Molecular identification, antimicrobial resistance and virulence gene profiling of Staphylococcus spp. associated with bovine sub-clinical mastitis in Bangladesh.

This study was conducted to investigate the diversity and antimicrobial resistance profiling of Staphylococcus species causing sub-clinical mastitis (SCM) in dairy herds in Bangladesh as well as putative risk factors associated with the infections. Individual quarter milk samples were collected from a total of 284 lactating cows from 30 dairy farms were screened by means of California mastitis test; 178 (62.7%) of them had at least of quarter affected by SCM. After conventional microbiological isolation procedures, PCR tests were used for Staphylococcus species identification and detection of antimicrobial resistance and virulence genes. S. chromogenes (65.7%) was the most predominant species followed by, S. epidermidis (20.2%), S. haemolyticus (19.1%), S. aureus (15.7%), and S. sciuri (5.6%). High levels of antimicrobial resistance to ampicillin and amoxicillin/clavulanic acid were observed in S. aureus (82.1% and 75%) and S. sciuri (80% and 70%), while resistance to cefepime was markedly higher in S. chromogenes (95.7%), S. haemolyticus (94.1%), and S. epidermidis (97.2%). Multidrug resistance isolates were identified in all five species. The mecA gene was detected in S. aureus (32.1%) and S. chromogenes (5.98%). In addition, 20% S. sciuri and 17.7% S. haemolyticus carried the cytotoxin (pvl) gene, while 14.3% S. aureus harbored the toxic shock syndrome toxin (tst) gene. Multivariable logistic regression analysis identified "Old aged" (OR [CI]: 3.5 [1-12.4]); "Early stage of lactation" (OR [CI]: 3.4 [1.2-9.7]) and, "Firm udder condition" (OR [CI]: 4.2 [1.2-14.6]) as risk factors associated with SCM caused by S. aureus, S. chromogenes, and S. haemolyticus, respectively. Moreover, "Use of antimicrobials" (OR [CI]: 10.4 [3.4-32.1] and "History of previous clinical mastitis" (OR [CI]: 4.9 [1.2-19.7] for the carriage of methicillin-resistant Staphylococcus spp.

Phenotypical Identification and Toxinotyping of Clostridium perfringens Isolates from Healthy and Enteric Disease-Affected Chickens.

Clostridium perfringens is a ubiquitous spore-forming anaerobic pathogen that is frequently associated with enteric disease in chickens. Moreover, enterotoxin-producing C. perfringens has high zoonotic potential as well as serious public health concerns due to the emanation of food-borne intoxication. The present study was designed to isolate, identify, and toxinotype C. perfringens from both healthy and cases of necrotic or ulcerative enteritis chickens. A total of 110 samples were collected from July 2019 to February 2021. Among the samples, 38 (34.5%, 95% CI: 26.39-43.83) were positive for C. perfringens and were obtained from broiler 21 (33.3%, 95% CI: 22.91-45.67), Sonali 9 (34.6%, 95% CI: 19.31-53.88), and layer 8 (38%, 95% CI: 20.68-59.20). C. perfringens was highly prevalent (35.7%, 95% CI: 25.48-47.44) in enteritis chickens compared with healthy ones. In multiplex PCR toxinotyping, 34 (89.4%) isolates were identified as C. perfringens type A by the presence of the alpha toxin gene (cpa). Moreover, in addition to the cpa gene, 3 (14.3%, 95% CI: 4.14-35.48) broiler and 1 (11.1%, 95% CI: 0.01-45.67) Sonali isolates harbored the enterotoxin gene (cpe) and were classified as type F. However, none of the isolates carried genes encoding beta (cpb), epsilon (etx), iota (iap), or beta-2 (cpb2) toxins. Multivariable logistic regression analysis identified the following variables such as; "previously used litter materials" (OR 21.77, 95% CI 2.22-212.66, p ≤ 0.008); intestinal lesions, "presence of ulceration" (OR 30.01, 95% CI 3.02-297.91, p ≤ 0.004); "ballooned with gas" (OR 24.74, 95% CI 4.34-140.86, p ≤ 0.001) and "use of probiotics" (OR 5.24, 95% CI 0.74-36.75, p ≤ 0.095) act as risk factors for C. perfringens colonization in chicken gut. This is the first study of molecular toxinotyping of C. perfringens from healthy and enteric-diseased chickens in Bangladesh, which might have a potential food-borne zoonotic impact on human health.

