Download Isolation, characterization and public health

Avian Pathology, 4:271-276,
1975
ISOLATION, CHARACTERIZATION AND PUBLIC HEALTH
ASPECTS OF VIBRIO CHOLERAE NAG ISOLATED
FROM A DANISH DUCK FARM
M. BISGAARD and K.K. KRISTENSEN
Institute of Avian Diseases
Dept. Langaa, Rypevej 1
DK-8870 Langaa, Denmark
Veterinary Faculty for FA O Fellows and Scholars,
Building A.B. 34, Copenhagen
DK-1870 V.Denmark
SUMMARY
The hygienic and pathogenic consequences of the isolation of Vibrio
cholerae NAG from the conjunctiva of two ducklings, from brackish
water samples in their surroundings, and from the intestinal contents
of a duckling, are discussed in relation to present knowledge of the
occurrence of Vibrio cholerae NAG in man and animals.
INTRODUCTION
In recent years, certain species of vibrio pathogenic to man have attracted increasing
attention. The reasons are several, one of them being that increased travel has caused
a wider spread of cholera, and at the same time other enteropathogenic vibrios, which
have previously been largely disregarded, are becoming more frequently incriminated
as the cause of cholera-like diseases or gastroenteritis.
The taxonomy of the so-called NAG vibrios is controversial; but they are now commonly classified as Vibrio cholerae NAG (Non Agglutinable) (Sakazaki, 1970), i.e.
they are not agglutinated in cholera antiserum 0 1. Apart from that difference, they
show great similarity to the classic Vibrio cholera type with respect to morphology,
biochemical properties, H antigens, and G-C percentages (Sakazaki etal, 1967;
Colwell et ai, 1968; Sakazaki, 1970). These similarities may consequently cause
differential diagnostic difficulties, as certain mucoid forms of V. cholerae may occur
which are not agglutinated in 0 1 serum (Sakazaki, 1970).
V. cholerae NAG may cause a cholera-like disease, giving the same symptom of ricewatery faeces as the classic vibrio type or the EL-TOR type. But often diarrhoea
will be mild (Me Intyre et al., 1965).
Chashi et al. (1972) state that antiserum against crude NAG toxin may completely
Received 14 April 1975
Accepted 5 August 1975
272
M. Bisgaaid and K.K. Kristensen
neutralize both purified and crude cholera enterotoxin. Zinnaka and Carpenter
(1972) mention that NAG vibrio isolated from patients causes accumulation of fluid
in ligated loops of ileum in rabbits, whereas similar vibrios isolated from water do not.
Since 1954 (Yajnik and Prasad, 1954) V. cholerae NAG has been known to cause
disease in man. The bacterium has been isolated from cases of diarrhoea in the
Phillippines, Thailand, East Pakistan, India, Iran, Sudan, Egypt, Czechoslovakia, West
Germany and Sweden (Gaines et al, 1964; Me Intyre and Feeley, 1965; Me Intyre et al, 1965;Aldova et al, 1968; Chatterjee and Neogy, 1971;Ohashieía¿, 1972;Ko
étal, 1973;Zafari et al, 1973; Back etal, 1974).
Apparently, the extent and occurrence of V. cholerae NAG in waste water, surface
water, and carriers, is not precisely known. An extensive investigation made in Iran
of approximately 22,000 samples of faeces showed that 160 (0.72%) were positive
(Zafari etal, 1973). However, the carrier percentage was much higher (about 16%)
in returned pilgrims.
Examinations in Thailand showed that 10% of patients with diarrhoea excreted
V. cholerae NAG, whereas it was only found in 0.6% of clinically healthy persons
(Gaines et al, 1964).
Examinations in Germany of waste water from a big city showed the surprising result
that all samples examined were positive. The bacterium was also isolated from 9 out
of 11 samples of river water (Ko etal, 1973).
MATERIAL AND METHODS
The institute received 13 live ducklings for examination from a farm, and 4 from a
hatchery which had no connection with the former.
Conjunctival scrapings from 13 birds were seeded on BA (Tryptose blood agar base,
Difco, with 5% calf s blood added) and TCBS-agar (Merck).
Intestinal contents from 10 of the ducklings were investigated for the presence of
V. cholerae NAG by mixing 20g of faeces and 300ml of V. cholerae enrichment
broth (Merck) containing 0.5% soluble starch and 0.3% alkylbenzene-sulphonate
(Lutensit A-BA). The broth was incubated for 18 hours at 37°C, and subcultured on
TCBS-agar. Suspect sucrose positive colonies were subcultured on BA.
The following environmental samples were collected at the farm: 7 water samples,
each of 250ml, were collected in sterile bottles (5 of the samples were taken from a
pool and 2 from drinking water troughs); 5 faeces-contaminated soil samples were
collected in sterile petri dishes.
A fortnight later the sampling procedure was repeated and 11 water samples and 10
faeces/soil samples were collected.
