Comparative pathogenicity of a genotype XXI.1.2 pigeon Newcastle disease virus isolate in pigeons and chickens.

Here, we performed molecular and pathogenic characterization of a Newcastle disease virus (NDV) isolate from pigeons in Bangladesh. Molecular phylogenetic analysis based on the complete fusion gene sequences classified the three study isolates into genotype XXI (sub-genotype XXI.1.2) together with recent NDV isolates obtained from pigeons in Pakistan (2014-2018). The Bayesian Markov Chain Monte Carlo analysis revealed that the ancestor of Bangladeshi pigeon NDVs and the viruses from sub-genotype XXI.1.2 existed in the late 1990s. Pathogenicity testing using mean embryo death time pathotyped the viruses as mesogenic, while all isolates carried multiple basic amino acid residues at the fusion protein cleavage site. Experimental infection of chickens and pigeons revealed no or minimum clinical signs in chickens, while a relatively high morbidity (70%) and mortality (60%) were observed in pigeons. The infected pigeons showed extensive and systemic lesions including hemorrhagic and/or vascular changes in the conjunctiva, respiratory and digestive system and brain, and atrophy in the spleen, while only mild congestion in the lungs was noticed in the inoculated chickens. Histologically, consolidation in the lungs with collapsed alveoli and edema around the blood vessels, hemorrhages in the trachea, severe hemorrhages and congestion, focal aggregation of mononuclear cells, and single hepatocellular necrosis in the liver, severe congestion, multifocal tubular degeneration, and necrosis, as well as mononuclear cell infiltration in the renal parenchyma, encephalomalacia with severe neuronal necrosis with neuronophagia were noticed in the brain in infected pigeons. In contrast, only slight congestion was found in lungs of the infected chickens. qRT-PCR revealed the replication of the virus in both pigeons and chickens; however, higher viral RNA loads were observed in oropharyngeal and cloacal swabs, respiratory tissues, and spleen of infected pigeons than the chickens. In conclusion, genotype XXI.1.2 NDVs are circulating in the pigeon population of Bangladesh since 1990s, produce high mortality in pigeons with pneumonia, hepatocellular necrosis, renal tubular degeneration, and neuronal necrosis in pigeons, and may infect chickens without overt signs of clinical disease and are likely to shed viruses via the oral or cloacal routes.

Comparative pathogenicity of infectious bursal disease viruses of three different genotypes.

Infectious bursal disease (IBD) is a highly immunosuppressive and often fatal viral disease of young chickens. The causal agent IBD virus (IBDV) is an avian Birnavirus having two genome segments that have evolved independently and contributed to the emergence of many genotypes with different pathogenic profile. The present study aimed at genetic and pathogenic characterization of IBDVs from Bangladesh. We performed phylogenetic analysis of 15 IBDV isolates recovered from field outbreaks in chickens during 2020-2021 and compared the pathogenicity of three selected isolates belonging to different genotypes on experimental infection in chickens. Out of 15 isolates, one was the typical vvIBDV of genotype A3B2, 13 were reassortant vvIBDV of genotype A3B3 having very virulent-like segment A and early Australian-like segment B, and the remaining one isolate was a classical virulent IBDV of A1aB1 genotype. A few amino acid substitutions were observed between the genotypes in four putative antigenic sites on VP2. In a comparative pathogenicity study, the typical vvIBDV isolate BD-25(A3B2) appeared to be the most virulent with 100% morbidity and 90% mortality, followed by the segment-reassortant vvIBDV isolate BD-28(A3B3) with 50% morbidity and 30% mortality. However, the gross and histopathological lesions in the bursa of Fabricius were similar. The classical virulent isolate BD-26(A1aB1) did not cause any clinical disease. In conclusion, three genotypes of IBDV are co-circulating in poultry of Bangladesh and the typical vvIBDV of A3B2 genotype was more virulent than the reassortant vvIBDV of A3B3 genotype. Further studies are required to assess the country-wide distribution of IBDV of different genotypes and the efficacy of the currently available vaccines in protecting chickens against different genotypes of IBDV in Bangladesh.

Genetic and biological characterization of Newcastle disease viruses circulating in Bangladesh during 2010-2017: further genetic diversification of class II genotype XIII in Southcentral Asia.

