Detecting Infectious Bursal Disease Using a VP1 Gene-Based RT-qPCR Assay Compared to Standard Methods of Virus Isolation, ELISA, and Histopathology.

Infectious bursal disease (IBD) is an immunosuppressive viral disease of chickens, associated with severe economic losses and major threats to poultry production worldwide. Disease prevention programs rely on unequivocal identification of the pathogen, as well as vaccination programs. This study developed a sensitive, one-step, real-time, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay using a hydrolysis probe system for infectious bursal disease virus (IBDV, VP1 gene) detection and quantification, which was compared to other routinely used diagnostic methods. The assay successfully detected IBD reference viruses and field isolates. The absence of cross-reactivity was detected with negative samples or with other avian viruses in the analytical specificity test. The detection limit of this assay was 70 RNA copies. RT-qPCR was more sensitive in the detection of serially diluted IBDV isolates compared to virus isolation. For clinical samples, the sensitivity and specificity values of RT-qPCR compared to enzyme-linked immunosorbent assay (ELISA) were 97.5% and 100%, respectively, and compared to histopathology, these values were 100% and 93.94%, respectively. RT-qPCR can provide a simple and reliable assay for IBDV surveillance programs and for evaluation of control strategies.

Population Dynamics of Thermotolerant Campylobacter in Broilers Reared on Reused Litter.

A study using sentinel broiler chickens was performed to address Campylobacter persistence in litter that was reused for successive flocks. Cloacal swabs, litter, drag swabs, darkling beetles, feed, and drinking water were weekly sampled and analyzed by standard microbiological procedures. Thermotolerant Campylobacter isolated strains were confirmed by polymerase chain reaction and subtyped by pulsed-field gel electrophoresis analysis. Campylobacter was not detected in samples collected immediately after downtime between broiler flocks. However, Campylobacter-positive samples were first detected at 21 d. After Campylobacter was initially isolated from the cloacal swabs, reused litter, drag swabs, or darkling beetles, these samples remained Campylobacter positive in the following weeks until the end of the rearing period. Campylobacter-positive cloacal swabs obtained from sentinel broilers ranged from 97.3% to 100% at 42 d. All isolated strains were identified as Campylobacter jejuni. Among the subtypes identified, an indistinguishable C. jejuni strain was predominant in sentinel broilers and was also detected in the other environmental samples analyzed, suggesting a common and persistent contamination source within the flocks. Sentinel broilers may have contributed to amplify the Campylobacter level, maintaining flock and broiler house contamination until the end of the production cycle.

Antimicrobial Resistance in Nontyphoidal Salmonella Isolated from Human and Poultry-Related Samples in Brazil: 20-Year Meta-Analysis.

Nontyphoidal Salmonella are one of the leading causes of foodborne diseases in the world. As poultry products are recognized as main sources of human salmonellosis, nontyphoidal Salmonella control has become a global issue for the poultry industry. The increasing antimicrobial resistance in poultry-related nontyphoidal Salmonella serovars is a global matter of concern. By monitoring the evolution of antimicrobial resistance, alternative treatments can be identified and possible restrictions in the treatment of systemic human salmonellosis foreseen. A meta-analysis was conducted to assess the profile and temporal evolution of the antimicrobial resistance of nontyphoidal Salmonella of poultry and human origin in Brazil, isolated in the period from 1995 to 2014. Four databases were researched; twenty-nine articles met the eligibility criteria and were included in the meta-analysis. In the nontyphoidal isolates of poultry origin, the highest levels of antimicrobial resistance were verified for sulfonamides (44.3%), nalidixic acid (42.5%), and tetracycline (35.5%). In the human-origin isolates, the resistance occurred mainly for sulfonamides (46.4%), tetracycline (36.9%), and ampicillin (23.6%). Twenty-two articles described results of antimicrobial resistance specifically for Salmonella Enteritidis, also enabling the individual meta-analysis of this serovar. For most antimicrobials, the resistance levels of Salmonella Enteritidis were lower than those found when considering all the nontyphoidal serovars. In the poultry-origin isolates, a quadratic temporal distribution was observed, with reduced resistance to streptomycin in Salmonella Enteritidis and in all nontyphoidal serovars, and a linear increase of resistance to nalidixic acid in Salmonella Enteritidis. In the human-origin isolates, a linear increase was identified in the resistance to nalidixic acid in Salmonella Enteritidis and in all the nontyphoidal isolates, and to gentamicin in Salmonella Enteritidis. Continuous monitoring of the development and spread of antimicrobial resistance could support the measurement of the consequences on poultry and human health.

