Celullitis in broilers
Cellulitis-associated bacterial virulence and resistance of broiler strains
Published: October 19, 2011
By: BG Brito1*, TM Tejkowski2, GA Oliveira2; BD Soares2, FRF Jaenisch3, VY Obara2, SMH Silva2, AR Torres2, J Domingues2, RO Melo2, KCT Brito4
1Instituto de Pesquisas Veterinárias Desidério Finamor. Rio Grande do Sul – Brazil; 2Bolsista CNPq- Brazil; 3Embrapa Suínos e Aves. Santa Catarina – Brazil; 4Ecolvet Laboratório. Paraná – Brasil.
Summary
Cellulitis-associated risk factors such as inoculum dose rate, strain source, and influence of the iss and iutA genes in the pathogenicity of Escherichia coli, and the resistance of two broiler strains were evaluated. The combination of these two genes results in worse lesions, which shows the characteristics of this multifactorial disease. No significant difference was found in the susceptibility to cellulitis in two broiler strains. Furthermore, we observed a correlation between challenge inoculum bacterial concentration and the magnitude of lesions in the chicken. We concluded that the iss and iutA genes serve as molecular markers for the virulence of cellulitis-producing E. coli isolates.
Key Words: Broilers, Cellulitis, Skin, Risk factors.
Introduction
Epidemiologic studies regarding the etiology of avian cellulitis, have shown that Escherichia coli (E. coli) is the main organism found in this pathology (Fallavena, 2000). E. coli has some genes responsible for its pathogenicity, including the iut A and iss genes (Brito et al., 2003). Besides, several authors state that this disease is multifactorial and that certain bird strains are more prone to cellulitis (Barnes et al., 2003). The objective of this study was to analyze the risk factors related to this pathology, such as the dose of inoculum, source of the strain, and influence of the iss and iutA genes in the pathogenicity of E. coli, and the resistance of two broiler strains.
Materials and Methods
Experiment 1
One hundred, 56-day-old, Cobb broilers were used. Birds were distributed into groups of 20 birds each. Groups were challenged with E. coli (CEL 49) at a concentration of 109 colony-forming units (CFU)/mL, 108 CFU/mL, 107 CFU/mL and 106 CFU/mL, a control group inoculated, just and only, with saline. Three days after, animals were evaluated for the occurrence cellulitis lesions.
Experiment 1
One hundred, 56-day-old, Cobb broilers were used. Birds were distributed into groups of 20 birds each. Groups were challenged with E. coli (CEL 49) at a concentration of 109 colony-forming units (CFU)/mL, 108 CFU/mL, 107 CFU/mL and 106 CFU/mL, a control group inoculated, just and only, with saline. Three days after, animals were evaluated for the occurrence cellulitis lesions.
Experiment 2
Ten strains of E. coli were used, 5 of them of a fecal origin and the other 5 were isolated from lesions of cellulitis. Each strain was characterized based on the presence or absence of virulence iss and iut A factors, by means of the Polymerase Chain Reaction (PCR). Moreover, 110 Cobb broilers, one day old, were challenged by means of a pathogenicity test. Ten birds were used for each strain, a control group inoculated with saline. A bacterial concentration of 106 CFU/mL was used in the inoculum. The evaluation of the animals regarding the occurrence of lesions of cellulitis took place three days after.
Ten strains of E. coli were used, 5 of them of a fecal origin and the other 5 were isolated from lesions of cellulitis. Each strain was characterized based on the presence or absence of virulence iss and iut A factors, by means of the Polymerase Chain Reaction (PCR). Moreover, 110 Cobb broilers, one day old, were challenged by means of a pathogenicity test. Ten birds were used for each strain, a control group inoculated with saline. A bacterial concentration of 106 CFU/mL was used in the inoculum. The evaluation of the animals regarding the occurrence of lesions of cellulitis took place three days after.
Experiment 3
The strains of the above mentioned experiment, were inoculated into 250 broilers, 35 days old, of a commercial strain. Birds were divided into 10 groups, 20 animals each and each group was challenged with different strains. Fifty birds were used as a control group, receiving saline solution inoculums. Groups were subcutaneously inoculated in the left side of the breast, with 107 CFU/mL of each strain and were observed during three days in search of the occurrence of the lesions. Immediately after, necropsy was performed and lesions were classified according to the area of the lesion.
The strains of the above mentioned experiment, were inoculated into 250 broilers, 35 days old, of a commercial strain. Birds were divided into 10 groups, 20 animals each and each group was challenged with different strains. Fifty birds were used as a control group, receiving saline solution inoculums. Groups were subcutaneously inoculated in the left side of the breast, with 107 CFU/mL of each strain and were observed during three days in search of the occurrence of the lesions. Immediately after, necropsy was performed and lesions were classified according to the area of the lesion.
Experiment 4
One hundred (50 Ross and 50 Cobb) broilers were used. Both groups were raised in the same way, up to 35 days of age, when they were challenged with a virulent strain of E. coli CEL 49) isolated from a lesion of cellulitis. Birds were divided into 3 treatment groups: group 1 - control, inoculated with saline solution , group 2 was challenged with a dose of 108 CFU/mL (high challenge) and group 3, received 10 5 CFU/mL (low challenge), subcutaneously in the breast region. Necropsy took place three days after challenge and lesions were classified according to the area and degree of the lesion.
