Toxigenic Escherichia coli in Spanish piggeries from 1986 to 1991

veterinary microbiology Veterinary

Microbiology

47 ( 1995) 17-25

Toxigenic Escherichia coli in Spanish piggeries from 1986 to 1991 J.I. Garabal, E.A. Gonzalez *, F. Vazquez, J. Blanco, M. Blanc0 Departamento de Microbioloxia e Parasitoloxia. Facultade de Veterinaria. Universidade de Santiago Campus de Lugo. Lugo-27002 Spain Received 1 November

1994; accepted

17 March 1995

Abstract Four-hundred and fourteen faecal samples from pigs with diarrhoea, oedema disease or healthy pigs, were collected from 65 piggeries located in different areas of Spain from 1986 to 1991. A total of 1334 Escherichiacoli cultures were isolated from the pigs and studied for production of heat-labile (LT) and heat-stable ( STa) enterotoxins, verotoxin (VT) and for type 1 ( CNFl ) and type 2 (CNF2) cytotoxic necrotizing factors. Strains producing enterotoxins (P < 0.001) or verotoxin (P < 0.05) were associated with enteric diseases of pigs. In the majority ( 82.3%) of piglets with strains, producing verotoxin the strains were also positive for production of STa enterotoxin. The most frequent toxin detected was STa. Although we isolated strains producing CNFl from 1.5% of sick pigs, they were not statistically associated (P < 0.7) with emetic disease. Pigs may constitute a natural reservoir of CNFl producing E. coli strains in Spain; their presence in the porcine intestine may be of significance in public health because such strains have been associated with human extraintestinal infections. Keywords:

Escherichia coli; Toxins; Diarrhoea;

Oedema disease

1. Introduction

Escherichia coli is a normal component of the lower gut bacterial flora, although some strains are pathogenic for humans and domestic animals (Levine, 1987; Holland, 1990). Diseases associated with E. coli infection include diarrhoea and toxemia caused by toxigenic strains and systemic colibacillosis caused by invasive strains (Wray and Morris, 1985; Fairbrother, 1993 ) . Enterotoxigenic (ETEC) and verotoxigenic (VTEC) E. coli constitute the major categories of toxigenic strains that cause colibacillosis in newborn and postweaning pigs (Hol* Corresponding

author, Tel: 82-252231

ext. 201 Fax: 82-252195

0378-l 135/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSD10378-1135(95)00107-7

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J.I. Garabal et al. /Veterinary

Microbiology 47 (1995) 17-25

land, 1990; Imberechts et al., 1992). Enterotoxigenic E. coli strains cause diarrhoea mainly in newborn and weaning piglets. ETEC strains synthesize heat-stable ( STa and/or STb) and heat-labile (LT) enterotoxins, and colonize the small intestine by means of speciesspecific fimbrial adhesins ( Gaastra and De Graaf, 1982; Fait-brother, 1993 ) . Oedema disease or E. coli-toxemia constitutes another important disease iyhich usually occurs in pigs shortly after weaning. The cause of oedema disease is complex and factors such as food composition, ambient temperature and genetic susceptibility, play important roles in the onset of the disease (Nielsen, 1986; Imberechts et al., 1992). However this disease has been associated with infection by verotoxigenic E. coli strains (Linggood and Thompson, 1987; Gannon et al., 1989; MacLeod and Gyles, 1990). VTEC strains belong to a limited number of serotypes, predominantly 0138:K81, 0139:K82 and 0141:K85 (Smith et al., 1983; Gonzalez and Blanco, 1985a), and synthesize the Edema Disease Principle (EDP) , also named oedema verocytotoxin (VTe) or Shiga-like toxin II variant (SLT-IIv), which is a toxic protein active on Vero cells (Blanc0 et al., 1983; Dobrescu, 1983; Marques et al., 1987). VTEC strains, like ETEC, require adhesive mechanisms with which to attach themselves to the intestinal epithelium in order to resist the washing flow of the intestine. Recently, F107 fimbriae have been described in VTEC strains isolated from pigs with oedema disease and postweaning diarrhoea (Bertschinger et al., 1990; Imberechts et al., 1994). During the last decade, in addition to LT, ST and VT, some E. coli strains of human and animal origin have been found to synthesize a toxin named cytotoxic necrotizing factor (CNF) (Caprioli et al., 1983; Gonzalez and Blanco. 1985a; De Rycke et al., 1987). These strains were named necrotizing E. coli (NTEC) and recently the existence of two different types of cytotoxic necrotizing factors (CNFl and CNF2) produced by human and animal isolates was established (De Rycke et al., 1990; Blanc0 et al., 1990). Nevertheless, the role of NTEC strains in animal infection and their prevalence in pigs remains unknown. The objective of the investigation reported here was to determine the association of ETEC, VTEC and NTEC in porcine colibacillosis in Spain.

