Bacterial Metabarcoding of Raw Palm Sap Samples from Bangladesh with Nanopore Sequencing.

The traditional practice of harvesting and processing raw date palm sap is not only culturally significant but also provides an essential nutritional source in South Asia. However, the potential for bacterial or viral contamination from animals and environmental sources during its collection remains a serious and insufficiently studied risk. Implementing improved food safety measures and collection techniques could mitigate the risk of these infections. Additionally, the adoption of advanced food analytical methods offers the potential to identify pathogens and uncover the natural bacterial diversity of these products. The advancement of next-generation sequencing (NGS) technologies, particularly nanopore sequencing, offers a rapid and highly mobile solution. In this study, we employed nanopore sequencing for the bacterial metabarcoding of a set of raw date palm sap samples collected without protective coverage against animals in Bangladesh in 2021. We identified several bacterial species with importance in the natural fermentation of the product and demonstrated the feasibility of this NGS method in the surveillance of raw palm sap products. We revealed two fermentation directions dominated by either Leuconostoc species or Lactococcus species in these products at the first 6 h from harvest, along with opportunistic human pathogens in the background, represented with lower abundance. Plant pathogens, bacteria with the potential for opportunistic human infection and the sequences of the Exiguobacterium genus are also described, and their potential role is discussed. In this study, we demonstrate the potential of mobile laboratory solutions for food safety purposes in low-resource areas.

Snapshot study of canine distemper virus in Bangladesh with on-site PCR detection and nanopore sequencing.

Canine distemper virus (CDV) is a highly contagious virus that affects domestic and wild animals, causing severe illness with high mortality rates. Rapid monitoring and sequencing can provide valuable information about circulating CDV strains, which may foster effective vaccination strategies and the successful integration of these into conservation programs. During two site visits in Bangladesh in 2023, we tested a mobile, deployable genomic surveillance setup to explore the genetic diversity and phylogenetic patterns of locally circulating CDV strains. We collected and analysed 355 oral swab samples from stray dogs in Rajshahi and Chattogram cities, Bangladesh. CDV-specific real-time RT-PCR was performed to screen the samples. Out of the 355 samples, 7.4% (10/135) from Rajshahi city and 0.9% (2/220) from Chattogram city tested positive for CDV. We applied a real-time RT-PCR assay and a pan-genotype CDV-specific amplicon-based Nanopore sequencing technology to obtain the near-completes. Five near-complete genome sequences were generated, with phylogenetic relation to the India-1/Asia-5 lineage previously identified in India. This is the first study to provide genomic data on CDV in Bangladesh and the first demonstration of a mobile laboratory setup as a powerful tool in rapid genomic surveillance and risk assessment for CDV in low resource regions.

Molecular Detection of Tetracycline-Resistant Genes in Multi-Drug-Resistant Escherichia coli Isolated from Broiler Meat in Bangladesh.

This study aimed to estimate the antimicrobial resistance (AMR) patterns and tetracycline-resistant gene profiles of Escherichia coli (E. coli) from broiler meat and livers sourced from live bird markets (LBMs) and supermarkets (SMs) in Chattogram, Bangladesh. In total, 405 samples were collected from SMs and LBMs, comprising muscle (n = 215) and liver (n = 190) samples. Disc diffusion tests were used to determine antimicrobial susceptibility profiles. PCR was used to identify E. coli and tetracycline-resistant genes. Over half (57%) of the chicken product samples were positive for E. coli. The AMR profiling of these isolates showed that the highest prevalence of resistance was against sulphamethoxazole-trimethoprim (89%), followed by tetracycline (87%), ampicillin (83%), and ciprofloxacin (61%). Among the antimicrobials listed by the World Health Organization as critically important, E. coli isolates were found to be resistant to cephalexin (37%), gentamicin (32%), and colistin sulfate (21%). A large proportion of E. coli demonstrated multi-drug resistance (MDR). Most (84%) of the tetracycline-resistant isolates encoded tetA. Of the remaining isolates, 0.5% encoded tetC, 6.0% encoded two genes, and 3.6% of isolates were tetD, which was newly identified by this study in Bangladesh. Broiler products in Bangladesh are frequently contaminated with multi-drug-resistant E. coli, with differential carriage of tetracycline genes. The prevalence of tetracycline resistance among E. coli indicates a concern for poultry health and welfare regarding the management of colibacillosis. It also indicates growing public health risks of AMR among broiler-associated pathogens, which can be transferred to humans via the food chain. Appropriate control measures should be developed and implemented, focused on the rational use of antimicrobials in poultry farming systems, to mitigate risk from this drug-resistant zoonotic pathogen from foods of animal origin and to protect public health.