A temporal study of Salmonella enterica serotypes from broiler farms in Brazil.

The present study analyzes the characteristics of Salmonella spp. from broiler chicken farms in Brazil. In total, 82 Salmonella spp. strains were characterized by serotyping, determining susceptibility to antimicrobials, and using pulsed-field gel electrophoresis (PFGE). Fifteen Salmonella serotypes were identified, among which Minnesota (40.24%), Infantis (14.63%), Heidelberg (7.31%), Senftenberg (6.09%), and Mbandaka (6.09%) were the most frequent. Salmonella Minnesota occurred mostly in the state of Mato Grosso do Sul and in one of the broiler companies surveyed. Approximately 60% of the strains were resistant to at least one of the antimicrobials tested. From these isolates, 17.07% were resistant to only one antimicrobial (tetracycline or streptomycin), and 9.75% were resistant to 3 or more antimicrobial classes. Thirteen resistance profiles were characterized, the most frequent of which were the resistance to tetracycline (15.85%); to the combination of trimethroprim with sulfamethoxazole, and tetracycline (10.97%); and to the combination of streptomycin and tetracycline (9.75%). Multiple correspondence analysis revealed that susceptibility or resistance of the analyzed strains and also particular Salmonella serotypes were associated with broiler-producing companies where the samples were collected. Strains presented high intraserotype genetic variability, as shown by the 64 PFGE profiles, suggesting the existence of several contamination sources in the surveyed farms.

Wild-type lytic bacteriophages against Salmonella Heidelberg: Further characterization and effect of prophylactic therapy in broiler chickens.

To characterize wild-type bacteriophages and their effect on Salmonella Heidelberg intestinal colonization in broilers, phages combined in a cocktail were continuously delivered via drinking water since the first day after hatching. After challenge with a field strain, broilers were evaluated at regular intervals for S. Heidelberg and bacteriophages in tissues and cecum, and gross and microscopic lesions in organs. Phages were highly virulent against S. Heidelberg by efficiency of plating. One-step growth curves exhibited eclipse period from 20 to 25 min, whereas the lowest latent period and higher burst size found were 45 min and 54 PFU/cell, respectively. Bacteriophage whole genomic sequencing analyses revealed a lack of genes related to lysogeny, antimicrobial resistance, and virulence factors. Relevant gross or microscopic lesions were absent in tissues analyzed from treated broilers. Although numerically stable bacteriophage concentrations were detected in the cecal contents of treated broilers, no significant difference was found for the S. Heidelberg cecal load in comparison to the untreated group and for the prevalence of positive tissues throughout the evaluated period. The phages produced turbid plaques against some S. Heidelberg re-isolated from treated broilers, suggesting the evolving of a resistant subpopulation. Overall, the results provide new evidence of the safety and in vitro replication of such phages in S. Heidelberg. Nevertheless, continuous administration of the phage suspension most likely induced the development of bacteriophage-resistant mutants, which might have affected the in vivo effect. Therefore, a putative administration protocol should be based on other strategies, such as short-term therapy at pre-harvest age.

Pathogenicity of GI-23 Avian Infectious Bronchitis Virus Strain Isolated in Brazil.

