460 TRANSACTIONS OFTHEROYALSOCIETY OFTROPICALMEDICINEANDH~~mm,V0~.73,N0.4,1979
Naegleria
fowlen
in the chick embryo*
THOMAS W. HOLBROOK
Division of Clinical Microbiology, Department of Laboratory Medicine, Medical University of South Carolina, Charleston, S. C. 29403, U.S.A.
Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis. The disease in man is usually fatal although occasional individuals have recovered from infection following intensive chemotherapeutic intervention (CARTER, 1972; CENTER FOR DISEASE CONTROL, 1978). The protozoan is infective for mammalian hosts under experimental conditions (CARTER, 1970; CERVA, 1971) but the susceptibility of other vertebrates to infection by N. fowleri has apparently not been studied. Avian embryos can be infected by a variety of parasitic protozoa of birds and mammals (PIPKIN, 1960). The present study demonstrates that chick embryos are susceptible to infection by N. fowleri and may represent a useful host for experimental studies of the protozoan. Materials and Methods N. fowleri was isolated from cerebrospinal fluid (CSF) of an eight-year-old Caucasian male. Details of the clinical course and post-mortem findings will be the subject of a subsequent report. Direct intranasal inoculation of CSF in which motile amoebae were seen produced infection in mice and the organism has since been maintained in mice by serial intranasal passage of minced, infected brain. White leghorn chick embryos were obtained commercially and maintained at 37°C. Infection of embryos in initial experiments was by implantation of a small portion ( < 0 *5 mg) of infected mouse brain on the chorioallantoic membrane. In experiments using calculated numbers of amoebae the procedure was as follows. Infected mouse brain was minced in sterile Medium 199, transferred to a test tube, and larger particulate matter was allowed to settle. The supernatant was transferred to another tube and numbers of amoebae were estimated by counting in a haemocytometer. Medium 199 was used for dilution to the desired concentration. In an experiment utilizing the intravenous route (EICHORN, 1940) for infection the amount of particulate matter from mouse brain was further reduced. A 0 *5 ml suspension of infected mouse brain was inoculated into a 25 cm2 culture flask containing 3.5 ml Medium 199 supplemented with 10 % foetal bovine serum. One day later the culture medium was gently decanted, replaced with fresh Medium 199 and the flask was agitated to suspend amoebae which were attached to the flask. The number of organisms was determined by counting in a haemocytometer and the suspension was diluted to the desired concentration with Medium 199.
In each experiment infected embryos were incubated at 37°C and candled daily. Dead embryos were removed from the shell and wet mount slides were made from chorioallantoic fluid, brain, liver, spleen and lung. Slides were examined for the presence of amoebae by phase contrast microscopy. Results (a) Infection by chorioallantoic membrane (CAM) implantation of infected mouse brain In an initial experiment seven embryos were infected on incubation day 7. All embryos were dead three days later and amoebae were seen in all organs examined. In a second experiment 1Zday chick embryos were used. Three days after implantation of mouse brain all six embryos were alive. One embryo was sacrificed at that time and amoebae were seen in brain, liver and lung. No amoeba was seen in chorioallantoic fluid or spleen. One embryo died on day 4 postinfection and the remaining four embryos were found dead on day 5. Amoebae were present in all organs examined. Of seven embrvos infected on incubation dav Il. three were dead four days later. Two living embryos were sacrificed at that time and amoebae were seen in all organs examined. The remaining two embryos were dead on the 5thpostinfection day. It should be noted that neither cysts nor flagellated forms were seen in embryos in this series of experiments nor in experiments described below. (b) Effect of inoculum size A total of 30 embryos were used in two experiments to determine the number of amoebae needed to initiate infection. Embryos were infected on incubation day 11 by CAM inoculation of amoebae from minced mouse brain. Results are presented in Table I. The effect of inoculum size on time of death of embrvos varied sliahtlv but dilution of a suspension to *an equivalent of only 10 amoebae resulted in deaths of all nine embryos in that group on the fifth day postinfection. (c) Route of inoculation of amoebae Grouos of four 13-day embryos received 6 x lo3 amoebae from cultures by inoculation on the CAM, bv intravenous (IV) injection or bv injection into the chorioallantoic cavity. Infection-by CAM or IV inoculation resulted in deaths of all embryos by * Supported by the South Carolina for Biomedical Research.