Newcastle disease virus (NDV) is endemic in Bangladesh and is a major threat to commercial poultry operations. While complete fusion (F) genes are recommended for molecular characterization and classification of NDV isolates, heretofore, only partial F gene data have been available for Bangladeshi NDVs. To this end, we obtained the full-length F gene coding sequences of 11 representative NDVs isolated in Bangladesh between 2010 and 2017. In addition, one of the viruses (MK934289/chicken/Bangladesh/C161/2010) was used in an experimental infection of chickens to establish the viral pathotype and study gross and microscopic lesions. Phylogenetic analysis provided evidence that all studied Bangladeshi isolates belong to genotype XIII.2 of class II NDVs. Six of the viruses were isolated between 2010 and 2017 and grouped together with isolates from neighbouring India during 2013-2016. Another four Bangladeshi isolates (2010-2016) formed a separate monophyletic branch within XIII.2 and showed high nucleotide distance from the isolates from India and the other six Bangladeshi viruses within the sub-genotype; however, none of these groups fulfils all classification criteria to be named as a separate sub-genotype. The eleventh Bangladeshi virus studied here (C162) was genetically more distant from the remaining isolates. It out-grouped the viruses from sub-genotypes XIII.2.1 and XIII.2.2 and showed more than 9.5 % nucleotide distance from all genotype XIII sub-genotypes. This isolate may represent an NDV variant that is evolving independently from the other viruses in the region. The experimental infection in chickens revealed that the tested isolate (C161) is a velogenic viscerotropic virus. Massive haemorrhages, congestion and necrosis in different visceral organs, and lymphoid depletion in lymphoid tissues, typical for infection with velogenic NDV, were observed. Our findings demonstrate the endemic circulation of sub-genotype XIII.2 in Southcentral Asia and further genetic diversification of these viruses in Bangladesh and neighbouring India. This constant evolution of the viruses may lead to the establishment of new genetic groups in the region. Additional historical and prospective virus and surveillance data from the region and neighbouring countries will allow a more detailed epidemiological inference.

Growth kinetics of a Vero cells adapted Bangladeshi strain of peste des petits ruminants (PPR) virus in cell culture.

Growth kinetics of a Vero cells adapted Bangladeshi strain of peste des petits ruminants virus was studied in Vero cells to determine maximum virus yield. One-step growth curve was formulated after determining virus in both supernatant (CFV) and cell lysate (CAV) at different time categories by microtitre plate titration in Vero cells and the viral presence was confirmed by real-time RT-PCR. The virus was first detected in both the supernatants and cell pellets at 12 hpi. The virus titre reached its plateau at 72 hpi. Maximum virus titre of CAV was 6.2 log10 TCID50/ml and that of CFV was 5.2 log10 TCID50/ml at 72 hpi. After that, the titer gradually declined, but maintained at 4.5 log10 TCID50/ml in case of CAV and 4.2 log10 TCID50/ml in case of CFV at 96 hpi. It was concluded that the optimum time point for harvesting Vero cell culture is 72 hpi.

Circulation of three genotypes and identification of unique mutations in neutralizing epitopes of infectious bronchitis virus in chickens in Bangladesh.

Infectious bronchitis (IB) is a highly contagious respiratory disease caused by a gammacoronavirus that has been circulating for many years in chickens in Bangladesh, resulting in significant economic losses. The aim of this study was to detect and characterize infectious bronchitis virus (IBV) from clinical outbreaks and surveillance samples. Real-time RT-PCR was used to detect IBV in pooled lung and tracheal tissue samples (n = 78), oropharyngeal swabs (n = 19), and pooled fecal samples (n = 13) from live-bird markets. Both respiratory and nephropathogenic forms of IB were suspected at necropsy (n = 7) from clinical outbreaks. Sequencing of hypervariable regions (HVR1-2 and HVR3) of the region of the spike gene (S) encoding the S1 subunit of five isolates revealed circulation of the Mass-like, QX-like, and 4/91-like genotypes of IBV in Bangladesh. Each genotype was extremely variable, as shown by separate clustering of the viruses in a phylogenetic tree and high nucleotide (nt) sequence divergence (38.8-41.2% and 25.7-37.4% in the HVR1-2 and HVR3 sequence, respectively). The unique mutation G65E was observed in each Mass-like isolate, and Y328S was observed in each 4/91-like Bangladeshi isolate. Three neutralizing epitope sites were predicted within the HVRs that differed significantly among the three genotypes. In addition, one Bangladeshi isolate carried fixed mutations at 294F and 306Y, like other pathogenic QX-like IBVs, which could affect epitopes involved in neutralization, facilitating virus circulation among vaccinated flocks. Therefore, continuous screening and genotype characterization will be necessary to track the epidemiology of IBV and control IB infection in Bangladesh.