Both water and faeces/soil samples were examined for vibrios according to the technique described for the intestinal contents. For practical reasons, two samples were
usually bulked for testing. In all cases, 20g faeces-contaminated soil or 30ml water
per 300ml broth were used.
Preliminary tests, colonial and bacterial morphology, haemolytic activity, citrate, Dtartrate, mucate and malonate utilisation, gelatin liquefaction, nitrate reduction,
KCN sensitivity, lysine and ornithine decarboxylation, arginine and esculine hydrolyzation, formation of acid and gas from carbohydrates, hydrogen sulphide, urease,
Isolation of Vibrio cholerae NAG
273
phenylalanine deaminase, indole, acetoin and phosphatase production were performed
as earlier described by Bisgaard (1975). ONPG and ability to reduce tellurite in concentrations of 0.001, 0.01 and 0.04 took place according to Cowan and Steel (1970).
Deoxyribonuclease, lipolytic and lecithinase activity was studied on bacto DNA's test
agar (Difco), tributyrin agar (Merck) and egg yolk medium (Nordic Committee on
Food Analysis, 1968), respectively. Haemagglutination of chicken red blood cells was
carried out according to Finkelstein and Mukerjee (1963), "string-test" and sensitivity to 2.4-diamino-6.7-di-isopropyl pteridine according to Smith (1970) and Caselitz
etal (1961) respectively.
Examination for growth on EBM, McConkey and BLSF agar was made on the respective Difco media.
Growth at different pH values was tested in peptone water. After adjustment of pH to
5 , 6 , 7 , 8 , 9 , 10, 11, and 12, respectively, the broth was seitz-filtered before inoculation.
The temperature limits for growth were determined in veal infusion broth (Difco).
The same medium was used to examine sodium chloride tolerance.
Hydrolysis of starch at different sodium chloride concentrations was examined on
agar plates. To obtain a sodium chloride concentration of 2,4 and 6% respectively,
sufficient sodium chloride was dissolved in tryptose blood agar base (Difco) containing 1% starch.
Pigment formation at 22°C was examined on Pseudomonas agar F and P (Difco).
Finally, the strains were inoculated into the yolk sack and allantoic cavity of 7 and 11
days old chick embryos, respectively, to determine their lethality. The inoculum used
was 0.1 ml of 2 hours veal infusion broth culture per egg (5 x 107 cells/ml).
Unless mentioned to the contrary, incubation took place at 37°C and the final result
was taken after a week.
RESULTS
After having spent the first 3 weeks of their lives in a heated house, all ducks were
moved to an open field in hilly grounds, without natural pools or streams. They had
free access to a commercial feed mixture and to running water from a drinking water
source. The running water formed artificial pools in the low parts of the area. Normally, however, the ducks had no access to these pools.
V. cholerae NAG was isolated from the conjunctival scrapings of 2 ducklings suffering from serous conjunctivitis, although not in pure culture, but mixed with Micrococcus — and Bacillus species, from 4 bulked water samples and from the intestines
of 1 out of 4 ducklings originating from the hatchery.
Description of the isolated V. cholerae NAG strains
Colonial morphology. Colonies were circular, 2-3mm in diameter, low convex or
slightly conical, with glistening surface, and entire edge; they were butyrous in consistency and easily emulsifiable. The colonies were surrounded by a zone of partial
or complete haemolysis, 4-6mm in diameter. The zone of haemolysis was surrounded
by a dark, lmm wide, band. Where the growth was confluent, the zone of partial
haemolysis showed a tendency to clear. Single colonies were centrally greyish, but
transparent at the periphery. After further incubation, the colonies became larger
and assumed a brown/greenish colour, especially where the growth was confluent. At
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M. Bisgaatd and K.K. Kristensen
the same time the agar showed a dark greenish discoloration.
Three of the strains isolated dissociated into R - and S forms.
Bacterial morphology. Gram-stained smears revealed slightly curved Gram-negative
rods, arranged singly and often resembling a comma. Size was variable, the axis generally being curved with parallel sides and rounded ends.
Biochemical reactions. All strains were positive in the following tests: catalase, citrate,
DNA'se, gelatin, haemolysis, índole, lipase, lysine, motility, nitrate, ornithine, oxidase,
pellicle formation, pteridine, Voges-Proskauer (22°C), fructose, galactose, glucose,
glycerol, maltose, mannitol, sucrose, starch, trehalose, hydrolysis of starch with 2 and
4%NaCladded, growth on McConkey, at pH 6, 7, 8,9, 10 and 11, at 15,40, 42 and
44°C and in 2, 4, 6 and 7%NaCl.
Negative reaction, was obtained in the other tests, as follows: arginine, D-tartrate, hydrogen sulphide, KCN, lecithinase, malonate, methyl red, mucate, phenylalanine,
phosphatase, pigment, urease, adonitol, amygdalin, arabinose, cellobiose, dulcitol,
gas from glucose, inositol, inulin, melezitose, melibiose, raffinose, rhamnose, salicin,
sorbitol, sorbose, xylose, esculin, hydrolysis of starch with 6%NaCl added, growth at
pH 5 and 12, at 10 and 45°C, in 8% NaCl and on EMB- agar.
