Prevalence and identification of caprine pasteurellosis in pneumonic goats in Bangladesh.

Objective: This research aimed to assess the prevalence of caprine pasteurellosis, isolate and identify pasteurellosis (Mannheimia haemolytica and Pasteurella multocida) in pneumonic goats, and discover the main bacterial cause of pneumonia. Materials and Methods: One hundred and five samples (94 nasal swabs and 11 lung tissues) from goats suspected of having pneumonia were taken and transferred aseptically to the laboratory. Following the processing of the collected samples, Pasteurella spp. was isolated with the aid of plate culture methods. Biochemical characteristics were used to identify all bacterial isolates, which were then verified by polymerase chain reaction (PCR). Antimicrobial susceptibility testing was also carried out to evaluate the sensitivity profiles of various antibiotics. The Pasteurella haemolytica serotype-specific antigen (PHSSA) gene was used to identify isolates of M. haemolytica, and the KMT1 gene was used to identify isolates of P. multocida. Results: From the 105 clinically suspicious samples, 51 (48.57%) were identified to be Pasteurella spp. through bacteriological testing and also by PCR targeting the 16S rRNA gene. Of these, 47.87% (45/94) were nasal swabs, and 54.55% (6/11) were lung tissues. Among confirmed samples, 70.59% (36/51) were identified as M. haemolytica, and 29.41% (15/51) were identified as P. multocida. Resistance to tetracycline, streptomycin, oxytetracycline, gentamicin, and ceftriaxone was found in 50%-83% of the isolates. In addition, PCR identified the PHSSA and KMT1 genes from isolates of P. multocida and M. haemolytica, respectively. Conclusion: The present study revealed that M. haemolytica and P. multocida primarily caused pasteurellosis in pneumonic goats in Bangladesh. However, when treating these animals, the proper choice of antimicrobials should be made to control this disease.

H9N2 avian influenza virus dispersal along Bangladeshi poultry trading networks.

Avian influenza virus subtype H9N2 is endemic in Bangladesh's poultry population. The subtype affects poultry production and poses a potential zoonotic risk. Insufficient understanding of how the poultry trading network shapes the dissemination of avian influenza viruses has hindered the design of targeted interventions to reduce their spread. Here, we use phylodynamic analyses of haemagglutinin sequences to investigate the spatial spread and dispersal patterns of H9N2 viruses in Bangladesh's poultry population, focusing on its two largest cities (Dhaka and Chattogram) and their poultry production and distribution networks. Our analyses suggest that H9N2 subtype avian influenza virus lineage movement occurs relatively less frequently between Bangladesh's two largest cities than within each city. H9N2 viruses detected in single markets are often more closely related to viruses from other markets in the same city than to each other, consistent with close epidemiological connectivity between markets. Our analyses also suggest that H9N2 viruses may spread more frequently between chickens of the three most commonly sold types (sunali-a cross-bred of Fayoumi hen and Rhode Island Red cock, deshi-local indigenous, and exotic broiler) in Dhaka than in Chattogram. Overall, this study improves our understanding of how Bangladesh's poultry trading system impacts avian influenza virus spread and should contribute to the design of tailored surveillance that accommodates local heterogeneity in virus dispersal patterns.

Exposure to Brucella spp. in Humans and Cows in a High Milk-Producing Area of Bangladesh.

