DTIC ADA286275: Oral Challenge with Aeromonas in Protein-Malnourished Mice

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2. REPORT DATE 3. REPORT TYPE AND DATES COVERED
1994 Journal article
4. TITLE AND SUETITLE S. FUNOINC"G NUMBERS
Oral challenge with Aeromonas in protein-malnourished mice
PE- N/A
PR -
6. AUTHOR(S) TA -
wur'i
Pazzaglia G, Winoto I, Jennings G -
7. PERFORMING ORGANIZATION NAME(S) AND ADORESS(ES) 8. PERFORtING ORGANIZATION
Naval Medical Research Institute REPORT NU;1,AER
Commanding Officer
8901 Wisconsin Avenue NIUU 94-57
Bethesda, Maryland 20SS9-5607
9. SPONSORING/IWONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORING /MONITORING
Naval Medical Research and Development Command AGENCY REPORT NUMBER
National Naval Medical Center
Building 1, Tower 12
8901 Wiscosin Avenue
Bethesda, Maxyland 20889-5606
11. SUPPLEMENTARY NOTES
Reprinted from: Journal of Diarrhoeal Diseases Research 1994 Vol.12 No.2 pp. 108-112
12a. DISTRIBUTION /AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE
Approved for public release; distribution is unlimited.
It. SU&JECT TERVMS 1s. 'K:MEER OF PAGES I
Aeromonas; animal model; malnutrition; protein deficiency; diarrhea. __
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OF REPORT OF THIS PAGE OF ABSTRACT
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NSN 754-0.1-280-5SS0O --. :or 298 (Rev 2-ES(cid:127)
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J Dmrrhocal Dis Res 1QW4J un:12(2): I0g-12 1994 International Centre for Diarrhocal
0253-8769194 S 1.Oo0ýi 10 Discase Research. Bangladesh
7~ccesion For
Oral Challenge with "omonasi n DTIC ,
Unalwollf~[ C,'
Protein-M alnour., .ý M ice JU........
G PAZZAGLIA, I WINOTO, AND G I- NINGS By..
U.S. Naval Medical Research Unit No. 2. Jakarta, Indonesia. Distribution/
AvaiLability Codes
ABSTRACT "--IAalad/o
The lack of an animal model for Aeromonas-associated diarrhoea has hindered progress Dist Special
toward understanding the pathogenesis of this potentially important enteric infection.
Protein-malnourished mice were challenged orally with Aeromonas strains to determine if
diminished levels of resistance would allow the induction of a diarrhoeal response. The 15
Aeromonas spp. faecal isolates used for challenge included 7 A. caviae, 4 A. hvdrophila, I A.
sobria by. sobria, and I A. sobrio by. veronii from patients with diarrhoea, and 2 A. caviae from
healthy volunteers. All had at least I known virulence marker, with the exception of I strain.
Mice on the protein deficient diet had lost an average of 23% of their initial body weight at the
time of challenge. Although mice consumed l01 cfu per day for a minimum of 4 days, none
became ill due to Aeromonas spp. ingestion. Aeromonas spp. were isolated from 75% of faecal
cultures obtained 7 days after initial challenge, indicating bowel colonization had occurred.
K(cid:127).. words: Aeromonas: Animal model; Malnutrition; Protein deficiency, Diarrhoea.
IN'TRODUCTION been reported for studies performed in developing
countries such as Bangladesh (16) and Peru (5), where
The more frequent faccal isolation of Aeromonas spp. children hospitalized with diarrhoea are frequently
from patients with diarrhoea compared to healthy suffering from malnutrition, especially due to
individuals (1-5) and the large proportion of Aeromonas protein-deficient diets (17). Protein and other nutritional
strains that produce numerous potential virulence factors deficiencies have been shown to have adverse effects on
(6,7) supports the conviction by many that some the host's ability to mount a successful defense following
phenotypes of Aeromonas are important enteropathogens. infection by common viral and bacterial agents. Among
However, several case-control studies (8-I1) and those defenses shown to be diminished by malnutrition are
conmunut,-based longitudinal studies (12-14) have shown mucosal barriers, serum and secretory antibody responses,
a weak or non-existent association with disease, and the cell-mediated immune response (17).