Variations between strains were seen in the following tests: haemagglutination, where
2 of the strains became strongly positive within 10 seconds, while the rest showed
weaker and delayed positive reactions within 1-2 minutes, the control still being negative; ONPG (4 strains positive); "string-test" (3 strains positive); Voges-Proskauer at
37°C (5 strains positive), lactose (4 strains positive) and mannose (3 strains positive).
A pronounced alkaline reaction was observed in meat extract peptone broth after a
few days incubation. With some strains the same reaction could be seen as well on
TCBS as on BLSF agar.
Tellurite was reduced at a concentration of 0.01% potassium tellurite even though
growth was greatly retarded. A concentration of 0.04% potassium tellurite completely
inhibited growth.
Serology. None of the strains isolated were agglutinated by V. cholera O-group I
serum. Serological investigation of 4 strains revealed that 3 of the strains belonged to
O-group 39 and the fourth to O-group 41.
Inoculation of chick embryos. All strains killed chick embryos after 18 hours.
DISCUSSION
Apart from negative fermentation reactions in d-tartrate, lactose and ONPG, the biochemical reactions were in accordance with those reported by Sakazaki (1970).
Because of the alkaline properties of these Vibrio, sugar fermentation results should
be taken daily, as the later readings may be affected by the concommitant production of alkaline substances.
Recent investigations have shown that V. cholerae NAG is widespread not only in
South East Asia but also in Europe. The realization that this species may cause severe
cases of diarrhoea has given rise to concern as to whether there may be a non-human
reservoir besides the human one. It has been reported that fish and molluscs form a
reservoir of importance for the spread and persistence of these vibrios in a given
region(Duttera/. ( 1971).
Isolation of Vibrio choleraeNA G
21S
Investigations in India of cattle, buffaloes, sheep and goats in the Ganges esturary
showed that V. cholerae NAG could be demonstrated in 5-38% of samples of rumen
liquor (Abou-Gareeb, 1960). Examination in India of dogs revealed the presence of
NAG Vibrio in 28 of 203 (14%) faeces samples. It was isolated from crows also
(Sack, 1973).
Unlike V. cholerae NAG, the normal type of Vibrio cholera seems exclusively to occur
in human populations (Sack, 1973).
The isolation, without any aetiological connection to pathological changes of V. cholerae NAG from the conjunctiva and gut contents of ducks in Denmark indicates that
the bacterium also can be associated with poultry, the living habits of which bring
them into close contact with soil and water.
Our present knowledge about the occurrence and significance of V. cholerae NAG in
Europe is based on very few publications. But the present observations in conjunction
with Swedish and German reports (Back et al, 1974; Ko et al, 1973) give reasons for
greater interest in this microorganism from the point of view of water and food hygiene. It should be pointed out that the bacterium was isolated from water samples
as late as the 21st of September, after a period of low temperatures.
The source of the initial contamination of the surroundings is not known. No large
stream to which the ducks had access passes by the farm. Hence the contamination
was unlikely to have originated from human sources through contaminated waste
water effluent. As waste water and contaminated surface water might be contaminated with V. cholerae NAG (Dutt et al, 1971;Ko etal, 1973) migratory birds foraging in these areas could be excreters of the organism, thus carrying the infection over
long distances. The results indicate that V. cholerae NAG can survive in the soil/
water environment. More knowledge is therefore considered to be necessary on the
role played by the animal reservoir in the dissemination and ecology of these vibrios
potentially pathogenic to humans.
Acknowledgement
The authors are greatly indebted to Dr. Riichi Sakazaki, chief of Enteric Bacteriology
Laboratory, National Institute of Health, 10-35 kamiasaki 2 - chôme, Shinagawa-ku,
Tokyo, Japan, for serotyping the strains.
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RESUME
Isolement et caractérisation de Vibrio cholerae NAG
provenant d'un élevage danois de canards. Aspects
hygiéniques de l'infection.
Dans cet article, les conséquences hygiéniques et pathogéniques de l'isolement de
Vibrio cholerae NAG de la muqueuse conjunctive de deux canetons, de quatre spécimens d'eau boueuse constituant l'environnement normal de ces canetons et enfin du
contenu intestinal d'un caneton sont discutée par rapport aux connaissances actuelles
sur Vibrio cholerae NAG chez les animaux et l'homme.
ZUSAMMENFASSUNG
Vibrio cholerae NAG in einer dänischen Entenfarm
— Isolierung, Charakterisierung und hygienische Aspekte
Es werden im Anschluβ an eine Isolierung von Vibrio cholerae NAG aus den Konjunktiven zweier Enten, aus Brackwasserproben in ihrer Umwelt und aus dem Darminhalt
eines Entchens die hygienischen und pathogenetischen Konsequenzen unter Bezug
auf unsere derzeitigen Kenntnisse über Vibrio cholerae NAG bei Mensch und Tier
diskutiert.