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EXPERIMENTAL CHOLERA A CANINE MODEL
206
without therapy. The diarrhoea
EXPERIMENTAL CHOLERA A CANINE MODEL ONE of the
major obstacles
to the
satisfactory study
of
pathogenetic and immunological mechanisms of cholera has been the lack of a suitable animal model. The infant rabbitand the isolated adult rabbit ileum2 are at present the only available models; but both have serious disadvantages because of their size and their lack of adaptability to long-term studies. We describe here our preliminary findings in the development of a canine model in which a disease simulating human cholera can predictably be produced. METHODS
Adult 8-20 kg. mongrel dogs of both sexes were fasted for two to five days before challenge. On the day of challenge an orogastric tube was inserted and 50 ml. of 6% sodium bicarbonate was instilled, followed immediately by 100 ml. of a five-hourSyncase ’3 broth culture of Vibrio cholera, containing 109 organisms per ml. The tube was then removed, and the dogs were placed in metabolic cages and observed for the development of diarrhoea. They were given water only ad libitum for another twenty-four hours, and then placed on a regular diet. Serum-samples were obtained immediately before challenge and again ten to fourteen days later. A rectal swab was taken immediately before challenge; thereafter stools were cultured daily for the first five days and then every other day until vibrio excretion was no longer found on three successive cultures. Several surviving dogs were rechallenged with vibrios after being maintained on a regular diet for up to three months. V. cholera strains Ogawa 385 and Ogawa 412 were isolated from patients with clinical cholera in Calcutta in the spring of 1964. The strains had been maintained in paraffin-sealed agar stabs at room temperature for two years until used in the present study, during which time they were maintained on parafilm-sealed agar slants in the refrigerator. V. cholera Ogawa B1307 (received from Dr. John Craig) was obtained from a patient with cholera in Dacca in 1964, and had been lyophilised until used in the present study. A lyophilised culture of V. cholera Inaba 569B, a strain originally obtained from Dr. N. K. Dutta, was received from Dr. R. A. Finkelstein. For use in the challenge, 5-0 ml. of an overnight growth of vibrios in peptone water (1% peptone Difco, 0-5% sodium chloride), pH 7-4, were inoculated into 500 ml. of syncase media in a 2-litre Erlenmeyer flask. This was incubated at 37°C with magnetic stirring for five hours. At this time, when the organisms were determined to be in the late logarithmic phase of growth, they were removed from the incubator, the viable-cell concentration was determined by the drop methodand the suspension was used for oral challenge within the next thirty to sixty minutes. Stool cultures were made on TCBS media5 (Eiken) by both streaking directly and again after overnight enrichment in alkaline peptone water (pH 9-0). V. cholera colonies were identified by their characteristic appearance on both TCBS and nutrient agar, and by their agglutination in group and type specific cholera antisera. Agglutinin titres were determined with the use of live vibrio suspensions,6 using strain Ogawa 385.
was
of short duration,
remaining 13 dogs had only exceeding days. normal or soft stools throughout the period of observation, The challenge procedure was then modified as follows: the dogs were fasted for five days, a different vibrio strain (Ogawa 395) was used, and 100 ml. of fresh syncase (without organisms) was given through the orogastric tube immediately following the broth culture containing vibrios. In this manner 22 dogs were challenfed, of which 7 (32%) developed massive watery diarrhoea within sixteen to thirty-six hours. The stool was init: ally brown and watery, but later became colourless and typically " rice-water ". These dogs were found to develop very poor skin turgor, sunken eyes, cyanosis of the tongue, tachycardia, and complete inability to stand; bloodstudies showed severe hxmoconcentration and metabolic acidosis. If untreated, the dogs invariably died within four to eighteen hours of the onset of collapse. 5 other dogs in this group had mild watery diarrhoea similar to the dogs in the first challenge group. The remaining 10 dogs (45%) showed no significant change in stool character. Using the identical challenge procedure, 15 dogs from the first group which had been challenged two to three months previously with Ogawa 412, were rechallenged with vibrio strain Ogawa 395. 6 of these dogs (40%) developed a clinical cholera syndrome identical to that described above, 1 had mild diarrhoea, and 8 had normal
not
two
The
TABLE IŁDIARRHCEAL DISEASE PRODUCED IN DOGS WITH
*These dogs had been challenged two to three months V. cholera Ogawa 412.