Brucellosis is a zoonotic disease, caused by some species within the Brucella genus. The primary and secondary objectives of this cross-sectional study were to determine the seroprevalence of Brucella antibodies in humans and cows and identify risk factors for exposure to Brucella spp. among people in Shahjadpur sub-district, Bangladesh. Twenty-five villages were randomly selected from the 303 milk-producing villages in the sub-district. We randomly selected 5% of the total households from each village. At each household, we collected demographic information and history of potential exposure to Brucella spp. in humans. In addition, we collected serum from household participants and serum and milk from cattle and tested to detect antibodies to Brucella sp. Univariate analysis was performed to detect associations between seropositivity and demographics, risk factors, and behaviors in households. We enrolled 647 households, 1313 humans, and 698 cows. Brucella antibodies were detected in sera from 27 household participants (2.1%, 95% confidence interval [95%CI]: 1.2-2.9%). Eleven (1.6%, 95%CI 0.6-2.4%) cows had detectable Brucella antibodies in either milk or serum. About half (53%) of the 698 cows exhibited more than one reproductive problem within the past year; of these, seven (2%) had Brucella antibodies. Households with seropositive individuals more frequently reported owning cattle (78% vs. 32%, P < 0.001). Despite a low prevalence of Brucella seropositivity in the study, the public health importance of brucellosis cannot be ruled out. Further studies would help define Brucella prevalence and risk factors in this region and nationally.

Potential risk factors of avian influenza virus infection in asymptomatic commercial chicken flocks in selected areas of Bangladesh during 2019.

OBJECTIVE: Avian influenza is a zoonotic disease with a pandemic potential that can infect avian and mammalian species, including humans. Studies aimed at investigating avian influenza virus (AIV) status in asymptomatic chickens and their shedding are uncommon in Bangladesh. Therefore, the current study aimed to examine the distribution of AIV subtypes in asymptomatic commercial chicken flocks and to identify the possible risk factors associated with this infection in two selected sub-districts of Bangladesh. MATERIALS AND METHODS: A total of 582 oropharyngeal swabs were collected from 23 chicken farms during 2019 and evaluated for the presence of AIV and its subtypes by real-time reverse transcription PCR assays. Risk factors associated with AIV infection were analyzed from questionnaire data. RESULTS: Overall, AIV prevalence was 7.73% (n = 45) with 7.39% and 7.92% in Dhamrai and Gazipur Sadar sub-districts, respectively. In AIV-positive samples, the prevalence of A/H5N1, A/H5N2, A/H9N1, and A/H9N2 was 31.11%, 28.89%, 6.67%, and 8.89%, respectively. None of the samples were positive for N6 and N8. The odds ratio (OR) of AIV infection was 1.15 in broiler versus layer and 2 in Sonali versus layer chickens. The OR was 1.95 for medium versus small, 2.6 for large versus small flock size, 1.5 for moderate versus good biosecurity, and 2.92 for poor versus good biosecurity practicing farms. CONCLUSION: The results demonstrated that A/H5N1, A/H5N2, A/H9N1, and A/H9N2 are circulating in asymptomatic chickens of selected areas. Strict farm biosecurity practices and avoiding higher flock density are recommended to prevent AIV spread in the study.

First Isolation and Molecular Characterization of Chicken Astrovirus and Avian Nephritis Virus in Chickens in Bangladesh.

Chicken astrovirus (CAstV) and avian nephritis virus (ANV) are enteric viruses of poultry and have infected a wide range of poultry species worldwide, causing runting-stunting syndrome (RSS), which requires virus screening and results in serious economic damage. No confirmed cases have been reported from Bangladesh. In the present study, CAstV and ANV were monitored in Bangladesh. We monitored samples for CAstV and ANV and compared their genomic sequences to other reference strains. We found 8/31 flocks (25.8%) were positive for CAstV, 6/31 flocks (19.3%) had mixed infection of CAstV and ANV, and 1 flock (3.2%) was positive for ANV. Only ANV and a combination of CAstV and ANV were found in broilers and broiler breeders, but CAstV was found in all types of chickens. We isolated two of each from CAstV and ANV through specific pathogen-free chicken embryonated eggs via the yolk sac route. Phylogenetic analysis based on the ORF1b conserved region of CAstV and ANV suggested that the locally circulating strain was closely related to the strains isolated from India and Brazil. This report is the first molecular characterization of CAstV and ANV in Bangladesh. This study highlights that CAstV and ANV are circulating in Bangladeshi poultry.

First report on the seroprevalence of avian encephalomyelitis virus antibody in Sonali (cross-bred) chickens in Bogura, Bangladesh.

