Characterization, histopathology and immunogenicity of the lumpy skin disease virus isolated during 2019-20 in Bangladesh.

Introduction: Lumpy skin disease (LSD) is a highly contagious vector-borne viral disease of cattle. LSD has emerged in Bangladesh in 2019, causing significant economic losses due to its high morbidity and mortality. This research was designed to isolate, identify, and assess the immunogenicity of LSD virus (LSDV) using nodular tissue samples obtained from affected cattle during the 2019-20 outbreak across nine districts of Bangladesh. Methods: To determine the presence of LSDV in nodular tissues, we initially used iiPCR and PCR, followed by histopathological examination. 151 were positive via iiPCR and PCR among the 180 collected samples. The PCR positive 151 samples were then inoculated into 10-day-old embryonated chicken eggs via the CAM route to isolate LSDV, confirmed through PCR. Subsequently, partial sequencing and phylogenetic analysis of the P32 gene were performed to determine the origin of the circulating LSDV strain. The immunogenicity of selected LSDV strains was assessed through an ELISA test. Results: The PCR results revealed a distinct positive band at 192 bp in both the nodular tissue samples and the LSDV isolated from chicken embryo inoculations. Microscopic analysis of the nodular lesions revealed thickening of the epidermis, ballooning degeneration of keratinocytes, and proliferation of follicular epithelia. Additionally, mononuclear infiltration was observed at the demarcation line between infected and healthy tissue, with necrosis of muscular tissues beneath the epidermis. The LSDV isolate from Bangladesh exhibited a close genetic relationship with LSDV strains isolated from neighboring and other regional countries including India, Myanmar, and Mongolia. This observation strongly suggests the possibility of a transboundary spread of the LSD outbreak in Bangladesh during 2019-2020. The results of the immunogenicity test showed that the serum antibody titer remained at a protective level for up to 18 months following secondary immunization with inactivated LSDV antigen. This finding suggests that the inactivated LSDV antigen could be a potential vaccine candidate to protect cattle in Bangladesh against LSDV. Conclusion: In conclusion, our research successfully isolated, identified, and characterized LSDV in cattle nodular tissues from the 2019-20 outbreak in Bangladesh. Furthermore, it provided insights into the probable origin of the circulating strain and investigated a potential vaccine candidate to protect cattle in the region from LSDV.

Complete genome sequence analysis of the multidrug resistant Aeromonas veronii isolated for the first time from stinging catfish (Shing fish) in Bangladesh.

Objective: Whole genome sequencing (WGS) of Aeromonas veronii Alim_AV_1000 isolated from ulcerative lesions of Shing fish (stringing catfish; Heteropneustes fossilis) was performed during the outbreak year 2021. Materials and Methods: Using next-generation sequencing (Illumina) technology, WGS was accomplished, resulting in the sequencing, assembly, and analysis of the entire genome of the A. veronii strain. Moreover, the genomic features, virulence factors, antimicrobial resistome, and phylogenetic analysis for the molecular evolution of this strain were also examined. Results: The genome size of the A. veronii Alim_AV_1000 strain was 4,494,515 bp, with an average G+C content of 58.87%. Annotation revealed the known transporters and genes linked to virulence, drug targets, and antimicrobial resistance. Conclusion: The findings of the phylogenetic analysis revealed that the strain of the present study has a close relationship with the China strain TH0426 and strain B56. This study provides novel information on A. veronii isolated from Shing fish in Bangladesh.

Serological study and risk factor analysis on Peste des Petits Ruminants in sheep in Bangladesh.

A cross-sectional study was conducted to determine the seroprevalence of the Peste des Petits Ruminant (PPR) virus (PPRV) in sheep populations and to determine the potential epidemiological risk factors associated with this infection. Between October 2014 and March 2017, 2420 sheep serum samples were collected from ten selected PPR outbreak-prone districts in Bangladesh. The collected sera were analysed by competitive enzyme-linked immunosorbent assay (cELISA) test to detect antibodies against PPR. A previously designed disease report form was used to gather data on important epidemiological risk factors, and a risk analysis was performed to ascertain their association with PPRV infection. By cELISA, 44.3 % (95 % confidence interval:42.4-46.4 %) of sheep sera were positive for PPRV antibodies against PPR. In univariate analysis, the Bagerhat district had significantly higher seropositivity (54.1 %, 156/288) than other districts. Moreover, significantly higher (p < 0.05) seropositivity was found in the Jamuna River Basin (49.1 %, 217/442) compared to other ecological zones, in crossbreeds (60 %; 600/1000) related to native sheep, in males (69.8 %, 289/414) associated with females, in imported sheep (74.3 %, 223/300) compared to other sources, and in winter (57.2 %, 527/920) than in other seasons. In the multivariate logistic regression model, six possible risk factors were identified: study location, ecological zone, breed, sex, source, and season. The high seroprevalence of PPRV is significantly associated with several risk factors, suggesting that PPR is epizootic throughout the country.