A unified genotypic classification of infectious bursal disease virus based on both genome segments.

Infectious bursal disease virus (IBDV) of chickens is a birnavirus with a bi-segmented double-stranded RNA genome, the segments designated as A and B. We performed phylogenetic analysis using a 366-bp fragment of segment A (nt 785-1150) and a 508-bp fragment of segment B (nt 328-835) of IBDV. A total of 463 segment A and 434 segment B sequences from GenBank, including the sequences of eight recent Bangladeshi isolates, were used in the analysis. The analysis revealed eight genogroups of segment A under serotype 1, designated as A1 (classical), A2 (US antigenic variant), A3 (very virulent), A4 (dIBDV), A5 (atypical Mexican), A6 (atypical Italian), A7 (early Australian) and A8 (Australian variant), and a single genogroup under serotype 2, designated as A0. On the other hand, segment B could be categorized into five genogroups irrespective of serotype, these being B1 (classical-like), B2 (very virulent-like), B3 (early Australian-like), B4 (Polish & Tanzanian) and B5 (Nigerian). Segment B of serotype 2 strains clustered within genogroup B1. With the bi-segmented genome of IBDV, these differences would allow for a total of 45 possible assortments. Based on the combinations of segment A and segment B genogroups observed in 463 IBDV strains, a total of 15 genotypes could be recognized. Recent Bangladeshi IBDV strains, isolated in 2016, appeared to be segment reassortants having segment A of genogroup A3 (very virulent) and segment B of genogroup B3 (early Australian-like). An extended system of nomenclature of IBDV strains is proposed.

Characterization of the mechanical properties of van der Waals heterostructures of stanene adsorbed on graphene, hexagonal boron-nitride and silicon carbide.

Stanene has revealed a new horizon in the field of quantum condensed matter and energy conversion devices but its significantly lower tensile strength limits its further applications and effective operation in these nanodevices. Van der Waals heterostructures have given substantial flexibility to integrate different two-dimensional (2D) layered materials over the past few years and have proven highly functional with exceptional features, appealing applications, and innovative physics. Considerable efforts have been made for the preparation, thorough understanding, and applications of van der Waals heterostructures in the fields of electronics and optoelectronics. In this paper, we have executed Molecular Dynamics (MD) simulations to predict the tensile strength of van der Waals heterostructures of stanene (Sn) adsorbed on graphene (Gr), hexagonal boron nitride (hBN), and silicon carbide (SiC) (Sn/Gr, Sn/hBN, and Sn/SiC, respectively) subjected to both armchair and zigzag directional loading at different strain rates for the first time, which has enticing applications in electronic, optoelectronic, energy storage and bio-engineered devices. Among all the van der Waals heterostructures, the Sn/SiC heterostructure exhibits the lowest tensile strength and tensile strain. Furthermore, it has been found that zigzag directional loading could endure more tensile strain before fracture. Besides, it has been disclosed that though the rule of mixtures may accurately reproduce the Young's modulus of these heterostructures, it has limitations to predict the tensile strength. Fracture analysis suggests that for the Sn/hBN heterostructure the fracture initiates from the stanene layer while for the Sn/Gr and Sn/SiC heterostructures the fracture initiates from the Gr and SiC layer, respectively, for both armchair and zigzag directional loading. Overall, this study would aid in the design and efficient operation of Sn/Gr, Sn/hBN, and Sn/SiC heterostructures when subjected to mechanical force.

Phonon thermal conductivity of the stanene/hBN van der Waals heterostructure.