OBJECTIVES: The study intended to detect the presence and distribution of avian encephalomyelitis virus (AEV)-specific antibodies in Sonali (cross-bred) parent chickens regarding farm location, flock size, and age in Bogura district of Bangladesh, a Sonali chicken belt. MATERIALS AND METHODS: A total of 275 Sonali parent chickens' blood samples were collected randomly from 39 flocks during laying age with a healthy and non-vaccination history against AEV. Blood samples were collected aseptically from the wing veins of chickens using 3-ml syringes and sera were separated. Then, the sera were transferred to the laboratory by maintaining a cool chain. Indirect enzyme-linked immunosorbent assay was used to detect the specific antibodies against AEV present in the sera samples. RESULTS: Overall, 70.18% of the chickens were found seropositive for AEV antibodies. Based on the location, the highest seropositivity was recorded in Bogura Sadar [91.30%, confidence intervals (CI) 79.21%-97.58%], and the lowest was in the Adomdighi sub-district (45.45%, CI 29.49%-63.08%). For flock size, AEV seropositivity was significantly (p < 0.05) higher in the large flock (82.22%, CI 72.74%-89.48%). Regarding age groups, the seropositivity of AEV was significantly (p < 0.05) increased with chickens' age. Higher seropositivity was noted in chickens aged >51 weeks (89.32%, CI 81.69%-94.55%). CONCLUSION: The results indicate that AEV is circulating in the environment, and chickens were exposed to the field strain of AEV. To the best of our knowledge, this is the first report on AEV in chickens in Bangladesh. Proper vaccination and standard farm biosecurity practice could minimize AEV infection in chickens. A detailed epidemiology study, detection, and characterization of the AEV would be essential for effective AEV infection control.

Molecular insights into peste des petits ruminants virus identified in Bangladesh between 2008 and 2020.

An in-depth knowledge of the molecular evolution of the peste des petits ruminants virus (PPRV) is critical for the success of the current global eradication program. For this reason, a molecular evolutionary analysis of PPRVs circulating in Bangladesh over a decade (2008-2020) was performed. The complete genome sequencing of three PPRV isolates from 2008 (BD2), 2015 (BD12) and 2017 (BD17) as well as full length nucleocapsid (N), matrix (M) and fusion (F) gene sequencing of seven more samples from 2015 to 2020 was performed. Phylogenetic analysis classified all ten PPRVs from Bangladesh as members of lineage IV and showed that they were closely related to PPRV strains detected in China and Tibet during 2007-2008, and India during 2014-2018. Time scale Bayesian Maximum Clade Credibility (MCC) phylogenetic analysis of the three complete genomes revealed a mean Time to Most Recent Common Ancestor (TMRCA) of 2000. Comparative deduced amino acid residue analysis at various functional motifs of PPRVs related to virus structure and function, virulence and host adaptation, receptor binding sites and polymerase activity revealed conserved residues among the PPRVs from Bangladesh. In total sixteen epitopes were predicted from four immunogenic proteins i.e. N, M, F and haemagglutinin (H). Interestingly, the predicted epitopes from the N and M proteins shared conserved epitopes with two vaccine strains currently being used, indicating that the strains from Bangladesh could be potentially used as alternative local vaccines.

Detection of an emerging novel sublineage Ind2001BD1 and lineage PanAsia of foot-and-mouth disease virus serotype O in cattle in Manikgonj district of Bangladesh, 2018.

