Molecular identification and genotyping of hepatitis E virus from Southern Punjab, Pakistan.

Hepatitis E is a global health concern. Hepatitis E virus (HEV) infection is endemic in Pakistan. HEV has four genotypes: HEV-1 through HEV-4. The genotypes HEV-1 and HEV-2 are associated with infection in humans, especially in countries with poor sanitation. The genotypes HEV-3 and HEV-4 are zoonotic and human infection takes place by consuming undercooked meat or being in contact with animals. The present study was designed to ascertain the presence of HEV in the Southern Punjab region of Pakistan. First, blood samples (n = 50) were collected from patients suspected of infection with the hepatitis E virus from the Multan District. The serum was separated and the samples were initially screened using an HEV IgM-ELISA. Second, the ELISA-positive samples were subjected to PCR and were genetically characterized. For PCR, the RNA extraction and complementary DNA synthesis were done using commercial kits. The HEV ORF2 (Open Reading Frame-2, capsid protein) was amplified using nested PCR targeting a 348 bp segment. The PCR amplicons were sequenced and an evolutionary tree was constructed using MEGA X software. A protein model was built employing the SWISS Model after protein translation using ExPASy online tool. The positivity rate of anti-HEV antibodies in serum samples was found as 56% (28/50). All Pakistani HEV showed homology with genotype 1 and shared common evolutionary origin and ancestry with HEV isolates of genotype 1 of London (MH504163), France (MN401238), and Japan (LC314158). Sequence analysis of motif regions assessment and protein structure revealed that the sequences had a similarity with the reference sequence. These data suggest that genotype 1 of HEV is circulating in Pakistan. This finding could be used for the diagnosis and control of HEV in the specific geographic region focusing on its prevalent genotype.

Utility of Feathers for Avian Influenza Virus Detection in Commercial Poultry.

The present study evaluated the potential utility of feather samples for the convenient and accurate detection of avian influenza virus (AIV) in commercial poultry. Feather samples were obtained from AIV-negative commercial layer facilities in Iowa, USA. The feathers were spiked with various concentrations (106 to 100) of a low pathogenic strain of H5N2 AIV using a nebulizing device and were evaluated for the detection of viral RNA using a real-time RT-PCR assay immediately or after incubation at -20, 4, 22, or 37 °C for 24, 48, or 72 h. Likewise, cell culture medium samples with and without the virus were prepared and used for comparison. In the spiked feathers, the PCR reliably (i.e., 100% probability of detection) detected AIV RNA in eluates from samples sprayed with 103 EID50/mL or more of the virus. Based on half-life estimates, the feathers performed better than the corresponding media samples (p < 0.05), particularly when the samples were stored at 22 or 37 °C. In conclusion, feather samples can be routinely collected from a poultry barn as a non-invasive alternative to blood or oropharyngeal-cloacal swab samples for monitoring AIV.

The immunogenicity and efficacy of acommercially available Infectious Bovine Rhinotracheitis (IBR) virus vaccine against a Pakistani field IBR strain.

Infectious bovine rhinotracheitis (IBR) is a highly communicable disease of cattle and wild ruminants that is caused by Bovine alphaherpesvirus 1 (BoHV­1). For IBR control, several developed countries have adopted the immunization and eradication programs focusing on IBR­positive animals. In Pakistan, livestock producers are importing commercially available vaccine of BoHV­1, but no studies on the efficacy of these commercial vaccines against local isolates are available. Therefore, the present study was aimed to evaluate the efficacy of a commercially available vaccine of BoHV­1 against local field isolates of virus. The rabbit model was used and the vaccine was evaluated for immunogenicity and protection after challenge with a highly virulent strain of a field virus. The immune response was measured by virus neutralization titers (VNT). This vaccine induced a humoral response in rabbits but that was not sufficient to completely protect the vaccinated animals against the wild­type BoHV­1 strain challenge. While a low virus titer compared to control rabbits was observed in the vaccinated rabbits (p<0.05), there was no sterilizing immunity or freedom from infection. However, complete freedom from disease, for example, the absence of pyrexia was noticed in the vaccinated group. In conclusion, the present study demonstrated that imported vaccine stock provoked only a partial protection against indigenous isolated of BoHV­1. However, tests performed on rabbits are preliminary, as only those performed on the source species can determine more reliable results.