Epidemiological Profile of a Human Hepatitis E Virus Outbreak in 2018, Chattogram, Bangladesh.

Hepatitis E virus (HEV) is a waterborne zoonotic disease that can result in a high fatality rate in pregnant women and infants. In 2018, a large HEV outbreak emerged in Chattogram, Bangladesh, resulting in 2800 cases and a significant public health response to mitigate the transmission. While the source of the outbreak remained poorly understood, authorities suggested that possible risk factors for HEV infection included contamination of water supply, exacerbated by concurrent severe flooding events in the community. A cross-sectional study was conducted to investigate the distribution and risk factors for HEV seroprevalence between January and December 2018 in the Chattogram city area. A total of 505 blood samples were collected from symptomatic patients of 10 hospitals who met the case definition for an HEV infection. Standard ELISA tests were performed in all patients to identify anti-HEV antibodies. The size and location of HEV seroprevalence clusters within Chattogram were investigated using SaTScan. We investigated the association between risk of HEV infection and individual and environmentally lagged risk factors using Bernoulli generalised linear regression models. Our results indicate an overall HEV seroprevalence of 35% with significant variation according to sex, source of drinking water, and boiling of drinking water. A positive cross-correlation was found between HEV exposure and precipitation, modified normalised difference water index (MNDWI), and normalised difference vegetation index (NDVI). Our model indicated that risk of infection was associated with sex, age, source of drinking water, boiling of water, increased precipitation, and increased MNDWI. The results from this study indicate that source and boiling of drinking water and increased precipitation were critical drivers of the 2018 HEV outbreak. The communities at highest risk identified in our analyses should be targeted for investments in safe water infrastructure to reduce the likelihood of future HEV outbreaks in Chattogram.

Major bat-borne zoonotic viral epidemics in Asia and Africa: A systematic review and meta-analysis.

Bats are the natural reservoir host for many pathogenic and non-pathogenic viruses, potentially spilling over to humans and domestic animals directly or via an intermediate host. The ongoing COVID-19 pandemic is the continuation of virus spillover events that have taken place over the last few decades, particularly in Asia and Africa. Therefore, these bat-associated epidemics provide a significant number of hints, including respiratory cellular tropism, more intense susceptibility to these cell types, and overall likely to become a pandemic for the next spillover. In this systematic review, we analysed data to insight, through bat-originated spillover in Asia and Africa. We used STATA/IC-13 software for descriptive statistics and meta-analysis. The random effect of meta-analysis showed that the pooled estimates of case fatality rates of bat-originated viral zoonotic diseases were higher in Africa (61.06%, 95%CI: 50.26 to 71.85, l2 % = 97.3, p < 0.001). Moreover, estimates of case fatality rates were higher in Ebola (61.06%; 95%CI: 50.26 to 71.85, l2 % = 97.3, p < 0.001) followed by Nipah (55.19%; 95%CI: 39.29 to 71.09, l2 % = 94.2, p < 0.001), MERS (18.49%; 95%CI: 8.19 to 28.76, l2 % = 95.4, p < 0.001) and SARS (10.86%; 95%CI: 6.02 to 15.71, l2 % = 85.7, p < 0.001) with the overall case fatality rates of 29.86 (95%CI: 29.97 to 48.58, l2 % = 99.0, p < 0.001). Bat-originated viruses have caused several outbreaks of deadly diseases, including Nipah, Ebola, SARS and MERS in Asia and Africa in a sequential fashion. Nipah virus emerged first in Malaysia, but later, periodic outbreaks were noticed in Bangladesh and India. Similarly, the Ebola virus was detected in the African continent with neurological disorders in humans, like Nipah, seen in the Asian region. Two important coronaviruses, MERS and SARS, were introduced, both with the potential to infect respiratory passages. This paper explores the dimension of spillover events within and/or between bat-human and the epidemiological risk factors, which may lead to another pandemic occurring. Further, these processes enhance the bat-originated virus, which utilises an intermediate host to jump into human species.