V. Choler6e
previously
with
stools. In the same manner and with the same results, vibrio strains Ogawa B1307 and Inaba 569B were used to challenge 5 dogs each. The results from all challenges are summarised in table i. The average total stool output measured at the time of death was 135::1:: 17 ml. per kg. (mean::l:: standard error in 11 dogs from all groups). V. cholera could be recovered in the stool from 47 of the 52 primarily challenged dogs, and in all dogs who had diarrhoea. Vibrios were profusely present in the stool of all dogs with clinical cholera. In the surviving dogs, vibrio excretion persisted up to nine days with a mean of three days. In the 15 rechallenged dogs, vibrio RESULTS excretion was of shorter duration, not exceeding three The first 20 dogs challenged were starved for two to days, and there were 4 dogs from whom no vibrios could be three days and then given V. choler,5e Ogawa 412. 7 dogs in recovered. this group had watery-brown diarrhoea, but none showed A four-fold or greater rise in agglutinating antibody evidence of obvious salt depletion, and all recovered titres has been seen in all 11 paired sera examined to date, 1. Dutta, N. K., Habbu, M. K. Br. J. Pharmac. 1955, 10, 153. The geometric mean of the pre-infection titres was le55 2. De, S. N., Chattarjee, D. N. J. Path. Bact. 1953, 66, 559. 3. Finkelstein, R. A. Proceedings of the Cholera Research Symposium, than 1/20 and of the convalescent titres was 1/200. Honolulu; p. 264. Washington, 1965. In 4 of the animals with severe diarrhoea and collapse. 4. Miles, A. A., Misrs, S. S. J. Hyg., Camb. 1938, 38, 732. intravenous fluid therapy (two parts isotonic saline solutioc 5. Kobayashi, T., Enomoto, S., Sakazaki, R., Kuwahera, S. Jap. J. Microbiol. 1963, 18, 387. to 1 part isotonic sodium lactate) was initiated and the 6 Barua, D., Neogy. K. N., Sack, R. Bull. Calcutta Sch. trop. Med. 1963, 11, 156. gastrointestinal tract was examined at laparotomy to
207 determine the site of origin of the fluid and electrolyte loss. Ligatures were placed around the distal duodenum and just above the ileocaecal valve to divide the gastrointestinal tract into three segments. For fluid collection, cannulas were placed just proximal to these ligatures, and a tube was placed in the rectum. The dogs were kept under nembutal anaesthesia for periods up to twelve hours for fluid collection, and then killed. In all of these dogs, virtually all the fluid came from the jejuno-ileal segment of the small bowel (table n). The electrolyte content
The canine model would
seem to provide a sound for determining the basic physiological preparation in cholera. alterations Current data indicate that most of the fluid and electrolyte loss originates in the jejunum and ileum. This model also provides a preparation in which the effects of various vibrio " toxins " might be evaluated, and the possibility of antitoxic immunity investigated.
R. B. SACK M.D.
Oregon
C. C.
TABLE II-GASTROINTESTINAL OUTPUT DURING EXPERIMENTAL
J. CARPENTER Johns Hopkins R. W. STEENBURG M.D.
CHOLERA IN THE DOG
The Johns Hopkins University Schools of Medicine and Hygiene and Public Health,
Baltimore, Maryland 21205, U.S.A.
M.D.
