Virulence characteristics of Escherichia coli isolates obtained from broiler breeders with salpingitis

Comparative Immunology, Microbiology & Infectious Diseases 28 (2005) 1–15 www.elsevier.com/locate/cimid

Virulence characteristics of Escherichia coli isolates obtained from broiler breeders with salpingitis Maria A.R. Monroya, Terezinha Kno¨blb, Jose´ A. Bottinob, Claudete S. Astolfi Ferreirab, Antonio J. Piantino Ferreirab,* a Facultad de Medicina Veterinaria, Universidad la Salle, Bogota´, Cundinamarca, Colombia Departamento de Patologia, Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Sa˜o Paulo, Av. Prof. Dr Orlando Marques de Paiva, 87, 05508-900 Sa˜o Paulo, SP, Brazil

b

Accepted 17 November 2003

Abstract Thirty isolates of Escherichia coli from broiler breeders with salpingitis were studied. Using the slide agglutination test, the isolates were found to belong to serogroups O1, O2, O5, O36, O45, O53 and O78. Pathogenicity for day-old chicks was determined by air sac inoculation and isolates were categorized as having high, intermediate or low virulence. Growth on iron starvation medium was observed together with aerobactin production. Based on the results of in vitro adherence tests, attachment to oviduct epithelium from old birds was found to be superior to that observed using corresponding material from young birds. DNA hybridization testing for type 1, P, and S fimbriae revealed predominant expression of type 1, correlating with mannose-sensitive hemagglutination using guinea-pig erythrocytes. In this study, P and S fimbriae were not considered to be important adherence factors. Study findings would suggest that, as far as salpingitis is concerned, type 1 fimbriae can play an important role in E. coli infection in breeders. An interesting result to emerge from the study was the observation that E. coli isolates were completely resistant to serum from young breeders, whereas they were completely sensitive using serum from older breeders. Based on serogroups involved, pathogenicity for day-old chicks and virulence indicators, the salpingitis isolates were similar to those from cases of chronic respiratory disease. q 2004 Elsevier Ltd. All rights reserved. Keywords: Escherichia coli; Virulence characteristics; Salpingitis; Chicken

Re´sume´ Caracte´ristiques de la virulence de Escherichia coli isole´es de reproductrices atteintes de salpingite. Trente e´chantillons d’Escherichia coli isole´s de reproductrices atteintes de salpingites ont * Corresponding author. Tel.: þ 55-11-3818-7706; fax: þ55-11-3818-7829. E-mail address: [email protected] (A.J.P. Ferreira). 0147-9571/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2004.03.001

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e´te´ e´tudie´s. L’e´tude par agglutination sur lame a permis d’identifier les se´rogroupes O1, O2, O5, O36, O45, O53 et O78. La pathoge´nicite´ des germes isole´s a e´te´ de´terminine´e par inoculation sur des poussins aˆge´s d’un jour par inoculation dans le sac ae´rien. La pathoge´nicite´ a e´te´ classifie´e de faible a` e´leve´e. La croissance bacte´rienne sur milieu appauvri en fer et la production d’ae´robactine ont aussi e´te´ e´tudie´es. Les tests d’adhe´rences ‘in vitro’ ont montre´ une meilleure adhe´rence a` l’e´pithe´lium des trompes ovariennes des vieux oiseaux par rapport aux jeunes oiseaux. L’hybridation d’ADN a mis en e´vidence une pre´dominance de fimbrie de type 1. Cette pre´dominance est confirme´e par le test d’hemagglutination manosensible (HAMS) re´alise´ avec des he´maties de cobaye. Dans cette e´tude, les fimbries P et S n’ont pas e´te´ conside´re´es comme des facteurs importants d’adhe´rence. Les re´sultats vont dans le sens d’un roˆle important du pili de type 1 dans la salpingite des oiseaux. Des re´sultats inte´ressants ont e´te´ obtenus lorsque les e´chantillons d’E. coli se sont montre´s comple`tement re´sistant au se´rum d’oiseaux jeunes et comple`tement sensible au se´rum d’oiseaux aˆge´s. Si l’on conside`re les serogroupes, le test de pathoge´nicite´ sur les poussins aˆge´s d’un jour et les indicateurs de virulence bacte´rienne, les e´chantillons de salpingite pre´sentent des caracte´ristiques semblables a` la maladie respiratoire chronique. q 2004 Elsevier Ltd. All rights reserved. Mots-cle´: Escherichia coli; Caracte´ristiques de virulence; Salpingite; Poule