Background: Foot-and-mouth disease (FMD) is an endemic disease of cloven-hoofed animals in Bangladesh and multiple outbreaks occur every year because of the FMD virus (FMDV). Aim: The aim of the present investigation was to determine the molecular characterization of the VP1 coding region of FMDV serotype O outbreak in cattle. Methods: A total of four tongue epithelial specimens were collected from clinically FMD-positive cattle during June 2018 in Manikgonj district of Bangladesh. Results: All four isolates were recorded positive for FMDV serotype O. The phylogenetic analysis showed that two isolates were clustered within an emerging novel sublineage Ind2001BD1 under lineage Ind2001 of FMDV serotype O, which was identified during 2012-2016 in Bangladesh. One isolate was clustered within the lineage PanAsia of FMDV serotype O and was closely related to an isolate identified in Nepal in 2009. The phylogenetic reconstruction revealed that all the four isolates belong to the Middle East-South Asia topotype. Conclusion: Therefore, multiple lineages of the FMDV serotype O are circulating among the cattle in the outbreak area, which make it more complex for the FMD control program in Bangladesh. A comprehensive study on the genetic characteristics of FMDV across the country is required for effective FMD prevention and control strategy.

Evaluation of potential risk of transmission of avian influenza A viruses at live bird markets in response to unusual crow die-offs in Bangladesh.

In response to unusual crow die-offs from avian influenza A(H5N1) virus infection during January-February 2017 in Dhaka, Bangladesh, a One Health team assessed potential infection risks in live bird markets (LBMs). Evidence of aerosolized avian influenza A viruses was detected in LBMs and in the respiratory tracts of market workers, indicating exposure and potential for infection. This study highlighted the importance of surveillance platforms with a coordinated One Health strategy to investigate and mitigate zoonotic risk.

Outbreak investigation, molecular detection, and characterization of foot and mouth disease virus in the Southern part of Bangladesh.

OBJECTIVE: The objective of the study was to investigate Foot and Mouth Disease virus (FMDV) outbreak in cattle in the Sarankhola Upazila under Bagerhat district of Bangladesh with isolation, identification, and molecular characterization of FMDV during April 2018. MATERIALS AND METHODS: This Upazila is located at southern border of Bangladesh and surrounded by mangrove forest Sundarban. The outbreak investigation team collected epidemiological data from outbreak location. In addition, the team collected a total of 30 (15 calves, 15 adult) tongue epithelial tissue samples from a clinically FMD-affected cattle. The confirmation of FMDV and its three serotypes (A, O, and Asia-1) was performed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). An amplified product of the VP1 region of FMDV genome was sequenced by Sanger sequencing method after cultivation and reconfirmation of FMDV into the BHK21 cell line. Genetic variability was studied by constructing a phylogenetic tree. RESULTS: The investigation survey was carried out in overall 8,393 (8,393/15,580; 53.89%) cases including 3,050 (3,050/8,393; 36.34%) cases in calf and 5,343 (5,343/8,393; 59.77%) cases in adult cattle. The overall case fatality rate (CFR) was recorded as 2.27% (354/15,580) with significantly highest CFR in the calf (71.46%; 253/354) compared to an adult. The collected all 30 samples found with FMDV positive and mixed infection of all samples with serotype Asia-1 and serotype O were observed. In BHK 21 cell line, the eight FMDV positive samples showed a typical cytopathic effect during the third passage. Finally, DNA sequence data of two isolates found closely related with the isolates of bordering country India and Myanmar. CONCLUSION: The investigation identified the risk factors involved in an outbreak of FMDV, namely, sharing the common paddy land after harvesting, no FMD vaccination, the interaction between cattle and wildlife, and cross bordering movement.

Prevalence and molecular characterization of infectious bronchitis virus isolated from chicken in Bangladesh.