Assessing the health hazard originated via pesticide chemicals in human through rabbit model in agricultural production system in Bangladesh.

BACKGROUND: The use of chemical pesticides in developing countries like Bangladesh and their impacts on human health and food security is a global concern. Bangladesh is an agricultural dependent country for the growing population demand for food security and food safety. We conduct this study to assess public health threats of commonly utilised pesticides including malathion and nitrobenzene in female rabbit model. METHODS: Thirty New Zealand White healthy rabbit was divided randomly into three groups; and subjected to distilled water as control, malathion@ 5 mg/kg body weight and nitrobenzene@ 5 mg/kg body i.p daily for the next 15 days. Hematology, serum biochemistry and hormonal assay were performed. RESULTS: Red blood cell (RBC) concentrations (TEC, Hb, PCV%) were reduced in rabbits exposed to malathion and nitrobenzene. The neutrophil and eosinophil percentage were increased in the malathion and nitrobenzene exposed juvenile rabbit group. We found that serum aspartate aminotransferase (AST) and creatinine were increased in the nitrobenzene exposed group in infants, whereas malathion exposure increased serum alanine aminotransferase (ALT). In contrast, the juvenile group exposed to malathion increased the ALT level. There was no change in AST or creatinine levels in juvenile rabbits exposed to malathion or nitrobenzene. Serum estradiol levels were significantly lower in rabbits exposed to malathion and nitrobenzene. Serum testosterone concentration was increased in juvenile rabbits exposed to malathion and nitrobenzene, but progesterone was decreased in malathion exposed juvenile rabbits. CONCLUSION: However, this study highlights the importance of rigorous monitoring and testing of agricultural products. In addition, strengthen research and extension in the fields of agro economy, organic farming, local universities and farmer associations.

Knowledge, Attitudes, and Common Practices of Livestock and Poultry Veterinary Practitioners Regarding the AMU and AMR in Bangladesh.

Current evidence indicates that more than half of all antimicrobials are used in the animal food-producing sector, which is considered a significant risk factor for the development, spread, and existence of antimicrobial resistance (AMR) pathogens in animals, humans, and the environment. Among other factors, clinical etiology and the level of knowledge, attitudes, and practices (KAP) of veterinarians are thought to be responsible for inappropriate prescriptions in the animal-source protein production sector in lower-resource settings like Bangladesh. We performed this cross-sectional study to assess factors associated with veterinarians' antimicrobial prescription behavior and their KAP on antimicrobial use (AMU) and AMR in Bangladesh. Exploratory and multivariate logistic models were used to describe an association between knowledge, attitudes, and practices of AMU and AMR and demographic characteristics of veterinarians. The results demonstrated that when selecting an antimicrobial, there was no to minimal influence of culture and susceptibility tests and patients' AMU history but moderate to high influence of the farmer's economic condition and drug instructions among the veterinarians. The results also demonstrated that more than half of the veterinarians had correct KAP regarding AMU and AMR, while the rest had moderate or lower levels of KAP. The factor score analysis revealed that age, level of education, years of experience, gender, and previous training on AMU and AMR were the key influencing factors in their level of KAP. Adjusted logistic regression analysis showed that respondents' age, current workplace, and previous training on AMU and AMR had a positive association with increased KAP. Considering the results, it is imperative to include AMR issues on vet curricula, and to provide post-education training, awareness campaigns, easy access to, and dissemination of AMR resources. Increasing the veterinary services to the outreach areas of the country and motivating veterinarians to follow the national AMR guidelines could be some other potential solutions to tackle the over-prescriptions of antimicrobials.

Knowledge, Attitude, and Practices on Antimicrobial Use and Antimicrobial Resistance among Commercial Poultry Farmers in Bangladesh.