Appropriation
T. W. HOLBROOK
Table I-Effect of numbers of N. fowleri used to initiate infection in chick embryos. Amoebae were harvested from minced mouse brain and inoculated on the chorioallantoic membrane of 11-day embryos
Group
Number of embryos
Amoebae in inoculum
Mean time to death Ways)
1
4
6000
5.2
z 4 5 6
43* 3” 5 9
3000 1500 600 100 10
i:; ;:z 5.0
* Excludes one embryo found dead one day after inoculation, probably due to traumatic injury during the procedure. of route of N. fowleriinoculation Table II-Effect of chick embryos. Embryos were inoculated on incubation day 13 with 6000 amoebae from one-day cultures from infected mouse brain. Mean time to death (days)
Group
Number of embryos
1
3”
Chorioallantoic membrane
4.3
2
4
Intravenous
3.5
3
4
Chorioallantoic cavity
7.5
Route
* Excludes one embryo found dead one day after inoculation, probably due to traumatic injury during procedure.
day 5 postinfection. Embryos survived longer after injections of amoebae into chorioallantoic fluid (CAF) but eventual death resulted (Table II). The longer period of survival of embryos in that group suggested that CAF may have an inhibitory effect on N. fowleri. However, CAF removed by syringe aspiration from an uninfected 13-day embryo had no apparent deleterious effect on amoebae. Neither motility nor morphology of amoebae from infected mouse brain was altered by in vitro exposure to CAF. (d) Serial passage in chick embryos N. fowleri is presently maintained by serial passage in chick embryos. Characteristics of infection in embryos infected with amoebae from embryo brain are similar to those produced by inoculation of amoebae from mice. Eleven to 14-day embryos which receive infected embryo brain or supernatant from minced brain on the CAM invariably die three to five days later and amoebae are seen in all
461
organs examined. On day 3 or 4 after infection an embryo is sacrificed to obtain amoebae for transfer. In vitro transformation of N. fowleri to the biflagellate form was induced following the fifth serial passage in chick embryos. Minced embryo brain was suspended in distilled water at 37°C and numerous flagellates and amoebae were seen 12 hours later. Discussion Although chick embryos have been experimentally infected with a variety of protozoan parasites (PIPKIN, 1960), studies of infectivity of amoebae in avian embryos are few. MEEROVITCH (1956) inoculated nine to 15-day chick embryos intravenously with Entamoeba invadens and demonstrated amoebae in the liver of 45 05 y0 of the embryos after one to seven davs at 30°C. CERVA (1970) studied a nathoaenic strain of Acanthamoeba‘ casteilanii in chick emb-qos. Injection of lo3 or lo4 amoebae into the yolk sac invariably resulted in death of five to eight-day embryos but none died when smaller inocula were used. Parasites were found in brain, liver and yolk sac wall following inoculation on the CAM but most embryos survived to hatch. Small foci of amoebae were seen in the CAM and in the brain and spine of infected embryos. The present study demonstrated that chick embryos are highly susceptible to infection with a pathogenic isolate of N. fowleri. Inoculation of as few as 10 amoebae (by dilution) on the chorioallantoic membrane invariably resulted in embryo death and amoebae were found in several organs and in chorioallantoic fluid. Embryos also succumbed to infection initiated via intravenous injection or inoculation of amoebae into the chorioallantoic cavity. The latter route allowed a longer survival time of embryos, probably due to a delay in entrance of amoebae into the circulatory system. The susceptibility of chick embryos to infection may present a sensitive method for isolation of N. fowleri in clinical situations, but whether the system is useful for differentiation of pathogenic from nonpathogenic isolates of N. fowleri or differentiation of N. fowleri from nonpathogenic species has not been tested. An advantage of this host-parasite system is that serial transfer of N. fowleri in chick embryos is easily managed. This relatively inexpensive and highly reproducible system may circumvent difficulties in possible loss of infectivity in culture and the more expensive, and less sensitive, maintenance of N. fowl&i by passage in mice. Further studies will examine infectivity of embrvopassaged N. fowleri for mice. References Carter, R. F. (1970). Description of a Naegleria sp. isolated from two cases of primary amoebic meningoencephalitis, and of the experimental nathological changes induced by it. Journal of i>athoZoiy, 100,21?-244. Carter, R. F. (1972). Primary amoebic meningoencephalitis. An appraisal of present knowledge. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 193-213.