Furthermore, challenges in adult humans failed to produce We hypothesized that a diminished level of resistance
disease (15). and there has been no outbreak in malnourished mice would allow the induction of a
unequivocally linked to an Aeromonas strain. A proven diarrhoea] response when challenged with Aeromonas
virulence marker for diarrhoeagenic strains of Aeromonas strains. Our objective was to test selected strains of
spp. has not been identified. Aeromonas for their ability to produce diarrhoea in mice
An animal diarrhoea model of Aeromonas spp. malnourished by a protein-deficient diet.
infection would help clarify these apparent contradictions
regarding this organism's role in human infectious MATERIALS.AND) METHODS
diarrhoeas and provide an understanding of the
pathogenesis of Aeromonas-associated disease. To date, Experimental design. Four identical experiments were
no model has been reported which produces diarrhoea in performed, each using 4 strains of Aeromonas spp. for
Aeromonas-challenged animals in vivo. challenge (15 strains total). For each experiment, mice
The highest prevalence of Aeromonas-associated were allocated into 4 challenge groups and 2
diarrhoea and the strongest association with disease have weight-control groups. Groups consisted of 4 to 6 mice
each. The 4 challenge groups were mice which had been
Correspondence and reprint requests should be addressed to: fed a protein-deficient diet for 7 days, and then challenged
Dr G Pazzaglia. Naval Medical Research Institute by placing Aeromonas-inoculated water in their water
8901 Wisconsin Avenue. Bethesda. MarYland 20889-560". U.S.A. bottles (Day 0). The 2 weight-conlrol groups served as
94-35043
U..- .. ... . ,-,. II IIIIIIIII!!lIll
~ ~~1.
Aermonnx in protein-mainounshed mice 109
controls for measuring the severity of malnourishment of Colony-forming units (cfu) per ml were calculated after
mice while on the protein-deficient diet. One weight- counting Aeromonas spp. colonies (lactose-negative
control group was provided a complete diet, and the other colonies; typical morphology) on each plate following
was given the same protein-deficient diet as the challenge overnight incubation at 36±1 °C. Titrations were also
groups. There was no normal diet challenge group. performed on challenge suspensions sampled on days 1, 2,
Challenged animals were observed for 7 days after and 5, although not all suspensions were titrated on every
switching their drinking water to Aeromonas strain occasion.
suspensions. All challenge groups consumed viable
bacterial suspensions for at least 4 days. Challenge procedure. Following weaning at 24 to 25
days of age, C3H mice were placed on a diet of
Animal use. The housing, treatment, and experimental commercially bottled water and formulated food. Two
use of animals complied with the Guidelines of the mice from each litter (normal diet weight control-group)
Committee on Care and Use of Laboratory Animals of the received a complete diet (Normal Protein Diet for Rat or
National Research Council. Mouse, ICN Biomedicals, Cleveland, OH), and the
remaining mice of each litter (challenge groups +
Source of challenge strains. All of the challenge strains malnourished weight control group) received a
were from previously reported population studies (5,14). protein-deficient diet (Protein Free Diet, ICN
Thirteen of the 15 challenge Aeromonas spp. faecal Biomedicals; a semi-synthetic rodent food composed of
isolates were from Peruvian children with diarrhoea who 67% sucrose, 15% corn starch, 8% non-nutritive bulk,
were less than 2 years of age. No other enteropathogens 5% corn oil, and 5% vitamin and mineral supplements).