2. Materials and methods 2. I. Animals and samples Faecal samples were collected from 65 piggeries located in different areas of Spain (Zone A and B) from 1986 to 1991. Zone A corresponds to Galicia in the North-west, and Zone B corresponds to other regions (Andalucia, Aragon, Castilla-Le6n, Castilla-La Mancha, Madrid and Murcia). Stool specimens were obtained using a rectal swab from 337 piglets with enteric disease (diarrhoea and oedema disease) and from 77 healthy piglets used as controls. The pigs were divided into two age groups for further studies: younger than fifteen and older than fifteen days. A single faecal sample was taken from each pig. 2.2. Microbiological methods Samples were streaked for colony isolation onto eosin-methylene blue agar and McConkey agar plates (Oxoid, U.K.). After incubatian (37°C for 24 h), typical lactose-

J.I. Garabal et al. /Veterinary Microbiology 47 (1995) 17-25

19

fermenting colonies were identified as E. coli by standard bacteriological methods and biochemical tests (API-20E and VITEK, bioM&-ieux, France). Three to five colonies per sample were identified and a total of 1334 E. coli cultures from 414 pigs were investigated for toxin production. Pure cultures were punctured in soft nutrient agar tubes (Difco, USA), with only 0.75% (w/v) agar, and kept at room temperature. 2.3. Toxin production Erlenmeyer flasks containing 5 ml tryptone soya broth (Oxoid, U.K.), pH 7.5, were inoculated and incubated in an orbital shaker (37”C/200 rpm for 20 h) . For production of CNFl and CNF2, 20 ~1 of mitomycin C solution (Sigma, USA) containing 2.5 pg of mitomycin C, was added to cultures 5 h after inoculation, as previously described (Blanc0 et al., 1990). After incubation, cultures were centrifuged (6000 g/4”C for 15 min) and supernatants were tested for presence of STa, LT, VT, CNFl and CNF2, or stored at 4°C for a maximum of a week. Haemolysin production was detected after 24 h growth on blood agar medium (Merck, Germany) containing 5% (v/v) washed sheep erytrocytes. 2.4. Detection of STa enterotoxin STa was demonstrated by the infant mouse test (IMT) with BALB/C mice. To sum up, 50 ~1 Evans blue dye (2% w/v in PBS) was added to 1 ml of heated ( 100°C for 15 min) culture supematant without mitomycin C, and 0.1 ml of the mixture per mouse was inoculated into three 3-4 day old mice as previously described (Gonzalez and Blanco, 1985b). A supematant was positive for STa when the IMT ratio (intestine weight/carcass weight) was >O.l. 2.5. Detection of LT, VT, CNFI and CNF2 on cell cultures Toxic activity of culture filtrates (with and without mitomycin C) was tested on Vero and HeLa cell monolayers in plastic plates 24 X 16 mm diameter wells (Soria Greiner, Spain), as previously reported (Gonzalez and Blanc0 1985a; Blanc0 et al., 1990). Cell monolayers were obtained by seeding 0.5 ml RPM1 growth medium with 10% foetal calf serum (Flow, U.K.) per well containing lo5 cells the day before inoculation. For the Vero cell assay, growth medium was substituted at the moment of the assay by fresh Eagle’s Minimum Essential Medium with Earle’s salts but without foetal calf serum and supplemented with polymyxin B sulphate (50 units/ml) and 0.05 mM methyl-isobutyl-xanthine (Sigma, USA) as a phosphodiesterase inhibitor. For the HeLa cell assay, the growth medium was changed at the moment of assay by fresh RPM1 without foetal calf serum and supplemented with polymyxin B sulphate. To each well containing HeLa or Vero cell monolayers with 0.5 ml fresh medium, 50 ~1 of culture filtrate passed through 0.22 pm pore size filters was added. The cells were incubated at 37°C in a 5% CO,/95% air atmosphere, and specific morphological changes in the cells were observed under a phase contrast inverted microscope after 24 h and 48 h of incubation. Morphological changes caused by LT, VT, CNFl and CNF2 on Vero and/or HeLa cells have been previously reported (Gonzalez and Blanco, 1985a; Blanco et al., 1990).