462
N.
fm.deri
IN THE CHICK
Cerva, L. (1970). Growth of the pathogenic A 1 strain of Acanthamoeba castellanii in the chick embryo. Folia Parasitologica(Praha), 17,315 318. Cerva. L. (1971). Exnerimental infection of laboratory animals by &e pathogenic Naegleria fowleri strain. Folia Parasitologica (Praha), 18, 171-176. Eichorn, E. A. (1940). A technique for the intravenous inoculation of chick embryos. Science, 92, 245-246. Meerovitch, E. (1956). Experimental infection of chick embryos with Entamoebainvadens. Canadian Journal of Microbiology, 2, 1-5. Center for Disease Control, U.S. Dept. of Health,
EMBRYO
Education and Welfare/Public Health Service (1978). Primary amebic meningoencephalitisCalifornia, Florida, New York. Morbidity and Mortality Weekly Report, 27, 343-344. Pipkin, A. C. (1960). Avian embryos and tissue culture in the study of parasitic protozqa. II. Protozoa other than Plasmodium. Experimental Parasitology, 9, 167-203.
Acceptedfor publication 7th December,1978.
Naegleria
fowlen
in the chick embryo*
THOMAS W. HOLBROOK
Division of Clinical Microbiology, Department of Laboratory Medicine, Medical University of South Carolina, Charleston, S. C. 29403, U.S.A.
Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis. The disease in man is usually fatal although occasional individuals have recovered from infection following intensive chemotherapeutic intervention (CARTER, 1972; CENTER FOR DISEASE CONTROL, 1978). The protozoan is infective for mammalian hosts under experimental conditions (CARTER, 1970; CERVA, 1971) but the susceptibility of other vertebrates to infection by N. fowleri has apparently not been studied. Avian embryos can be infected by a variety of parasitic protozoa of birds and mammals (PIPKIN, 1960). The present study demonstrates that chick embryos are susceptible to infection by N. fowleri and may represent a useful host for experimental studies of the protozoan. Materials and Methods N. fowleri was isolated from cerebrospinal fluid (CSF) of an eight-year-old Caucasian male. Details of the clinical course and post-mortem findings will be the subject of a subsequent report. Direct intranasal inoculation of CSF in which motile amoebae were seen produced infection in mice and the organism has since been maintained in mice by serial intranasal passage of minced, infected brain. White leghorn chick embryos were obtained commercially and maintained at 37°C. Infection of embryos in initial experiments was by implantation of a small portion ( < 0 *5 mg) of infected mouse brain on the chorioallantoic membrane. In experiments using calculated numbers of amoebae the procedure was as follows. Infected mouse brain was minced in sterile Medium 199, transferred to a test tube, and larger particulate matter was allowed to settle. The supernatant was transferred to another tube and numbers of amoebae were estimated by counting in a haemocytometer. Medium 199 was used for dilution to the desired concentration. In an experiment utilizing the intravenous route (EICHORN, 1940) for infection the amount of particulate matter from mouse brain was further reduced. A 0 *5 ml suspension of infected mouse brain was inoculated into a 25 cm2 culture flask containing 3.5 ml Medium 199 supplemented with 10 % foetal bovine serum. One day later the culture medium was gently decanted, replaced with fresh Medium 199 and the flask was agitated to suspend amoebae which were attached to the flask. The number of organisms was determined by counting in a haemocytometer and the suspension was diluted to the desired concentration with Medium 199.