1. Introduction Throughout the world, the poultry industry suffers significant economic losses from pathogenic Escherichia coli infection. This can result in decreased productivity, increased condemnation of carcasses, and elevated medication costs. In general, the bacteria act as opportunistic agents in infections caused by viruses or mycoplasmas, or where there are environmental problems or poor management [5]. E. coli infection is associated with several kinds of disease [5] and is considered to be a major etiological cause of salpingitis in chickens [6,8,26,33]. In adult birds, E. coli can infect the reproductive tract, resulting in salpingitis and, occasionally, peritonitis. Early infection in breeders may be clinically asymptomatic, although it can be responsible for the occurrence of opacities, and an ensuing increase in embryonic mortality in the hatchery [5]. Generally, bacterial salpingitis is characterized by a subacute suppurative process, and this is the most common sequel to infection. The inflammatory process is characterized by the formation of a layer of exudate that compresses the mucosa. The migration of granulocytes throughout the mucosa into the oviduct lumen has been reported. Coliform bacteria are the most commonly isolated agents from cases of the disease [49]. Several virulence mechanisms have been identified mainly with chronic respiratory disease (CRD) related strains. They have been associated with type 1 or type 1-like pili and P fimbriae [17,54]. Furthermore, it has been suggested that the expression of type 1 fimbriae is involved in the initial stages of colonization of the upper respiratory tract, whereas the P fimbriae are involved in colonization of the internal organs and the development of septicemia [18,47]. The ability of a bacterium to grow in an iron-deficient medium can be attributed to the production of iron-chelating substances called siderophores. Avian E. coli strains (APEC) usually produce aerobactin [16,19,23,61]. Bacterial resistance to the action of complement

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enables the organism circumvent the host’s defenses [32,46]. Several authors have credited this resistance to the presence of the capsular antigen K-1, as well as to the presence of traT and iss genes [46,66]. Notwithstanding these findings, the virulence mechanisms of avian E. coli have not yet been fully elucidated [19], especially in respect of establishment of infection [59]. In Brazil, as in some major poultry producing regions of the world, there is insufficient information on the virulence characteristics of E. coli strains isolated from salpingitis in breeders. The aim of this study was to identify of the virulence properties of E. coli isolated from salpingitis in broiler breeders.

2. Materials and methods 2.1. Bacterial isolates and strains A total of 30 E. coli isolates were obtained from the oviducts of in-production broiler breeders—ranging from 35 to 45 weeks in age—all with salpingitis. Each isolate was collected from a different bird during seven separate outbreaks of the disease in different poultry-rearing areas in Brazil over a 6-month period in 1998. After isolation and identification, strains of E. coli were stored at 2 20 8C in a Brain Heart Infusion (BHI-Difco) broth containing 15% glycerol. E. coli K12 strain C600 was used as a negative control in all tests, while the LG 1522 and LG 1315 strain were used as a reference in the aerobactin system, as negative and positive control, respectively. E. coli O78 strain DRC 1.5 was used as a positive control in investigating serum resistance and for the performance of the pathogenicity test in 1-day-old chicks [23]. E. coli strain LG 1522 and LG 1315 were kindly supplied by P.H. Williams, Department of Microbiology and Immunology, University of Leicester, Leicester, UK. Both, E. coli K12 strain C600 and E. coli O78 strain DRC 1.5 were isolated in our laboratory. 2.2. Determining the serogroups Serogroups were identified using the slide agglutination test [22]. The complete commercial kit for E. coli serotyping was kindly provided by Dr J. Blanco, Department of Microbiology and Parasitology, University of Santiago of Compostela, Lugo, Spain, and Dr D.S. Leite, University of Campinas, Brazil. 2.3. Pathogenicity test in day-old chicks Ten, 10-day-old male chicks obtained from a commercial source were used to determine the pathogenicity of each isolate. For this purpose, E. coli isolates were cultivated in BHI broth for 1 h at 37 8C under stationary condition. Chicks were challenged with 0.1 ml of the culture containing approximately 107 CFU/ml by inoculation into the left thoracic air sac of each chick. Chicks inoculated with culture