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3. Results

Among the 1334 E. coli isolates investigated for toxin production, we found that 280 were toxigenic, 267 of which were positive for STa and/or LT, 51 were positive for VT (the majority of them also enterotoxigenic) and only 7 were positive for CNFl . No E. coli culture was positive for CNF2. Toxigenic E. coli strains were isolated from 78 ( 18.8%) piglets distributed as indicated in Table 1. Of the 78 piglets with toxigenic strains, 70 ( 16.9%) harboured ETEC strains, 17 (4.1%) VTEC strains and 5 ( 1.2%) NTEC strains. VTEC strains isolated from 14 (82.3%) of 17 piglets also synthesized STa toxin. Coinfection by two different toxigenic strains showing two different toxic phenotypes was detected in six diarrhoeic piglets: three pigs harboured STa and LT strains, two STa and STa VT strains, and one STa and STa LT strains. In the remaining piglets with toxigenic strains, all the strains isolated from a single piglet showed a single toxin phenotype. Enterotoxigenic E. coli (ETEC) strains were isolated from 64 (20.2%) piglets with diarrhoea, 5 (25%) piglets with oedema disease and from 1 (1.3%) healthy piglet (Table 1) . ETEC were statistically associated (P < 0.001) with piglets with diarrhoea or oedema disease. According to geographic distribution, ETEC were isolated from 35 Table 1 Geographical distribution of enterotoxigenic in piglets with colibacillosis Origin”

DIARRHOEA (Zone A) Subtotal (Zone B) Subtotal SUBTOTAL

Age

< 15 days > 15 days < 15 days > 15 days

DLARBIIOEA < 15 days > 15 days TOTAL DIARRIIOEA OEDEMA DISEASE (Zone A) > 30 days > 30 days (Zone B) TOTAL OEDEMA DIS. IIEALTIIY (Zone A) < 15 days > 15 days TOTAL HEALTHY TOTAL STUDY

(ETBC), verotoxigenic

Sampling

(VTBC) and necrotizing E. coli (NTEC)

W piglets with

piglets

cultures

ETBC (%)

VTBC (%)

Nmc

96 125 221 37 59 96

341 449 790 133 197 330

10 25 35 10 19 29

(10.4) (20.0) (15.8) (27.0) (32.2) (30.2)

3 (3.1) 10 (8.0) 13 (5.9) 0 0 0

3 (3.1) 1 (0.8) 0 0 0 0

133 184 317

474 646 1120

20 (15.0) 44 (23.9) 64 (20.2)

3 (2.2) 10 (5.4) 13 (4.1)

3 (2.2) 1 (0.5) 4 (1.3)

15 5 20

34 17 51

3 (20.0) 2 (40.0) 5 (25.0)

1 (6.6) 2 (40.0) 3 (15.0)

0

16 61 77 414

32 131 163 1334

0

0

0

l(l.6) l(l.3) 70 (16.9)

l(l.6) l(l.3) 17 (4.1)

0 0 5 (1.2)

(%)

1 (20.0) 1 (5.0)

“Geographical regions of Spain: Zone A = Galicia; Zone B = Andaluc2ta. Arag&& Castilla-Leon, Castilla-La Mancha, Madrid and Murcia

J.I. Garabal et al. /Veterinary

Microbiology

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47 (1995) 17-25

( 15.8%) diarrhoeic piglets from Zone A, and from 29 (30.2%) diarrhoeic piglets from Zone B (P
STa LT VT CNFl CNF2 a-haemolysm Total piglets with toxigenic strains

N” (%) piglets

x2 cona

< 15 days (n = 149)

> 15 days (n=265)

Total (n=414)

19 (12.7) 1 (0.7) 3 (2.0) 3 (2.0) 0 16 (10.7) 38 (25.5)

41 (15.5) 18 (6.8) 14 (5.3) 2 (0.7) 0 35 (13.2) 57 (21.5)

60 ( 14.5) 19 (4.6) 17 (4.1) 5 (1.2) 0 51 (12.3) 95 (22.9)

a p calculated with respect to piglets younger than 15 days versus piglets older than 15 days.

P
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J.I. Garabal et al. /Veterinary