DOCUMENTATION OF TRYPANOSOMA EVANSI IN CAPTIVE TIGERS AND LIONS IN PUNJAB (2016-2018), PAKISTAN.

Trypanosoma evansi is an important hemoparasite of a variety of animal species worldwide. This parasite is a threat to the health of domestic animals as well as wild animals, particularly those managed in captivity. The current study investigated the presence of T. evansi in captive tigers (Panthera tigris tigris) and lions (Panthera leo) in Pakistan. In total, 24 blood samples from 11 tigers and 3 lions (n = 14) were collected during the course of roughly 3 yr (2016-2018). Eighteen samples were subjected to both microscopic and molecular evaluation for the presence of T. evansi; the remaining 6 samples were processed for PCR only. Of the 18 samples tested by both methods, 3 (16%) and 8 (44%) were positive by microscopy and PCR, respectively. This highlights the higher sensitivity of PCR over microscopy for detection of trypanosomes. Of the 24 total samples evaluated by PCR, 12 (50%) were positive. The three sequences obtained showed 99% identity with variant surface glycoprotein genes of the different isolates of T. evansi. The sensitivity, specificity, positive predictive value, and negative predictive value of microscopy in identifying T. evansi was 37.5, 100, 100, and 66.7%, respectively, considering PCR as the gold standard. We recommend rigorous monitoring of captive tigers and lions for hemoparasites, particularly in winter and early spring in areas with high infection rate of this parasite, preferably via PCR.

Evaluation of PCR-based hemagglutinin subtyping as a tool to aid in surveillance of avian influenza viruses in migratory wild birds.

The surveillance of migratory wild birds (MWBs) for avian influenza virus (AIV) allows detecting the emergence of highly pathogenic AIV that can infect domestic poultry and mammals, new subtypes, and antigenic/genetic variants. The current AIV surveillance system for MWBs in the United States is based on virus isolation (VI) followed by sequencing isolates. This system primarily focuses on the early detection of H5 and H7 AIVs. However, it is suboptimal in assessing diverse AIV subtypes at any given time because of the low VI success rate. To improve such a shortfall, a SYBR® Green-based real-time reverse transcription-polymerase chain reaction (rtRT-PCR) panel was developed for direct HA subtyping of AIVs in oropharyngeal-cloacal (OPC) swabs from MWBs. Under optimal conditions, the PCR panel detected AIVs of all 16 different HA subtypes with an average limit of detection of 102.6 copies/reaction (2 µl of extract). In testing 90 OPC swabs from 13 MWB species, the PCR provided a significantly faster turnaround of results and demonstrated the presence of more subtypes and concurrent infection among MWBs compared to what the current surveillance testing algorithm showed. In conclusion, newly developed SYBR® Green rtRT-PCR panel can be a useful tool for monitoring MWBs for AIVs.

Evaluation of Feedstuffs as a Potential Carrier of Avian Influenza Virus between Feed Mills and Poultry Farms.