were detected in their stools. All of the 10 children had a Weight measurements (pooled) were made at least every- 2
secretory type diarrhoea, 5 had mucus in their stools days. When the protein-malnourished mice demonstrated
(strain no. 1, 3. 4. 11, 14), 6 had associated fevers (strain a >20% pooled weight loss compared to their initial body
no. 1, 4, 6, 10, 11, 14), but none had bloody stools. Two weight (day 7), the diet deficient mice were allocated into
of the challenge strains were from healthy control children separately caged groups (4 to 6 mice of the same sex in
(strain no. 7, 12). each group) for challenge with Aeromonas strains, or
continued observation as an unchallenged weight-control
Challenge strain phenotypes. Because no individual group. At the time of challenge, bedding was removed
strain, biochemical phenotype, or virulence marker and the floor of the cage was left bare (absorbent paper
phenotype of Aeromonas spp. has been proven to be was not used because the animals shredded the material
diarrhoeagenic in animals or humans, cha!lenge strains overnight, even when it was firmly taped to the cage
were selected which represented a variety of bottom). Animals and the cage bottom were observed
characteristics an4 phenotypic profiles (Table 1), as daily for signs of diarrhoea (faecal material on the
determined previously (18). In total, there were 9 A. anal-rectal area, or signs of liquid stool on the cage
caioae, 4 A. hydrophila, I A. sobria by. sobria, and I A. bottom, respectively). The animals were placed into clean
sobria by. veronii (metabolically phenotyped by the cages each day, with special precautions taken to prevent
method of .landa [6D); 10 strains were reactive in more cross-contamination. When challenge suspensions were
than I cell culture type, 4 were haemolytic, 3 depleted (usually on day 4 or 5), drinking bottles were
spontaneously pelleted in broth (SP+ [7]), 2 precipitated replaced with containers of their regular water.
after boiling (PAS+- 17]). 3 were positive in the infant
mouse assay for enterotoxin (19,20), 3 were positive in Challenge dosages. Serial dilutions of samples from
the rabbit ileal loop assay for enterotoxin (21,22), and 6 challenge inocula yield 9.3 x 10' to 6.0 x 109 cfu per ml,
were lethal to mice when injected intraperitoneally with at with the average inoculum being 8.9 x 108 cfu per ml. As
least 107 organisms, mice consumed approximately 10 mn of the bacterial
suspension per day per mouse, the average challenge dose
Preparation of challenge inoculum. For preparation of for each mouse on the first day of challenge (day 0) was
each challenge inoculum, 3 to 5 Aeromoras colonies were approximately 9.0 x 109 cfu. All challenge groups
picked from fresh growth on MacConkey agar and consumed the bacterial suspensions for at least 4 days.
suspended in 50 ml brain heart infusion broth. Broth Die-off of Aeromonas spp. organisms in the drinking
cultures were incubated 18 h at 36+1 C in a shaking water was slow; by days 1, 2, and 5 post-challenge,
incubator (200 rpm). Overnight cultures were centrifuged Aeromonas spp. average log viable counts had declined by
(2,000 x g for 30 min at 5°C), the supernatant decanted. 0.40, 0.62, and 1.00, respectively. This indicated the
and the pellets resuspended in 50 ml sterile phosphate mice were being continuously challenged through day 4.
buffered saline (PBS) by vonexing. Bacterial suspensions
were again centrifuged and decanted, and the pellet was Faecal cultures. For determinations of mouse
resuspended in 200 ml sterile unchlorinated water. These colonization by Aeromonas strains, faeces were obtained
suspensions served as the challenge inocula and were from the bottoms of cages on days 1. 2, 5, 6, and 7. Not
placed in individual feeding bottles which had been rinsed all groups were cultured on all days. Faeces were
with 70% ETOH and dried before filling. vortexed in I m) of sterile PBS. then 0.01 ml of the
At the time challenge suspensions were prepared. each suspension was directly inoculated onto MacConkey agar
suspension was tirated by serially diluting 1.0 ml and and incubated overnight at 36±÷IC. To enrich for
inoculating dilutions onto MacConkey agar plates. Aeromonas spp., 10 mnl of alkaline peptone water was
110 J Diarrtocal Dis Res Jun 1994 Pazzalhia ei al.
added to the remaining suspension, incubated overnight at Virulence of challenge strains. Four separate challenge
ambient temperature, then subcultured onto MacConkey experiments were performed, using 4 Aeromonas strains
agar and incubated overnight at 36 ± 1 C. Direct cultures in each experiment (1 strain was repeatedl. There were
and enriched subcultures were inspected for suspect no illnesses or deaths in challenged mice which could be
Aeromonas spp. colonies. Lactose-negative, oxidase- attributed to Aeromonas challenge strains.