Antimicrobial resistance (AMR) has become an emerging health issue globally, posing a threat to zoonotic pathogens and foodborne diseases. In Bangladesh, the poultry sector supplies the majority of the demand for animal-source protein. The irrational and excessive use of antimicrobials (AMU) has been observed in the poultry sector. The development of AMR is associated with many factors, including the knowledge and attitudes of poultry farmers. Therefore, AMR reduction requires intervention from all the stockholders, including the farmers who are considered as end users of antimicrobials. This current research conducted a cross-sectional study to assess the knowledge, attitudes, and practices (KAP) of poultry farmers on AMU and AMR in Bangladesh. We determined the KAP of poultry farmers (broiler and layer farmers) of some selected districts of the country using a tested and paper-based questionnaire. The results demonstrated that most of the respondents have insufficient KAP regarding AMU and AMR. The respondents used a variety of antimicrobials primarily in the treatment of various diseases in poultry. One-third of the farmers did not seek antimicrobials from registered vets. Instead, they depended on others or themselves. The factor score analysis further revealed that the farmers' demographic and socioeconomic variables were significant factors influencing the KAP. An adjusted logistic regression analysis showed that older farmers with 9-12 years of farming experience and graduate-level education, engaging in medium-sized layer farming, were more likely to have correct KAP on AMU and AMR. Further, farmers from the Cox's Bazar region showed correct knowledge, whereas farmers of the Chattogram region showed a correct attitude towards AMU and AMR. A Spearman's rank-order correlation revealed a positive association between knowledge-attitudes and knowledge-practices. The findings of the current investigation provide baseline evidence about the KAP of poultry farmers from low-income resources and offer insights into designing interventions and policies for the use of AMU and AMR in Bangladesh.

Knowledge, Attitude, and Practices on Antimicrobial Use and Antimicrobial Resistance among Poultry Drug and Feed Sellers in Bangladesh.

Poultry production has boomed in Bangladesh in recent years. The poultry sector has contributed significantly to meet the increased demand for animal source proteins in the country. However, increased use of antimicrobials appeared to be a significant threat to food safety in the poultry sector. The poultry drug and feed sellers are at the frontline position involving selecting and delivering the antimicrobials to the poultry farmers. Studies assessing the poultry drug and feed sellers' knowledge, attitudes, and practices (KAPs) are limited. The current study aimed to assess the community poultry drug and feed sellers' KAPs of antimicrobial use (AMU) and antimicrobial resistance (AMR) in some selected areas of Bangladesh. We determined the respondents' (drug and the feed sellers) KAPs of AMU and AMR using a tested and paper-based questionnaire. The study demonstrated that most respondents have insufficient knowledge, less positive attitudes, and inappropriate practices regarding AMU and AMR. The factor score analysis further showed that the type of respondents and their years of experience, level of education, and training on the drug were the significant factors impacting the current knowledge, attitudes, and practices of AMU and AMR. The adjusted logistic regression analysis revealed that the drug sellers who completed their education up to 12th grade and had training on the drug had adequate knowledge of AMU and AMR. The data also showed that the drug sellers belong to the age group 31-35 and 36-40 years and who completed 12th grade had good attitudes on the same. Likewise, the analysis further determined that drug sellers belonging to the age category 18-25 and 26-30 years, and interestingly, the respondents who completed education up to 12th grade, had better practices. Spearman's rank-order correlation revealed a positive association between each pair of the KAPs scores for the respondents. The correlation was fair between knowledge-attitudes, knowledge-practices, and attitudes-practices. Based on the current study results, we recommend educational interventions and appropriate training for the poultry drug and feed sellers to raise awareness and to upgrade their current knowledge on the appropriate use of antimicrobials. This will ultimately lead to reducing the chances of developing AMR in the poultry sectors of the country.

Residual antimicrobial agents in food originating from animals.