We use classical non-equilibrium molecular dynamics (NEMD) simulations to investigate the phonon thermal conductivity (PTC) of hexagonal boron nitride (hBN) supported stanene. At first, we examine the length dependent PTCs of bare stanene and hBN, and the stanene/hBN heterostructure and realize the dominance of the hBN layer to dictate the PTC in the heterostructure system. Afterward, we assess the length-independent bulk PTCs of these materials. The bulk PTCs at room temperature are found as ∼15.20 W m-1 K-1, ∼550 W m-1 K-1, and ∼232 W m-1 K-1 for bare stanene and hBN, and stanene/hBN, respectively. Moreover, our simulations reveal that bare stanene exhibits a substantially lower PTC compared to bare hBN, and the predicted PTC of stanene/hBN lies between those of stand-alone stanene and hBN. We also found that the PTC obtained for the stanene/hBN system from NEMD simulations nicely agrees with the theoretical formula developed to predict the PTC of heterostructures of two distinct materials. Temperature studies suggest that the PTC of the stanene/hBN heterostructure system follows a decreasing trend with increasing temperature. Additionally, corresponding phonon density of states (PDOS) and phonon dispersion data are provided to comprehensively understand the phonon properties of bare stanene and hBN, and stanene/hBN. Overall, this NEMD study would offer a deep understating towards the PTC of the stanene/hBN heterostructure and would widen the scope of its successful operations in future nanoelectronic, spintronic, and thermoelectric devices.

Active virological surveillance in backyard ducks in Bangladesh: detection of avian influenza and gammacoronaviruses.

Domestic waterfowl play an important role in the perpetuation and transmission of avian pathogens including avian influenza viruses (AIV) of low and high pathogenicity, which pose severe economic and public health concerns in Bangladesh. This study focused on active surveillance of several avian viral pathogens with a special reference to AIV in selected backyard duck populations in Bangladesh. A total of 500 pooled oropharyngeal and cloacal samples from individual ducks of four districts were tested by real time PCRs for the presence of AIV, avian avulavirus-1, anatid herpesvirus-1, avian parvovirus, avian bornavirus and avian coronavirus. The investigation identified 27 (5.4%) ducks positive for AIV and 12 (2.4%) positive for avian coronavirus. In 13 samples, RNA specific for AIV H4N6 was detected. Phylogenetic analysis of the AIV haemagglutinin H4 and neuraminidase N6 genes suggested a clustering of Bangladeshi AIV H4N6 in Eurasian lineage group 2. Other AIV positive samples had very low virus loads (Cq > 36) and were not subtyped. Coronaviral sequences of a fragment of the polymerase gene were related to Eurasian-Australian duck gamma-coronaviruses. Our current active surveillance in free-range domestic backyard ducks in Bangladesh failed to detect highly pathogenic (HP) AIV in contrast to our previous passive monitoring study. Nevertheless, active monitoring of domestic duck populations may be important to highlight presence and transmission dynamics of economically less important AIV that still may serve as reassortment partners for the generation of new HP and zoonotic AIV. RESEARCH HIGHLIGHTS Active surveillance for viral pathogens in domestic free-range backyard ducks. Detection of avian influenza virus subtype H4N6. First identification of avian gammacoronavirus in ducks in Bangladesh.

Atomic-scale analysis of the physical strength and phonon transport mechanisms of monolayer ß-bismuthene.

Bismuthene has opened up a new avenue in the field of nanotechnology because of its spectacular electronic and thermoelectric features. The strong spin-orbit-coupling enables its operation as the largest nontrivial bandgap topological insulator and quantum spin hall material at room temperature, which is unlikely for any other 2D material. It is also known to be the most promising thermoelectric material due to its remarkable thermoelectric properties, including a substantially high power factor. However, an in-depth understanding of the mechanical and thermal transport properties of bismuthene is crucial for its practical implementation and efficient operation. Employing the Stillinger-Weber potential, we utilized molecular dynamics simulations to inspect the mechanical strength and thermal conductivity of the monolayer ß-bismuthene for the first time. We analyzed the effect of temperature on the tensile mechanical properties along the armchair and zigzag directions of bismuthene nanosheets and found that increasing temperature causes a significant deterioration in these properties. The material shows superior fracture resistance with zigzag loading, whereas the armchair direction exhibits an improved elasticity. Next, we showed that increasing vacancy concentration and crack length notably reduce the fracture stress and strain of ß-bismuthene. Under all these conditions, ß-bismuthene showed a strong chirality effect under tensile loading. We also explored the fracture phenomena of a pre-cracked ß-bismuthene, which reveal that the armchair-directed crack possesses a higher fracture resistance than the zigzag-directed crack. Interestingly, branching phenomena occurred during crack propagation for the armchair crack; meanwhile, the crack propagates perpendicular to loading for the zigzag crack. Afterward, we investigated the effect of loading rate on the fracture properties of bismuthene along the armchair and zigzag directions. Finally, we calculated the thermal conductivity of bismuthene under the influence of temperature and vacancy and recorded a substantial decrement in thermal conductivity with increasing temperature and vacancy. The obtained results are comprehensively discussed in the light of phonon density of states, phonon dispersion spectrum, and phonon group velocities. It is also disclosed that the thermal conductivity of ß-bismuthene is considerably lower than that of other analogous honeycomb structures. This study can add a new dimension to the successful realization of bismuthene in future (opto)electronic, spintronic, and thermoelectric devices.