positive colonies were confirmed Aeromonas spp. using In one experiment, 3 of 4 mice challenged with strain
conventional biochemical tests (24). no. 14 became ruffled and lethargic on day 4, but showed
no signs of diarrhoea. Two moribund
Table 1. Phenotypic characteristics of 15 Aeromonas strains used for mice in this group were sacrificed and a
oral challenge of protein-malnourished mice. necropsy revealed no fluid accumulation
in the bowel or other abnormalities.
Aggiuti. Invasive- Samples of heart blood were cultured and
Strai nation Lb CvtStoxinS' Enterotoxin" ness., found to be sterile. The 2 surviving mice
number Aeromonas specie; SP PAB SRBC Y1 CHO Vero mouse rabbit mouse died on day 6. A necropsy on I of these
Il + rni mice again showed no abnormalities and a
I hydrophila - +" culture of post-monem heart blood was
2 hydrophila - " * - sterile. Strain no. 14 was retested in a
4 hydrophila - - - later experiment; all animals survived with
4 sobhabsabria +. no evidence of illness.
6 sobria by. veronii nt ft -
7 cavuae - + + DISCUSSION
8 cavaae - + - * ft
9 cavise - + - Although several animal assays in vivo
10 v - - .. * have been used to identify Aeromonas
II caviae strains with potential virulence markers
12 ,.*v,= + (6). there have been no reports of
13 cavia- + successfully inducing diarrhoea through
14 cavvf oral challenges of animals not surgically
15 cavi,. manipulated. Challenges using the
removable intestinal tie adult rabbit
AbbrvanUo: SP. spontUaeous pelleting. PAB, precipitaion after boiling. SRBC. sheep diarrhoea (RITARD) model (25) have
red blood cells (hmolysis): Yt. adrenal cell culture; CHO. Chinese hamster ovary cell demonstrated some strains to be highly
culture: Vero. monkey kidney cell culture: nt. not tested. virulent, but only after invasion of the gut
b Agglutination phenotypes as de(cid:127)ribed bn Jand8 (7) mucosa, bacteraemia, and death due to
Causes cytotnic or lytic responm. sepsis (26). In the same investigation,
d Caue inueinna fluid sommulatn. RITARD challenges of rabbits with
Lta ifinjected inaperitoneally at a dose of IOe cfu (23). invasive strains of Aeromonas were shown to infrequently
produce diarrhoea. When diarrhoea did occur, it was not
R.ESULTS voluminous and contributed little to the outcome of the
challenged rabbits.
Effects of protein-deficient diet. The figure illustrates The current investigation, using a mouse protein
the average weight loss in 23 control mice fed a 0% deficiency model, provided no evidence that a murine host
protein diet compared to the weight gains of 21 mice fed a can be made more susceptible to Aeromonas spp. infection
complete diet (combined results of 4 experiments). After through a dietary-induced state of malnutrition. The deaths
7 days, unchallenged control mice fed the protein-free in mice which had been challenged with strain no. 14
food had lost an average 23% of their original body were considered to have resulted from an unrelated cause.
weight, whereas unchallenged mice fed a complete diet Necropsies of moribund mice failed to demonstrate
had gained an average 35.9% of their original body evidence of infection from the Aeromonas challenge
weight. By day 18. mice fed the protein-free diet had lost strain, and a second experiment, using the same strain,
31.6% of their body weight, and normal-diet mice had failed to produce illness in challenged mice. Considering
gained 106.1%. that all of the mice that became ill were housed in a single
cage, we hypothesize that undetected microenvironmental
Colonization. Ten of the 15 (75.0%) challenged groups changes may have led to the diminished physiological and
yielded Aeromonas-positive faecal specimens, even though health status of these animals.