The present study was conducted to assess the potential vector role of feedstuffs for the area spreading of avian influenza virus (AIV). Firstly, feed samples were collected from commercial poultry facilities that experienced highly pathogenic avian influenza (H5N2) in 2014−2015 for AIV testing by a real-time RT−PCR specific for the viral matrix gene. Secondly, feed materials obtained from an AIV-negative farm were spiked with various concentrations of a low pathogenic AIV H5N2. Virus-spiked cell culture media were prepared in the same manner and used for comparison. The spiked feed and media samples were tested by a multiplex real-time RT−PCR ran in a quantitative manner, either immediately or after incubation at −20, 4, 22, and 37 °C for 24, 48, and 72 h. Some of the feedstuffs collected from the poultry facilities or feed mills were positive for AIV RNA but negative by the virus isolation (VI) test, while all the formaldehyde-treated feedstuffs were PCR-negative. In the spiked feeds, the AIV titer was 1−3 logs lower than that in the corresponding media, even when tested immediately after spiking, suggesting that feed might have a negative impact on the virus or PCR detection. The half-life of AIV RNA was shorter at a higher temperature. A significant decay in the viral RNA over time was noted at 37 °C (p < 0.05), suggesting that feedstuffs should be maintained in the cold chain when testing is desired. Furthermore, the thermal degradation of AIV suggests that the heat treatment of feeds could be an alternative to chemical treatment when contamination is suspected. Collectively, the study observations indicate that AIV survivability in feed is relatively low, thus rendering it a low risk.

Rate of Multiple Viral and Bacterial CoInfection(s) in Influenza A/H9N2-Infected Broiler Flocks.

Repeated cases of low pathogenic influenza A/H9N2 virus (IAV/H9N2) have been reported in commercial chickens since its emergence in 1998 in Pakistan. However, recently increased mortality and severe respiratory complications under field conditions have been noticed, suggesting concomitant influenza infections with respiratory viral and/or bacterial pathogens. Therefore, the present study aimed to investigate the presence of IAV/H9N2 coinfecting with multiple viral and bacterial pathogens in broiler chicken flocks. We surveyed 60 broiler flocks with respiratory signs from March through July 2019 in Punjab, Pakistan. Suspected flocks were screened for the presence of IAV using a lateral-flow device. Tracheal, cloacal, and bone marrow samples were collected and further tested for seven viral agents (chicken anemia; Newcastle disease; infectious bronchitis; infectious laryngeotracheitis [ILT]; and IAV subtypes H9, H7, and H5) and three bacterial agents (Mycoplasma gallisepticum; Mycoplasma synovae; Ornithobacterium rhinotracheale [ORT]) using PCR assays. Upon initial screening for IAV, 35/60 (58.3%) flocks tested positive. The coinfection of IAV/H9N2 with other pathogens was detected in 25 (71.4%) flocks and only IAV/H9N2 was detected in 10 (28.6%) flocks out of total positive IAV flocks (n = 35). IAV subtypes H5 and H7, ILT, and ORT were not detected throughout the study period. The detection rate of double, triple, and quadruple combinations of coinfections with IAV/H9N2 were 37% (13 flocks), 26% (9 flocks), 9% (3 flocks), respectively. Higher average mortality (28.5%) was found in broiler chicken flocks coinfected with viral and/or bacterial pathogens than in flocks where only H9 low pathogenic IAV/H9N2 was detected (20.8%). In conclusion, higher circulation of IAV/H9N2 with other viral and bacterial pathogens may contribute to higher production and economic losses at the farm level.