Colistin Resistance in Multidrug-Resistant Escherichia coli Isolated from Retail Broiler Meat in Bangladesh.

The emergence of colistin resistance in Escherichia coli is a global public health concern. Contaminated food can accelerate the spread of colistin-resistant E. coli to humans. This study aimed to detect and characterize colistin-resistant E. coli from broiler meat in Bangladesh. We analyzed 136 pooled broiler meat samples from 240 carcasses collected from 40 live bird markets in urban and rural areas and 8 metropolitan supermarkets. The mean count of E. coli in broiler meat samples collected from rural retail shops, metropolitan supermarkets, and urban retail shops was 5.3 ± 1.1, 4.1 ± 1.4, and 3.9 ± 0.8 log10 colony-forming unit per gram, respectively. Colistin-resistant E. coli (minimum inhibitory concentration >2 mg/L) was found in 78% (95% confidence interval 70.2-84.1%) of the samples. All colistin-resistant isolates harbored the mcr-1 gene, while the rest of the mcr genes (mcr-2 to mcr-9) were not detected. Most colistin-resistant E. coli isolates (98%) showed coresistance to tetracycline, sulfamethoxazole/trimethoprim followed by ciprofloxacin (95%). Alarmingly, all of the colistin-resistant isolates were found to be multidrug resistant. Phylogenetic analysis showed close similarities of the mcr-1 gene sequences of this study with many strains of Enterobacterales isolated from humans, animals, and the environment. This study detected colistin-resistant E. coli contamination in broiler meat, which can pose a serious public health threat.

Prevalence of coagulase-positive methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in dogs in Bangladesh.

BACKGROUND: The emergence of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have a significant health impact on people with direct or supportive occupations in veterinary medicine including veterinarians, animal handlers, laboratory personnel and pet owners. OBJECTIVES: This cross-sectional survey was conducted to determine the prevalence of and risk factors for S. aureus, S. pseudintermedius, MRSA and MRSP in dogs in Bangladesh. METHODS: A total of 358 swab samples were collected from different body sites of 150 dogs attending a university teaching veterinary hospital between January and June 2018. Standard bacteriological procedures were followed to isolate Staphylococcus, and identification was confirmed to the species level by PCR to detect the nuc gene. MRSA and MRSP were confirmed by the presence of the mecA gene. RESULTS: The prevalence of coagulase-positive S. aureus and S. pseudintermedius in dogs were 16% and 45.3%, respectively. S. aureus and S. pseudintermedius isolates displayed the highest resistance against nalidixic acid (95.2% and 91%, respectively) and erythromycin (89.3% and 84.7%, respectively). Notably, all the staphylococcal isolates showed resistance to ≥3 antimicrobial classes. The prevalence of MRSA and MRSP in dogs was 8.7% and 6%, respectively. Multivariable logistic regression analysis identified the following variables as risk factors for MRSA colonisation in dogs: dogs with dermatitis (odds ratio [OR], 12.24, 95% CI: 3.12-57.33; p < 0.001) and history of antibiotic use (OR 8.73, 95% CI: 2.23-43.10; p < 0.001). Presence of otitis (OR 14.22; 95% CI: 1.64-103.58; p = 0.008) and oral lesions (OR 9.48, 95% CI: 1.14-64.82; p = 0.002) were identified as the significant risk factors for the carriage of MRSP. CONCLUSIONS: The circulation of multidrug-resistant S. aureus and S. pseudintermedius is a serious concern to dogs and humans. To our knowledge, this is the first report of S. pseudintermedius and MRSP affecting dogs in Bangladesh.

Antimicrobial resistance profiling and burden of resistance genes in zoonotic Salmonella isolated from broiler chicken.