Pathology of clade 2.3.2.1 avian influenza virus (H5N1) infection in quails and ducks in Bangladesh.

We performed pathological and molecular virological investigation of three outbreaks of highly pathogenic avian influenza (HPAI) in a quail farm and two duck farms of Mymensingh and Netrokona districts of Bangladesh in 2011. HPAI viruses of subtype H5N1 were detected from all three outbreaks and phylogenetic analysis of HA gene sequence placed the viruses into clade 2.3.2.1. The outbreak in the quail farm was characterized by acute death with 100% mortality within two days. Marked haemorrhages and congestion with necrotic and inflammatory lesions in the respiratory tract, liver, pancreas and kidneys were the major gross and histopathological lesions. In the case of ducks, nervous signs were the remarkable clinical manifestations and the mortality was around 10%. No significant gross lesions were observed at necropsy. Non-purulent encephalitis with gliosis and neuronal degeneration was observed on histopathological examination. By immunohistochemistry, viral antigen could be detected in different organs of both quails and ducks. This study records varying clinical and pathological manifestations of HPAI in ducks and quails following natural infection with the same strain of the virus. RESEARCH HIGHLIGHTS HPAIV of clade 2.3.2.1 was detected from clinical outbreaks in quails and ducks Sudden death with severe haemorrhages in various organs was found in quails Pronounced nervous signs with non-purulent encephalitis were observed in ducks Viral antigen could be localized in different organs by immunohistochemistry.

Review analysis and impact of co-circulating H5N1 and H9N2 avian influenza viruses in Bangladesh.

Almost the full range of 16 haemagglutinin (HA) and nine neuraminidase subtypes of avian influenza viruses (AIVs) has been detected either in waterfowl, land-based poultry or in the environment in Bangladesh. AIV infections in Bangladesh affected a wide range of host species of terrestrial poultry. The highly pathogenic avian influenza (AI) H5N1 and low pathogenic AI H9N2 were found to co-circulate and be well entrenched in the poultry population, which has caused serious damage to the poultry industry since 2007. By reviewing the available scientific literature, the overall situation of AIVs in Bangladesh is discussed. All Bangladeshi (BD) H5N1 and H9N2 AIV sequences available at GenBank were downloaded along with other representative sequences to analyse the genetic diversity among the circulating AIVs in Bangladesh and to compare with the global situation. Three different H5N1 clades, 2.2.2, 2.3.2.1 and 2.3.4.2, have been detected in Bangladesh. Only 2.3.2.1a is still present. The BD LP H9N2 viruses mostly belonged to the H9 G1 lineage but segregated into many branches, and some of these shared internal genes with HP viruses of subtypes H7N3 and H5N1. However, these reassortment events might have taken place before introduction to Bangladesh. Currently, H9N2 viruses continue to evolve their HA cleavage, receptor binding and glycosylation sites. Multiple mutations in the HA gene associated with adaptation to mammalian hosts were also observed. Strict biosecurity at farms and gradual phasing out of live-bird markets could be the key measures to better control AIVs, whereas stamping out is not a practicable option in Bangladesh. Vaccination also could be an additional tool, which however, requires careful planning. Continuous monitoring of AIVs through systematic surveillance and genetic characterisation of the viruses remains a hallmark of AI control.

Phylogenetic analysis of Newcastle disease viruses from Bangladesh suggests continuing evolution of genotype XIII.

