Avian Cellulitis Research Articles

Molecular characterization of Escherichia coli isolates recovered from broilers with cellulitis

Avian cellulitis in broilers, caused by avian pathogenic Escherichia coli, is a major cause for carcass rejections during meat inspection, resulting in significant economic losses. In this study, we analysed E. coli isolates obtained from broiler chickens affected by cellulitis for their genetic relatedness and antimicrobial resistance phenotype and genotype. The objective was to determine whether there is a clonal spread or whether these clinical isolates differ. For this purpose, E. coli was isolated from swab samples collected from diseased broilers across 77 poultry farms in Germany, resulting in 107 isolates. These isolates were subjected to serotyping, PCR-based phylotyping and macrorestriction analysis with subsequent pulsed-field gel-electrophoresis for typing purposes. In addition, the presence of virulence genes associated with avian pathogenic E. coli (APEC) was investigated by PCR. Antimicrobial susceptibility of the isolates was examined by the disk diffusion method according to CLSI guidelines and subsequently, the presence of corresponding resistance genes was investigated by PCR. Typing results revealed that a significant proportion of the isolates belonged to serotype O78:K80, which is one of the major APEC serotypes. Phylogenetic grouping showed that phylogenetic group D was most commonly represented (n = 49). Macrorestriction analysis showed overall heterogenous results, however, some clustering of closely related isolates was observed. The level of antimicrobial resistance was high, with 83.8% of isolates non-susceptible to at least one class of antimicrobial agents and 40% of isolates showing resistance to at least three classes. The most frequently observed resistance was to ampicillin, mediated by blaTEM (n = 56). However, few isolates were non-susceptible to ciprofloxacin (n = 8) and none of the isolates was resistant to 3rd generation cephalosporins or carbapenems. Overall, the results show that genetically diverse APEC associated with avian cellulitis can be found among and within German poultry farms. While most isolates were antimicrobial resistant, resistance levels to high(est) priority critically important antimicrobials were low.

Avian cellulitis: a skin affection associated with economic losses in broiler chickens

This review was designed for focusing on cellulitis condition in broiler chickens regarding causes, clinical picture, and prevention measures of this condition. Cellulitis is an acute diffuse inflammation of subcutaneous tissues and muscles especially on the skin of thighs and abdomen. This condition is more common in broilers than others and it is usually associated with economic losses. At processing, low grade chicken carcasses and high incidences of condemnation are the sequels of cellulitis. Skin integrity, stocking density, and litter conditions are predisposing factors for induction of cellulitis. However, other infectious bacterial and immunosuppressive viral pathogens are associated with cellulitis. Affected birds display areas of yellow skin along with a plaque of pus underneath the skin and the underlying muscles show hemorrhages. Presence of caseous, yellowish to green, dark red, or brown fetid gangrenous exudate could also be observed in the advanced cellulitis cases. Prevention and control of cellulitis are based on application of hygienic practices, vaccination, antibiotic therapy, genetic selection, and nutrition.

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts.

Background Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.

Extended-Spectrum Beta-Lactamase Producing Strains of Escherichia Coli Isolated from Avian Cellulitis Lesions

ABSTRACT Avian cellulitis causes significant losses to the poultry industry. Avian-pathogenic Escherichia coli (APEC) is the etiological agent of that disease. This microorganism has zoonotic potential and may act as reservoir of antimicrobial-resistance genes. In this context, the production of extended-spectrum B-lactamase (ESBL) is one of the main antimicrobial resistance mechanisms. The objective of this study was to determine the production of ESBL in an Escherichia coli (E. coli) strain isolated from avian cellulitis lesions. Twenty-two E. Coli isolates were harvested from cellulitis lesions in chicken carcasses in a commercial processing plant. Isolates were then submitted to virulence genotypic profile (iutA, hlyF, iss, ironN, ompT) analysis, antimicrobial susceptibility test, and detection of ESBL production. The results showed that 22.7% of the isolates presented five virulence genes, 9.1% four genes, 36.4% three genes, 13.6% two genes, and 18.2% one gene. The tested isolates showed resistance to ampicillin (90.9%), ceftiofur (54.5%), gentamicin (45.5%), tetracycline (72.1%), sulfamethoxazole/trimethoprim (54.5%), and enrofloxacin (54.5%). Furthermore, 77.3% of the isolates presented multidrug resistance (MDR) profile and 72.7% were positive for ESBL production. This study is the first description of ESBL-producing APEC isolated from avian cellulitis lesions, which suggests the need to establish efficient APEC control measures and programs to prevent flock productivity losses due to colibacillosis and public health risks.