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medium (BHI) alone, or with E. coli K12 strain C600 at the same concentration, served as negative controls. Inoculated chicks were observed daily for a period of 10 days and, based on mortality rates, isolates were classified as follows: highly pathogenic (mortality . 80%), intermediate pathogenicity (mortality . 50% but , 80%), low pathogenicity (mortality , 50%), and non-pathogenic (zero mortality). All birds were necropsied, either after they were found dead or at the end of each experiment [35]. 2.4. ‘In vitro’ adherence to chicken oviduct epithelium Oviducts were obtained from broiler breeders between 12 and 50 weeks of age with no clinical history of disease. The birds were killed, the oviducts removed and washed three times with 0.85% NaCl in order to remove any secretions. Segments approximately 5 cm in length were taken from parts of the magnum and infundibulum and placed in 24-well microplates (Nunc, Roskilde, Denmark) containing 3.0 ml of Eagle’s medium with 5% fetal bovine serum. Using an inverted light microscope, this material was examined for evidence of ciliary motility, which was considered indicative of cell viability. The E. coli isolates from cases of salpingitis and control cultures were grown on Minca agar [27], incubated at 37 8C for 24 h. The adherence test was carried out by depositing 100 ml of bacterial suspension (diluted to contain approximately 106 CFU/ml) into each well of the microplate, which was then incubated at 37 8C for 3 h. 2.5. Colony hybridization for type 1, P, and S fimbriae The isolates were examined by colony blot hybridization [39], using specific DNA probes labeled with [a-d-32P]-dATP by nick translation. The DNA probe employed to detect fim B-H genes was a 9.6 kb HindIII –Sal I fragment from recombinant plasmid, pIB254 [34]. Detection of pap and sfa genes was determined using oligonucleotide fragments obtained by the polymerase chain reaction as described by Le Bouguenec et al. [38]. 2.6. Hemagglutination assay The presence of fimbriae was determined based on the ability of strains to agglutinate erythrocytes from guinea-pig, human and sheep in the presence or absence of 2% D -mannose, i.e. mannose-resistant hemagglutination (MRHA) and mannose-sensitive hemagglutination (MSHA), respectively, as described by Evans et al. [20]. E. coli isolates were cultured on Minca agar [27] at 37 8C for 24 h. 2.7. Growth in iron-deficient medium E. coli isolates were cultured in Luria Broth (LB) containing the iron chelator 2,20 -dipyridyl (Sigma) at a final concentration of 200 mM. Cultures were incubated at 37 8C for 24 h. After the second subculture, isolates were cultured on LB agar with 2,20 dipyridyl, to identify the strains that produced siderophores [58].

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2.8. Aerobactin production assay The detection of aerobactin was carried out according to the procedures of Carbonetti and Williams [12]. Isolates were cultured in a M9 minimal medium containing 200 mM of 2,20 -dipyridyl [58]. The test for aerobactin was conducted using E. coli K12 strain LG 1522, an organism whose phenotype is aerobactin-deficient [65] together with E. coli K12 strain LG 1315 [65] and E. coli K12 strain C600 [55] as positive and negative controls, respectively. 2.9. Serum resistance Determination of resistance to the bactericidal action of serum was conducted as described by Nilius and Savage [45]. Blood was obtained from 12-week-old and 50-weekold broiler breeders with no history of the disease. Optical density of serum mixed with bacterial suspension was measured on an hourly basis for 5 h using a spectrophotometer at a wavelength of 550 nm. E. coli K12 strain C600 and E. coli O78 strain DRC 1.5 were used as a negative and as a positive control, respectively. 2.10. Statistical analysis The significance of any differences observed was determined using differentiation test between proportions ðp , 0:05Þ [57].