AIM: The present study was aimed to determine the prevalence of infectious bronchitis virus (IBV) as well as virus isolation, identification, and molecular characterization of various strains circulating in Bangladesh. MATERIALS AND METHODS: A total of 371 swabs and organ samples were collected from four types of chicken including layer, Sonali (local), broiler, and broiler breeder under eight districts (Rangpur, Bogura, Tangail, Dhaka, Gazipur, Mymensingh, Jamalpur, and Cumilla) during 2014-2016 in Bangladesh. RESULTS: Out of 371 samples, 65 samples were positive in reverse transcriptase polymerase chain reaction (RT-PCR) for molecular identification of IBV. The overall prevalence was 17.52% recorded and among the selected types of chicken, the highest prevalence of IBV was found in layer that was 42.22% followed by 17.24% in Sonali, 14.93% in broiler breeder, and lowest prevalence was 11.94% in broiler chicken, respectively. Moreover, the prevalence of IBV was recorded highest in aged chicken at 41-60 weeks, which was 54.55% in layer, 27.27% in Sonali, and, afterward, 14.68% was found in broiler breeder, respectively. Frequency of IBV more frequently in winter (22.67%) followed by rainy (15.87%) and summer season (11.58%). The highest prevalence of IBV was found Tangail district (41.67%) followed by Mymensingh (24.42%), Gazipur (19.32%), Dhaka (15.38%), Jamalpur (16.67%), Bogura (13.68%), Cumilla (5.88%), and Rangpur (9.26%), respectively. Samples that were found high positive in IBV RT-PCR (Ct value below 30) were subjected to inoculation into chicken egg embryo to observe characteristic changes in chicken embryo. Swabs and organ samples were processed and passaged in 9-day-old embryonated chicken eggs through allantoic cavity route. IBV virus suspected samples inoculated into chicken egg embryos after 3-5 passages showed dwarfing and curling of the embryos which are characteristic lesions of IBV. Allantoic fluid was collected from all inoculated eggs and performed partial sequencing of S1 gene for three isolates. After sequencing, the phylogenetic tree was constructed from the nucleotide sequences of IBV isolates. Two of the isolates are 4/91 IBV and another one matched with QX-like IBV. CONCLUSION: The results revealed that the three isolates from different places in Bangladesh were identified for the 1st time as which will help for IBV control strategy.

Seroprevalence of major avian respiratory diseases in broiler and sonali chicken in selected areas of Bangladesh.

OBJECTIVE: This study was conducted to investigate different respiratory diseases in broiler and sonali birds in some selected districts of Bangladesh. MATERIALS AND METHODS: We were collected a total of 460 blood samples from 46 farms with 36 broiler farms and 10 sonali farms (cross-breed) from 2015 to 2017. All the collected serum samples were tested for determining specific antibodies of avian rhinotracheitis (ART) virus, infectious laryngotracheitis (ILT) virus, infectious bronchitis (IBV) virus, and Ornithobacterium rhinotracheale (ORT) infection using commercially available enzyme-linked immunosorbent assay kits. RESULTS: The overall seropositivity was highest in ORT (45.9%), followed by IBV (37.6%), ART (2.6%), and ILT (0.4%). Out of 360 broiler samples, highest seropositivity was recorded in ORT (43.3%) and lowest in IBV (31.4%). Surprisingly, no broiler samples were found positive for ART and ILT. In case of sonali, the seropositivity was highest in IBV (60%) and lowest in ILT (2%). With respect to types of birds and age groups, the seropositive percentage of all four pathogens was found higher in sonali than broiler. Between two age groups of sonali, the seropositive percentage of ART (12%), ORT (55%), ILT (2%), and IBV (60%) was highest at 21-60 weeks of age compared to 5-20 weeks of age. However, based on location, the seropositive of ORT and IBV was highest in Jamalpur (63.3%) and Fulbariya and Trishal (50%) and lowest in Sreepur (16.7%) and Jamalpur (3.3%). CONCLUSION: The four pathogens are ubiquitous in nature for the sonali chickens, and the prevalence of ORT and IBV was the most prevalent viruses in the study areas. This study indicates a need for improved surveillance and characterization of ORT and ART circulating in all types of poultry in Bangladesh.

Development and validation of BLRI Mastitis Test Kit at Bangladesh Livestock Research Institute Regional Station, Sirajganj.

