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The recent trend of global warming poses a significant threat to ecosystems worldwide. This global climate change has also impacted the pollution levels in aquatic ecosystems, subsequently affecting human health. To address these issues, an experiment was conducted to investigate the mitigating effects of iron nanoparticles (Fe-NPs) on arsenic and ammonia toxicity as well as high temperature stress (As+NH3+T). Fe-NPs were biologically synthesized using fish waste and incorporated into feed formulations at 10, 15, and 20 mg kg-1 diet. A total of 12 treatments were designed in triplicate following a completely randomized design involving 540 fish. Fe-NPs at 15 mg kg-1 diet notably reduced the cortisol levels in fish exposed to multiple stressors. The gene expressions of HSP 70, DNA damage-inducible protein (DDIP), and DNA damage were upregulated by stressors (As+NH3+T) and downregulated by Fe-NPs. Apoptotic genes (Cas 3a and 3b) and detoxifying genes (CYP 450), metallothionein (MT), and inducible nitric oxide synthase (iNOS) were downregulated by Fe-NPs at 15 mg kg-1 diet in fish subjected to As+NH3+T stress. Immune-related genes such as tumor necrosis factor (TNFα), immunoglobulin (Ig), and interleukin (IL) were upregulated by Fe-NPs, indicating enhanced immunity in fish under As+NH3+T stress. Conversely, Toll-like receptor (TLR) expression was notably downregulated by Fe-NPs at 15 mg kg-1 diet in fish under As+NH3+T stress. Immunological attributes such as nitro blue tetrazolium chloride, total protein, albumin, globulin, A:G ratio, and myeloperoxidase (MPO) were improved by dietary Fe-NPs at 15 mg kg-1 diet in fish, regardless of stressors. The antioxidant genes (CAT, SOD, and GPx) were also strengthened by Fe-NPs in fish. Genes associated with growth performance, such as growth hormone regulator (GHR1 and GHRβ), growth hormone (GH), and insulin-like growth factor (IGF 1X and IGF 2X), were upregulated, enhancing fish growth under stress, while SMT and MYST were downregulated by Fe-NPs in the diet. Various growth performance indicators were improved by dietary Fe-NPs at 15 mg kg-1 diet. Notably, Fe-NPs also enhanced arsenic detoxification and reduced the cumulative mortality after a bacterial infection. In conclusion, this study highlights that dietary Fe-NPs can effectively mitigate arsenic and ammonia toxicity as well as high temperature stress by modulating gene expression in fish.

The study was carried out to investigate the isolation of Escherichia coli from tracheal and oropharyngeal swab of clinically sick chickens. The antibiotic susceptibility patterns of the isolates to several antimicrobials were determined with a striking emphasis on oxytetracycline. The PCR technique was applied to detect tetA, tetB, and tetC in the tetracycline-resistant isolates. The isolates were initially screened for their resistance patterns against 6 antimicrobials of six different groups using the disc diffusion technique. The results showed that 41% tracheal, 51% oropharyngeal, and 34% samples from both sites were E. coli positive respectively. Antimicrobial resistance profiling of the isolates revealed that all the isolates were resistant to oxytetracycline and sulphamethoxazole-trimethoprim, and also 90 %, 82.9%, 63.4%, and 39% resistant to ciprofloxacin, amoxicillin, gentamicin, and colistin respectively. Notably, 82.9% isolates (95% CI 68.4%–91.8%) showed resistance to ≥3 groups of antimicrobials that means these were multi-drug resistant. Among the tetracycline-resistant isolates, 85.4% (95% CI 71.2%–93.5%), 29.3% (7.5%–44.6%), and 7.3% (1.8% - 20.1) were positive for tetA, tetB, and tetC genes respectively. The frequency of the isolation of E. coli is greater in oropharyngeal than tracheal and both kinds of samples. Commercial poultry with E. coli strains has acquired extensive resistance to oxytetracycline. This study suggests a possible association between the tetA gene and oxytetracycline resistance in E. coli isolates, but further investigations like knockdown, whole-genome sequencing, and rescue experiments are needed to establish a direct causal relationship.

