Escherichia coli producing CNF1 and CNF2 cytotoxins in animals with different disorders

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Escherichia coli producing CNF1 and CNF2 cytotoxins in animals with different disorders

P. Pohl, E. Oswald, K. van Muylem, E. Jacquemin, P. Lintermans, Jacques Mainil

To cite this version:

P. Pohl, E. Oswald, K. van Muylem, E. Jacquemin, P. Lintermans, et al.. Escherichia coli producing

CNF1 and CNF2 cytotoxins in animals with different disorders. Veterinary Research, BioMed Central,

1993, 24, pp.311-315. �hal-02711771�

Short note

Escherichia coli producing CNF1 and CNF2

cytotoxins ⁱⁿ animals with different disorders

P Pohl E Oswald K Van Muylem ^E Jacquemin

P Lintermans J Mainil

1 Institut National de Recherches Vétérinaires, Groeselenberg 99, ¹¹⁸⁰ Brussels;

2 Department ^of Microbiology, ^Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda MD 20814-4799, USA;

3 Faculté de Médecine Vétérinaire, Université de Liège,

Sart-Tilman-Bâtiment B43, ⁴⁰⁰⁰ Liège, Belgium

(Received ^{28 October} 1992; accepted ^{12 March} 1993)

Summary ― ^{Two DNA} probes ^were used for the detection of CNF1- and CNF2-positive ^{E coli}

strains in a collection of 553 E coli isolates from cattle, sheep, goats, pigs, horses, dogs, ^{cats and} poultry. CNF-positive ^{E coli were} frequently associated with septicaemia ⁱⁿ cattle, dogs, ^and cats, with diarrhoea in calves, cats and dogs, and with abortion in bovine and porcine species. ^CNF2- positive ^{strains were} observed among adult healthy ^cattle. They ^were also found in cases of pneu- monia, metritis, ^{mastitis in} cattle and in 1 case of metritis of a mare. The physiopathology ^induced by CNF-positive E coli strains remains to be elucidated. However, ^the impact of CNF strains on veteri- nary pathology is clear and the diagnosis ^of CNF-producing E coli should become routine in veteri- nary practice.

animal species ^{I CNF I DNA} probe ^{I disorder}

Résumé ― Présence d’Escherichia coli productrices ^des cytotoxines ^{CNF1 et} CNF2 chez des animaux atteints de pathologies diverses. Deux sondes d’ADN ont été utilisées pour détecter la

production des toxines CNF parmi ^une collection de 553 E coli provenant ^de bovins, de moutons, ^de chèvres, de porcs, de chevaux, ^de chiens, de chats et de volailles. Les E coli CNF+ sont fréquem-

ment observés dans des cas de septicémies ^{chez les} veaux, les chiens et les chats dans des cas de diarrhée chez les veaux, les chats et les chiens, dans des cas d’avortement chez les bovins et les porcs. On observe également des E coli CNF2+ dans les fèces de bovins adultes sains. On trouve

également des E coli CNF+ dans des cas de pneumonies, ^de métrites, de mammites chez les bo- vins et dans un cas de métrite chez une jument. ^La physiopathologie provoquée par les E coli pro- ductrices des toxines CNF reste à élucider Toutefois, ^leur impact ^en pathologie vétérinaire est clair et elles devraient être recherchées au cours des diagnostics de routine.

espèces animales / CNF / sondes d ADN / maladies

*

Correspondence ^and reprints

INTRODUCTION

In the early seventies, ^Smith described E coli strain S5 isolated from a case of septi-

caemia in a lamb (Smith, 1974). ^{This strain} contains a plasmid encoding the so-called Vir toxin that is lethal for hens, mice and rabbits.

In 1980 and later, Caprioli demonstrated that E coli strains isolated from babies with enteritis produce ^a toxin that induces nec-

rotising ^{lesions in} the rabbit skin; ^{this toxin} also induces morphological changes ⁱⁿ

HeLa and Vero cell lines. The toxin was

designated cytotoxic necrotizing ^{factor or}

CNF (Caprioli ef al, 1983).

The Vir toxin possesses biological prop- erties (necrotising ^{lesions in rabbit} skin, morphological changes in HeLa and Vero cell lines) ^{similar to} those of the CNF toxin and both are serologically ^related (De Rycke ^et al, 1987, 1990). However, ^{the 2}

toxins are not identical. The toxin de- scribed by Caprioli ^{has been} designated CNF1 and the Vir toxin has been called CNF2 (Oswald ^{and De} Rycke, 1990).

CNF1-producing ^{E coli were isolated}

from cases of enteritis, septicaemia ^and nephritis in humans (Caprioli ^et al, 1983;

Alonso et al, 1987), of diarrhoea in calves and pigs (De Rycke ^et al, 1987; Blanco et

al, 1988; ^{Gonzalez and} Blanco, 1985;

McLaren and Wray, 1986); ^of septicaemia

in bulls (Contrepois ^and Ribot, 1987; ^Pohl

et al, 1992), ^and finally ^{from cases of} septi-

caemia and urinary infections in cats and

dogs (Prada et al, 1991 ).

CNF2-producing ^{E coli were observed}

in cases of septicaemia ^{and enteritis in}

calves and lambs (Smith, 1974, 1975;

Blanco et al, 1988; ^De Rycke ^et al, 1987;

Oswald et al, 1991 ).

CNF1- and CNF2-producing ^{E coli are} clearly associated to pathological process-

es in humans and domestic animals, ^but

their prevalence ^{in various} species ^{is not}

well established.