BACKGROUND: The agricultural food products industry in Bangladesh depends on utilizing antimicrobials indiscriminately as growth promoters and for controlling infectious diseases. Thus, there is always a risk of antimicrobial agent accumulation in food sources that originate from agricultural production. METHODS: In the present study, we collected data from published articles between January, 2013 and December, 2019 on antimicrobial residues in human food sources such as meat, milk, eggs, and fishes. RESULTS: Liver contained the highest percentage of antimicrobial residues (74%; 95% CI: 59.66-85.37) against the in vitro enteric pathogen Escherichia coli in layer chickens. Similar results were demonstrated in liver (68%; 95% CI: 53.30-80.48) and kidney (66%, 95% CI: 51.23-78.79) of layer chickens against Bacillus cereus and Bacillus subtilis. Amongst all antibiotics, the highest concentrations of ciprofloxacin were detected in kidney (48.57%; 95% CI: 31.38-66.01), followed by liver (47.56; 95% CI: 40.88-54.30) of broiler chickens. Ciprofloxacin was also present in liver (46.15%; 95% CI: 33.70-58.96) of layer chickens. The percentage of ciprofloxacin in thigh and breast meat in broiler bird were 41.54% (95% CI: 34.54-48.79) and 37.95% (95% CI: 31.11-45.15) respectively. Enrofloxacin was the second most dominant antimicrobial agent and was present in the liver of both types of poultry (Broiler and Layer chickens: 41.54%; 95% CI: 29.44-54.4 and 437.33%; 95% CI: 30.99-44.01). The prevalence rates of enrofloxacin in thigh and breast meat of broiler chickens were 24.10% (95% CI: 18.28-30.73) and 20.51% (95% CI: 15.08-26.87), respectively. Tetracycline, a commonly used antibiotic in livestock, was present in the liver (49.23%; 95% CI: 36.60-61.93) of layer chickens. In case of aquaculture food products, the highest amount of amoxicillin (683.2 mg/kg) was detected in Tilapia fish (Oreochromis niloticus), followed by 584.4 mg/kg in climbing perch (Anabas testudineus) and 555.6 mg/kg in Rui fish (Labeo rohita). Among the five types of fishes, Rui fish (0.000515 mg/kg) contained the highest concentrations of chloramphenicol antibiotic residues. CONCLUSIONS: The presence of antimicrobial residues in meat, milk, egg, and fish is a serious public health threat due to the potential induction of antimicrobial resistance. It can negatively impact the food supply chain, especially with the current strain that it is already facing with the current COVID-19 pandemic. The findings of the present study highlight the ongoing risk of residual antimicrobial agents in food of animal origin in Bangladesh and countries with similar practices. This can draw the attention of public health officials to propose plans to mitigate or stop this practice.

Understanding the Community Perceptions and Knowledge of Bats and Transmission of Nipah Virus in Bangladesh.

Bats are known reservoirs of Nipah virus (NiV) and some filoviruses and also appear likely to harbor the evolutionary progenitors of severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV). While bats are considered a reservoir of deadly viruses, little is known about people's knowledge, attitudes, and perceptions of bat conservation and ecology. The current study aimed to assess community people's knowledge, attitudes, and perceptions of bat ecology, myths, and the role of bats in transmitting NiV in Bangladesh. Since 2001, NiV has been a continuous threat to public health with a mortality rate of approximately 70% in Bangladesh. Over the years, many public health interventions have been implemented to raise awareness about bats and the spreading of NiV among the community peoples of Nipah outbreak areas (NOAs) and Nipah non-outbreak areas (NNOAs). We hypothesized that people from both areas might have similar knowledge of bat ecology and myths about bats but different knowledge regarding their role in the spreading of NiV. Using a four-point Likert scale-based questionnaire, our analysis showed that most people lack adequate knowledge regarding the role of bats in maintaining the ecological balance and instead trust their beliefs in different myths about bats. Factor score analysis showed that respondents' gender (p = 0.01), the outbreak status of the area (p = 0.03), and their occupation (p = 0.04) were significant factors influencing their knowledge of bat ecology and myths. A regression analysis showed that farmers had 0.34 times the odds of having correct or positive knowledge of bat ecology and myths than businesspersons (odds ratio (OR) = 0.34, 95% confidence interval (95% CI) = 0.15-0.78, p = 0.01). Regarding the spreading of NiV via bats, people had a lower level of knowledge. In NOAs, age (p = 0.00), occupation (p = 0.00), and level of education (p = 0.00) were found to be factors contributing to the amount of knowledge regarding the transmission of NiV, whereas in NNOAs, the contributing factors were occupation (p = 0.00) and level of education (p = 0.01). Regression analysis revealed that respondents who were engaged in services (OR = 3.02, 95% CI = 1.07-8.54, p = 0.04) and who had completed primary education (OR = 3.06, 95% CI = 1.02-9.17, p < 0.05) were likely to have correct knowledge regarding the spreading of NiV. Based on the study results, we recommend educational interventions for targeted groups in the community, highlighting the ecosystem services and conservation of bats so as to improve people's current knowledge and subsequent behavior regarding the role of bats in ecology and the spreading of NiV in Bangladesh.