BACKGROUND: Salmonella is frequently found in poultry of which only motile serovars have zoonotic significance due to their potential to induce human gastrointestinal infections. Antimicrobial resistance, being a public health concern, the emergence of multidrug-resistant (MDR) Salmonella serotypes affecting food chain has greater impact worldwide. AIM: Information on circulation of zoonotic Salmonella strains in commercial poultry farm level is limited in many parts of the world. This cross-sectional study was aimed to investigate the zoonotic Salmonella strains circulating in the broiler farm environment with their detailed antimicrobial resistance profiling. METHODS: Pooled faecal samples were collected randomly from commercial broiler farms of Chattogram district, Bangladesh. Standard bacteriological procedure was followed to isolate Salmonella, and identification was confirmed by genus specific polymerase chain reaction (PCR). After phenotypic characterisation of resistance profile against eight antimicrobials by disc diffusion technique, all strains were screened by PCR for some selected resistance genes. RESULTS: Out of the 350 samples, Salmonella was isolated and identified from 86 samples. In antimicrobial sensitivity testing, more than 98.8% isolates showed resistance to ampicillin and 94.2% to tetracycline followed by enrofloxacin (56%) and ciprofloxacin (50%). Notably, 94% isolates were found to be MDR. The results of PCR assays revealed that 81.4% of the isolates were carrying the tetA gene, 19.8% the tetB and 10.47% the tetC gene. The prevalence of the isolates bearing the blaTEM , blaCTX-M and Sul-I gene were 95.4%, 7.0 % and 37.2 %, respectively. CONCLUSION: There is a great risk to secure healthy poultry products due to the circulation of these MDR zoonotic Salmonella.

Isolation and antimicrobial resistance of motile Salmonella enterica from the poultry hatchery environment.

Salmonella is a globally distributed major food-borne pathogen and poultry is one of the predominant sources of salmonellosis in humans. To investigate the presence of motile Salmonella in the poultry hatchery environment, we collected 97 fluff samples from four selected broiler breeder chicken hatcheries from Chattogram, Bangladesh during July-December 2015. To isolate motile Salmonella enterica, we used conventional bacteriological techniques followed by serological verification using anti-Salmonella Poly A-E serum and species confirmation by conventional PCR assay. Antimicrobial susceptibility testing by Kirby-Bauer disc diffusion method for 10 commonly used antibiotics was performed on all isolates. Isolates displaying phenotypic resistance to ampicillin were tested by PCR for blaTEM gene, whereas those resistant to tetracycline were tested for the presence of tetA, tetB and tetC genes. A total of 24 samples (24.7%; 95% CI: 16.5-34.5, N = 97) from 3 hatcheries were positive for motile Salmonella. Of them, 21 (87.5%) and 12 (50.0%) were resistant to ampicillin and tetracycline, respectively, 9 (37.5%) to nalidixic acid and sulphamethoxazole/trimethoprim. No resistance was detected to ceftriaxone, cefoxitin, gentamicin, neomycin, ciprofloxacin and colistin. Ten (42%) of 24 isolates from 2 hatcheries were multi-drug resistant (i.e. resistant to ≥ 3 antimicrobial classes). Six of 21 ampicillin resistant isolates contained blaTEM gene and 10 of 12 tetracycline resistant isolates contained tetA gene. This study highlights the circulation of multi-drug resistant motile Salmonella in the hatchery environment for the first time in Bangladesh. Further epidemiological and molecular studies are therefore needed to identify the serotypes and source of the bacteria in hatcheries.

Abundance of Mobilized Colistin Resistance Gene (mcr-1) in Commensal Escherichia coli from Diverse Sources.