Pharmacophore-based virtual screening and in-silico study of natural products as potential DENV-2 RdRp inhibitors.

Dengue fever is a significant public health concern throughout the world, causing an estimated 500,000 hospitalizations and 20,000 deaths each year, despite the lack of effective therapies. The DENV-2 RdRp has been identified as a potential target for the development of new and effective dengue therapies. This research's primary objective was to discover an anti-DENV inhibitor using in silico ligand- and structure-based approaches. To begin, a ligand-based pharmacophore model was developed, and 130 distinct natural products (NPs) were screened. Docking of the pharmacophore-matched compounds were performed to the active site of DENV-2 RdRp protease . Eleven compounds were identified as potential DENV-2 RdRp inhibitors based on docking energy and binding interactions. ADMET and drug-likeness were done to predict their pharmacologic, pharmacokinetic, and drug-likeproperties . Compounds ranked highest in terms of pharmacokinetics and drug-like appearances were then subjected to additional toxicity testing to determine the leading compound. Additionally, MD simulation of the lead compound was performed to confirm the docked complex's stability and the binding site determined by docking. As a result, the lead compound (compound-108) demonstrated an excellent match to the pharmacophore, a strong binding contact and affinity for the RdRp enzyme, favourable pharmacokinetics, and drug-like characteristics. In summary, the lead compound identified in this study could be a possible DENV-2 RdRp inhibitor that may be further studied on in vitro and in vivo models to develop as a drug candidate.Communicated by Ramaswamy H. Sarma.

Epidemiological investigation of lumpy skin disease outbreaks in Bangladeshi cattle during 2019-2020.

Lumpy skin disease (LSD) is an acute infectious viral disease of cattle with a wide distribution that emerged in Bangladesh in 2019, causing huge economic losses. This study was undertaken to investigate the epidemiological features of LSD emergence in nine districts of Bangladesh between December 2019 and December 2020. A total of 8215 cattle from 603 herds were investigated and LSD was diagnosed based on the characteristic clinical findings. A standard questionnaire was administered to collect herd-level data including location, herd size, number of LSD-infected cattle, number died due to LSD, farm type, season, house type, vector presence, sanitation and fly repellent use. Similarly, data on clinical signs, sex, age, animal class and breed of the LSD-infected cattle were also recorded. The herd-level attack risk (%) and mortality risk (%) were calculated based on the number of infected and dead cattle, respectively, as a proportion of total cattle. The herd-level risk factors for LSD were identified using a multivariable Poisson regression model. The most common clinical signs were skin nodules (100%), fever (97.9%) and depression with anorexia and weight loss (97.9%). Crossbred (84.9%) and female (72.2%) cattle were mostly affected by LSD. The overall LSD attack risk, mortality risk and case fatality were 26.5%, 0.26% and 0.97%, respectively. The LSD attack risk was significantly higher in small herds (risk ratio: [RR] 1.39; 95% CI: 1.27; 1.53) than large herds. In addition, significantly higher LSD attack risk was observed in semi-intensive management systems (RR = 1.29; 95% CI: 1.01; 1.64) than intensive management systems. Moreover, it was also significantly higher in hut (RR = 1.81; 95% CI: 1.12; 2.92), temporary (RR = 1.62; 95% CI: 1.21; 2.17) and tin-shed houses (RR = 1.29; 95% CI: 1.11; 1.51) than in semi-building houses. To the best of our knowledge, this is the first detailed epidemiological study of LSD emergence in South Asia. Female crossbred cattle in small herds under semi-intensive management should be prioritized for LSD surveillance and vaccination to prevent further outbreaks and control the impact of the disease in Bangladesh.