Antimicrobial resistance pattern in domestic animal - wildlife - environmental niche via the food chain to humans with a Bangladesh perspective; a systematic review.

BACKGROUND: Antimicrobial resistance (AMR) is a growing concern globally, but the impact is very deleterious in the context of Bangladesh. Recent review article on the AMR issue demonstrates the scenario in human medicine; unfortunately, no attempt was taken to address this as One Health issue. The antimicrobial resistance bacteria or genes are circulating in the fragile ecosystems and disseminate into human food chain through direct or indirect ways. In this systematic review we are exploring the mechanism or the process of development of resistance pathogen into human food chain via the domestic animal, wildlife and environmental sources in the context of One Health and future recommendation to mitigate this issue in Bangladesh. RESULTS: Tetracycline resistance genes were presenting in almost all sample sources in higher concentrations against enteric pathogen Escherichia coli. The second most significant antibiotics are amino-penicillin that showed resistant pattern across different source of samples. It is a matter of concerns that cephalosporin tends to acquire resistance in wildlife species that might be an indication of this antibiotic resistance gene or the pathogen been circulating in our surrounding environment though the mechanism is still unclear. CONCLUSIONS: Steps to control antibiotic release and environmental disposal from all uses should be immediate and obligatory. There is a need for detailed system biology analysis of resistance development in-situ.

Vaccination of koalas (Phascolarctos cinereus) against Chlamydia pecorum using synthetic peptides derived from the major outer membrane protein.

Chlamydia pecorum is a mucosal infection, which causes debilitating disease of the urinary tract, reproductive tract and ocular sites of koalas (Phascolarctos cinereus). While antibiotics are available for treatment, they are detrimental to the koalas' gastrointestinal tract microflora leaving the implementation of a vaccine as an ideal option for the long-term management of koala populations. We have previously reported on the successes of an anti-chlamydial recombinant major outer membrane protein (rMOMP) vaccine however, recombinant protein based vaccines are not ideal candidates for scale up from the research level to small-medium production level for wider usage. Peptide based vaccines are a promising area for vaccine development, because peptides are stable, cost effective and easily produced. In this current study, we assessed, for the first time, the immune responses to a synthetic peptide based anti-chlamydial vaccine in koalas. Five healthy male koalas were vaccinated with two synthetic peptides derived from C. pecorum MOMP and another five healthy male koalas were vaccinated with full length recombinant C. pecorum MOMP (genotype G). Systemic (IgG) and mucosal (IgA) antibodies were quantified and pre-vaccination levels compared to post-vaccination levels (12 and 26 weeks). MOMP-peptide vaccinated koalas produced Chlamydia-specific IgG and IgA antibodies, which were able to recognise not only the genotype used in the vaccination, but also MOMPs from several other koala C. pecorum genotypes. In addition, IgA antibodies induced at the ocular site not only recognised recombinant MOMP protein but also, whole native chlamydial elementary bodies. Interestingly, some MOMP-peptide vaccinated koalas showed a stronger and more sustained vaccine-induced mucosal IgA antibody response than observed in MOMP-protein vaccinated koalas. These results demonstrate that a synthetic MOMP peptide based vaccine is capable of inducing a Chlamydia-specific antibody response in koalas and is a promising candidate for future vaccine development.

Immunization of a wild koala population with a recombinant Chlamydia pecorum Major Outer Membrane Protein (MOMP) or Polymorphic Membrane Protein (PMP) based vaccine: New insights into immune response, protection and clearance.