Aims: Antimicrobial resistance (AMR) spreads not only by pathogenic but also by commensal bacteria, and the latter can become a reservoir for resistance genes. This study was aimed to investigate the AMR patterns along with the presence of mobilized colistin resistance (mcr) genes in commensal Escherichia coli circulating in chickens, farm environments, street foods, and human patients. Materials and Methods: By a cross-sectional survey, isolates obtained from 530 samples were tested for their AMR profiles against 9 antimicrobials. Minimum inhibitory concentration (MIC) of the phenotypically colistin-resistant isolates was determined and screened for a set of mcr genes followed by sequencing of mcr-1 gene in the multidrug-resistant (MDR) isolates. Results: A total of 313 E. coli strains were isolated and confirmed by polymerase chain reaction. Antimicrobial susceptibility testing revealed that about 98% (confidence interval [95% CI] 95-99) of the isolates were MDR, and 58% (95% CI 52-63) isolates exhibited resistance to colistin. MIC values of colistin against the isolates ranged from 4 to 64 mg/L. Except for human patients, 20.4% colistin-resistant isolates from other sources of isolation had mcr-1 gene. Conclusions: There is abundance of commensal MDR E. coli strains with the acquisition of mcr-1 gene circulating in chickens and farm environments in Bangladesh.

Molecular epidemiology and pathogenicity of H5N1 and H9N2 avian influenza viruses in clinically affected chickens on farms in Bangladesh.

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.

Coagulase-positive methicillin-resistant Staphylococcus aureus circulating in clinical mastitic goats in Bangladesh.

BACKGROUND AND AIM: Staphylococcus aureus is argued as one of the principal organisms responsible for mammary gland infection in lactating goats, causing both clinical and subclinical mastitis. Being highly zoonotic potential, pathogen emergence of methicillin-resistant S. aureus (MRSA) has a significant clinical impact on treatment and management of clinical mastitis. We conducted a cross-sectional study to investigate the prevalence of coagulase-positive S. aureus (CoPS), antimicrobial resistance profile of Staphylococcus spp., prevalence of MRSA, and association between different clinical parameters with CoPS. MATERIALS AND METHODS: A total of 67 clinical mastitic goats were sampled based on clinical examination and California mastitis test. Standard bacteriological methods were performed to isolate and identify Staphylococcus spp. CoPS were confirmed by nuc gene using polymerase chain reaction (PCR). All staphylococcal isolates were further examined for antimicrobial susceptibility testing by disk diffusion method. MRSA was confirmed based on mecA gene-based PCR. RESULTS: Here, 49 (73.13%; 95% confidence interval [CI], 61.41-82.35) samples were positive for Staphylococcus spp., of which 17 (34.69%; 95% CI, 22.88-48.73) isolates were CoPS and rest of the isolates (32; 65.30%; 95% CI, 51.27-77.12) were identified as coagulase-negative Staphylococcus spp. (coagulase-negative staphylococci [CNS]). Both, CoPS and CNS isolates displayed the highest resistance against tetracycline (76.47% and 75%, respectively) and oxacillin (70.58% and 68.75%, respectively). Notably, all staphylococcal isolates were multidrug-resistant (showed resistance to ≥3 classes of antimicrobials). mecA gene was found in 6 (8.96%; 95% CI, 3.84-18.52) CoPS isolates indicating MRSA strains. Among different clinical parameters, presence of high body temperature (p<0.05), firm udder consistency (p<0.01), bloodstained milk (p<0.00), and pus in milk (p<0.00) were significantly associated with the presence of CoPS in mastitic caprine milk. CONCLUSION: To the best of our knowledge, this is the first report of MRSA isolated from clinical caprine mastitis cases in Bangladesh. The findings of this study would help in cautious selection as well as administration of antimicrobials for therapeutic management of mastitic goats.

Circulation of oxytetracycline- and ciprofloxacin-resistant commensal Escherichia coli strains in broiler chickens and farm environments, Bangladesh.

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.

Acquisition of Plasmid-Mediated Colistin Resistance Gene mcr-1 in Escherichia coli of Livestock Origin in Bangladesh.