Celulite em codornas japonesas alimentadas com extrato de orégano nas dietas e inoculadas com Escherichia coli

Resumo: Com o objetivo de avaliar a utilização do extrato de orégano nas dietas de codornas japonesas (Coturnix coturnix japonica) e desafiadas com cepas de Escherichia coli, sobre as características de desempenho, a incidência de celulite aviária e titulação de anticorpos específicos contra antígenos de E. coli, foram utilizadas 360 codornas japonesas, com 90 dias de idade, distribuídas em gaiolas de arame galvanizado em galpão convencional. O delineamento experimental foi inteiramente casualizado, em esquema fatorial 5x2 (extrato de orégano x desafiado ou não com E. oli), totalizando dez tratamentos com seis repetições de seis aves por gaiola. Os níveis do extrato de orégano (EO) avaliados foram: 0,00; 0,025; 0,050; 0,100 e 0,150%. Foram avaliadas características de desempenho produtivo, lesões macroscópicas da celulite após períodos pós-inoculação das cepas e amostras de soro foram colhidas para verificar a titulação de anticorpos nas aves. Os dados obtidos foram submetidos à análise de variância e as médias comparadas pelo Teste T. Foi observado efeito de E. coli sobre todas as características produtivas, independentemente dos níveis de EO avaliados, onde grupos desafiados apresentaram piores resultados de desempenho. As lesões macroscópicas, características da celulite, observadas somente nas aves desafiadas com E. coli foram classificadas como grau leve e sem presença de hemorragias. Para a titulação de anticorpos específicos, houve maior quantificação para aves desafiadas com as cepas de E. coli em relação às não desafiadas. Pode-se concluir que o extrato de orégano suplementado nas rações não se mostrou eficaz frente ao desafio com E. coli em codornas na fase de postura e as aves desafiadas com E. coli apresentaram maiores respostas imunes humoral e celular, em relação às não desafiadas, caracterizadas pelo aumento na titulação de anticorpos e pela lesão macroscópica peitoral, independentemente dos níveis de extrato de orégano avaliados.

Antimicrobial resistance of bacterial strains isolated from avian cellulitis

Avian cellulitis is an inflammatory process in the subcutaneous tissue, mainly located in the abdomen and thighs. This problem is commonly observed in poultry at slaughter and it is considered one of the major causes of condemnation of carcasses in Brazil. The aim of this study was to perform the microbial isolation of lesions of avian cellulitis from a processing plant located in the State of Goias in order to analyze antimicrobial resistance by antibiogram test and to detect resistance genes by polymerase chain reaction. A total of 25 samples of avian cellulitis lesions were analyzed, from which 30 bacterial strains were isolated. There were eleven (44%) strains of Escherichia coli, nine (36%) strains of Staphylococcus epidermidis, seven (28%) strains of Proteus mirabilis and three (12%) strains of Manheimiahaemolytica. The antibiogram test showed that all strains were resistant to at least one antimicrobial. The gene of antimicrobial resistance tetB was detected in E. coli, S. epidermidis and P. mirabilis strains, and was the most frequently observed gene. The gene of antimicrobial resistance Sul1 was detected in all bacterial species, while tetA was found in E. coli and S. epidermidis strains, SHV in E. coli strains, S. epidermidis and P. mirabilis,and cat1 in one P. mirabilis strain. The results suggest a potential public health hazard due to the ability of these microorganisms to transmit antimicrobial resistancegenes to other microorganisms present in the intestinal tract of humans and animals, which may affect clinical-medical usage of these drugs.