Efficacy of bi-valent whole cell inactivated bacterial vaccine against Motile Aeromonas Septicemia (MAS) in cultured catfishes (Heteropneustes fossilis, Clarias batrachus and pangasius pangasius) in Bangladesh.

The Motile Aeromonas Septicemia (MAS) is an important disease of cultured catfishes (Heteropneustes fossilis, Clarias batrachus and Pangasius pangasius), caused by different species of Aeromonas bacteria which have been documented to be higher death rates (≤70%) in Bangladesh since 2016. Present study was conducted to develop bi-valent vaccine using A. hydrophila and A. veronii, and to validate their efficacy via intra-muscular (IM) and oral-routes of immunization in selected species of fishes. Brood fishes of the three species were immunized with three doses of inactivated vaccine (107 CFU /2.3 mg/ml). Hematological parameters of brood fishes and antibody levels (IgM) of broods, their larvae and eggs were determined by ELISA. Additionally, Relative Percent Survivability (RPS) and the IgM levels of the larvae after challenge with virulent A. hydrophila and A. veronii were also evaluated. Findings of this study showed that the lymphocytes, monocytes, granulocytes counts and antibody (IgM) titre of brood fishes, larvae and eggs from the vaccinated fishes were found significantly higher (p< 0.05) compared to the un-vaccinated control groups. Alternatively, antibody levels (IgM) in the larvae of vaccinated group of brood fishes fed with antigen coated feed was exhibited to be remarkably higher (p< 0.05) than the antigen non-fed group. The RPS of larvae of Shing (91.24 ± 2.00%), Magur (88.09 ± 2.88%) and Pangas (93.17 ± 1.52%) was found to be higher in the larvae at 20-day age of vaccinated group compared to non-vaccinated brood fishes group. Findings of this study indicated that the active immunization of brood fishes followed by oral immunization of their larvae feeding with antigen coated feed showed synergistic effect in protecting cultured Shing, Magur and Pangas fishes from frequent attack with Aeromonas spp at any age of their lifetime.

Serological survey on the prevalence of chicken infectious anemia virus in broiler breeder and layer farms in some selected areas of Bangladesh.

OBJECTIVE: Chicken infectious anemia virus (CIAV) is an economically important emerging infection of poultry as it causes immunosuppression and reduces egg production. Although it is worldwide distributed and first reported (single case) in Bangladesh in 2002, no epidemiological and serological investigations have been conducted. The current study aimed to conduct a serological investigation on the prevalence of CIAV infection in broiler breeder and layer farms in some selected areas of Bangladesh. MATERIALS AND METHODS: A total number of 460 sera samples were randomly collected from unvaccinated broiler breeder and layer flocks, of which 276 were from 11 broiler breeder farms and 184 from 12 layer farms. The sera samples were subjected to a commercially available enzyme-linked immunosorbent assay kit to observe antibodies induced by CIAV. RESULTS: Results demonstrated that the overall prevalence of CIAV was 83.6% among a total of 460 samples. In broiler breeder birds, the prevalence was 89.9%, whereas it was 78.3% in layer birds. A higher number of female birds was found to be seropositive than male birds. However, chickens of all age groups were found to be susceptible to the virus. CONCLUSIONS: These results indicate the presence of CIAV in Bangladesh, which may be the sequel of naturally occurring either vertical or horizontal infection in all bird flocks tested without clinical symptoms of the disease. A further epidemiological investigation will be required, followed by molecular isolation and characterization of the virus for suitable vaccine candidate selection and/or preparation.

Molecular characterization of duck plague virus from selected Haor areas of Bangladesh.