A total of 23 Newcastle disease virus (NDV) isolates from Bangladesh taken between 2010 and 2012 were characterized on the basis of partial F gene sequences. All the isolates belonged to genotype XIII of class II NDV but segregated into three sub-clusters. One sub-cluster with 17 isolates aligned with sub-genotype XIIIc. The other two sub-clusters were phylogenetically distinct from the previously described sub-genotypes XIIIa, XIIIb and XIIIc and could be candidates of new sub-genotypes; however, that needs to be validated through full-length F gene sequence data. The results of the present study suggest that genotype XIII NDVs are under continuing evolution in Bangladesh.

Serological and virological surveillance of avian influenza virus in domestic ducks of the north-east region of Bangladesh.

BACKGROUND: Wild waterfowl are considered as the natural reservoir for avian influenza (AI) viruses. Bangladesh has been experiencing highly pathogenic avian influenza (HPAI) outbreaks since 2007, mostly in chickens and occasionally in ducks. Ducks play an important role in the persistence and genetic recombination of AI viruses. This paper presents the results of serological and virological monitoring of AI in domestic ducks in 2013 in the north-east region of Bangladesh. RESULTS: A total of 871 and 662 serum samples and 909 and 302 pairs of cloacal and oropharyngeal swabs from domestic ducks of Mymensingh and Sylhet division, respectively, were analysed. Antibodies to type A influenza virus were detected by blocking ELISA in 60.73 and 47.73% serum samples of Mymensingh and Sylhet division, respectively. On haemagglutination-inhibition (HI) test 17.5% of ELISA positive serum samples were found to be seropositive to H5 avian influenza virus. Five cloacal swabs and one oropharyngeal swab were positive for M gene of type A influenza virus by real time RT-PCR (rRT-PCR), but all of them were negative for H5 influenza virus. Three of the six viruses were successfully characterized as H1N5, H2N5 and H7N5 subtype of AI virus, the other three remained uncharacterized. On sequencing and phylogenetic analysis the HA and NA genes were found to be of Eurasian avian lineage. The H7 virus had cleavage site motif of low pathogenic virus. CONCLUSIONS: Low pathogenic avian influenza viruses were detected from apparently healthy domestic ducks. A small proportion of domestic ducks were found seropositive to H5 AI virus.

Natural peste des petits ruminants virus infection in Black Bengal goats: virological, pathological and immunohistochemical investigation.

BACKGROUND: Peste des Petits Ruminants (PPR), also known as Goat Plague, occurs in goats, sheep and related species. It is caused by a morbillivirus in the family Paramyxoviridae. In Bangladesh PPR is endemic and it causes serious economic losses. Pathology of PPR has been reported in different goat and sheep breeds from natural and experimental infections. Field results are better indicators of pathogenicity of the circulating virus. The severity of the disease varies with species, breed and immune status of the host. Pathological investigations of natural outbreaks of PPR in Balck Bengal goats are very limited. The current investigation was aimed at describing pathology and antigen localization in natural PPR infections in Black Bengal goats. RESULTS: A total of 28 outbreaks were investigated clinically and virologically. Average flock morbidity and mortality were 75% and 59%, respectively, with case fatality rate of 74%. Necropsy was conducted on 21 goats from 15 outbreaks. The major gross lesions were congestion of gastrointestinal tract, pneumonia, engorged spleen, and oedematous lymphnodes. Histopathological examination revealed severe enteritis with denudation of intestinal epithelium, severe broncho-interstitial pneumonia with macrophages within lung alveoli and extensive haemorrhages with depletion of lymphoid cells and infiltration of macrophages in the sinuses of spleen. In lymph nodes, the cortical nodules were replaced by wide sinusoids with severe depletion of lymphocytes, infiltration of mononuclear cells and some giant cells in sub-capsular areas and medullary sinuses. PPR virus antigen was found in pneumocytes and alveolar macrophages in lungs. Viral RNA could be detected by RT-PCR in 69 out of 84 nasal swab, 59 out of 84 blood and 21 out of 21 lymph node samples. Sequence analyses revealed closeness of Bangladeshi strains with other recent Asian isolates. CONCLUSION: Natural outbreaks of PPR in Black Bengal goats in Bangladesh resulted in 75% and 59% flock morbidity and mortality, respectively, with a case fatality rate of 74%. The striking histo-morphologic diagnosis of PPR was acute pneumonia and severe gastro-enteritis. A detailed experimental pathological study on Black Bengal goats infected with recent isolates is required.