Intercropping offers greater scope to introduce new crops. Cultivation of crops with diverse root architecture and different durations enhances the productivity of scarce resources like land and water. This study aimed to determine the effect of intercrop competition and irrigation regimes on yield, competition, land usage, irrigation water use efficiency (IWUE), and fatty acids of chia. The field experiment was conducted in semi-arid India during 2020–2022 with full (I100) and deficit irrigation (I50) and six intercrops. Results demonstrated that chia + fenugreek intercropping in I50 improved the crops’ competitiveness, land equivalent ratio (LER) (1.77), land use efficiency (142.5%), and the IWUE of chia (23.2%). Notably, a chia + radish/spinach system in I50 reduced the seed yield (42.6–45.0%) of chia over I100 monocropping. A chia + fenugreek system in I50 resulted in a higher seed yield (196.2 kg ha−1) than chia monocropping in I100. Further, chia + fenugreek intercropping resulted in higher omega-3 content (56.68%) under I100. Therefore, a chia + fenugreek system under I100 may be suggested over monocropping for better yield and oil quality. However, during water scarcity situations, growers can adopt a chia + fenugreek system under I50 which can give a similar chia equivalent yield and a higher LER and IWUE compared to chia monocropping under I100.

Abstract 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.

Abstract 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

SUMMARY: Brassica carinata (BBCC) commonly referred to as Ethiopian mustard is a natural allotetraploid containing the genomes of Brassica nigra (BB) and Brassica oleracea (CC). It is an oilseed crop endemic to the northeastern regions of Africa. Although it is under limited cultivation, B. carinata is valuable as it is resistant/highly tolerant to most of the pathogens affecting widely cultivated Brassica species of the U's triangle. We report a chromosome‐scale genome assembly of B. carinata accession HC20 using long‐read Oxford Nanopore sequencing and Bionano optical maps. The assembly has a scaffold N50 of ~39.8 Mb and covers ~1.11 Gb of the genome. We compared the long‐read genome assemblies of the U's triangle species and found extensive gene collinearity between the diploids and allopolyploids with no evidence of major gene losses. Therefore, B. juncea (AABB), B. napus (AACC), and B. carinata can be regarded as strict allopolyploids. We cataloged the nucleotide‐binding and leucine‐rich repeat immune receptor (NLR) repertoire of B. carinata and, identified 465 NLRs, and compared these with the NLRs in the other Brassica species. We investigated the extent and nature of early‐generation genomic interactions between the constituent genomes of B. carinata and B. juncea in interspecific crosses between the two species. Besides the expected recombination between the constituent B genomes, extensive homoeologous exchanges were observed between the A and C genomes. Interspecific crosses, therefore, can be used for transferring disease resistance from B. carinata to B. juncea and broadening the genetic base of the two allotetraploid species. Significance Statement: We report a highly contiguous genome assembly of allotetraploid mustard Brassica carinata HC20 (BBCC) which contains resistance to many diseases affecting the other extensively cultivated Brassica species of the U's triangle. Comparative analyses of the long‐read assemblies of the six Brassica species revealed high gene collinearity between the three diploid and their allotetraploid species. We tracked the early‐generation genomic interactions between the subgenomes of B. carinata (BBCC) and B. juncea (AABB) in their interspecific crosses using molecular karyotyping and proposed strategies to utilize this information for genomic introgressions. [ABSTRACT FROM AUTHOR]

The strength and durability properties of the recycled aggregate concrete (RAC) have been affected by the cracks and the weak interfacial transition zone (ITZ) of the recycled coarse aggregates (RCA). However, the mechanical and physical features of RCA can be improved by microbially induced calcite precipitation (MICP). Therefore, immobilization techniques were used to protect and maintain the high efficiency of Bacillus bacteria for the formation and precipitation of calcium carbonates in self‐healing concrete over a period of time. The objective of the present study was to show the viability of the immobilized bacterial consortium‐enhanced RCA to form self‐healing cracks. Further, the self‐healing capability of enhanced RCA was investigated along with two other immobilization methods, that is, RCA and hydrated lime and brick powder (HBr)‐immobilized bacteria. The experimental results show that the increase in the bio‐deposition time improved the physical and mechanical properties of the RCA. Further, subsequently 56 days of the healing incubation period, the immobilized consortium‐enhanced RCA concrete specimens completely healed the cracks of width 0.58 mm. However, the equivalent cracks of width 0.56 mm were also recovered by the HBr immobilized bacterial cultures. Furthermore, the field emission scanning electron microscope (FESEM), energy dispersive spectroscopy (EDS) and X‐ray diffraction (XRD) analysis revealed that the existence of precipitation at the crack surface was calcium carbonate with a regular cubic‐shaped and lamellar layer morphology. The outcomes of the current study show that consortium‐enhanced RCA has promising potential to develop self‐healing concrete with self‐repaired and improved durability properties in the concrete construction field. [ABSTRACT FROM AUTHOR]