Background: Duck viral enteritis, commonly known as duck plague (DP), is an acute and contagious fatal disease in ducks, geese, and swans caused by the DP virus (DPV). It poses a serious threat to the growth of duck farming in the Haor (wetland) areas of Bangladesh. Aim: This study aimed to detect the circulating DPV by molecular characterization, followed by phylogenetic analysis, targeting the UL30 gene in infected ducks from five Haor districts in Bangladesh and to observe the variation in the genome sequence between the field virus and vaccine strain of DPV. Methods: A total of 150 samples (liver, 50; intestine, 50; and oropharyngeal tissue, 50) were collected from DP-suspected sick/dead ducks from 50 affected farms in Kishoreganj, Netrokona, B. Baria, Habiganj, and Sunamganj districts in Bangladesh. For the identification of DPV in collected samples, polymerase chain reaction (PCR) was utilized. Nucleotide sequences of the amplified UL30 gene were compared with those of other DPV strains available in GenBank. Results: Of the 150 samples, 90 (60%) were found to be positive for DPV, as confirmed by PCR. Organ-wise prevalence was higher in the liver (72%), followed by the intestine (64%) and oropharyngeal tissue (44%). Regarding areas, the highest and lowest prevalence in the liver and intestine was observed in Habiganj and B. Baria, respectively, whereas the highest and lowest prevalence in the oropharyngeal tissue was observed in B. Baria and Habiganj, respectively. Two isolates, BAU/KA/DPV(B1)/2014 from Kishoreganj and BAU/KA/DPV(B4)/2014 from Sunamganj were sequenced, and phylogenetic analysis revealed that these isolates are evolutionarily closely related to Chinese isolates of DPV. Additionally, the isolates of DPV BAU/KA/DPV(B1)/2014 and BAU/KA/DPV(B4)/2014 showed the highest (98%) similarity to each other. The nucleotide sequence of the isolate BAU/KA/DPV(B1)/2014 exhibited higher nucleotide variability (246 nucleotides) than that of the vaccine strain (accession no. EU082088), which may affect protein function and additional drug sensitivity. Conclusion: Based on the findings of the molecular study, it can be assumed that the Bangladeshi isolates and all Chinese isolates of DPV may have a common ancestry.

Isolation and molecular detection of Streptococcus agalactiae from popped eye disease of cultured Tilapia and Vietnamese koi fishes in Bangladesh.

OBJECTIVE: Present research aims to isolate, identify, and determine the virulence of the Streptococcus agalactiae (group B Streptococcus; GBS), isolated from popped eye disease affected Tilapia and Vietnamese Koi (V. Koi) fishes. MATERIALS AND METHODS: A total of 330 fish samples were collected, of which Tilapia (n = 180) and V. Koi (n = 150), were collected from 35 affected ponds of four selected districts of Bangladesh. Isolation of the bacterium was done using different culture media (Nutrient broth, Plate count agar, Tryptic Soy Agar, and Blood agar), and identification by using various biochemical tests (conventional and using API 20 Strep kit) and polymerase chain reaction (PCR) using primers against 16S rRNA gene of S. agalactiae. Antibiotic susceptibility of the bacteria was performed using seven different antibiotics disc (Tetracycline, Oxytetracycline, Chlortetracycline, Streptomycin, Ciprofloxacin, Gentamicin, and Neomycin). Virulence of the isolated S. agalactiae was determined by infecting healthy Tilapia and V. Koi fishes through experimental infection. RESULTS: Isolated bacteria were found Gram-positive paired and chained cocci, ß-hemolytic and non-motile. Findings of biochemical and serological tests indicate that the isolated bacterium belongs to Group B Streptococcus of Lancefield classification. PCR result also confirmed that the bacteria were S. agalactiae. The bacterial isolates possessed resistance property against all the seven antibiotics used in this study. The isolated GBS was found highly virulent and showed 80%-90% mortality for Tilapia and V. Koi fishes in experimental infection within 1-6 days of post-infection. CONCLUSION: From the findings of this study, it may be concluded that isolated GBS from the Tilapia and V. Koi fishes were highly virulent and possessed multidrug-resistance properties.

Microbial risk assessment of ready-to-eat mixed vegetable salads from different restaurants of Bangladesh Agricultural University campus.