OBJECTIVE: The objective of this study was to develop a low-cost kit for the detection of subclinical mastitis (SCM) and to check its validity, reproducibility, and efficacy at the field level. MATERIALS AND METHODS: A total of 550 quarter milk samples from crossbred dairy cows were collected, of which 400 milk samples were used to validate the newly developed BLRI mastitis test (BMT) kit to justify its efficacy as an individual test kit in detecting SCM based on somatic cell count (SCC) by direct microscopic count (DMC). The efficacy of the newly developed BMT was compared with the California Mastitis Test (CMT) kit. Another 150 milk samples were subjected to SCC determined by DMC and DCC (De Laval cell counter®) categorized by CMT and BMT scores. RESULTS: A SCM test kit, namely, BMT kit was successfully developed in this study. The percentage accuracy of CMT and BMT were 76.75% and 75.75%; sensitivity 69.36% and 67.56%; specificity 85.95% and 85.85%; positive predictive value 86.03% and 85.71%; negative predictive value 69.23% and 68%, respectively. A p value of 0.001 was found for both CMT and BMT. However, CMT and BMT had no significant difference in sensitivity (p = 0.778). Average SCCs (cells/ml) determined by DCC and DMC, respectively, were mostly corresponded to the SCC ranges of both CMT and BMT scores. CONCLUSION: The newly developed BMT kit is an independent, cheap, farmer-friendly, first country made, and reliable SCM diagnostic test kit that can be used at field condition.

The spread of highly pathogenic avian influenza (subtype H5N1) clades in Bangladesh, 2010 and 2011.

Since the global spread of highly pathogenic avian influenza H5N1 during 2005-2006, control programs have been successfully implemented in most affected countries. HPAI H5N1 was first reported in Bangladesh in 2007, and since then 546 outbreaks have been reported to the OIE. The disease has apparently become endemic in Bangladesh. Spatio-temporal information on 177 outbreaks of HPAI H5N1 occurring between February 2010 and April 2011 in Bangladesh, and 37 of these outbreaks in which isolated H5N1 viruses were phylogenetically characterized to clade, were analyzed. Three clades were identified, 2.2 (21 cases), 2.3.4 (2 cases) and 2.3.2.1 (14 cases). Clade 2.2 was identified throughout the time period and was widely distributed in a southeast-northwest orientation. Clade 2.3.2.1 appeared later and was generally confined to central Bangladesh in a north-south orientation. Based on a direction test, clade 2.2 viruses spread in a southeast-to-northwest direction, whereas clade 2.3.2.1 spread west-to-east. The magnitude of spread of clade 2.3.2.1 was greater relative to clade 2.2 (angular concentration 0.2765 versus 0.1860). In both cases, the first outbreak(s) were identified as early outliers, but in addition, early outbreaks (one each) of clade 2.2 were also identified in central Bangladesh and in northwest Bangladesh, a considerable distance apart. The spread of highly pathogenic avian influenza H5N1 in Bangladesh is characterized by reported long-distance translocation events. This poses a challenge to disease control efforts. Increased enforcement of biosecurity and stronger control of movements between affected farms and susceptible farms, and better surveillance and reporting, is needed. Although the movement of poultry and equipment appears to be a more likely explanation for the patterns identified, the relative contribution of trade and the market chain versus wild birds in spreading the disease needs further investigation.

Molecular epidemiology of influenza A (H5N1) viruses, Bangladesh, 2007-2011.

To investigate the origins, evolution and patterns of spread of HPAI H5N1 outbreaks in Bangladesh, we performed a phylogenetic reconstruction analysis using Bayesian methods. The analysis was conducted using 81 hemagglutinin (HA) gene sequences from the H5N1 viruses isolated in Bangladesh from 2007 to 2011, together with 264 publicly available HA sequences of clade 2.2, 2.3.2 and 2.3.4 retrieved from GenBank. Our study provides evidence that clade 2.2.2 viruses that caused outbreaks in Bangladesh were lineages independent from the viruses introduced earlier into India. Furthermore, the Bangladesh clade 2.2.2 descendents subsequently spread to India and Bhutan. This has implications for avian influenza control in southern Asia suggesting multiple routes of entry of the virus including one pathway that spread to neighboring countries via Bangladesh.