Abstract Live animal markets are known hotspots of zoonotic disease emergence. To mitigate those risks, we need to understand how networks shaped by trading practices influence disease spread. Yet, those practices are rarely recorded in high-risk settings. Through a large cross-sectional study, we assessed the potential impact of live poultry trading networks’ structures on avian influenza transmission dynamics in Bangladesh. Networks promoted mixing between chickens sourced from different farming systems and geographical locations, fostering co-circulation of viral strains of diverse origins in markets. Viral transmission models suggested that the observed rise in viral prevalence from farms to markets was unlikely explained by intra-market transmission alone, but substantially influenced by transmission occurring in upstream network nodes. Disease control interventions should therefore alter the entire network structures. However, as networks differed between chicken types and city supplied, standardised interventions are unlikely to be effective, and should be tailored to local structural characteristics.

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.

AbstractA sustainable and reliable methodology to strengthen the mortar adhered to recycled concrete aggregate (RCA) is crucial for optimizing its utilization in construction. This research proposes a mortar strengthening method by soaking RCA in different dosages (weight percentages) of pozzolanic slurries, i.e., fly ash and silica powder (F-S), fly ash and cement (F-C), cement and silica powder (C-S) for various durations. The influence of each slurry dosage and soaking duration on RCA's physical and mechanical properties and surface morphology were examined. Further, a modified two-stage mixing approach (M-TSMA) was employed to produce recycled aggregate concrete (RAC) incorporating 100% untreated and surface-treated RCA. The impact of M-TSMA and treated RCA on concrete was demonstrated regarding its mechanical and durability properties, such as compressive strength, ultrasonic pulse velocity, rebound hammer, sorptivity, and resistance to chloride ions. Results show that soaking RCA in 30% C-S slurry for 6 h enhances RCA's overall properties by increasing the calcium silicate hydrate (C-S-H) content. Moreover, employing M-TSMA and incorporating treated RCA improves the mechanical and durability properties of RAC by reducing microcracks and densifying the microstructure. Overall, the proposed surface treatment and M-TSMA can potentially enhance RAC performance with complete RCA utilization. [ABSTRACT FROM AUTHOR]

Summary: Rapid intelligent transportation systems (ITS) innovations need a reliable MAC protocol to enable massive nonsafety message delivery and high‐priority safety broadcasts. The significant rise in spectrum need is regulated by collaboration in cognitive vehicular networks (CVNs). For QoS improvement, a reliable cooperative MAC for CVNs (CCVN‐MAC) is presented in this study. CCVN allows vehicles to collaborate and share channel status information, allowing for proactive channel switching in case of a legacy user (LU) appearance. To enable transmission mode selection, additional control signals are included. Using the suggested cooperative makeup technique, the helper nodes resend failed transmission. A Markov chain represents the protocol, and NS‐2 is used to evaluate it for several performance characteristics. Compared with conventional MAC techniques, the proposed protocol depicts improved performance, including depreciation of 70% in average latency and an increment of 42.4% in throughput. [ABSTRACT FROM AUTHOR]

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.