Escherichia coli from cellulitis lesions in broilers

Samples of chicken broiler lesions were collected at a poultry slaughterhouse in the Bahia Reconcavo region to recover Escherichia coli for phenotypic and genotypic characterization. Poultries used in current research came from four poultry houses in the same region. Weight of cellulitis lesions ranged between 0.9 and 1.7 g, of which 85 % (34/40) were on the left side of the broiler. E. coli was isolated in 82.5 % (33/40) of samples with cellulitis lesions. Gene iss was identified in 87.9 % (29/33) of the samples. Polymerase chain reaction (PCR) verified iss genes to serum resistance, identifying avian pathogenic E. coli (APEC). Iss was detected in 87.9 % of isolates. Results suggest that the partial removal of cellulitis lesions merely decrease the carcass’s repugnant aspect, or rather, it is more aesthetic than hygienic. A re-evaluation of the criteria used is required to notify avian cellulitis cases so that safety in the consumption of broiler-derived products may be guaranteed.

Cellulitis lesions in broiler chickens are induced by Escherichia coli Vacuolating Factor (ECVF)

Escherichia coli Vacuolating Factor (ECVF) is a heat-labile, vacuolating cytotoxin produced by avian pathogenic E. coli (APEC) isolated from avian cellulitis lesions. In this report, we intend to demonstrate that purified ECVF induces the inflammatory process of cellulitis. Our group is the first to demonstrate the effect of ECVF in a histological analysis by in situ inoculation of broiler chickens with purified ECVF. The animals were inoculated with the APEC AC53 and with purified ECVF subcutaneously on their ventral surface (in the sternum region). The histological analysis showed different grades of an acute inflammatory response in the epidermis, dermis and panniculus. An increase in mRNA expression of the proinflammatory cytokine TNF-α was also demonstrated in the inflamed tissue. When ECVF was systemically administered, increased levels of TNF-α and IL-10 were observed in the serum. These results suggest that ECVF plays a key role in the inflammatory process associated with cellulitis that is mainly mediated by TNF-α. In addition, this inflammation can be downregulated by the anti-inflammatory cytokine IL-10.

Virulence genotyping of Escherichia coli isolates from avian cellulitis in relation to phylogeny

Coliform cellulitis in broilers is caused by certain avian pathogenic Escherichia coli strains. Sixty-four E. coli samples were isolated in a slaughterhouse from broiler carcasses with cellulitis. The isolates were examined for the presence of selected virulence genes and phylogenetic determination by PCR assays. E. coli isolates belonged to A (51.56%), B1 (18.75%) and D (29.68%) phylogenetic groups. All of the isolates were positive for at least two virulence genes. The three most prevalent genes were csgA (92.18%), fimH (85.93%) and iucD (76.56%). F17 fimbrial family (f17A), P (papC) and S (sfa) fimbriae encoding genes were detected in one, nine and two isolates, respectively. The highest and the lowest number of virulence genes were found in D and A phylogenetic groups, respectively. Several combination of the virulence genes were detected; csgA, fimH, iucD was the most prevalent pattern. None of the isolates harboured the afa E-VIII, afaI B-C, eaeA, hly, stx1 and stx virulence genes.

Virulence factors and clonal relationships among Escherichia coli strains isolated from broiler chickens with cellulitis.

In this study, we compared Escherichia coli isolates from chickens with avian cellulitis with those from feces of healthy chickens. Cellulitis-derived strains presented phenotypic and genotypic characteristics of greater virulence than did the fecal isolates. Phylogenetic analysis by repetitive extragenic palindromic-PCR showed that, in agreement with their virulence characteristics, the cellulitis isolates form two clonal groups distinct from the fecal isolates.