Aims: To investigate plasmid-borne colistin resistance mechanism (plasmid-mediated colistin resistance [mcr-1]) in Escherichia coli of human, veterinary, and environmental origin in Bangladesh. Materials and methods: A total of 810 samples were collected from different sources. Isolation and identification of E. coli was performed using classical bacteriology and then tested for antimicrobial susceptibility. Colistin-resistant isolates were further analyzed for mcr-1 gene using PCR. Minimum inhibitory concentration (MIC) was determined using microbroth dilution technique. After sequencing of mcr-1 gene, phylogenetics was conducted to see the relationship with other mcr-1 gene sequences. Results: A total of 358 E. coli were isolated from 810 samples of humans, animals, environment, and food in Bangladesh. Of them 49 (15.9%) isolates were phenotypically resistant to colistin and 254 (70.9%) were resistant to multiple antimicrobials. mcr-1 gene was detected in three E. coli isolates of poultry source. For the three mcr-1 positive isolates the MIC of colistin sulfate was 4, 8, and 128 µg/mL. Gene sequencing of two of the three mcr-1 positive isolates and phylogenetic analysis showed close similarities of one isolate to other mcr-1 sequences available at GenBank while the other appeared to have evolved locally. Conclusion: First-ever report on circulation of mcr-1 E. coli of livestock origin in Bangladesh.

Risk for infection with highly pathogenic avian influenza virus (H5N1) in backyard chickens, Bangladesh.

To evaluate risk factors for infection with highly pathogenic avian influenza A virus (H5N1) in backyard chickens in Bangladesh, we conducted a matched case-control study. We enrolled 25 case farms (cases March-November 2007) and 75 control farms (June-November 2007). We used a questionnaire to collect farm data, which were analyzed by matched-pair analysis and multivariate conditional logistic regression. Factors independently associated were offering slaughter remnants of purchased chickens to backyard chickens (odds ratio [OR] 13.29, 95% confidence interval [CI] 1.34-131.98, p = 0.027), having a nearby water body (OR 5.27, 95% CI 1.24-22.34, p = 0.024), and having contact with pigeons (OR 4.47, 95% CI 1.14-17.50, p = 0.032). Separating chickens and ducks at night was protective (OR 0.06, 95% CI 0.01-0.45, p = 0.006). Reducing these risks and taking protective measures might reduce the risk for influenza (H5N1) infection in backyard chickens.

Antibiotic resistant zoonotic bacteria in Irrawaddy squirrel (Callosciurus pygerythrus).

Irrawaddy squirrel (Callosciurus pygerythrus) may play an important role in the transmission of zoonotic bacteria, but little is known about the carriage of zoonotic bacteria in this common frugivorous rodent in Bangladesh. We aimed to investigate the presence of common zoonotic bacterial pathogens in Irrawaddy squirrel in the southeast part of Bangladesh. A total of 27 rectal and 27 oro-nasal swabs were collected from 27 healthy wild Irrawaddy squirrels. Four common zoonotic bacteria were isolated following routine laboratory procedures, and were identified based on colony morphology, and biochemical and staining properties. The pathogenic potential of the identified bacteria was confirmed by detection of virulence genes by PCR. All isolates were subjected to antimicrobial susceptibility test against seven antibiotics from six generic groups which are commonly used in human and veterinary medicine in Bangladesh. The prevalence of Escherichia coli, Salmonella spp., Yersinia spp. and Staphylococcus spp. was 44.4% (95% CI, 32.0-57.6), 13% (95% CI, 6.1-24.7), 44.4% (95% CI, 32.0-57.6), and 72.2% (95% CI, 59.0-82.5), respectively. We identified potential zoonotic virulence genes in all of these four bacterial species. Antimicrobial susceptibility testing revealed the presence of several multidrug resistant bacterial strains in squirrels. To the best of our knowledge, this is the first report in Bangladesh of the detection of antibiotic resistant zoonotic bacteria in Irrawaddy squirrels. The findings underpin the role of Irrawaddy squirrel as a source of pathogenic antibiotic resistant bacteria, consequently, fruit rejected because of squirrel consumption and squirrel-bites deserve more concern than previously.

Avian influenza outbreaks in chickens, Bangladesh.

To determine the epidemiology of outbreaks of avian influenza A virus (subtypes H5N1, H9N2) in chickens in Bangladesh, we conducted surveys and examined virus isolates. The outbreak began in backyard chickens. Probable sources of infection included egg trays and vehicles from local live bird markets and larger live bird markets.

Validation of real-time reverse transcription polymerase chain reaction to detect virus titer and thermostability of Newcastle disease live virus vaccine.

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.

Poultry as a possible source of non-typhoidal Salmonella enterica serovars in humans in Bangladesh.