One hundred (50 Ross and 50 Cobb) broilers were used. Both groups were raised in the same way, up to 35 days of age, when they were challenged with a virulent strain of E. coli CEL 49) isolated from a lesion of cellulitis. Birds were divided into 3 treatment groups: group 1 - control, inoculated with saline solution , group 2 was challenged with a dose of 108 CFU/mL (high challenge) and group 3, received 10 5 CFU/mL (low challenge), subcutaneously in the breast region. Necropsy took place three days after challenge and lesions were classified according to the area and degree of the lesion.
Results and Discussion
Experiment 1
Birds challenged with the concentration of E. coli of 108 CFU/mL y 109 CFU/mL presented lesions of cellulitis of a medium size of 2.8 and 26.8 cm2, respectively. Chicken receiving inoculums of 106 CFU/mL and 107 CFU/mL showed no cellulitis lesions.
Experiment 1
Birds challenged with the concentration of E. coli of 108 CFU/mL y 109 CFU/mL presented lesions of cellulitis of a medium size of 2.8 and 26.8 cm2, respectively. Chicken receiving inoculums of 106 CFU/mL and 107 CFU/mL showed no cellulitis lesions.
Experiment 2
When compared with fecal E. coli isolates, the E. coli, strains isolated from lesions of cellulitis, showed a greater ability to kill one-day-old birds. PCR analysis proved that low pathogenicity E. coli isolates, or those without pathogenicity, did not feature the iss or iut A genes. Delicato et al. (2003), observed that iut A, iss, cva C, tsh, pap C and pap G and fel A genes, were detected more frequently in the isolates obtained from colibacillosis than from those from healthy bird feces. This shows that virulence genes with different potentials participate in the pathogenesis of colibacillosis.
When compared with fecal E. coli isolates, the E. coli, strains isolated from lesions of cellulitis, showed a greater ability to kill one-day-old birds. PCR analysis proved that low pathogenicity E. coli isolates, or those without pathogenicity, did not feature the iss or iut A genes. Delicato et al. (2003), observed that iut A, iss, cva C, tsh, pap C and pap G and fel A genes, were detected more frequently in the isolates obtained from colibacillosis than from those from healthy bird feces. This shows that virulence genes with different potentials participate in the pathogenesis of colibacillosis.
Experiment 3
We analyzed the capacity of the bacteria carrying iss and iut A genes of causing lesions in 35-day-old chickens. Table 1 shows that when E. coli strains possess the two virulence factors major, more severe lesions are present; this does not happen when the genes act independently.
We analyzed the capacity of the bacteria carrying iss and iut A genes of causing lesions in 35-day-old chickens. Table 1 shows that when E. coli strains possess the two virulence factors major, more severe lesions are present; this does not happen when the genes act independently.
Table 1. Area of lesions of cellulitis in broilers, after inoculation with E. coli, with or without the iss and iut A virulence factors
|
Virulence
Profile
|
Cellulitis Area (cm²)
|
|
iss+ / iut A +
|
9.25
|
|
iss+ / iut A -
|
5.96
|
|
iss - / iut A +
|
5.05
|
|
iss - / iut A -
|
2.76
|
Factors such as the increased occurrence of trauma, higher stocking density, delayed feathering, litter caking, etc., are correlated with the increase of cellulitis lesions. These factors, together with broiler strain susceptibility, may cause more or less lesions of cellulitis (Barnes et al., 2003).
Experiment 4
When challenged with high or low E. coli. concentrations, both broiler strains showed cellulitis lesions of a similar intensity. Furthermore, it was possible to observe linearity regarding the area of the lesion as challenge inoculum concentration was decreased (Table 2).
When challenged with high or low E. coli. concentrations, both broiler strains showed cellulitis lesions of a similar intensity. Furthermore, it was possible to observe linearity regarding the area of the lesion as challenge inoculum concentration was decreased (Table 2).
Table 2: Average area of the cellulitis lesions (cm2) in the two broiler strains subjected to challenge with different concentrations of E. coli
|
Challenge
|
CFU/mL
|
AVERAGE AREA OF THE LESIONS (cm²)
|
|
|
ROSS
|
COBB
|
||
|
High
|
10⁸
|
16.55
|
17.10
|
|
Low
|
10⁵
|
4.29
|
4.05
|
Conclusions
The models for the experimental reproduction of cellulitis developed in this research allow for the differentiation of the pathogenicity of E. coli strains involved in cases of cellulitis.
The iss and iut A genes serve as molecular markers for the virulence of cellulitis-associated E. coli isolates. The combination of these two genes, promotes lesions of a higher intensity, which shows the multifactorial nature of this pathology.
There was no significant difference in the comparison of the two broiler strains regarding their susceptibility to cellulitis lesions. Correlation exists between inoculum bacterial concentration and lesion size in birds.
Support
National Council for Scientific and Technological Development - Rhae and Doux Program.
Bibliography
Barnes HJ, Vaillancourt JP, Gross WB. 2003. Colibacillosis. pp. 631-644. In: Diseases of poultry. Calnek BD (ed.). University Press, Ames, USA.
Brito BG, Gaziri LCJ, Vidotto MC. 2003. Virulence factors and clonal relationships among Escherichia coli strains isolated from broiler chickens with cellulitis. Infect. Immun. 4175-4177.
Delicato ER, Brito BG, Gaziri LCJ, Vidotto MC. 2003. Virulence-associated genes in Escherichia coli isolates from poultry with colibacillosis. Vet. Microbiol. 94(2):97-103.
Fallavena LCB. 2000. Enfermidades da pele e das penas. pp. 239-252. In: Doenças das aves, Berchieri Júnior A & Macari M (eds.). FACTA. Campinas.
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