We assessed the effects of two different single-dose anti-Chlamydia pecorum (C. pecorum) vaccines (containing either Major Outer Membrane Protein (3MOMP) or Polymorphic Membrane Protein (Pmp) as antigens) on the immune response of a group of wild koalas. Both vaccines elicited a systemic humoral response as seen by the production of anti-chlamydial IgG antibodies in more than 90% of vaccinated koalas. A mucosal immune response was also observed, with an increase in Chlamydia-specific mucosal IgG and/or IgA antibodies in some koalas post-vaccination. Both vaccines elicited a cell-mediated immune response as measured by the production of the cytokines IFN-γ and IL-17 post-vaccination. To determine the level of protection provided by the vaccines under natural conditions we assessed C. pecorum infection loads and chlamydial disease status of all vaccinated koalas pre- and post-vaccination, compared to a non-vaccinated cohort from the same habitat. The MOMP vaccinated koalas that were infected on the day of vaccination showed significant clearance of their infection at 6 months post-vaccination. In contrast, the number of new infections in the PMP vaccine was similar to the control group, with some koalas progressing to disease. Genotyping of the ompA gene from the C. pecorum strains infecting the vaccinated animals, identified genetic variants of ompA-F genotype and a new genotype ompA-O. We found that those animals that were the least well protected became infected with strains of C. pecorum not covered by the vaccine. In conclusion, a single dose vaccine formulated with either recombinant PmpG or MOMP can elicit both cell-mediated and humoral (systemic and mucosal) immune responses, with the MOMP vaccine showing clearance of infection in all infected koalas. Although the capability of our vaccines to stimulate an adaptive response and be protective needs to be fully evaluated, this work illustrates the necessity to combine epitopes most relevant to a large panel of variable strains with an efficient adjuvant.

Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus) Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP) with Two Different Adjuvants.

Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus). In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP) antigen-based vaccine, combined with immune stimulating complex (ISC) adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri-adjuvant formula that comprises polyphosphazine based poly I: C and host defense peptides, with the same antigen. This formulation also produced strong cellular and humoral immune responses in captive koalas. In this current study, we directly compared the host immune responses of two sub-groups of wild Chlamydia negative koalas in one population vaccinated with the rMOMP protein antigen and adjuvanted with either the ISC or tri-adjuvant formula. Overall, both adjuvants produced strong Chlamydia-specific cellular (IFN-γ and IL-17A) responses in circulating PBMCs as well as MOMP-specific and functional, in vitro neutralising antibodies. While the immune responses were similar, there were adjuvant-specific immune differences between the two adjuvants, particularly in relation to the specificity of the MOMP epitope antibody responses.

Humoral immune responses in koalas (Phascolarctos cinereus) either naturally infected with Chlamydia pecorum or following administration of a recombinant chlamydial major outer membrane protein vaccine.

The development of a vaccine is a key strategy to combat the widespread and debilitating effects of chlamydial infection in koalas. One such vaccine in development uses recombinant chlamydial major outer membrane protein (rMOMP) as an antigen and has shown promising results in several koala trials. Previous chlamydial vaccine studies, primarily in the mouse model, suggest that both cell-mediated and antibody responses will be required for adequate protection. Recently, the important protective role of antibodies has been highlighted. In our current study, we conducted a detailed analysis of the antibody-mediated immune response in koalas that are either (a) naturally-infected, and/or (b) had received an rMOMP vaccine. Firstly, we observed that naturally-infected koalas had very low levels of Chlamydia pecorum-specific neutralising antibodies. A strong correlation between low IgG total titers/neutralising antibody levels, and higher C. pecorum infection load was also observed in these naturally-infected animals. In vaccinated koalas, we showed that the vaccine was able to boost the humoral immune response by inducing strong levels of C. pecorum-specific neutralising antibodies. A detailed characterisation of the MOMP epitope response was also performed in naturally-infected and vaccinated koalas using a PepScan epitope approach. This analysis identified unique sets of MOMP epitope antibodies between naturally-infected non-protected and diseased koalas, versus vaccinated koalas, with the latter group of animals producing a unique set of specific epitope-directed antibodies that we demonstrated were responsible for the in vitro neutralisation activity. Together, these results show the importance of antibodies in chlamydial infection and immunity following vaccination in the koala.

A Prototype Recombinant-Protein Based Chlamydia pecorum Vaccine Results in Reduced Chlamydial Burden and Less Clinical Disease in Free-Ranging Koalas (Phascolarctos cinereus).