IBV variants belonging to the GI-23 lineage have circulated since 1998 in the Middle East and have spread to several countries over time. In Brazil, the first report of GI-23 occurred in 2022. The study aimed to evaluate the in vivo pathogenicity of exotic variant GI-23 isolates. Biological samples were screening by real-time RT-PCR and classified in to GI-1 or G1-11 lineages. Interestingly, 47.77% were not classified in these lineages. Nine of the unclassified strains were sequenced and showed a high similarity to the GI-23 strain. All nine were isolated and three, were studied for pathogenicity. At necropsy, the main observations were the presence of mucus in the trachea and congestion in the tracheal mucosa. In addition, lesions on the tracheas showed marked ciliostasis, and the ciliary activity confirmed the high pathogenicity of isolates. This variant is highly pathogenic to the upper respiratory tract and can cause severe kidney lesions. This study confirm a circulation of GI-23 strain in the country and report, to first time, the isolation of an exotic variant of IBV in Brazil.

Research Note: A baseline survey of thermotolerant Campylobacter in retail chicken in southern Brazil.

Chicken is a leading source of thermotolerant Campylobacter, which triggers human foodborne enteritis. This study evaluated thermotolerant Campylobacter contamination of retail chicken in southern Brazil, using qualitative and quantitative analyses. Selective enrichment in Bolton broth for 24 and 48 h after plating onto modified charcoal-cefoperazone-deoxycholate (mCCD) agar and Preston agar was assessed. The combined results of the detection and enumeration methods revealed a frequency of 70% occurrence of thermotolerant Campylobacter in chicken samples. Campylobacter was enumerated in 60% of the samples, whereas 46% of the samples were positive in the qualitative analysis. Quantitative analysis showed average counts of 3.10 ± 0.15 log10 CFU/sample. Higher numbers of Campylobacter-positive samples were found using 24-h enrichment before plating onto Preston agar (46%) than onto mCCD agar (2%). The majority of isolated strains were identified as Campylobacter jejuni, and Campylobacter coli was also found but to a lesser extent. Subtyping revealed a clear distinction between strains isolated from different chicken sources. The enriched samples plated onto mCCD agar showed extensive spreading of nonproducing extended-spectrum ß-lactamases Proteus mirabilis that hampered the identification of Campylobacter colonies. P. mirabilis strains showed resistance to cefoperazone, trimethoprim, and polymyxin B present in broth and plate media used and were inhibited by rifampicin present in Preston agar. The results underline the effect of the spread of contaminant strains on Campylobacter cultures, which might be prevented using a recently revised International Organization for Standardization method for qualitative analysis of chicken.

Effect of time of therapy with wild-type lytic bacteriophages on the reduction of Salmonella Enteritidis in broiler chickens.

Salmonella Enteritidis remains a leading cause of human foodborne disease, mostly associated with the consumption of contaminated poultry products. To more strategically implement a phage therapy scheme for S. Enteritidis control in broilers, a cocktail containing three wild-type lytic bacteriophages (LBs) previously isolated from chickens was evaluated shortly and later after a challenge. Genomic characterization, lytic spectrum and in vitro efficacy were determined for each studied LB. In independent trials, broilers challenged with S. Enteritidis on day of hatch received phage therapy from 6 to 10 days of age (early treatment), and from 31 to 35 days of age (later treatment). S. Enteritidis analyses were performed before treatment and at 1, 4, 7 and 10 days post-treatment (dpt) in both trials. Partial DNA sequence analysis of each LB revealed close similarity to the Ackermannviridae family. LBs lysed different Salmonella enterica serovars, while other tested bacteria were refractory. An in-vitro reduction of 1.49, 0.65 and 0.58 log10 CFU/mL in S. Enteritidis number was obtained after co-incubation for 3 h with each LB. Both in vivo trials showed a significant reduction in the average number of intestinal S. Enteritidis calculated after phage therapy compared with controls. However, the highest efficiency was found in the later therapy, which resulted in a reduction of 1.08 log10 CFU/g in the average from 4 to 10 dpt, showing potential for future use as a pre-harvest strategy to reduce the S. Enteritidis intestinal colonization in broilers on farms.

Survey of Campylobacter in foods implicated in foodborne diseases in Southern Brazil.