Dried fluid spots for peste des petits ruminants virus load evaluation allowing for non-invasive diagnosis and genotyping.

BACKGROUND: Active surveillance of peste des petits ruminants (PPR) should ease prevention and control of this disease widely present across Africa, Middle East, central and southern Asia. PPR is now present in Turkey at the gateway to the European Union. In Bangladesh, the diagnosis and genotyping of PPR virus (PPRV) may be hampered by inadequate infrastructures and by lack of proper clinical material, which is often not preserved under cold chain up to laboratories. It has been shown previously that Whatman® 3MM filter paper (GE Healthcare, France) preserves the nucleic acid of PPRV for at least 3 months at 32°C. RESULTS: In this study, we demonstrate the performances of filter papers for archiving RNA from local PPRV field isolates for further molecular detection and genotyping of PPRV, at -70°C combined with ambient temperature, for periods up to 16 months. PPR-suspected live animals were sampled and their blood and nasal swabs were applied on filter papers then air dried. Immediately after field sampling, RT-PCR amplifying a 448-bp fragment of the F gene appeared positive for both blood and nasal swabs when animals were in febrile stage and only nasal swabs were detected positive in non-febrile stage. Those tested positive were monitored by RT-PCR up to 10 months by storage at -70°C. At 16 months, using real time RT-PCR adapted to amplify the N gene from filter paper, high viral loads could still be detected (~2 x 10(7) copy numbers), essentially from nasal samples. The material was successfully sequenced and a Bayesian phylogenetic reconstruction achieved adequate resolution to establish temporal relationships within or between the geographical clusters of the PPRV strains. CONCLUSIONS: This clearly reveals the excellent capacity of filter papers to store genetic material that can be sampled using a non-invasive approach.

Seroprevalence of peste des petits ruminants disease in Indonesian buffaloes may be an emerging threat to small ruminants.

Background and Aim: The peste des petit ruminants (PPR) is a disaster-class virus that causes catastrophic drawbacks to small ruminant industries in affected countries. As PPR disease has been reported in neighboring countries, Indonesia, which has a large population of sheep and goats, has become prone to the emerging threat of infection. Because the virus can also infect other animals with subclinical manifestations, large ruminants, such as buffaloes, may play an important role in spreading the virus in the environment. Therefore, the main objective of this study was to identify PPR seroprevalence in the buffalo population of Indonesia. Materials and Methods: A competitive enzyme-linked immunosorbent assay was performed to identify the specific antibody for PPR viruses in the buffalo population using serum bank collection from the National Research and Innovation Agency, Indonesia. Results: PPR virus seroprevalence was detected in buffalo from Central Java, East Java, and East Nusa Tenggara Province in Indonesia. Although seroprevalence was low in the population, the antibody titer was relatively high in the positive samples. Sex and age were identified as determinant factors in the seroprevalence distribution of the buffalo population. Conclusion: The presence of antibodies against the PPR virus in buffaloes may indicate that PPR virus is circulating in the buffalo population of Indonesia.

Effects of varying doses of conjugated estrogen on body weight, hormonal and histological alterations of reproductive organs in adult swiss albino female mice.

Estrogens are a group of hormones that have diverse effects on both reproductive and non-reproductive organs. Conjugated estrogens are medicine that contains a mixture of estrogen hormones. The study was conducted to observe the effects of varying doses of conjugated estrogen on body weight, hormonal and histological alterations of reproductive organs in adult swiss albino female mice. In this study, 60 female swiss albino mice (Mus musculus) aged 28-30 days with an average body weight of 28.2 ± 1 g were used. At first, the mice were randomly divided into 4 groups each containing 15 mice. Group A was served as vehicle control and fed on standard mice pellet and fresh drinking water. While, groups B, C and D were administered with conjugated estrogen orally at the daily dose rate of 125 µg, 250 µg and 500 µg/kg body weight respectively with 1 mL sesame oil as a vehicle by mixing with feed. The experiment was carried out for 90 days. After humanly euthanized, blood was collected and serum was prepared and organs were collected for histopathology. The results revealed that higher doses of conjugated estrogen resulted in weight loss in premenopausal female mice compared to lower doses. Serum estrogen and thyroxine concentration was increased significantly following the doses of conjugated estrogen. Ovarian histotexture showed congested blood vessels and cystic space with degeneration of follicles and corpus luteum. Uterine lesions included massive macrophage infiltration in endometrium and hyperplasia of glandular epithelium at a lower dose; hyperplasia and hypertrophy of glandular epithelium (pleomorphism) with normal macrophage infiltration in endometrium at a higher dose. Therefore, it can be concluded that oral conjugated estrogen therapy at high dose has more detrimental impacts on body weight and reproductive function compared to lower dose in female adult mice.