Harvard
N. F. PIERCE M.D. Michigan
EFFECT OF CURARISATION ON BREATH-HOLDING TIME
lung volumes the distress which limits breathholding greatly reduced and the breath-holding time is prolonged by vagal block.l This observation implies AT low
is
(mEq. per litre) of the jejuno-ileal effluent (mean values, 4 dogs) was: potassium, 10; bicarbonate, 54; sodium, 138; and chloride, 95. The gut appeared pink and healthy throughout the period of fluid loss. Ileal biopsies showed entirely intact mucosa, including the brush border, minimal focal polymorphonuclear infiltration of the lamina propria, dilatation of the lacteals and congestion of the capillaries of the lamina propria. DISCUSSION
A cholera
syndrome has been experimentally produced in approximately 35% of mongrel dogs challenged orally with 3 strains of V. cholerae. The disease resembles human cholera in all aspects so far examined. The massive outpouring of isotonic alkaline rice-water stools, the rapid development of vascular collapse, severe salt depletion, metabolic acidoses and death, and the development of agglutinating antibodies in the surviving dogs all indicate the disease is very similar in the two hosts. The predisposing factors essential to the establishment of the infection are as yet not delineated. Current studies indicate that an adequate period of fasting, followed by alkalinisation of the stomach, and the administration of large numbers of vibrios in the logarithmic phase of growth are probably important, but the role of any one of them has not yet been clearly defined. Whether differences in vibrio strains or the change in protocol accounted for the different results obtained in the two primary challenge groups is not known at this time. Further studies are in progress to determine the relative importance of these factors, in the hope of developing a model in which lethal disease occurs in all animals inoculated. Since dogs have a serological response to the vibrio infection which is similar to that in man, this model should also provide a means of evaluating the possible role of humoral antibodies in protecting against cholera. In the group of re-challenged dogs, the infection-rate was identical with the dogs challenged for the first time, suggesting that no immunity " had been produced, as measured in this model. The period of vibrio excretion is very similar to that found in patients with cholera, and would indicate that mechanisms leading to a carrier state might be studied. "
that the distress is due to consciousness either of afferent pulmonary drive or of frustration of motor response to this drive. Curarisation of the respiratory muscles might distinguish between these two possibilities because it would not remove the afferent drive but would prevent the development of muscular tension. The breath-holding time of two male volunteers was measured when they were atropinised and breathing 60-80° oxygen; they were then fully paralysed by d-tubocurarine chloride given intravenously (48 mg. in one and 39 mg. in the other; the total being achieved in increments of 3 or 6 mg. over 1 hour). Arterial occlusion in one arm for 3 minutes starting at the time of each dose preserved sufficient power in the forearm for communication by hand signals. The tolerable duration of apnoea at
resting lung-volume
was:
The sensation at breaking-point when paralysed was vague and less distressing than it was in the control
period. These results suggest that frustration of muscular contraction is essential to the distress of breath-holding and are compatible with the suggestion that the sensation is an extreme example of what has been called length!’/ tension inappropriatenessbut is better called, simply,
inappropriateness.3 E. J. M. CAMPBELL Lond., F.R.C.P. FREEDMAN S. M.B., B.SC. Lond., M.R.C.P. T. J. H. CLARK M.B., B.SC., Lond., M.R.C.P. J. G. ROBSON M.B. Glasg., F.F.A. R.C.S. J. NORMAN M.B. Leeds, F.F.A. R.C.S. M.D.,
Department of Medicine and the
Department of Anaesthetics, Postgraduate Medical School, Hammersmith Hospital, London W.12. 1.
2. 3.
PH.D.