Campylobacter is not routinely tested in foodborne disease investigations in Brazil. Here, we studied the occurrence of Campylobacter among other food-related bacteria commonly found in foodborne disease outbreaks reported in Rio Grande do Sul State, Southern Brazil. Seventy-two food samples were analyzed by using culture-based detection methods during the 18-month investigation of 36 foodborne disease outbreaks. The sampled foods from the foodborne disease outbreaks were all negative for Campylobacter . However, at least one of other routinely investigated foodborne-related bacteria was detected in 29.17% (21/72) of the samples. Taken together, these results suggest the need to monitor Campylobacter in foodborne diseases to detect sporadic cases caused by Campylobacter that might go unnoticed in Rio Grande do Sul.

Simple, Low-Cost and Long-Lasting Film for Virus Inactivation Using Avian Coronavirus Model as Challenge.

The COVID-19 infection, caused by SARS-CoV-2, is inequitably distributed and more lethal among populations with lower socioeconomic status. Direct contact with contaminated surfaces has been among the virus sources, as it remains infective up to days. Several disinfectants have been shown to inactivate SARS-CoV-2, but they rapidly evaporate, are flammable or toxic and may be scarce or inexistent for vulnerable populations. Therefore, we are proposing simple, easy to prepare, low-cost and efficient antiviral films, made with a widely available dishwashing detergent, which can be spread on hands and inanimate surfaces and is expected to maintain virucidal activity for longer periods than the current sanitizers. Avian coronavirus (ACoV) was used as model of the challenge to test the antivirus efficacy of the proposed films. Polystyrene petri dishes were covered with a thin layer of detergent formula. After drying, the films were exposed to different virus doses for 10 min and virus infectivity was determined using embryonated chicken eggs, and RNA virus quantification in allantoic fluids by RT-qPCR. The films inactivated the ACoV (ranging from 103.7 to 106.7 EID50), which is chemically and morphologically similar to SARS-CoV-2, and may constitute an excellent alternative to minimize the spread of COVID-19.

Longitudinal study reveals persistent environmental Salmonella Heidelberg in Brazilian broiler farms.

The vast capacity for maintenance and dissemination in the environment are major challenges for the control of Salmonella spp. in poultry farms. The aim of this study was to assess environmental contamination by non-typhoidal Salmonella in successive broiler flocks in nine commercial broiler farms integrated with three companies in the south of Brazil, for a twelve-month production period. Recycled broiler litter, feed and swabs from the evaporative cooling system pads were analyzed, and the total enterobacteria count in the litter samples was ascertained. Positive broiler houses were identified in two of the three broiler companies studied, in which non-typhoidal Salmonella were detected for the first time in the first or second flock, and recurred in the recycled litter of subsequent flocks. Feed and evaporative cooling pad swab samples were also positive in at least one of the assessed flocks. The majority of the isolates (87.5%) originating from different flocks, broiler houses and companies that were sampled were identified as S. Heidelberg, with the prevalence of one single genotype. The total enterobacteria levels in the litter diminished as the flocks progressed, but the presence of Salmonella spp. was constant over the course of time, indicating that the litter management procedures were not capable of interrupting the cycle of residual contamination. The predominance of S. Heidelberg highlights its emergence and dissemination in this region, as well as its resistance and maintenance in the environment, and reinforces the need to improve prevention and recycled litter management measures.

Impact of treatments for recycled broiler litter on the viability and infectivity of microorganisms.

The microbiological risk of recycled litter depends on the efficacy of the management system applied to inactivate residual microorganisms and preserve the health of the successive broiler flock. This study aimed to assess the viability and infectivity of the Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus (IBDV) and Salmonella Heidelberg in recycled litter exposed to different treatments. The litter was contaminated with microorganisms and submitted to the treatments (T): T1: shallow fermentation; T2: quicklime (calcium oxide); T3: shallow fermentation followed by addition of quicklime; T4: no treatment. Sentinel chicks housed on the treated litter showed that T1 and T3 inactivated residual IBDV. Analysis of the litter subjected to T1 also showed reduced levels of total enterobacteria. T2 was not able to reduce the microorganisms assessed and its association with T1 (T3) failed to enhance the effect of the treatment. NDV did not survive in the broiler litter, regardless of the treatment applied, and it was also not detected in the sentinel chicks. S. Heidelberg remained viable in the litter submitted to all studied treatments, being isolated from the sentinel chicks of all the experimental groups. The antimicrobial activity of T1 and T3 was associated to higher ammonia contents in the broiler litter. The results indicate that the shallow fermentation treatment is efficient for controlling residual IBDV and total enterobacteria in the recycled litter.

