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Avian influenza virus (AIV) subtypes H5N1 and H9N2 co-circulate in poultry in Bangladesh, causing significant bird morbidity and mortality. Despite their importance to the poultry value chain, the role of farms in spreading and maintaining AIV infections remains poorly understood in most disease-endemic settings. To address this crucial gap, we conducted a cross-sectional study between 2017 and 2019 in the Chattogram Division of Bangladesh in clinically affected and dead chickens in farms with suspected AIV infection. Viral prevalence of each subtype was approximately 10% among farms for which veterinary advice was sought, indicating high levels of virus circulation in chicken farms despite the low number of reported outbreaks. Co-circulation of both subtypes was common in farms, with our findings suggest that in the field, the co-circulation of H5N1 and H9N2 can modulate disease severity, which could facilitate an underestimated level of AIV transmission in the poultry value chain. Finally, using newly generated whole-genome sequences, we investigate the evolutionary history of a small subset of H5N1 and H9N2 viruses. Our analyses revealed that for both subtypes, the sampled viruses were genetically most closely related to other viruses isolated in Bangladesh and represented multiple independent incursions. However, due to lack of longitudinal surveillance in this region, it is difficult to ascertain whether these viruses emerged from endemic strains circulating in Bangladesh or from neighbouring countries. We also show that amino acids at putative antigenic residues underwent a distinct replacement during 2012 which coincides with the use of H5N1 vaccines.

Background and Aim: Newcastle disease is one of the most common diseases affecting poultry in Bangladesh. The disease can cause up to 100% mortality but is preventable if birds are timely and properly vaccinated with a vaccine of standard virus titer. Different live vaccines are commercially available in the country - most, if not all, are produced using lentogenic strains of the virus with variable virulence. One of the disadvantages of these vaccines is that they are not stable at high environmental temperature, and therefore, a proper cold chain must be maintained during transportation and storage. Information on how long these vaccine viruses can withstand environmental temperature, which is near the vicinity of 37°C in the summer season in Bangladesh, is scanty. The aim of this research was to measure the effect of temperature on virus titer of live ND virus vaccines and to develop a real-time reverse transcription polymerase chain reaction (rRT-PCR) standard curve to indirectly determine hemagglutination (HA) titer of virus by this highly sensitive method. Materials and Methods: In this study, thermostability of five commercial live vaccines containing LaSota, F, Clone 30, and B1 type LaSota strains was observed for up to 35 days keeping them at 37°C. From the most thermostability yielding sample, two rRT-PCR standard curves were developed: (1) By plotting the cycle threshold (CT) values as obtained from 10-fold serial dilutions up to 10-3 against their corresponding log (to the base 10) dilutions and (2) by plotting the CT values obtained from serial HA dilutions up to 2-4 against their corresponding HA titer dilutions. The PCR efficiencies based on which the graphs were fitted were also evaluated. Results: The vaccine from the LaSota strain withstood 37°C for 35 days with a gradual declination of HA titer over time, and this vaccine also had the highest initial HA titer, which was 211. The vaccine made from F strain was inactivated quickly, and it had the lowest HA titer at the beginning of the study. The first standard curve developed can be used to assess the level of virus titer in a diluted sample compared with the titer in the original undiluted vaccine preparation by plotting the CT value obtained from the dilution by rRT-PCR. The second standard curve can be used to calculate the HA titer of a vaccine dilution by plotting the CT value as obtained from the dilution by rRT-PCR. Conclusion: The regression equations for the first and second graphs were y=–3.535x+14.365 and y=–1.081x+13.703, respectively, suggesting that, for every 3.53 cycles, the PCR product would have increased 10 times and 2 times for every 1.08 cycles, respectively, indicating nearly (but not exactly) 100% PCR efficiency. Keywords: LaSota strain, Newcastle disease, real-time reverse transcription polymerase chain reaction, thermostability, vaccine, validation.

Background and Aim: The emergence of antimicrobial resistance (AMR) in commensal organism, such as Escherichia coli of food animals, is an alarming issue for global health. It increases the possibility of transmitting AMR determinant(s) to human bacterial pathogens by transferable genetic materials, particularly by plasmids. Hence, it is important to know which resistant genes are being carried by commensal organisms in food chain in a country and their level of temporal loads. As a result, pre-emptive measures can be advocated with an aim to reduce their risks in their primary source of circulation which consequently would benefit the public health. Materials and Methods: Commensal E. coli strains from broiler chickens on randomly selected 30 farms and the farm environments were examined for the frequencies of isolation of resistant strains to oxytetracycline and ciprofloxacin. Five birds were randomly selected from each farm to collect cloacal swab samples (total of 150 samples). Furthermore, a total of 150 environmental samples comprising one each from feed, water, soil, litter, and litter damping site of each farm were screened for the isolation of commensal E. coli strains. Strains thus obtained were initially tested for their resistance to oxytetracycline and ciprofloxacin by Kirby–Bauer disk diffusion method. Oxytetracycline-resistant strains were further screened for the presence of resistance determining genes, namely, tetA, tetB, and tetC by uniplex polymerase chain reactions. Risks associated with the isolation frequency of oxytetracycline- and ciprofloxacin-resistant E. coli were also assessed by univariable logistic regression analysis. Results: The results revealed that all E. coli isolates, regardless of the source of origin, were resistant to oxytetracycline, while 78.4% (95% confidence interval [CI] 69.1-85.5%) showed resistance to ciprofloxacin. All the randomly selected (20) oxytetracycline-resistant strains harbored the tetA gene, whereas tetB and tetC were reported in three and two isolates, respectively. After univariable analysis, only one variable, that is, strain 1 of broiler chickens compared to two other strains was found to be positively associated with the isolation of ciprofloxacin-resistant E. coli (odds ratio 12.75 [95% CI 1.0-157.1], p=0.047). Conclusion: Resistance emerged against oxytetracycline and ciprofloxacin in commensal E. coli strains circulating in live poultry and farm environments in Bangladesh seems to be very high. Thus, human infection with drug-resistant E. coli strains through food chain will critically compromise the therapeutic measures currently available.