Experimental Pathogenicity of H9N2 Avian Influenza Viruses Harboring a Tri-Basic Hemagglutinin Cleavage Site in Sonali and Broiler Chickens.

Low-pathogenic avian influenza (LPAI) H9N2 virus is endemic in Bangladesh, causing huge economic losses in the poultry industry. Although a considerable number of Bangladeshi LPAI H9N2 viruses have been molecularly characterized, there is inadequate information on the pathogenicity of H9N2 viruses in commercial poultry. In this study, circulating LPAI H9N2 viruses from recent field outbreaks were characterized, and their pathogenicity in commercial Sonali (crossbred) and broiler chickens was assessed. Phylogenetic analysis of currently circulating field viruses based on the hemagglutinin (HA) and neuraminidase (NA) gene sequences revealed continuous circulation of G1 lineages containing the tri-basic hemagglutinin cleavage site (HACS) motif (PAKSKR*GLF) at the HA protein. Both the LPAI susceptible Sonali and broiler chickens were infected with selected H9N2 isolates A/chicken/Bangladesh/2458-LT2/2020 or A/chicken/Bangladesh/2465-LT56/2021 using intranasal (100 µL) and intraocular (100 µL) routes with a dose of 106 EID50/mL. Infected groups (LT_2-So1 and LT_56-So2; LT_2-Br1 and LT_56-Br2) revealed no mortality or clinical signs. However, at gross and histopathological investigation, the trachea, lungs, and intestine of the LT_2-So1 and LT_56-So2 groups displayed mild to moderate hemorrhages, congestion, and inflammation at different dpi. The LT 2-Br1 and LT 56-Br2 broiler groups showed nearly identical changes in the trachea, lungs, and intestine at various dpi, indicating no influence on pathogenicity in the two commercial bird species under study. Overall, the prominent lesions were observed up to 7 dpi and started to disappear at 10 dpi. The H9N2 viruses predominantly replicated in the respiratory tract, and higher titers of virus were shed through the oropharyngeal route than the cloacal route. Finally, this study demonstrated the continuous evolution of tri-basic HACS containing H9N2 viruses in Bangladesh with a low-pathogenic phenotype causing mild to moderate tracheitis, pneumonia, and enteritis in Sonali and commercial broiler chickens.

Poultry waste management practices in Bangladesh: Farmer's perceptions, and food and environmental hazards.

Objective: The poultry industry plays a key role in developing socio-economic and health sectors in Bangladesh. Poultry waste is a potential environmental threat as untreated poultry waste is used in vegetable gardens. The study aimed to investigate the current situation of small-scale poultry farms and their waste management practices in selected areas of Bangladesh and detect Escherichia coli and Salmonella in vegetables from farms using untreated poultry waste as fertilizer. Materials and Methods: A structured questionnaire-based survey was conducted in 86 small-scale poultry farms from different upazilas of Mymensingh and Khulna districts. 104 samples, including vegetables, poultry litter, water, and soil, were collected from vegetable gardens, ponds, fields, and wet markets in Mymensingh district to detect microbial contamination. Bacteria were identified based on their growth and colony morphology on selective media and motility tests. The presence of E. coli and Salmonella was confirmed by polymerase chain reaction (PCR) using a commercial PCR kit. Results: The survey revealed that mostly middle-aged males were involved in poultry farming. Most of the farmers had primary education and engaged in farming for about 5 years without training. In the study area, 37% of farmers collected droppings daily in the morning and used them as organic fertilizer. About 58% of farmers did not know the hygienic handlings of droppings and faced health problems. In PCR, either E. coli or Salmonella or both were confirmed in vegetables, litter, soil, and pond water. Conclusion: Appropriate poultry waste management practices can reduce the possible contamination of microbial agents in the human food chain.