Guz, A., Noble, M. I. M., Widdicombe, J. G., Trenchard, D., Mushin, W. W., Makey, A. R. Clin. Sci. 1966, 30, 161. Campbell, E. J. M., Howell, J. B. L. Br. med. Bull. 1963, 19, 36. Campbell, E. J. M. in Breathlessness (edited by J. B L. Howell and E. J. M. Campbell); p. 55. Oxford, 1966.
without therapy. The diarrhoea
EXPERIMENTAL CHOLERA A CANINE MODEL ONE of the
major obstacles
to the
satisfactory study
of
pathogenetic and immunological mechanisms of cholera has been the lack of a suitable animal model. The infant rabbitand the isolated adult rabbit ileum2 are at present the only available models; but both have serious disadvantages because of their size and their lack of adaptability to long-term studies. We describe here our preliminary findings in the development of a canine model in which a disease simulating human cholera can predictably be produced. METHODS
Adult 8-20 kg. mongrel dogs of both sexes were fasted for two to five days before challenge. On the day of challenge an orogastric tube was inserted and 50 ml. of 6% sodium bicarbonate was instilled, followed immediately by 100 ml. of a five-hourSyncase ’3 broth culture of Vibrio cholera, containing 109 organisms per ml. The tube was then removed, and the dogs were placed in metabolic cages and observed for the development of diarrhoea. They were given water only ad libitum for another twenty-four hours, and then placed on a regular diet. Serum-samples were obtained immediately before challenge and again ten to fourteen days later. A rectal swab was taken immediately before challenge; thereafter stools were cultured daily for the first five days and then every other day until vibrio excretion was no longer found on three successive cultures. Several surviving dogs were rechallenged with vibrios after being maintained on a regular diet for up to three months. V. cholera strains Ogawa 385 and Ogawa 412 were isolated from patients with clinical cholera in Calcutta in the spring of 1964. The strains had been maintained in paraffin-sealed agar stabs at room temperature for two years until used in the present study, during which time they were maintained on parafilm-sealed agar slants in the refrigerator. V. cholera Ogawa B1307 (received from Dr. John Craig) was obtained from a patient with cholera in Dacca in 1964, and had been lyophilised until used in the present study. A lyophilised culture of V. cholera Inaba 569B, a strain originally obtained from Dr. N. K. Dutta, was received from Dr. R. A. Finkelstein. For use in the challenge, 5-0 ml. of an overnight growth of vibrios in peptone water (1% peptone Difco, 0-5% sodium chloride), pH 7-4, were inoculated into 500 ml. of syncase media in a 2-litre Erlenmeyer flask. This was incubated at 37°C with magnetic stirring for five hours. At this time, when the organisms were determined to be in the late logarithmic phase of growth, they were removed from the incubator, the viable-cell concentration was determined by the drop methodand the suspension was used for oral challenge within the next thirty to sixty minutes. Stool cultures were made on TCBS media5 (Eiken) by both streaking directly and again after overnight enrichment in alkaline peptone water (pH 9-0). V. cholera colonies were identified by their characteristic appearance on both TCBS and nutrient agar, and by their agglutination in group and type specific cholera antisera. Agglutinin titres were determined with the use of live vibrio suspensions,6 using strain Ogawa 385.
was
of short duration,
remaining 13 dogs had only exceeding days. normal or soft stools throughout the period of observation, The challenge procedure was then modified as follows: the dogs were fasted for five days, a different vibrio strain (Ogawa 395) was used, and 100 ml. of fresh syncase (without organisms) was given through the orogastric tube immediately following the broth culture containing vibrios. In this manner 22 dogs were challenfed, of which 7 (32%) developed massive watery diarrhoea within sixteen to thirty-six hours. The stool was init: ally brown and watery, but later became colourless and typically " rice-water ". These dogs were found to develop very poor skin turgor, sunken eyes, cyanosis of the tongue, tachycardia, and complete inability to stand; bloodstudies showed severe hxmoconcentration and metabolic acidosis. If untreated, the dogs invariably died within four to eighteen hours of the onset of collapse. 5 other dogs in this group had mild watery diarrhoea similar to the dogs in the first challenge group. The remaining 10 dogs (45%) showed no significant change in stool character. Using the identical challenge procedure, 15 dogs from the first group which had been challenged two to three months previously with Ogawa 412, were rechallenged with vibrio strain Ogawa 395. 6 of these dogs (40%) developed a clinical cholera syndrome identical to that described above, 1 had mild diarrhoea, and 8 had normal
not
two
The
TABLE IŁDIARRHCEAL DISEASE PRODUCED IN DOGS WITH
*These dogs had been challenged two to three months V. cholera Ogawa 412.