Evaluation of the adhesive capacity of Escherichia coli isolates associated with avian cellulitis.

It has been shown that Escherichia coli isolates from lesions of cellulitis belong to a limited number of clonal groups distinct from those of isolates found in the environment of these birds. In this study, different in vitro methods were used to evaluate adherence properties of E. coli isolates from cellulitis lesions and environments of high- and low-cellulitis prevalence broiler flocks. One hundred isolates were tested by hemagglutination. Adherence to frozen sections of chicken skin and binding to soluble fibronectin were examined for 40 of these 100 isolates by immunofluorescence and by immunocytofluorometry, respectively. Localization of bacterial adherence to skin tissues was confirmed by immunohistochemistry. It was demonstrated that O78:K80 isolates from cellulitis lesions adhered to skin sections to a much greater extent in deeper than in superficial tissue layers. A greater bacterial adherence following growth in TSB at 37 C was demonstrated for isolates from flocks with high prevalence of cellulitis than for isolates from flocks with low prevalence of cellulitis. MANOVA analysis results showed a significant difference between superficial and deep tissue layers only for one set of isolates from flocks with high prevalence of cellulitis. Hemagglutinating activity was variable among the O78:K80 isolates obtained from flocks with high prevalence of cellulitis. The results obtained for some O78:K80 isolates following growth in TSB suggest a role for type 1 fimbriae or F1 in adherence to skin sections. This was reinforced by the finding that adherence was inhibited by D-mannose. Poultry E. coli isolates that express F1 had no affinity for soluble fibronectin, although localization of the adherence in skin sections suggested a role for extracellular matrix components such as collagen and insoluble fibronectin.

Shiga toxin genes in avian Escherichia coli

The purpose of this study was to determine the presence of stx genes in avian pathogenic Escherichia coli (APEC). We examined 97 APEC isolates: 34 from lesions of avian cellulitis, 31 from avian septicemia, 13 from swollen head syndrome (SHS) in chickens, and 19 from diseased turkeys. We also examined five isolates from the feces of healthy chickens. All 102 E. coli isolates were tested for the presence of stx genes by PCR amplification and by colony blots using probes specific for stx1 and stx2. Fifty-three percent (52) of the 97 APEC carried stx gene sequences: one isolate carried stx2 sequences, two carried both stx1 and stx2 sequences, and the remaining 49 isolates carried only stx1 sequences. Twenty-six isolates were positive by both hybridization and PCR amplification, 10 were positive by PCR only, and 16 were positive by hybridization only. All the stx-positive isolates were negative by PCR for the eae and E -hlyA genes. The five isolates from healthy chickens were all negative for stx. All 13 SHS isolates were positive for the stx1 gene and had low titres for cytotoxicity in the Vero cell assay (VCA). Other stx-positive isolates were negative in the VCA. The stx1 gene from one SHS E. coli isolate was cloned and sequenced and shown to be identical to that of the stx gene of Shigella dysenteriae . The observations indicate that stx1 gene sequences are widespread among APEC but that cytotoxicity on Vero cells is uncommon.

Selection bias in epidemiological studies of infectious disease using Escherichia coli and avian cellulitis as an example.

In epidemiological studies of infectious disease, researchers often rely on specific cues of the host, such as clinical signs, as surrogate indicators of pathogen presence. A selection bias would manifest if the specific visual cues used in sampling for the pathogen were not representative of the full range of signs caused by the strains of that pathogen. In our molecular epidemiological studies of Escherichia coli associated with avian cellulitis in broilers, we collect carcasses at the processing plant based on visual cues of lesion morphology. Therefore, the objectives of this study were to: (1) explore the potential impacts of selection bias in an application of infectious disease epidemiology, and (2) utilize a validation protocol to assess the potential for selection bias in our molecular epidemiological studies of E. coli and avian cellulitis. In two different trials, E. coli DNA fingerprints were compared between birds that our observers collected and the birds that the observers missed. Using Fisher's exact tests and simulation models, we determined that the isolates collected by the observers were not significantly different from the isolates missed by the observers (P > 0.60 in both trials). Our method of selecting birds suspected of having cellulitis did not significantly bias our inferences about the population of E. coli associated with cellulitis in the flock. We encourage more investigators to critically assess the relationship of the sample to the target population in epidemiological studies of infectious disease.

