4 and 6 were detected in sheep, 2, 6 and 11 in goat, and 2 and 16 in buffalo. The study concludes a much frequent exposure of different serotypes of Bluetongue virus (BTV) in small and large ruminants and indicates its expansion to enzootic range worldwide.

Genomic and biological characterization of Newcastle disease viruses isolated from migratory mallards (Anas platyrhynchos).

Given the global evolutionary dynamics of Newcastle disease viruses (NDVs), it is imperative to continue extensive surveillance, routine monitoring and characterization of isolates originating from natural reservoirs (waterfowls). In this report, we isolated and characterized two virulent NDV strains from clinically healthy mallard (Anas platyrhynchos). Both isolates had a genome of 15,192 nucleotides encoding six genes in an order of 3´-NP-P-M-F-HN-L-5´. The biological characteristics (mean death time: 49.5-50 hr, EID50108.5 ml-1) and presence of a typical cleavage site in the fusion (F) protein (112R-R-Q-K-R↓F117) confirmed the velogenic nature of these isolates. Phylogenetic analysis classified both isolates as members of genotype VII within class-II. Furthermore, based upon the hypervariable region of the F gene (375 nt), isolates showed clustering within sub-genotype VIIi. Similarity index and parallel comparison revealed a higher nucleotide divergence from commonly used vaccine strains; LaSota (21%) and Mukteswar (17%). A comparative residues analysis with representative strains of different genotypes, including vaccine strains, revealed a number of substitutions at important structural and functional domains within the F and hemagglutinin-neuraminidase (HN) proteins. Together, the results highlight consistent evolution among circulating NDVs supporting extensive surveillance of the virus in waterfowl to better elucidate epidemiology, evolutionary relationships and their impacts on commercial and backyard poultry.

Sero-epidemiology of bluetongue virus (BTV) infection in sheep and goats of Khyber Pakhtunkhwa province of Pakistan.

Bluetongue virus (BTV) infection is an emerging hazard in small ruminants having socio-economic impacts on animals and associated people. The current study was aimed to estimate the sero-prevalence and associated risk factors in sheep and goat from Khyber Pakhtunkhwa (KP) province of Pakistan. Three distinct zones (northern, central and southern) with four districts (Mansehra, Abbottabad, Swabi, and Kohat) with a higher population of small ruminants were selected. A total of n = 408 sera originating from sheep (n = 212) and goats (n = 196) were randomly collected for detection of BTV group specific antibodies through competitive ELISA (c-ELISA). Univariable and multiple logistic regressions were applied to assess the potential risk factors associated with the occurrence of this disease. Results showed an overall prevalence of 50.00% (CI = 44.17-54.83) of BTV in both sheep and goats with a significant difference (p < 0.05) among different districts. The prevalence of BTV in sheep was found higher (56.60%, CI = 49.6-63.4) than goats (42.86%, CI = 35.8-50.1). The risk factors identified based on chi-square test were; 1-2 year of animals, herd size and location in sheep while, milking status, ticks infestation, location and herd size for goats (p < 0.05). On the basis of univariable analysis, 1-2 year of animals, and location for sheep while, ticks infestation and location for goats (OR > 1). Multiple logistic regressions conferred only herd size and location as potential risk factors (OR > 1) for BTV in sheep and goats. The study concluded higher prevalence of BTV in sheep than the goats, the risk factors were significantly associated with the occurrence of disease, and together ascertaining the needs to design appropriate disease management and control strategies in sheep and goats.

Phylogenomics and Infectious Potential of Avian Avulaviruses Species-Type 1 Isolated from Healthy Green-Winged Teal (Anas carolinensis) from a Wetland Sanctuary of Indus River.

Given the importance of Avian avulaviruses (AAvVs) in commercial poultry, continuous monitoring and surveillance in natural reservoirs (waterfowls) is imperative. Here, we report full genomic and biologic characterization of two virulent AAvVs isolated from apparently asymptomatic green-winged teal (Anas carolinensis). Genetic characterization (genome length, coding potential, and presence of typical cleave motif [112RRQKR| F117]) and biologic assessment (HA, log 29; mean death time, 49.2-50 hr; 10-6.51 50% egg infective dose [EID50]/0.1 mL; and 1.5 intracerebral pathogenicity index [ICPI] value) revealed virulence of both isolates. Phylogenetic analysis of the complete genome and hypervariable region of the fusion (F) gene revealed clustering of both isolates within class II strains in close association with domestic poultry-origin AAvVs representing genotype VII and subgenotype VIIi. The inferred residue analysis of F and hemagglutinin-neuraminidase genes showed a number of substitutions in critical domains compared with reference strains of each genotype (I-XVIII). The isolates showed a high nucleotide resemblance (99%) with strain isolated previously from backyard poultry; however, they also showed a variable similarity (16.1% to 19.3%) with the most commonly used vaccine strains, Mukteswar (EF201805) and LaSota (AF077761). In accordance with pathogenicity assessment and horizontal transmission, the clinical and histopathologic observations in experimental chickens indicated the velogenic viscerotropic nature of AAvV 1 isolates. Taken together, this study confirms the evolutionary nature of AAvVs and their potential role in disease occurrence, necessitating continuous surveillance of migratory/aquatic fowls to better elucidate infection epidemiology and potential impacts on commercial poultry.

Análisis filogenético y potencial infeccioso de avulavirus aviares de tipo 1 aislados de cercetas americanas (Anas carolinensis) de un santuario en los humedales del río Indo Dada la importancia de los avulavirus aviares en la avicultura, es imperativo tanto el monitoreo como la vigilancia continuos en los reservorios naturales (aves acuáticas). En este artículo se describe la caracterización genética completa y las características biológicas de dos avulavirus aviares virulentos aislados de cercetas americanas (Anas carolinensis) aparentemente asintomáticas. La caracterización genética (longitud del genoma, potencial de codificación y presencia del motivo típico de disociación [112RRQKR| F117]) y la evaluación biológica (ensayo de hemaglutinación [HA], log 29; tiempo promedio de mortalidad, 49.2­50 horas; 10­6.51 dosis infectantes50% [EID50] /0.1mL y el índice de patogenicidad intracerebral [ICPI] de 1.5, revelaron la virulencia de ambos aislamientos. El análisis filogenético del genoma completo y la región hipervariable del gene de fusión (F) revelaron la agrupación de ambos aislamientos con cepas de la clase II en estrecha asociación con los avulavirus de origen avícola que representan el genotipo VII y el subgenotipo VIIi. El análisis de residuos deducidos de los genes F y de la hemaglutininaneuraminidasa mostró varias sustituciones en los dominios críticos en comparación con las cepas de referencia de cada genotipo (IXVIII). Los aislamientos mostraron una gran semejanza en la secuencia de nucléotidos (99%) con una cepa aislada previamente de aves de traspatio; sin embargo, también mostraron similitudes variables (de 16.1% a 19.3%) con las cepas de vacunas más utilizadas, Mukteswar (EF201805) y LaSota (AF077761). De acuerdo con la evaluación de patogenicidad y la transmisión horizontal, las observaciones clínicas e histopatológicas en los pollos experimentales indicaron la naturaleza velogénica viscerotrópica de estos aislamientos de avulavirus del tipo 1. En conjunto, este estudio confirma la naturaleza evolutiva de los avulavirus aviares y su posible papel en la aparición de enfermedades, lo que requiere una vigilancia continua de las aves migratorias acuáticas para dilucidar mejor la epidemiología de la infección y el posible impacto en las aves comerciales.