The highly pathogenic avian influenza A virus subtype H5N1 (HPAI H5N1) is a deadly zoonotic pathogen. Its persistence in poultry in several countries is a potential threat: a mutant or genetically reassorted progenitor might cause a human pandemic. Its world-wide eradication from poultry is important to protect public health. The global trend of outbreaks of influenza attributable to HPAI H5N1 shows a clear seasonality. Meteorological factors might be associated with such trend but have not been studied. For the first time, we analyze the role of meteorological factors in the occurrences of HPAI outbreaks in Bangladesh. We employed autoregressive integrated moving average (ARIMA) and multiplicative seasonal autoregressive integrated moving average (SARIMA) to assess the roles of different meteorological factors in outbreaks of HPAI. Outbreaks were modeled best when multiplicative seasonality was incorporated. Incorporation of any meteorological variable(s) as inputs did not improve the performance of any multivariable models, but relative humidity (RH) was a significant covariate in several ARIMA and SARIMA models with different autoregressive and moving average orders. The variable cloud cover was also a significant covariate in two SARIMA models, but air temperature along with RH might be a predictor when moving average (MA) order at lag 1 month is considered.

Contaminated poultry and poultry products are a major source of motile Salmonellae for human salmonellosis worldwide. Local circulation of any motile Salmonella serovar in poultry has a wider public health impact beyond its source of origin for being dispersed elsewhere through poultry trades or human travels. To investigate the status of motile Salmonella serovars in breeder farms in Bangladesh, multiple flocks of two breeder farms were observed for a period of six months. In addition, a cross-sectional survey was carried out to determine the prevalence and serovar distribution of motile Salmonella by randomly selecting 100 commercial broiler poultry farms. Five pooled faecal samples representing an entire housed flock of breeders or broilers were screened for presence of motile Salmonella following conventional bacteriological procedures. The Salmonella isolates obtained were subsequently serotyped, and characterized by plasmid profiling and pulsed-field gel electrophoresis (PFGE). The results revealed that both the breeder farms were positive with three Salmonella serovars: S. Virchow, S. Paratyphi B var Java (S. Java) and S. Enteritidis. Eleven of the 100 broiler farms investigated were positive for motile Salmonella, giving a farm-level prevalence of 11% (95% confidence interval 5–17%). S. Virchow and S. Kentucky were the two predominant serovars isolated from the broiler farms. The PFGE genotyping demonstrated that the isolates belonging to the same serovars were closely related due to variation in only 1–4 bands. All the S. Virchow and S. Java isolates, irrespective of breeder or broiler farm origin, were plasmid-free, except for one S. Virchow isolate from a broiler farm that harboured a 9.7 kb-sized plasmid. The S. Kentucky isolates belonged to three plasmid profiles having plasmids of four different sizes, ranging from 2.7 to 109 kb. This is the first report of any motile Salmonella serovars from breeder and commercial broiler poultry farms in Bangladesh.

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

Background: The agro-ecology and poultry husbandry of the south Asian and south-east Asian countries share common features, however, with noticeable differences. Hence, the ecological determinants associated with risk of highly pathogenic avian influenza (HPAI-H5N1) outbreaks are expected to differ between Bangladesh and e. g., Thailand and Vietnam. The primary aim of the current study was to establish ecological determinants associated with the risk of HPAI-H5N1 outbreaks at subdistrict level in Bangladesh. The secondary aim was to explore the performance of two different statistical modeling approaches for unmeasured spatially correlated variation. Methodology/Principal Findings: An ecological study at subdistrict level in Bangladesh was performed with 138 subdistricts with HPAI-H5N1 outbreaks during 2007-2008, and 326 subdistricts with no outbreaks. The association between ecological determinants and HPAI-H5N1 outbreaks was examined using a generalized linear mixed model. Spatial clustering of the ecological data was modeled using 1) an intrinsic conditional autoregressive (ICAR) model at subdistrict level considering their first order neighbors, and 2) a multilevel (ML) model with subdistricts nested within districts. Ecological determinants significantly associated with risk of HPAI-H5N1 outbreaks at subdistrict level were migratory birds' staging areas, river network, household density, literacy rate, poultry density, live bird markets, and highway network. Predictive risk maps were derived based on the resulting models. The resulting models indicate that the ML model absorbed some of the covariate effect of the ICAR model because of the neighbor structure implied in the two different models. Conclusions/Significance: The study identified a new set of ecological determinants related to river networks, migratory birds' staging areas and literacy rate in addition to already known risk factors, and clarified that the generalized concept of free grazing duck and duck-rice cultivation interacted ecology are not significant determinants for Bangladesh. These findings will refine current understanding of the HPAI-H5N1 epidemiology in Bangladesh.