Extracellular Proteins of Mycoplasma synoviae.

Mycoplasma synoviae is a Gram positive bacteria lacking of cell wall that affects chickens and turkeys causing infection in the upper respiratory tract and in some cases arthritis, with economical impact to broiler breeders. Treatment and prevention of avian synovitis depend on knowledge of the infectious process. Secreted or surface-exposed proteins play a critical role in disease because they often mediate interactions between host and pathogen. In the present work, we sought to identify possible M. synoviae secreted proteins by cultivating the bacteria in a modified protein-free Frey medium. Using this approach, we were able to detect in the cell-free fraction a number of proteins that have been shown in other organisms to be secreted, suggesting that they may also be secreted by M. synoviae.

Antibacterial activity of vegetal extracts against serovars of Salmonella / Atividade antibacteriana de extratos vegetais sobre sorovares de Salmonella

in vitro antibacterial activity of 21 hydroethanolic vegetal extracts was assessed against 20 serovars of Salmonella. Regarding the tested extracts, 85.7 percent of them presented antibacterial activity. The six active extracts which showed activity on the largest number of serovars and the extract of Eucalyptus sp. were submitted to the determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). Of these, six extracts showed bacteriostatic and bactericidal activity with MIC and MBC for Punica granatum (pomegranate) from 20 and 60mg mL-1, for Eugenia jambolana (rose apple) from 40 and 240mg mL-1, Eugenia uniflora (surinam cherry) from 80 and 240mg mL-1, Caryophyllus aromaticus (clove) from 10 and 60mg mL-1, Psidium araca from 30 and 320mg mL-1 and Eucalyptus sp. from 40 and 160mg mL-1. Achyrocline satureioides (macela) presented only bacteriostatic potential and MIC from 160mg mL-1. Caryophyllus aromaticus, Eucalyptus sp., and Psidium araca presented the best results for bactericidal activity, inhibiting, respectively, 84.2 percent, 42.1 percent, and 17.6 percent of Salmonella's serovars. The activity of each extract varied for different serovars; S. London presented resistance to the six extracts in MBC, while S. Pullorum was the most susceptible serovar.

A atividade antibacteriana de 21 extratos hidroetanólicos vegetais foi avaliada in vitro frente a 20 sorovares de Salmonella. Dos extratos testados, 85,7 por cento apresentaram atividade antibacteriana. Os seis extratos que evidenciaram atividade sobre o maior número de sorovares e Eucalyptus sp. foram submetidos à determinação da Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM). Destes, seis extratos apresentaram atividade bacteriostática e bactericida com MIC para Punica granatum (romã) a partir de 20 e 60mg mL-1, Eugenia jambolana (jambolão) de 40 e 240mg mL-1, Eugenia uniflora (pitanga) de 80 e 240mg mL-1, Caryophyllus aromaticus (cravo) de 10 e 60mg mL-1, Psidium araca (araçá) 30 e 320mg mL-1 e Eucalyptus sp. (eucalipto) de 40 e 160mg mL-1. Achyrocline satureioides (macela) apresentou apenas atividade bacteriostática e MIC a partir de 160mg mL-1. Caryophyllus aromaticus, Eucalyptus sp. e Psidium araca apresentaram os melhores resultados para a atividade bactericida, inativando, respectivamente, 84,21 por cento, 42,1 por cento e 17,64 por cento dos sorovares de Salmonella. A atividade de cada extrato variou para diferentes sorovares. Nenhum dos seis extratos avaliados evidenciou atividade bactericida frente a S. London, enquanto S. Pullorum foi o sorovar mais sensível.