Diseases associated with Chlamydia pecorum infection are a major cause of decline in koala populations in Australia. While koalas in care can generally be treated, a vaccine is considered the only option to effectively reduce the threat of infection and disease at the population level. In the current study, we vaccinated 30 free-ranging koalas with a prototype Chlamydia pecorum vaccine consisting of a recombinant chlamydial MOMP adjuvanted with an immune stimulating complex. An additional cohort of 30 animals did not receive any vaccine and acted as comparison controls. Animals accepted into this study were either uninfected (Chlamydia PCR negative) at time of initial vaccination, or infected (C. pecorum positive) at either urogenital (UGT) and/or ocular sites (Oc), but with no clinical signs of chlamydial disease. All koalas were vaccinated/sampled and then re-released into their natural habitat before re-capturing and re-sampling at 6 and 12 months. All vaccinated koalas produced a strong immune response to the vaccine, as indicated by high titres of specific plasma antibodies. The incidence of new infections in vaccinated koalas over the 12-month period post-vaccination was slightly less than koalas in the control group, however, this was not statistically significant. Importantly though, the vaccine was able to significantly reduce the infectious load in animals that were Chlamydia positive at the time of vaccination. This effect was evident at both the Oc and UGT sites and was stronger at 6 months than at 12 months post-vaccination. Finally, the vaccine was also able to reduce the number of animals that progressed to disease during the 12-month period. While the sample sizes were small (statistically speaking), results were nonetheless striking. This study highlights the potential for successful development of a Chlamydia vaccine for koalas in a wild setting.

Vaccination of koalas (Phascolarctos cinereus) with a recombinant chlamydial major outer membrane protein adjuvanted with poly I:C, a host defense peptide and polyphosphazine, elicits strong and long lasting cellular and humoral immune responses.

Chlamydial infections are wide spread in koalas across their range and a solution to this debilitating disease has been sought for over a decade. Antibiotics are the currently accepted therapeutic measure, but are not an effective treatment due to the asymptomatic nature of some infections and a low efficacy rate. Thus, a vaccine would be an ideal way to address this infectious disease threat in the wild. Previous vaccine trials have used a three-dose regimen; however this is very difficult to apply in the field as it would require multiple capture events, which are stressful and invasive processes for the koala. In addition, it requires skilled koala handlers and a significant monetary investment. To overcome these challenges, in this study we utilized a polyphosphazine based poly I:C and a host defense peptide adjuvant combined with recombinant chlamydial major outer membrane protein (rMOMP) antigen to induce long lasting (54 weeks) cellular and humoral immunity in female koalas with a novel single immunizing dose. Immunized koalas produced a strong IgG response in plasma, as well as at mucosal sites. Moreover, they showed high levels of C. pecorum specific neutralizing antibodies in the plasma as well as vaginal and conjunctival secretions. Lastly, Chlamydia-specific lymphocyte proliferation responses were produced against both whole chlamydial elementary bodies and rMOMP protein, over the 12-month period. The results of this study suggest that a single dose rMOMP vaccine incorporating a poly I:C, host defense peptide and polyphosphazine adjuvant is able to stimulate both arms of the immune system in koalas, thereby providing an alternative to antibiotic treatment and/or a three-dose vaccine regime.

A survey of gastro-intestinal parasitic infection in domestic and wild birds in Chittagong and Greater Sylhet, Bangladesh.

A survey of gastrointestinal parasitic infection as determined by faecal examination was conducted among domestic and wild birds in Bangladesh. Birds were sampled from households, wet markets and wetlands in Chittagong and Greater Sylhet districts during April 2012 to February 2013. Mist nets were used to catch resident wild and migratory birds. The overall prevalence of parasitic infection ranged among locations from 25 to 55% in indigenous domestic ducks (live bird samples=304), 20% in resident wild birds (environmental faecal samples=40) and 40% in migratory birds (live bird samples=35). The prevalence of parasitic infection was significantly higher in indigenous domestic ducks collected during summer (39%) than winter (22%) (p=0.04). In domestic indigenous ducks and Muscovy ducks, both single and multiple types of parasitic infections were found. However, other domestic birds and wild birds often had a single type of parasitic infection. Ascaridia spp. with an average egg load of 50-900, was commonly detected in faecal samples of domestic and wild birds in this study. Other identified parasites were Capillaria spp. and Heterakis spp. both in domestic and wild birds. Improvement of biosecurity measures for household duck farms through educating and motivating household farmers could help mitigate the effects of parasitic infection on production.

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.