The conventional diagnosis ^{of CNF-} producing ^{E coli is} time-consuming. ^{It is}

based on the observation of multinucleat-

ing effects of potential ^{toxins on Vero and}

HeLa cells and their seroneutralization or on ELISA (Tabouret ^{and De} Rycke, 1990).

These assays ^can be carried out only ^{in a}

small number of laboratories. These prob-

lems hamper ^the study ^{of the} role of these bacteria in veterinary pathology.

Recently, ^{2 DNA} probes ^{were devel-} oped, enabling the identification of CNF-

producing ^{E coli} by ^DNA colony hybridiza-

tion (Oswald ^et al, 1992; and manuscript ⁱⁿ preparation). ^These tools allow the rapid screening ^{of a} large collection of strains isolated from various pathological ^condi-

tions in domestic animals.

MATERIALS AND METHODS

Five hundred and fifty-three E coli strains were

studied; 483 were isolated from cattle, ^{6 from} small ruminants, ^{1 from a} mare, 23 from cats, 30 from dogs, ^{5 from} pigs and 5 from poultry (ta- ble I). All strains were isolated in Belgium ^be-

tween 1977 and 1992.

Most strains were selected from the collec- tion on the basis of the haemolysis ^{on blood}

agar plates. Indeed, all CNF1- and some CNF2-

producing ^{E coli} (9%) ^{excrete an} a-haemolysin (Oswald et al, 1991; Pohl et al, 1992).

The probes ^{used are} designated pEOS1 ^and pEOS3 ^{and are} described elsewhere in detail

(Oswald ^et al, 1992; and manuscript ⁱⁿ prepara- tion). Briefly, they ^were derived from one CNF2-

producing strain cloned DNA fragment, ^which

conferred to a K12 E coli strain the ability of pro-

ducing ^CNF2. According ^{to the} comparison ^be-

tween colony hybridization ^and phenotypic ^as-

say results ^{on more} than 150 CNF1-positive, CNF2-positive ^or CNF-negative E coli isolates

(Oswald ^et al, 1992; ^and manuscript in prepara-

tion), ^the CNF1-producing ^isolates hybridize weakly (grey spots) ^{with the} pEOS1 probe

whereas the CNF2-producing ^isolates hybridize strongly (black spots) ^{with both} probes (fig 1 ).

DNA hybridizations ^{were carried out as de-}

scribed in Mainil et al (1990).

RESULTS

From of the 553 isolates, ^{27% were} posi-

tive with pEOS1 probe ^{and 13% were} posi-

tive with pEOS1 ^and pEOS3. ^{The former}

were considered as potential producers ^of

CNF1 and the latter as potential producers

of CNF2.

The CNF1-positive E coli strains were

associated with septicaemia, ^diarrhoea, pneumonia, mastitis, ^{abortions and} urinary

tract infections in cattle, goats, cats, dogs

and pigs (table I). ) .

The CNF2-positive ^{strains were isolated}

from cattle, goats, horses, ^{and cats, but}

not from dogs ^or pigs. ^{These strains were}

associated with metritis in addition to the

pathologies described for CNF1 strains.

CNF2-positive ^{strains were} also isolated from healthy adult cattle.

DISCUSSION

Our findings ^{confirm and} extend data al-

ready published ^{on the} association of

CNF-producing E coli with several disor- ders in different animal species. Moreover, CNF-positive ^{strains were} reported ^{for the}

first time in goats, horses and deer. In as

much as the choice of the strains that were

studied was restricted to haemolytic ^E coli, the values in table I are certainly ^overesti-

mated and most probably ^{do not} reflect the actual prevalence ^of CNF-positive ^{E coli in}

the different animal species and disorders:

100% E coli CNF1+ + are Hly+ (Pohl ^et al, 1992) ^but only 9% E coli CNF2 are Hly+

(Oswald et al, 1991 ).

The exact role of the CNF-producing ^E

coli strains in these different disorders could only ^be clearly proved using experi-

mental infections. Arguments ^to support

their role are:

-

in cases of septicaemia: a) ^{most CNF}

strains are resistant to complement ^and

secrete aerobactin (Oswald ^et al, 1991;

Pohl et al, 1992); b) ^a CNF2-producing

strain induces septicaemia and death in

colostrum-deprived ^calves (Smith, 1975);

-

in cases of diarrhea: a) ^{the CNF2 toxin}

has an enterotoxic activity (De Rycke ^et al, 1990); b) ^{CNF2 strains can harbour a} gene

homologous ^{to the LT-Ila} enterotoxin-

encoding gene (Oswald ^{et al,} 1991); c)

several CNF1 and CNF2 strains adhere to calf villi using ^{adhesins which are different}

from the classical adhesins (F5, F17, Att111) (Oswald ^et al, 1991; Pohl et al, 1992). CNF-positive ^{strains are} also isolat- ed from cases of pneumonia, metritis, ^mas- titis, abortion. The physiopathology ^{of the in-}

fection remains unclear in those cases.

The presence ^of CNF2-positive ^{E coli}

strains in healthy adult cattle also remains unclear.

However, ^{it is clear from this} study ^that

the CNF1- and CNF2-positive ^{E coli}

strains _may play a significant ^{role in animal}

diseases and that an adequate diagnostic

test should be developed. ^Since specific

DNA probes ^{and ELISA are still} impractical

in numerous laboratories, ^we try ^{to deter-}

mine a phenotype ^{which can} reveal CNF

toxin producers.

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