V. Choler6e
previously
with
stools. In the same manner and with the same results, vibrio strains Ogawa B1307 and Inaba 569B were used to challenge 5 dogs each. The results from all challenges are summarised in table i. The average total stool output measured at the time of death was 135::1:: 17 ml. per kg. (mean::l:: standard error in 11 dogs from all groups). V. cholera could be recovered in the stool from 47 of the 52 primarily challenged dogs, and in all dogs who had diarrhoea. Vibrios were profusely present in the stool of all dogs with clinical cholera. In the surviving dogs, vibrio excretion persisted up to nine days with a mean of three days. In the 15 rechallenged dogs, vibrio RESULTS excretion was of shorter duration, not exceeding three The first 20 dogs challenged were starved for two to days, and there were 4 dogs from whom no vibrios could be three days and then given V. choler,5e Ogawa 412. 7 dogs in recovered. this group had watery-brown diarrhoea, but none showed A four-fold or greater rise in agglutinating antibody evidence of obvious salt depletion, and all recovered titres has been seen in all 11 paired sera examined to date, 1. Dutta, N. K., Habbu, M. K. Br. J. Pharmac. 1955, 10, 153. The geometric mean of the pre-infection titres was le55 2. De, S. N., Chattarjee, D. N. J. Path. Bact. 1953, 66, 559. 3. Finkelstein, R. A. Proceedings of the Cholera Research Symposium, than 1/20 and of the convalescent titres was 1/200. Honolulu; p. 264. Washington, 1965. In 4 of the animals with severe diarrhoea and collapse. 4. Miles, A. A., Misrs, S. S. J. Hyg., Camb. 1938, 38, 732. intravenous fluid therapy (two parts isotonic saline solutioc 5. Kobayashi, T., Enomoto, S., Sakazaki, R., Kuwahera, S. Jap. J. Microbiol. 1963, 18, 387. to 1 part isotonic sodium lactate) was initiated and the 6 Barua, D., Neogy. K. N., Sack, R. Bull. Calcutta Sch. trop. Med. 1963, 11, 156. gastrointestinal tract was examined at laparotomy to
207 determine the site of origin of the fluid and electrolyte loss. Ligatures were placed around the distal duodenum and just above the ileocaecal valve to divide the gastrointestinal tract into three segments. For fluid collection, cannulas were placed just proximal to these ligatures, and a tube was placed in the rectum. The dogs were kept under nembutal anaesthesia for periods up to twelve hours for fluid collection, and then killed. In all of these dogs, virtually all the fluid came from the jejuno-ileal segment of the small bowel (table n). The electrolyte content
The canine model would
seem to provide a sound for determining the basic physiological preparation in cholera. alterations Current data indicate that most of the fluid and electrolyte loss originates in the jejunum and ileum. This model also provides a preparation in which the effects of various vibrio " toxins " might be evaluated, and the possibility of antitoxic immunity investigated.
R. B. SACK M.D.
Oregon
C. C.
TABLE II-GASTROINTESTINAL OUTPUT DURING EXPERIMENTAL
J. CARPENTER Johns Hopkins R. W. STEENBURG M.D.
CHOLERA IN THE DOG
The Johns Hopkins University Schools of Medicine and Hygiene and Public Health,
Baltimore, Maryland 21205, U.S.A.
M.D.