In each experiment infected embryos were incubated at 37°C and candled daily. Dead embryos were removed from the shell and wet mount slides were made from chorioallantoic fluid, brain, liver, spleen and lung. Slides were examined for the presence of amoebae by phase contrast microscopy. Results (a) Infection by chorioallantoic membrane (CAM) implantation of infected mouse brain In an initial experiment seven embryos were infected on incubation day 7. All embryos were dead three days later and amoebae were seen in all organs examined. In a second experiment 1Zday chick embryos were used. Three days after implantation of mouse brain all six embryos were alive. One embryo was sacrificed at that time and amoebae were seen in brain, liver and lung. No amoeba was seen in chorioallantoic fluid or spleen. One embryo died on day 4 postinfection and the remaining four embryos were found dead on day 5. Amoebae were present in all organs examined. Of seven embrvos infected on incubation dav Il. three were dead four days later. Two living embryos were sacrificed at that time and amoebae were seen in all organs examined. The remaining two embryos were dead on the 5thpostinfection day. It should be noted that neither cysts nor flagellated forms were seen in embryos in this series of experiments nor in experiments described below. (b) Effect of inoculum size A total of 30 embryos were used in two experiments to determine the number of amoebae needed to initiate infection. Embryos were infected on incubation day 11 by CAM inoculation of amoebae from minced mouse brain. Results are presented in Table I. The effect of inoculum size on time of death of embrvos varied sliahtlv but dilution of a suspension to *an equivalent of only 10 amoebae resulted in deaths of all nine embryos in that group on the fifth day postinfection. (c) Route of inoculation of amoebae Grouos of four 13-day embryos received 6 x lo3 amoebae from cultures by inoculation on the CAM, bv intravenous (IV) injection or bv injection into the chorioallantoic cavity. Infection-by CAM or IV inoculation resulted in deaths of all embryos by * Supported by the South Carolina for Biomedical Research.
Appropriation
T. W. HOLBROOK
Table I-Effect of numbers of N. fowleri used to initiate infection in chick embryos. Amoebae were harvested from minced mouse brain and inoculated on the chorioallantoic membrane of 11-day embryos
Group
Number of embryos
Amoebae in inoculum
Mean time to death Ways)
1
4
6000
5.2
z 4 5 6
43* 3” 5 9
3000 1500 600 100 10
i:; ;:z 5.0
* Excludes one embryo found dead one day after inoculation, probably due to traumatic injury during the procedure. of route of N. fowleriinoculation Table II-Effect of chick embryos. Embryos were inoculated on incubation day 13 with 6000 amoebae from one-day cultures from infected mouse brain. Mean time to death (days)
Group
Number of embryos
1
3”
Chorioallantoic membrane
4.3
2
4
Intravenous
3.5
3
4
Chorioallantoic cavity
7.5
Route
* Excludes one embryo found dead one day after inoculation, probably due to traumatic injury during procedure.