Nota de investigación- Tasa de coinfecciones virales y bacterianas múltiples en parvadas de pollos de engorde infectadas con virus influenza A/H9N2. Se han reportado varios casos del virus de influenza A de baja patogenicidad H9N2 (IAV/H9N2) en pollos comerciales desde su aparición en 1998 en Pakistán. Sin embargo, recientemente se ha observado un aumento de la mortalidad y complicaciones respiratorias graves en condiciones de campo, lo que sugiere infecciones concomitantes de influenza con patógenos respiratorios virales y/o bacterianos. Por lo tanto, el presente estudio tuvo como objetivo investigar la presencia del virus de influenza aviar H9N2 coinfectando con múltiples patógenos virales y bacterianos en parvadas de pollos de engorde. Se evaluaron 60 parvadas de pollos de engorde con signos respiratorios desde marzo hasta julio del año 2019 en Punjab, Pakistán. Las parvadas sospechosas fueron analizadas para detectar la presencia del virus de influenza aviar utilizando un dispositivo de flujo lateral. Se recolectaron muestras traqueales, cloacales y de médula ósea y se analizaron para detectar siete agentes virales (anemia infecciosa aviar, enfermedad de Newcastle, bronquitis infecciosa, laringeotraqueítis infecciosa [ILT] y subtipos H9, H7 y H5 de influenza aviar) y tres agentes bacterianos (Mycoplasma gallisepticum ; Mycoplasma sinovae; Ornithobacterium rhinotracheale [ORT]) utilizando ensayos de PCR. Tras la detección inicial del virus de la influenza aviar, 35/60 (58.3 %) parvadas resultaron positivas. La coinfección del virus de la influenza H9N2 con otros patógenos se detectó en 25 (71.4 %) parvadas y el virus de influenza aviar H9N2 fue detectado solo en 10 (28.6 %) parvadas del total de parvadas positivas (n = 35). Los subtipos H5 y H7 del virus de influenza, ILT y ORT no se detectaron durante el período de estudio. La tasa de detección de combinaciones dobles, triples y cuádruples de coinfecciones con el virus de influenza H9N2 fue del 37 % (13 parvadas), del 26% (9 parvadas), del 9 % (3 parvadas), respectivamente. Se encontró una mortalidad promedio más alta (28.5 %) en lotes de pollos de engorde coinfectados con patógenos virales y/o bacterianos que en lotes donde solo se detectó al virus de influenza H9 de baja patogenicidad (20.8%). En conclusión, una mayor circulación del virus de influenza aviar H9N2 con otros patógenos virales y bacterianos puede contribuir a mayores pérdidas en la producción y económicas a nivel de granja.

Lumpy skin disease is expanding its geographic range: A challenge for Asian livestock management and food security.

In recent years, lumpy skin disease virus has extended its geographical range outside of endemic sub-Saharan countries to the Middle East and Asia indicating transboundary spread. Recently, lumpy skin disease (LSD) outbreaks have been reported in Asian countries such as Bangladesh, India, China, Nepal, Bhutan, Vietnam, Myanmar, Sri Lanka, Thailand, Malaysia, Laos and for the first time and represent a cause of serious concern for their livestock and dairy industries. This report summarizes information on the recent outbreaks of LSD in southern Asia and emphasizes the threat it poses to neighbouring countries. Various strategies and actions needed to control outbreaks of this emerging disease in Asia are also suggested.

Environmental Sampling for Avian Influenza Virus Detection in Commercial Layer Facilities.

The present study was designed to evaluate the utility of environmental samples for convenient but accurate detection of avian influenza virus (AIV) in commercial poultry houses. First, environmental samples from AIV-negative commercial layer facilities were spiked with an H5N2 low pathogenic AIV and were evaluated for their effect on the detection of viral RNA immediately or after incubation at -20 C, 4 C, 22 C, or 37 C for 24, 48, or 72 hr. Second, Swiffer pads, drag swabs, and boot cover swabs were evaluated for their efficiency in collecting feces and water spiked with the H5N2 LPAIV under a condition simulated for a poultry facility floor. Third, environmental samples collected from commercial layer facilities that experienced an H5N2 highly pathogenic AIV outbreak in 2014-15 were evaluated for the effect of sampling locations on AIV detection. The half-life of AIV was comparable across all environmental samples but decreased with increasing temperatures. Additionally, sampling devices did not differ significantly in their ability to collect AIV-spiked environmental samples from a concrete floor for viral RNA detection. Some locations within a poultry house, such as cages, egg belts, house floor, manure belts, and manure pits, were better choices for sampling than other locations (feed trough, ventilation fan, and water trays) to detect AIV RNA after cleaning and disinfection. Samples representing cages, floor, and manure belts yielded significantly more PCR positives than the other environmental samples. In conclusion, environmental samples can be routinely collected from a poultry barn as noninvasive samples for monitoring AIV.