Microbiology 47 (1995) 17-25

4. Discussion E. coli infections are one of the major causes of mortality and sickness in neonatal and postweaning pigs (Nielsen, 1986; Holland, 1990). Diarrhoea induced by enterotoxigenic E. coli (ETEC) depends on two major factors namely colonization of the small intestine and production of enterotoxins (Gaastra and De Graaf, 1982; Fairbrother, 1993). Similarly, verotoxigenic E. coli (VTEC) causing oedema disease need also to multiply and colonize the small intestine, where verotoxin causes vascular lesions followed by oedema and neurological symptoms (Smith and Halls, 1968; Gannon et al., 1989; Bertschinger et al., 1990). Therefore, the final cause of colibacillosis is the presence of pathogenic E. coli strains with specific virulence attributes in the intestine of animals. As was expected, the most important toxigenic E. coli groups as causative agents of porcine colibacillosis in Spain were ETEC and VTEC. These bacteria were isolated at higher rates from sick piglets than from healthy piglets, ETEC being statistically associated with porcine colibacillosis (diarrhoea and oedema disease) (P < 0.001) and VTEC with oedema disease (P cO.05). ETEC strains were isolated from 70 ( 17%) out of the 414 piglets investigated. ETEC incidence found in this work is comparable to that reported by Oliveira et al. ( 198 1) in Brazil (25%)) Patamaroj et al. ( 1983) in Thailand (32%) and Shimizu et al. ( 1984) in Japan (34%). With respect to previous studies carried out in Spain, we have obtained a ETEC incidence slightly different from that previously reported in Galicia (Zone A in this study) (Gonzalez and Blanco, 1986) and in regions from Zone B (Blanc0 et al., 1988; Suarez et al., 1987,198s). Variations in ETEC incidence may be also found in studies carried out in Canada (Lariviere and Lallier, 1976; Hare1 et al., 1991)) Japan (Shimizu et al., 1984; Nakazawa et al., 1987) and Sweden (Hand1 et al. 1992). Only two (STa and LT) of the three enterotoxins synthesized by ETEC were studied, and thus the real ETEC incidence in porcine colibacillosis in Spain may be higher bearing in mind the production of STb enterotoxin. Several publications have reported the frequent isolation of ETEC strains producing STb enterotoxin alone or in combination with STa or LT at ratios as high as 48% to 90% of isolates (Wilson and Francis, 1986; Hand1 et al., 1992; Osek and Truszczynski, 1992). Of the enterotoxin types studied, our results indicate that STa was the most frequently produced by porcine ETEC isolates in Spain, whereas the production of LT occurred at lower frequency. This observation has been also reported in previous studies on porcine colibacillosis carried out in Spain (Gonzalez and Blanco, 1986; Blanc0 et al. 1988; Suarez et al., 1987,198s). Verotoxigenic E. coli (VTEC) strains were usually detected at incidence rates ranging from 60% to 100% of pigs with oedema disease (Gannon et al., 1988; Nagy et al., 1990; Wasteson et al., 1992). In this study, we found VTEC strains in only 3 ( 15%) out of 20 pigs with oedema disease. The presumptive diagnosis of oedema disease may be complex and we can not exclude some kind of error in veterinary diagnostics. Smith and Halls ( 1968) proved that before developing the typical signs of eodema disease, infected pigs may manifest a profuse diarrhoea. We found VTEC strains in 13 (4.1%) out of the 317 piglets with diarrhoea investigated, an incidence which was similar to that reported in other studies (Kashiwazaki et al., 1980: Smith et al., 1983; Nagy et al., 1990; Hare1 et al., 1991). Our results showed VTEC strains may play an important role in postweaning diarrhoea with an 8% incidence in diarrhoeic pigs. These VTEC strains were detected in 5 ( 11.6%) out of 43

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23

piggeries with diarrhoeic piglets from Zone A (data not shown). We have recently determined that 77% of VTEC strains isolated in this study belonged to serotype 0138:K81, which synthesized both VT and STa toxins, and some of them expressed F107 fimbriae (manuscript in preparation). Our results support earlier reports on the isolation of VTEC strains from diarrhoeic piglets (Linggood and Thompson, 1987; Gannon et al., 1988; Nagy et al., 1990; Hare1 at al., 1991). Isolation of VTEC strains from diarrhoeic piglets from piggeries in Zone A supports the Linggood and Thompson ( 1987) hypothesis that enterotoxins may induce diarrhoea in infected piglets and thus eliminate the oedema-diseaseprovoking agent from the intestine. On the other hand, we have found that VTEC were not frequently isolated from the faeces of healthy pigs, whereas Gannon et al. ( 1988) reported the isolation of VTEC from 18 ( 14%) out of 129 clinically normal weaned piglets. Finally, this is the first report on the prevalence of CNFl and CNF2 producing NTEC strains in pigs, although the presence of NTEC in sick piglets has been previously reported (Gonzalez and Blanco, 1985a; Wray et al., 1993). Our results indicate that NTEC were not frequently isolated from the faeces of pigs and that they were not statistically associated with porcine colibacillosis. Among NTEC strains isolated, CNFl toxin but not CNF2 toxin was synthesized. After testing 1334 E. coli isolates, we may hypothesize that porcine NTEC synthesize only CNFl toxin, although this will have to be confirmed in further studies. In any case, having isolated CNFl strains from porcine, and bearing in mind the implication of these NTEC strains in human extraintestinal infections, we speculate that the porcine intestine could be a natural reservoir of pathogenic E. coli strains for humans.

Acknowledgements

This work was supported by grants from Xunta de Galicia (XUGA 84301188 and 8430489)) FiSs (90/0447-2) and from DGCICYT (PM89-0142 and PTR 91-0050). Our grateful thanks to S. Femandez, M.L. Lopez, I. Ferreiro and C. Fem6ndez for skilfull technical assistance. JIG, FV and MB acknowledge the receipt of the FPI research fellowships from the Ministerio de Education y Ciencia.

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