day 5 postinfection. Embryos survived longer after injections of amoebae into chorioallantoic fluid (CAF) but eventual death resulted (Table II). The longer period of survival of embryos in that group suggested that CAF may have an inhibitory effect on N. fowleri. However, CAF removed by syringe aspiration from an uninfected 13-day embryo had no apparent deleterious effect on amoebae. Neither motility nor morphology of amoebae from infected mouse brain was altered by in vitro exposure to CAF. (d) Serial passage in chick embryos N. fowleri is presently maintained by serial passage in chick embryos. Characteristics of infection in embryos infected with amoebae from embryo brain are similar to those produced by inoculation of amoebae from mice. Eleven to 14-day embryos which receive infected embryo brain or supernatant from minced brain on the CAM invariably die three to five days later and amoebae are seen in all
461
organs examined. On day 3 or 4 after infection an embryo is sacrificed to obtain amoebae for transfer. In vitro transformation of N. fowleri to the biflagellate form was induced following the fifth serial passage in chick embryos. Minced embryo brain was suspended in distilled water at 37°C and numerous flagellates and amoebae were seen 12 hours later. Discussion Although chick embryos have been experimentally infected with a variety of protozoan parasites (PIPKIN, 1960), studies of infectivity of amoebae in avian embryos are few. MEEROVITCH (1956) inoculated nine to 15-day chick embryos intravenously with Entamoeba invadens and demonstrated amoebae in the liver of 45 05 y0 of the embryos after one to seven davs at 30°C. CERVA (1970) studied a nathoaenic strain of Acanthamoeba‘ casteilanii in chick emb-qos. Injection of lo3 or lo4 amoebae into the yolk sac invariably resulted in death of five to eight-day embryos but none died when smaller inocula were used. Parasites were found in brain, liver and yolk sac wall following inoculation on the CAM but most embryos survived to hatch. Small foci of amoebae were seen in the CAM and in the brain and spine of infected embryos. The present study demonstrated that chick embryos are highly susceptible to infection with a pathogenic isolate of N. fowleri. Inoculation of as few as 10 amoebae (by dilution) on the chorioallantoic membrane invariably resulted in embryo death and amoebae were found in several organs and in chorioallantoic fluid. Embryos also succumbed to infection initiated via intravenous injection or inoculation of amoebae into the chorioallantoic cavity. The latter route allowed a longer survival time of embryos, probably due to a delay in entrance of amoebae into the circulatory system. The susceptibility of chick embryos to infection may present a sensitive method for isolation of N. fowleri in clinical situations, but whether the system is useful for differentiation of pathogenic from nonpathogenic isolates of N. fowleri or differentiation of N. fowleri from nonpathogenic species has not been tested. An advantage of this host-parasite system is that serial transfer of N. fowleri in chick embryos is easily managed. This relatively inexpensive and highly reproducible system may circumvent difficulties in possible loss of infectivity in culture and the more expensive, and less sensitive, maintenance of N. fowl&i by passage in mice. Further studies will examine infectivity of embrvopassaged N. fowleri for mice. References Carter, R. F. (1970). Description of a Naegleria sp. isolated from two cases of primary amoebic meningoencephalitis, and of the experimental nathological changes induced by it. Journal of i>athoZoiy, 100,21?-244. Carter, R. F. (1972). Primary amoebic meningoencephalitis. An appraisal of present knowledge. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 193-213.
462
N.
fm.deri
IN THE CHICK
Cerva, L. (1970). Growth of the pathogenic A 1 strain of Acanthamoeba castellanii in the chick embryo. Folia Parasitologica(Praha), 17,315 318. Cerva. L. (1971). Exnerimental infection of laboratory animals by &e pathogenic Naegleria fowleri strain. Folia Parasitologica (Praha), 18, 171-176. Eichorn, E. A. (1940). A technique for the intravenous inoculation of chick embryos. Science, 92, 245-246. Meerovitch, E. (1956). Experimental infection of chick embryos with Entamoebainvadens. Canadian Journal of Microbiology, 2, 1-5. Center for Disease Control, U.S. Dept. of Health,
EMBRYO
Education and Welfare/Public Health Service (1978). Primary amebic meningoencephalitisCalifornia, Florida, New York. Morbidity and Mortality Weekly Report, 27, 343-344. Pipkin, A. C. (1960). Avian embryos and tissue culture in the study of parasitic protozqa. II. Protozoa other than Plasmodium. Experimental Parasitology, 9, 167-203.
Acceptedfor publication 7th December,1978.