Salmonella is a globally widespread food-borne pathogen having major impact on public health. All motile serovars of Salmonella enterica of poultry origin are zoonotic, and contaminated meat and raw eggs are an important source to human infections. Information on the prevalence of Salmonella at farm/holding level, and the zoonotic serovars circulating in layer poultry in the South and South-East Asian countries including Bangladesh, where small-scale commercial farms are predominant, is limited. To investigate the prevalence of Salmonella at layer farm level, and to identify the prevalent serovars we conducted a cross-sectional survey by randomly selecting 500 commercial layer poultry farms in Bangladesh. Faecal samples from the selected farms were collected following standard procedure, and examined for the presence of Salmonella using conventional bacteriological procedures. Thirty isolates were randomly selected, from the ninety obtained from the survey, for serotyping and characterized further by plasmid profiling and pulsed-field gel electrophoresis (PFGE). Results of the survey showed that the prevalence of motile Salmonella at layer farm level was 18% (95% confidence interval 15-21%), and Salmonella Kentucky was identified to be the only serovar circulating in the study population. Plasmid analysis of the S. Kentucky and non-serotyped isolates revealed two distinct profiles with a variation of two different sizes (2.7 and 4.8 kb). PFGE of the 30 S. Kentucky and 30 non-serotyped isolates showed that all of them were clonally related because only one genotype and three subtypes were determined based on the variation in two or three bands. This is also the first report on the presence of any specific serovar of Salmonella enterica in poultry in Bangladesh.

Background: Escherichia coli O157 (EcO157) infection has been recognized as an important global public health concern. But information on the prevalence of EcO157 in cattle at the global and at the wider geographical levels is limited, if not absent. This is the first meta-analysis to investigate the point prevalence of EcO157 in cattle at the global level and to explore the factors contributing to variation in prevalence estimates. Methods: Seven electronic databases- CAB Abstracts, PubMed, Biosis Citation Index, Medline, Web of Knowledge, Scirus and Scopus were searched for relevant publications from 1980 to 2012. A random effect meta-analysis model was used to produce the pooled estimates. The potential sources of between study heterogeneity were identified using metaregression. Principal findings: A total of 140 studies consisting 220,427 cattle were included in the meta-analysis. The prevalence estimate of EcO157 in cattle at the global level was 5.68% (95% CI, 5.16-6.20). The random effects pooled prevalence estimates in Africa, Northern America, Oceania, Europe, Asia and Latin America-Caribbean were 31.20% (95% CI, 12.35-50.04), 7.35% (95% CI, 6.44-8.26), 6.85% (95% CI, 2.41-11.29), 5.15% (95% CI, 4.21-6.09), 4.69% (95% CI, 3.05-6.33) and 1.65% (95% CI, 0.77-2.53), respectively. Between studies heterogeneity was evidenced in most regions. World region (p

Background The number of outbreaks of HPAI-H5N1 reported by Bangladesh from 2007 through 2011 placed the country among the highest reported numbers worldwide. However, so far, the understanding of the epidemic progression, direction, intensity, persistence and risk variation of HPAI-H5N1 outbreaks over space and time in Bangladesh remains limited. Methodology/principal findings To determine the magnitude and spatial pattern of the highly pathogenic avian influenza A subtype H5N1 virus outbreaks over space and time in poultry from 2007 to 2009 in Bangladesh, we applied descriptive and analytical spatial statistics. Temporal distribution of the outbreaks revealed three independent waves of outbreaks that were clustered during winter and spring. The descriptive analyses revealed that the magnitude of the second wave was the highest as compared to the first and third waves. Exploratory mapping of the infected flocks revealed that the highest intensity and magnitude of the outbreaks was systematic and persistent in an oblique line that connects south-east to north-west through the central part of the country. The line follows the Brahmaputra-Meghna river system, the junction between Central Asian and East Asian flyways, and the major poultry trading route in Bangladesh. Moreover, several important migratory bird areas were identified along the line. Geostatistical analysis revealed significant latitudinal directions of outbreak progressions that have similarity to the detected line of intensity and magnitude. Conclusion/significance The line of magnitude and direction indicate the necessity of mobilizing maximum resources on this line to strengthen the existing surveillance.