3. Results Of the 30 E. coli isolates examined, 6 (20%) belonged to serogroup O2, 5 (16.66%) to serogroup O78, 4 (13.33%) to serogroup O5, 2 (6.66%) to serogroup O53, 1 (3.33%) to serogroup O1, 1 (3.33%) to serogroup O36 and 1 (3.33%) to serogroup O45. Ten isolates (33.33%) were not typeable. As illustrated in Table 1, pathogenicity tests in 1-day-old chicks revealed that 12 (40%), 15 (50%), and 3 (10%) isolates were of high, intermediate and low virulence, respectively. In instances where no mortality occurred after 4 days, lesions were observed on necropsy involving one or both air sacs. Occasionally, pericarditis and perihepatitis with fibrin deposits were also detected. In relation to adherence tests, 19 of the isolates (63.33%) adhered to the oviduct epithelia of both the young and the old birds. A total of 7 isolates (23.33%) adhered only to the epithelia of old birds, and 4 isolates (13.33%) did not adhere to either epithelia. There were no significant differences between the results of adherence to the epithelia of either young or adult birds ðp , 0:05Þ: As far as bacterial adherence to chicken oviducts is concerned, no differences were observed between the different regions of the organ studied, i.e. infundibulum and magnum (Fig. 1A – C). Results from the DNA hybridization tests revealed that 24 isolates (80%) were positive for type 1 fimbriae and agglutinated guinea-pig erythrocytes in the absence of D -mannose. However, 2 isolates (6.67%) were positive in the guinea-pig hemagglutination test but

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Table 1 Results of pathogenicity tests in day-old chicks of strains of E. coli isolated from adult broiler breeders with salpingitis Salpingitis outbreaka

E. coli isolates and designation

Serogroups ‘O’

Mortality index

Pathogenicity classification

Number of birds dead/challenged

Mortality (%)

1

BV1 BV2 BV3 BV4

2 2 2 2

10/10 10/10 10/10 10/10

100 100 100 100

2

584-1 584-2 584-3

2 36 2

5/10 6/10 5/10

50 60 50

Low Intermediate Low

3

598-1 598-2 598-3 598-4

NTb NT NT NT

7/10 8/10 7/10 4/10

70 80 70 40

Intermediate High Intermediate Low

4

582-1 582-2 582-3 582-4

53 5 45 5

7/10 6/10 7/10 7/10

70 60 70 70

Intermediate Intermediate Intermediate Intermediate

5

3720-1 3720-2 3720-3 3720-4

78 78 78 78

7/10 7/10 7/10 7/10

70 70 70 70

Intermediate Intermediate Intermediate Intermediate

6

C1-1 C1-2 C2-1 C2-2 C2-3 C3-1 C3-2 C4-1 C4-2

78 53 5 5 NT NT NT NT NT

10/10 10/10 8/10 6/10 7/10 7/10 10/10 8/10 7/10

100 100 80 60 70 70 100 80 70

High High High Intermediate Intermediate Intermediate High High Intermediate

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PLO-1 PLO-2 E. coli DRC 1.5c E. coli K12 C600d

1 NT 78

10/10 10/10 10/10 0/10

100 100 100 0

High High High Non-pathogenic

a b c d

The number of E. coli isolates from each salpingitis outbreak, designated one to seven. NT, these isolates were not typable. E. coli O78 strain DRC 1.5 was used as a positive control. E. coli K12 C600 was used as a negative control.

High High High High

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Fig. 1. Adherence of E. coli to oviduct epithelium of young and adult chickens. (A) Adherence of E. coli O1 strain PLO-1 to ciliated epithelium of chicken oviduct, magnum (arrow). (B) Adherence of E. coli O2 strain BV-4 to ciliated epithelium from chicken oviduct and to secreted mucus, infundibulum (*, arrow). (C) Adherence of E. coli O78 strain 3720-2 (arrow) to ciliated oviduct epithelium from an adult chicken, magnum (arrow). (A, B, and C, Giemsa staining, 360 £ ).