The Effect of Vitamin E on Cellulitis in Broiler Chickens Experiencing Scratches in a Challenge Model

Two experiments are described; each experiment contained five treatments with each treatment consisting of a specific diet and vitamin E at 8.82 mg, 41.89 mg, 74.96 mg, 108.03 mg, or 141.10 mg vitamin E per kilogram of feed. Birds were raised with continuous feed containing the various levels of vitamin E available throughout the experiment. At 4 wk of age, the birds were scratched on the breast and placed onto avian cellulitis Escherichia coli-seeded litter. One week later, the birds were euthanatized and lesion presence was noted. There appeared to be a positive correlation between vitamin E and the inhibition of cellulitis formation when the birds were fed a diet containing 74.96 mg vitamin E/kg feed. Conflicting results were seen in the two experiments when the birds were fed 41.89 and 108.03 mg vitamin E/kg feed. Both experiments had a high incidence of cellulitis in birds whose diets consisted of 141.10 mg vitamin E/kg feed.

Persistence of cellulitis-associated Escherichia coli DNA fingerprints in successive broiler chicken flocks

Avian cellulitis in broiler chickens is primarily caused by Escherichia coli. Previous research found that the E. coli isolates of cellulitis origin were unique to each ranch, suggesting that these E. coli were endemic within the ranch environment. To test the hypothesis that the E. coli associated with cellulitis are endemic in the litter of the broiler house, we designed a study to determine whether E. coli DNA fingerprints associated with cellulitis persist over successive flocks that are grown in the same house. In addition, we assessed the impact of different cleaning and disinfection strategies on this persistence. Two broiler houses were followed on each of five farms over 3–4 flocks. A total of 353 E. coli isolates from cellulitis lesions were analyzed in this study, and 314 of these isolates (89%) were DNA fingerprinted by PFGE. In each ranch, there were several DNA fingerprint patterns that were present over successive flocks, regardless of the cleaning and disinfection strategy utilized. Isolates persisted as long as 191 days, implying that these E. coli are capable of persisting in the broiler house environment for long periods of time. In addition, these E. coli isolates were associated with cellulitis lesions in successive flocks. Thus, the isolates of E. coli that are associated with cellulitis in broiler chickens appear to be endemic in the litter environment of the broiler house.

A statistical model for assessing sample size for bacterial colony selection: a case study of Escherichia coli and avian cellulitis.

A general problem for microbiologists is determining the number of phenotypically similar colonies growing on an agar plate that must be analyzed in order to be confident of identifying all of the different strains present in the sample. If a specified number of colonies is picked from a plate on which the number of unique strains of bacteria is unknown, assigning a probability of correctly identifying all of the strains present on the plate is not a simple task. With Escherichia coli of avian cellulitis origin as a case study, a statistical model was designed that would delineate sample sizes for efficient and consistent identification of all the strains of phenotypically similar bacteria in a clinical sample. This model enables the microbiologist to calculate the probability that all of the strains contained within the sample are correctly identified and to generate probability-based sample sizes for colony identification. The probability of cellulitis lesions containing a single strain of E. coli was 95.4%. If one E. coli strain is observed out of three colonies randomly selected from a future agar plate, the probability is 98.8% that only one strain is on the plate. These results are specific for this cellulitis E. coli scenario. For systems in which the number of bacterial strains per sample is variable, this model provides a quantitative means by which sample sizes can be determined.