We investigated Salmonella enterica isolates from human clinical cases of gastroenteritis to determine the distribution of non-typhoidal Salmonella serovars in the human population, and compared them to isolates originating from poultry by serotyping, phage typing, plasmid profiling, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) to evaluate the potential role of poultry in human non-typhoidal salmonellosis in Bangladesh. Nine different serovars were identified among the human isolates of which Salmonella Paratyphi B var Java (S. Java), S. Kentucky, S. Enteritidis, S. Virchow and S. Weltevreden also were commonly isolated from poultry. The poultry isolates belonging to S. Java, S. Kentucky and S. Enteritidis were indistinguishable from human isolates or genetically closely related, based on PFGE profiles and MLST. S. Kentucky clone ST198 and S. Java clone ST43 both well-known cause of human infections were also isolated from poultry.

Molecular characterization of motile serovars of Salmonella enterica from breeder and commercial broiler poultry farms in Bangladesh.

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 vitro and in vivo investigation on genomic stability of Salmonella enterica Typhimurium DT41 obtained from broiler breeders in Denmark.

Salmonella enterica serovar Typhimurium phage type DT41 has previously been identified from salmonella-positive broiler breeder flocks in Denmark and isolates obtained from different flocks have demonstrated major diversity by multiple-locus variable-number tandem-repeats analysis (MLVA) typing. To elucidate whether the high diversity observed by MLVA was related to multiple independent introductions at farm level or genetic instability of markers, we investigated the genomic stability of different clones of S. Typhimurium DT41. In the in vitro genomic stability experiment, feed pellet- and dust samples inoculated with four strains of DT41 were kept at three different temperatures. The in vitro genomic stability was also assessed by conducting a serial passage experiment. In a subsequent in vivo experiment, broiler breeders of three different age groups were challenged with a strain of poultry and human origin, respectively. The in vitro experiment demonstrated that DT41 survived more than 6 months in feed-pellets at 20 °C whereas the survival in dust was less than 4 weeks. Infection pattern and excretion varied for the poultry and human strain and birds of different age groups as revealed by the in vivo experiment. Genetic stability of cultures obtained from the in vitro and in vivo survival/passage was investigated by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and MLVA. The results of plasmid profiling and PFGE demonstrated genomic stability of all but one strain kept in dust at 20 °C for 3 weeks. Minor genetic changes were observed in isolates from the in vitro experiment as revealed by MLVA. The epidemiological impact of these findings is briefly discussed.

The global establishment of a highly-fluoroquinolone resistant Salmonella enterica serotype Kentucky ST198 strain.

While the spread of Salmonella enterica serotype Kentucky resistant to ciprofloxacin across Africa and the Middle-East has been described recently, the presence of this strain in humans, food, various animal species (livestock, pets, and wildlife) and in environment is suspected in other countries of different continents. Here, we report results of an in-depth molecular epidemiological study on a global human and non-human collection of S. Kentucky (n = 70). We performed XbaI-pulsed field gel electrophoresis and multilocus sequence typing, assessed mutations in the quinolone resistance-determining regions, detected ß-lactam resistance mechanisms, and screened the presence of the Salmonella genomic island 1 (SGI1). In this study, we highlight the rapid and extensive worldwide dissemination of the ciprofloxacin-resistant S. Kentucky ST198-X1-SGI1 strain since the mid-2000s in an increasingly large number of contaminated sources, including the environment. This strain has accumulated an increasing number of chromosomal and plasmid resistance determinants and has been identified in the Indian subcontinent, Southeast Asia and Europe since 2010. The second substitution at position 87 in GyrA (replacing the amino acid Asp) appeared helpful for epidemiological studies to track the origin of contamination. This global study provides evidence leading to the conclusion that high-level resistance to ciprofloxacin in S. Kentucky is a simple microbiological trait that facilitates the identification of the epidemic clone of interest, ST198-X1-SGI1. Taking this into account is essential in order to detect and monitor it easily and to take rapid measures in livestock to ensure control of this infection.

Prevalence and characterization of motile Salmonella in commercial layer poultry farms 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.