negative for type 1 fimbriae. Additional isolates that were negative for type 1 fimbriae in the hybridization test were also negative in the hemagglutination test. Based on the results of hemagglutination tests conducted in the presence of D -mannose, 5 isolates (16.67%) agglutinated both human and sheep erythrocytes; 5 isolates (16.67%) agglutinated only human erythrocytes; 7 isolates (23.33%) agglutinated only sheep erythrocytes and, finally, 13 isolates (43.33%) did not agglutinate either human or sheep erythrocytes. Only 2 E. coli isolates (BV1 and BV4) hybridized with the pap operon and showed MRHA activity with human erythrocytes. All isolates examined were negative for the sfa operon. When cultured in iron-deficient medium, 19 isolates (63.33%) produced siderophores and aerobactin. Two isolates (6.67%) produced only siderophores, and 9 isolates (30%) neither grew in the iron-deficient medium nor produced any aerobactin (Table 2). Of the 30 isolates studied, 27 (90%) were resistant in the presence of 12.5% serum from young birds, while one isolate (3.33%) showed intermediate resistance. Two others (6.67%) were completely sensitive under the same experimental conditions. Using serum from adult birds the same experimental conditions, all isolates (100%) showed growth inhibition (Table 2). There were significant statistical differences ðp , 0:05Þ between the results using two the types of serum. Results of the tests conducted on the 30 strains are summarized in Tables 1 and 2.

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Table 2 Analysis of virulence factors associated with Escherichia coli isolates from broiler breeders with salpingitis E. coli isolates and controls

DNA hybridization

Hemagglutination activity

Young birds (12-week-old)

F1

MSHA Gp

Older birds (50-week-old)

P

S

Growth on iron-deficient medium

Aerobactin production

MRHA Hu

Sh

Serum resistancea (12.5%)

Young birds (12-week-old)

Older birds (50-week-old)

BV1 BV2 BV3 BV4

þ þ þ þ

þ þ þ þ

þ þ þ þ

þ 2 2 þ

2 2 2 2

þ þ þ þ

þ 2 þ þ

2 þ þ 2

þ þ þ þ

þ þ þ þ

R R R R

S S S S

584-1 584-2 584-3

þ þ þ

þ þ þ

þ þ þ

2 2 2

2 2 2

þ þ þ

þ 2 2

þ 2 2

2 2 2

2 2 2

R R R

S S S

598-1 598-2 598-3 598-4

2 þ 2 2

þ þ þ þ

þ þ þ þ

2 2 2 2

2 2 2 2

þ þ þ þ

þ 2 þ 2

2 þ þ 2

þ þ 2 2

þ þ 2 2

R R R S

S S S S

582-1 582-2 582-3 582-4

2 þ 2 þ

þ þ þ þ

2 þ þ þ

2 2 2 2

2 2 2 2

2 þ þ þ

2 2 2 2

2 þ 2 2

þ þ þ þ

þ þ þ þ

S R R R

S S S S

3720-1 3720-2 3720-3 3720-4

þ þ þ þ

þ þ þ þ

þ þ 2 þ

2 2 2 2

2 2 2 2

þ þ 2 þ

þ þ 2 2

þ 2 2 þ

þ þ þ þ

þ þ þ þ

R R R R

S S S S

C1-1 C1-2 C2-1 C2-2

2 þ þ þ

þ þ þ þ

2 2 þ 2

2 2 2 2

2 2 2 2

2 þ þ þ

2 2 2 2

2 2 þ þ

þ þ þ 2

þ þ þ 2

R R R I

S S S S

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‘In vitro’ adherence to oviduct epithelium

2 þ þ 2 2

2 þ þ 2 2

þ þ þ þ 2

2 2 2 2 2

2 2 2 2 2

þ þ þ þ 2

2 2 2 þ 2

þ 2 2 2 2

2 þ þ 2 2

2 2 þ 2 2

R R R R R

S S S S S

PLO-1 PLO-2

2 2

þ 2

þ þ

2 2

2 2

þ þ

2 2

2 þ

þ þ

þ þ

R R

S S

Total of positives

19

26

24

2

0

26

9

12

21

20

S ¼ 30

E. coli DRC 1.5b E. coli K12 C600c

ND 2

ND 2

2 ND

2 2

2 2

ND 2

ND 2

ND 2

þ 2

þ 2

R ¼ 27, I ¼ 1, S¼2 R S

a

ND S

Serum resistance was determined and designed as: R, resistant; S, sensitive; I intermediate. E. coli O78 strain DRC 1.5 was used as a positive control. c E.coli K12 strain C600 was used as a negative control. ND, not determined. þ , growth or adherence; 2, no growth or adherence. Gp, guinea-pig; Hu, human; Sh, sheep. b