Scratches as a component in the pathogenesis of avian cellulitis in broiler chickens exposed to cellulitis origin Escherichia coli isolates collected from different regions of the US

Cellulitis was induced in broiler chickens in two experiments. Birds were placed on used pine-shaving litter at day of hatch and raised to 28 days of age, at which time one-half the birds in each pen were scratched and the litter was treated with either one of seven cellulitis origin Escherichia coli (collected from various locations in the US) or sterile saline. Although minor differences could be detected in the association of specific regional isolates with differing rates of cellulitis, all isolates were capable of inducing cellulitis in a preponderance of the scratched birds. These same isolates were not capable of producing cellulitis in birds that were not scratched by design, confirming the importance of scratches in the pathogenesis of cellulitis. Those birds in the unscratched groups placed on litter inoculated with either a cellulitis E. coli isolate (EC-AR1) or sterile saline that did develop cellulitis lesions showed the remnants of healed scratches which had occurred naturally during the course of the growout, again providing evidence as to the importance of scratches in the development of cellulitis.

Spatial Heterogeneity of Escherichia coli DNA Fingerprints Isolated from Cellulitis Lesions in Chickens

Avian cellulitis in broiler chickens is characterized by subcutaneous lesions that result in economic losses because of the partial or complete condemnation of the carcasses at processing. Escherichia coli is the primary causative agent of this condition. Previous research with a biotyping system found that the E. coli of cellulitis origin were unique to each ranch, suggesting that these E. coli were endemic within the ranch environment. The objective of our study was to analyze the genetic variability of E. coli isolates associated with cellulitis. We analyzed the genetic relatedness of the isolates in relation to the houses, ranches, and complexes in which the broilers were grown. This analysis enabled us to assess the spatial heterogeneity, or genetic diversity on a spatial scale, of the isolates. Forty-nine broilers with cellulitis lesions were necropsied. These broilers came from six houses on four ranches on three complexes that had been placed with chicks from the same hatchery within a 2-wk period. Isolates of E. coli from the lesions were DNA fingerprinted by pulsed-field gel electrophoresis. Relatedness among isolates was determined with the Dice coefficient and an unweighted pair group method with average linkages cluster analysis. The complexes possessed isolates with a variety of DNA fingerprints, yet each complex appeared to have isolates with a unique set of DNA fingerprints. Isolates from the same complex tended to form clusters with similarity coefficients greater than 90%. Isolates from different complexes were genetically distinct. This heterogeneity at the level of the complex suggests that isolates were not disseminated from a source common to the complexes. The spatial heterogeneity of the E. coli isolates in this study implies an endemic population of cellulitis-associated E. coli exists in the broiler house environment.

Evaluation of Scratches as an Essential Element in the Development of Avian Cellulitis in Broiler Chickens

Currently, the published cellulitis models do not adequately address the actual pathogenesis as seen in the commercial broiler industry. In this model, small dermal scratches were made on the skin of broiler chickens, which were then placed on litter seeded with avian cellulitis-associated Escherichia coli. The research confirms scratches are required for the induction of avian cellulitis. The research also confirms that "type I" cellulitis lesions or those previously thought to be due to hatchery-borne infections can be induced with scratches. The described methods provide a realistic model for cellulitis development that will improve the reliability of prophylactic and therapeutic-regimen efficacy testing data, thereby providing information more directly useful to the commercial broiler industry.

Avian cellulitis

In recent years the incidence of avian cellulitis (inflammatory process, IP) has risen dramatically so that today it follows septicaemia-toxaemia as the second leading cause of broiler carcass condemnation. Affected birds frequently appear as large, healthy and normally growing, but exhibit caseous mats called plaques under the skin at processing. Affected flocks increase costs to processors from direct product loss, downgrading and yield loss due to trimming and the slowing of processing lines. Although a number of bacterial species have been isolated from cellulitis lesions, Escherichia coli is most consistently recovered. This review examines past and present scientific and popular press literature on cellulitis and attempts to separate opinion from scientifically verified and tested fact.