some groups were only cultured once (data not shown). Although infant mice (3 to 5 days of age) have
Aeromonas-positive faeces from the bottoms of cages previously been used for detection of Aeromonas spp.
demonstrated that Aeromonas strains are able to transit the enterotoxin (13,20), we looked for a diarrhoeic response
gastric-acid barrier without the use of a feeding tube or in older mice (24 to 25 days). Weanling mice were used
buffered liquid vehicle. The majority of mice cultured because the day of weaning is the earliest age at which
were still shedding the organism I to 3 days after the mice can easily be placed on a protein-free diet. It is
Aeromonas challenge suspensions had been replaced possible that some of the orally fed strains of Aeromonas
(Table I1). spp. might have caused diarrhoea had they been fed to
Aeromoi(cid:127)O. in pmilten-malnnounshed mice Ill
younger protein-malnourished mice, but a protein- state did not seem to affect the susceptibility of mice to
deficient substitute for colostrum was not available. Aeromonas spp. infection, but there may be other
The challenge experiments were unsuccessful in enteropathogens which could be used in similar models to
inducing Aeromonas-associated diarrhoea using a protein- study the interaction of malnutrition and diarrhoea] disease
malnourished mouse model. However, there were several in humans.
observations which may prove beneficial to diarrhoeal
disease researchers. First, it is possible to at least Table II. Colonization of Aeromonas strains in groups of
temporarily zrolonize mice with Aeromonas strains using orally challenged mice (4 to 6 mice per group): mice
oral inocula. Asymptomatic carriage of Aeromonas were fed Aeromonas strain suspensions in drinking
strains in humans is common in some developing water on days I through 5.
countries (5,10,27), and transient colonization in
challenged rabbits has been previously reported (26). Dav of experiment
Although some species of laboratory animals have been g uI 2 5 6 7 Overall
fN. groups cultured 8 4 4 4 28
found to be naturally colonized, others have not (28). A Ng roups Aeromonas-positive 3 6 2 2 3 16
mouse- colonization model may be useful in determining % groups Aeromonas-positive 37.5 75.0 50.0 50.0 75.0 57.1
what factors lead to intestinal carriage in some human and
animal populations, but not others.
The current study was planned as the first in a
2 eigh I;) series of protein-deficiency experiments, and so did
26-
not include challenged normal. diet control groups.
comp0%illp rDoliina ialThe determination that none of the 15 Aeromona~s
strains tested caused diarrhoea in protein-
20 -malnourished mice precluded the expenditure of
additional mice to test other strains, or for use as
additional controls. Future investigations using
murine models for studies of other enteropathogens
in malnutrition-associated diarrhoea will require
the use of such controls.
The lack of an animal model for Aeromonas-
associated diarrhoea continues to hinder progress in
studies of pathogenic mechanisms for this newly
recognized and potentially important
enteropathogen. Despite the current study's
outcome, it is likely that future investigations using
S1 _ _ _ _ 6 7._ *,_ _ _ 1 3,_ _ 1_5ro mutine 17 understanmdiondge lso fw tihlle pcaotnhtorgibeuntees ist owofa rAde rao mboentatesr-
Days associated diarrhoea in humans, and an improved
ability to distinguish virulent strains of these
Figure. Average individual weight loss of mice fed a 0% organisms.
protein diet (n - 23) compared to the weight gains of mice (n -
21) fed a complete diet at the time of weaning; combined ACKNOWLEDGEMENTS
results of 4 experiments.
This work was supported by the Indonesian Ministry of Health and
Aeromonas spp. are known to be aquatic organisms the US Naval Medical Research and Development Command. Bethesda.
(29) and have been shown to survive as long as 2 weeks Maryland, work unit no. 62787A 3M)627871870- ARS. The opinions
on a moist surface in a humid environment (28). As and assertions contained herein are those of the authors and are not to he
expected, the challenge strains were capable of surviving construed as reflecting the views of the US Navy or the Indonesian
for relatively long periods in drinking water with no added Ministry of Health.
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