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C2-3 C3-1 C3-2 C4-1 C4-2

9

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4. Discussion The E. coli serogroups identified in this study have already been reported from cases of avian colibacillosis [5] and been associated with salpingitis [4,7,8,25,37,51]. Fundamental to the pathogenesis of the disease is the ability of the bacteria to adhere to and colonize the host’s tissues [28,43,48]. The results of this study, which examined the adherence properties of E. coli strains in vitro, showed that there was greater adherence to ciliated epithelium in the oviducts removed from adult breeders (50-week-old) than was observed using the same tissue from young breeders (12-week-old). Salpingitis afflicts adult birds in production [5] when cytodifferentiation of the oviduct from hormone production has already taken place [9,31,36,53]. The observed difference in adherence properties may be associated with this differentiation, especially since mature epithelium comprises different types of epithelial cells [36,53]. This being the case, it is likely that certain proteins present on the cellular membrane allow for binding with E. coli. The presence of cilia in the differentiated mucosa [9,53] could also be involved in bacterial adherence. Dho and Lafont [15] have associated the virulence of avian E. coli with its ability to adhere to tracheal epithelial cells. However, in another study, a comparison was made between pathogenic isolates from broilers with CRD and non-pathogenic isolates from the cecum of healthy broilers. Both types adhered in vitro to ciliated tracheal epithelium [23]. Another possibility is that adherence occurs via proteins that are secreted. Protein secretion is different from one epithelium to the other, i.e. the oviduct of the immature chick does not secrete egg-white proteins [9]. When progesterone was administered to chickens after a short estrogen pretreatment, the production of secretory proteins on the surface epithelium [31] and of induced mucus producing goblet cells in the surface epithelium was noted [52]. Because there is greater secretion of proteins on the epithelium of adult chickens, these proteins could play an essential role in the adherence of E. coli. The presence of mucus on this epithelium could represent another factor involved in greater bacterial adherence. Since previous studies have shown that certain E. coli strains isolated from chickens with colisepticemia adhere to the mucus layer [63], we suspect that this phenomenon can also occur with respect to salpingitis isolates. In this study, 80% of E. coli isolates were found to hybridize with fim operon and exhibited MSHA with guinea-pig erythrocytes. Also, the 21 isolates (70%) that agglutinated guinea-pig erythrocytes and hybridized with the fim operon, were shown to adhere to the epithelium of adult chickens, indicating the importance of pili type 1 in adherence of E. coli to the oviduct. This adherence may be linked to the greater secretory capacity of these cells, since it is known that E. coli type-1 fimbriae can bind to various non-cellular body constituents, including soluble glycoproteins in body fluids and extra cellular matrix proteins [1]. Likewise, pili type 1 can also adhere to epithelial cells [41]. Studies with Salmonella spp. have demonstrated that adherence to epithelial cells is a complex process involving different fimbrial operons [30]. The simultaneous gene deletion involved in the biosynthesis of four distinct fimbrial adhesins results in marked attenuation of Salmonella typhimurium [60]. Other fimbriae besides type 1 must be directly involved in adherence of E. coli isolates to the epithelium of the adult chicken