Irrational and inappropriate use of antibiotics in commercial chicken production can contribute to the development of antimicrobial resistance. We aimed to assess antibiotic usage in commercial chicken production in Bangladesh, and identify factors associated with this practice. We conducted a large-scale cross-sectional study to collect information on antibiotic usage in commercial chickens from January to May 2021. Structured interviews were conducted with 288 broiler, 288 layer and 192 Sonali (locally-produced cross-bred) farmers in 20 sub-districts across Bangladesh. The frequency of antibiotic usage, the types of antibiotics and purpose of usage were estimated for each production type. Adjusted odds ratios (aOR) were calculated to measure the association between antibiotic usage and factors related to the characteristics of the farms and farmers using multivariable logistic regression models. The proportion of farms, irrespective of their production type, reporting usage of antibiotics in the 24 hours preceding the interview was 41% (n = 314, 95% CI: 37-44%). Forty-five percent (n = 344, 41-48%) reported antibiotic usage in the last 72 hours, 86% (n = 658, 83-88%) in the last 14 days, and almost all farms, 98% (n = 753, 97-99%), had used antibiotics since the start of their production cycle. Use of antibiotics in the 24 hours preceding an interview was more frequently reported in broiler (OR 1.91, 95% CI: 1.36-2.69) and Sonali (OR 1.94, 95% CI: 1.33-2.33) than layer farms. Oxytetracycline (23-31%, depending on production type), doxycycline (18-25%), ciprofloxacin (16-26%) and amoxicillin (16-44%) were the most frequently used antibiotics. Antibiotics were reported to be used for both treatment and prophylactic purposes on most farms (57-67%). Usage of antibiotics in the 24h preceding an interview was significantly associated with the occurrence of any illnesses in chickens (aOR broiler: 41.22 [95% CI:13.63-124.62], layer: aOR 36.45[9.52-139.43], Sonali: aOR 28.47[4.97-162.97]). Antibiotic usage was mainly advised by veterinary practitioners (45-71%, depending on production type), followed by feed dealers (21-40%) and farmers (7-13%). Improvement of chicken health through good farming practices along with changes in key stakeholders (feed dealers and practitioners) attitudes towards antibiotic recommendations to farmers, may help to reduce the levels of antibiotic usage and thus contribute to mitigate antimicrobial resistance.

Abstract Performance of a low temperature polymer electrolyte membrane fuel cell (PEMFC) is highly dependent on the kind of catalysts, catalyst supports, ionomer amount on the catalyst layers (CL), membrane types and operating conditions. In this work, we investigated the influence of membrane types and CL compositions on MEA performance. MEA performance increases under all practically relevant load conditions with reduction of the membrane thickness from 50 to 15 μm, however further decrease in membrane thickness from 15 to 10 μm leads to reduction in cell voltage at high current loads. A thick anode CL is found to be beneficial under wet operating conditions assuming more pore space is provided to accommodate liquid water, whereas under dry operating conditions, an intermediate thickness of the anode CL is beneficial. When studying the impact of catalyst layer thickness, too thin a catalyst layer again shows reduced performance due to increased ohmic resistance ruled out the performance of the MEAs which have identical Pt crystallite sizes on the cathode CLs i. e. the thinnest the cathode CL, the highest the voltage were achieved at a defined current load. Adaptation of the operating conditions is highly anticipated to achieve the highest MEA performance.

Durum wheat (Triticum turgidum var. durum Desf.) is an important cereal crop in many regions of the world. In March of 2018 and 2019, symptoms typical of blast were frequently observed on durum wheat plants under field conditions in Jashore, Bangladesh. The putative causal pathogen was isolated from infected wheat spike specimens onto potato dextrose agar and oatmeal agar, and was identified from mono-conidium cultures as Magnaporthe oryzae, based on morphological features. The pathotype of the fungus was identified as Triticum, based on comparative molecular analyses of ITS sequences and MoT3 specific markers. BLAST analysis revealed >99.8% similarity with M. oryzae/P. oryzae, retrieved from the NCBI Genebank. This was confirmed through amplification of the predicted products with MoT3 primers in PCR analysis. Pathogenicity was confirmed by inoculating healthy durum wheat seedling leaves and spikes with a conidium suspensions of M. oryzae isolate DuBWMRI1901.2A. The fungus produced similar symptoms on inoculated leaves and spikes as those observed in the field, and was subsequently re-isolated, fulfilling Koch’s postulates. This is the first report of blast of durum wheat caused by Magnaporthe oryzae pathotype Triticum in Bangladesh.

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.