OBJECTIVE: The study was aimed to analyze the microbiological quality of mixed vegetable salads and to understand the risk related with its consumption from different restaurants around Bangladesh Agricultural University campus in Mymensingh. MATERIALS AND METHODS: Sixty (60) samples of mixed vegetable salads were taken from twelve (12) different restaurants in five different time points from each restaurant. In parallel, restaurant workers were asked for handling practices while the consumers were interviewed about their salad consumption pattern and whether they had experienced any health-related problems. Microbial risk assessment of Staphylococcus spp., Salmonella spp., and Escherichia coli was estimated by Monte Carlo simulation (10,000 iterations), an exponential model. RESULTS: Aerobic plate count was ranged from 7.73 ± 0.61 to 9.04 ± 0.26 log cfu/gm, Staphylococcus spp. from 4.64 ± 0.61 to 6.42 ± 0.53 log cfu/gm, Salmonella spp. from 4.75 ± 0.08 to 5.27 ± 0.53 log cfu/gm, and E. coli from 4.98 ± 0.20 to 6.66 ± 0.80 log cfu/gm. From the survey, it was found that total consumers had 18% chances where the male had 13% and the female had 30% chances of being infected with salads. Again frequent, average, and occasional consumers had 31%, 13%, and 0% chances, respectively, of being infected with those salads. From the Monte Carlo simulation, the calculated mean annual risks of Staphylococcus spp., Salmonella spp., and E. coli infection for the three exposure scenarios were found to be about 100%. CONCLUSION: The study actually revealed the potential microbial contamination in mixed vegetable salads which may impact on food safety and human health. So, the findings suggest that following hygienic measures during processing and handling the microbiological quality of mixed vegetables salads can be improved.

Serological detection of avian reovirus in different poultry flocks of Gazipur and Mymensingh districts of Bangladesh.

BACKGROUND AND AIM: Avian reovirus (ARV) is a constraint to poultry industry in Bangladesh as a cause of several diseases in chickens, especially in broiler. However, the actual status of the viral infection is not known because the large-scale study is not conducted in this country. Therefore, this study aimed to check the presence and distribution of ARV-specific antibody in respect to area, types of chickens (broiler breeder, broiler, and layer), vaccination status, and age of chickens in Gazipur and Mymensingh districts of Bangladesh. MATERIALS AND METHODS: A total of 276 chickens' blood samples were collected from two well-organized broiler breeder stock, seven broiler farms, and five layer farms located at two districts, namely Gazipur and Mymensingh of Bangladesh. Blood samples were collected from wing vein of the apparently healthy chickens using 3 ml of syringe and serum was harvested by keeping the syringe at room temperature in slanting position. The sera were transferred to the laboratory by maintaining the cool chain and further processing was performed by indirect enzyme-linked immunosorbent assay using ARV antibody test kit. RESULTS: The results of serological test revealed that an average of 39.5% seropositive against ARV was recorded in chickens of Gazipur and Mymensingh districts. Among these, chickens of Gazipur district had the highest seropositivity of 50.5% than Mymensingh (30.7%). With respect to vaccination status, the seropositivity of vaccinated chickens in both areas was 100% and non-vaccinated chickens was 50.5% in Gazipur and 30.7% in Mymensingh district, respectively. However, regarding age groups, the seropositivity was higher in the age of 4-6 weeks (64.5%). CONCLUSION: The present serological findings showed a higher prevalence of ARV-specific antibodies in broiler birds. It indicates that the poultry industries of Bangladesh are contaminated with ARV which may naturally be transmitted to chickens either vertically or horizontally.

Molecular characterization of two Bangladeshi infectious bursal disease virus isolates using the hypervariable sequence of VP2 as a genetic marker.

Two Bangladeshi infectious bursal disease virus (IBDV) isolates collected in 2007, termed GB1 and GB3, were subjected to comparative sequencing and phylogenetic analyses. Sequence analysis of a 474-bp hypervariable region in the VP2 gene revealed that among four major amino acid substitutions observed in the strains, two were unique to GB1 and GB3 (Ser217Leu and Ala270Thr) while one substitution was only found in GB1 (Asn299Ser). Among IBDVs from Bangladesh including GB1 and GB3, the rate of identity and homology was around 97~99%. The amino acid sequences of GB1 and GB3 differ from those of previous Bangladeshi IBDV isolates and contain amino acid substitutions Pro222Ala and Asn299Ser (in GB3 only). Phylogenetic analysis revealed that GB1 and GB3 are grouped with other very virulent IBDVs of European and American origin in contrast to two previously isolated Bangladeshi IBDV strains (GenBank accession Nos. AF362776 and AF260317), which belong to the Asian group. It was concluded that GB1 and GB3 belong to a very virulent group of IBDVs. However, amino acid sequences of GB1 and GB3 differ from those of the other Bangladeshi IBDVs by one or two amino acids encoded in the hypervariable region of the VP2 gene.