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since three of the isolates (16.67%) in this study were negative for the fim, pap, and sfa operons and also were negative in the hemagglutination assays. Although some isolates agglutinated human erythrocytes (10 of the isolates) or sheep erythrocytes (12 of the isolates) in the presence of D -mannose, only isolates BV-1 and BV-4 hybridized with the pap DNA probe. This finding would suggest that further investigation is needed to establish the relevance of adherence in the oviduct of both young and older birds in the pathogenesis of salpingitis caused by E. coli, as well as its possible significance with respect to other infectious agents. The results of this study confirm the ability of different E. coli isolates to produce siderophores when cultured in an iron-deficient medium. Closer examination of the relationship between pathogenicity and the ability to grow in an iron-deficient medium revealed that the 11 pathogenic isolates studied (91.67%) produced siderophores and aerobactin, and that 10 (66.67%) other strains characterized as having intermediate virulence also produced siderophores. Likewise, three isolates of low pathogenicity did not grow at all in the iron-deficient medium. The results of the study have shown that there is indeed a strong correlation between the ability of E. coli strains to grow in an irondeficient medium and pathogenicity. The salpingitis isolates studied behaved in a similar manner to strains of E. coli isolated from cases of CRD and colisepticemia [14,16,40,44, 61,62]. Based on a correlation among pathogenicity, iron-sequestration and adherence to epithelium of adult breeders, 83.33% of the highly pathogenic isolates and 66.67% of intermediately pathogenic isolates conformed to this phenotype. On the other hand, it was not possible to establish such a correlation for any of the isolates of E. coli of a low pathogenicity. These results are consistent with those with of Dho and Lafont [16], who observed an association between adherence and iron-sequestration properties with 52% of the most lethal strains they studied. Results from this study have led us to hypothesize that strains of E. coli may compete with ovotransferrin for the iron present in the oviduct, chelating and using this source of iron for their metabolism and growth. This highlights the importance of the production of both siderophores and aerobactin in the pathogenesis of salpingitis. It can be concluded that iron is essential for bacterial growth, and that the ability of an organism to chelate iron from host tissues can increase its pathogenicity [11]. By comparing the bactericidal activity of sera from young breeders with that from adult breeders, it was possible to verify that adult serum is a better inhibitor of bacterial growth when used at a 12.5% concentration. Significant differences ðp , 0:05Þ were found between the two sources of serum, with all the isolates studied sensitive to the serum obtained from adult breeders. Greater susceptibility of avian isolates of E. coli to adult chicken serum has also been reported [23]. In the latter study, serum from 28-day-old chickens was compared with that from 1-year-old birds when used at a 5% concentration. This study demonstrated that serum from adult birds increased the profile of bacterial susceptibility. Some authors have suggested that this can be related to the antibody repertoire stimulated by enteric microbiota, subclinical infection and other factors, including the production of defensins by heterophils [10,21,29]. Both IgG and IgM immunoglobulins against E. coli are commonly found in the serum of animals without any reported history of clinical disease [32,42].

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The significance of serum resistance and presence of the capsular antigen K-1 is that the latter has low immunogenic properties and does not activate the alternative route of complement [24,32,64]. In birds, virulent strains of the O1:K1, O2:K1, and O50:K1 serogroups are found in cases of colibacillosis [56,61]. The membrane protein known as traT could function as a mechanism for evasion of complement [2]. A comparison between pathogenicity and serum resistance led to the discovery that all virulent strains of E. coli were resistant to serum from breeder birds. Among isolates of intermediate virulence, 13 (43.3%) were resistant to serum from young birds, 1 (3.3%) showed intermediate resistance, and 1 (3.3%) was completely sensitive to it. Out of the three low virulence isolates, two (Isolates 584-1 and 584-3) belonging to serogroup O2, were resistant to serum, whereas the other isolate (Isolate 598-4) was serum sensitive. Other workers have previously reported the correlation between pathogenicity and serum resistance observed in this study [13,46,66]. Although strains of E. coli characterized in this study were isolated from different outbreaks, an analysis of the results of the parameters studied, only two isolates (6,67%) could be considered to be from a single clone. The findings of the study help gain a better understanding of the virulence properties of E. coli that play an important role in the pathogenesis of salpingitis. These properties were found to be similar to those of strains of E. coli isolated from cases of CRD in terms of the serogroups they belong to, virulence indicators and pathogenicity for day-old chicks [3,16,17,23,50,56]. Although greater adherence to oviductal epithelium from adult chickens was observed, 19 isolates (63.33%) adhered also to the epithelium from young chickens, confirming that these isolates could cause salpingitis in chicks whose epithelium had not yet fully matured. The virulence characteristics of E. coli isolates recovered from salpingitis in broiler breeders needs further study, especially in respect of adherence properties and in vivo pathogenicity.

Acknowledgements We are grateful to CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico) for financial support—grant 404869/90-4VT and to CAPES for fellowship to M.A.R. Monroy.

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