Harvard
N. F. PIERCE M.D. Michigan
EFFECT OF CURARISATION ON BREATH-HOLDING TIME
lung volumes the distress which limits breathholding greatly reduced and the breath-holding time is prolonged by vagal block.l This observation implies AT low
is
(mEq. per litre) of the jejuno-ileal effluent (mean values, 4 dogs) was: potassium, 10; bicarbonate, 54; sodium, 138; and chloride, 95. The gut appeared pink and healthy throughout the period of fluid loss. Ileal biopsies showed entirely intact mucosa, including the brush border, minimal focal polymorphonuclear infiltration of the lamina propria, dilatation of the lacteals and congestion of the capillaries of the lamina propria. DISCUSSION
A cholera
syndrome has been experimentally produced in approximately 35% of mongrel dogs challenged orally with 3 strains of V. cholerae. The disease resembles human cholera in all aspects so far examined. The massive outpouring of isotonic alkaline rice-water stools, the rapid development of vascular collapse, severe salt depletion, metabolic acidoses and death, and the development of agglutinating antibodies in the surviving dogs all indicate the disease is very similar in the two hosts. The predisposing factors essential to the establishment of the infection are as yet not delineated. Current studies indicate that an adequate period of fasting, followed by alkalinisation of the stomach, and the administration of large numbers of vibrios in the logarithmic phase of growth are probably important, but the role of any one of them has not yet been clearly defined. Whether differences in vibrio strains or the change in protocol accounted for the different results obtained in the two primary challenge groups is not known at this time. Further studies are in progress to determine the relative importance of these factors, in the hope of developing a model in which lethal disease occurs in all animals inoculated. Since dogs have a serological response to the vibrio infection which is similar to that in man, this model should also provide a means of evaluating the possible role of humoral antibodies in protecting against cholera. In the group of re-challenged dogs, the infection-rate was identical with the dogs challenged for the first time, suggesting that no immunity " had been produced, as measured in this model. The period of vibrio excretion is very similar to that found in patients with cholera, and would indicate that mechanisms leading to a carrier state might be studied. "
that the distress is due to consciousness either of afferent pulmonary drive or of frustration of motor response to this drive. Curarisation of the respiratory muscles might distinguish between these two possibilities because it would not remove the afferent drive but would prevent the development of muscular tension. The breath-holding time of two male volunteers was measured when they were atropinised and breathing 60-80° oxygen; they were then fully paralysed by d-tubocurarine chloride given intravenously (48 mg. in one and 39 mg. in the other; the total being achieved in increments of 3 or 6 mg. over 1 hour). Arterial occlusion in one arm for 3 minutes starting at the time of each dose preserved sufficient power in the forearm for communication by hand signals. The tolerable duration of apnoea at
resting lung-volume
was:
The sensation at breaking-point when paralysed was vague and less distressing than it was in the control
period. These results suggest that frustration of muscular contraction is essential to the distress of breath-holding and are compatible with the suggestion that the sensation is an extreme example of what has been called length!’/ tension inappropriatenessbut is better called, simply,
inappropriateness.3 E. J. M. CAMPBELL Lond., F.R.C.P. FREEDMAN S. M.B., B.SC. Lond., M.R.C.P. T. J. H. CLARK M.B., B.SC., Lond., M.R.C.P. J. G. ROBSON M.B. Glasg., F.F.A. R.C.S. J. NORMAN M.B. Leeds, F.F.A. R.C.S. M.D.,
Department of Medicine and the
Department of Anaesthetics, Postgraduate Medical School, Hammersmith Hospital, London W.12. 1.
2. 3.
PH.D.
Guz, A., Noble, M. I. M., Widdicombe, J. G., Trenchard, D., Mushin, W. W., Makey, A. R. Clin. Sci. 1966, 30, 161. Campbell, E. J. M., Howell, J. B. L. Br. med. Bull. 1963, 19, 36. Campbell, E. J. M. in Breathlessness (edited by J. B L. Howell and E. J. M. Campbell); p. 55. Oxford, 1966.