Muestreo ambiental para la detección del virus de la influenza aviar en instalaciones de aves de postura comerciales. El presente estudio fue diseñado para evaluar la utilidad de las muestras ambientales para la detección rápida pero precisa del virus de la influenza aviar (AIV) en casetas avícolas comerciales. Primero, muestras ambientales de las instalaciones comerciales de aves de postura negativas para influenza aviar se inocularon con un virus de la influenza de baja patogenicidad (LPAIV) H5N2 y se evaluaron para determinar su efecto en la detección de ARN viral inmediatamente o después de la incubación a -20 C, 4 C, 22 C o 37 C durante 24 hr, 48 hr o 72 horas. En segundo lugar, se evaluaron las esponjas marca Swiffer, los hisopos de arrastre y los cubre botas para muestreo ambiental para determinar su eficiencia en la recolección de heces y agua inoculada con el virus de influenza aviar de baja patogenicidad H5N2 en una condición simulada de piso de una instalación avícola. En tercer lugar, muestras ambientales recolectadas de instalaciones comerciales de ponedoras que experimentaron un brote de influenza aviar altamente patógena H5N2 en 2014-15, se evaluaron para determinar el efecto de la ubicación de muestreo en la detección de influenza aviar. La vida media del virus de la influenza aviar fue comparable en todas las muestras ambientales, pero disminuyó con el aumento de la temperatura. Además, los dispositivos de muestreo no difirieron significativamente en su capacidad para recolectar muestras ambientales inoculadas con influenza aviar de un piso de concreto para la detección de ARN viral. Algunas ubicaciones dentro de la caseta aviar, como jaulas, bandas transportadoras de huevo, piso de la caseta, bandas transportadoras de gallinasa y fosas de gallinasa, fueron las mejores opciones para el muestreo en comparación con otras ubicaciones (comederos, ventiladores y bandejas de agua) para detectar el ARN del virus de influenza después de la limpieza y desinfección. Las muestras que representan jaulas, piso y bandas transportadoras de gallinasa arrojaron significativamente más resultados positivos de PCR que las otras muestras ambientales. En conclusión, las muestras ambientales se pueden recolectar rutinariamente de uns granja avícola como muestras no invasivas para monitorear al virus de influenza aviar.

Evaluating the role of wild songbirds or rodents in spreading avian influenza virus across an agricultural landscape.

BACKGROUND: Avian influenza virus (AIV) infections occur naturally in wild bird populations and can cross the wildlife-domestic animal interface, often with devastating impacts on commercial poultry. Migratory waterfowl and shorebirds are natural AIV reservoirs and can carry the virus along migratory pathways, often without exhibiting clinical signs. However, these species rarely inhabit poultry farms, so transmission into domestic birds likely occurs through other means. In many cases, human activities are thought to spread the virus into domestic populations. Consequently, biosecurity measures have been implemented to limit human-facilitated outbreaks. The 2015 avian influenza outbreak in the United States, which occurred among poultry operations with strict biosecurity controls, suggests that alternative routes of virus infiltration may exist, including bridge hosts: wild animals that transfer virus from areas of high waterfowl and shorebird densities. METHODS: Here, we examined small, wild birds (songbirds, woodpeckers, etc.) and mammals in Iowa, one of the regions hit hardest by the 2015 avian influenza epizootic, to determine whether these animals carry AIV. To assess whether influenza A virus was present in other species in Iowa during our sampling period, we also present results from surveillance of waterfowl by the Iowa Department of Natural Resources and Unites Stated Department of Agriculture. RESULTS: Capturing animals at wetlands and near poultry facilities, we swabbed 449 individuals, internally and externally, for the presence of influenza A virus and no samples tested positive by qPCR. Similarly, serology from 402 animals showed no antibodies against influenza A. Although several species were captured at both wetland and poultry sites, the overall community structure of wild species differed significantly between these types of sites. In contrast, 83 out of 527 sampled waterfowl tested positive for influenza A via qPCR. DISCUSSION: These results suggest that even though influenza A viruses were present on the Iowa landscape at the time of our sampling, small, wild birds and rodents were unlikely to be frequent bridge hosts.