120 TRANSACTIONS
OF THE ROYAL
SOCIETY OP TROPICAL
MEDICINE
AND HYGIENE,
VOL.
73, No.
1, 1979
Correspondence To the Editor
Toxins of Entamoeba histolytica SIR-A long-standing controversy exists on the ability of Entamoeba histolytica to kill cells at a distance. JARUMILINTA & KRADOLFER (1964) demonstrated a contact-dependent lysis of mammalian white blood cells caused by the amoeba, but could not observe this phenomenon when using extracts of amoebic cultures. Others, e.g. KNIGHT, BIRD & MCCAUL (1975) described progressively appearing defects in monolayers of rabbit kidney cells during interaction with E. histolytica but failed to demonstrate damage caused by sonicated amoebae. During the 7th Seminar on Amoebiasis, held in Mexico Citv from 3rd to 5th November 1977. LUSBAUGH and co-workers from Charleston Medical University (South Carolina, USA) presented an interesting paper on the cytotoxicity of a cell-free extract of E. histolytica. They suggested that the addition of 5”/ foetal calf serum (FCS) inhibited the cytopathogenic effect (CPE) of amoebic sonicates. Using monolavers of babv hamster kidney cells, growing in Nunc microplates on L-15 medium, I found a clear CPE of amoebic sonicates, some of which were keot lvoohilized at 4°C for several years. Indeed, the preshnce of FCS largely inhibited cell damage. Cells were washed with L-15 medium without serum prior to incubation with antigens. CPE was measured after 20 hours and titrated. Six axenically cultivated strains of E. histolytica and E. invadens were tested, both the living amoebae and their sonicates. The sonicate of one strain produced marked CPE at a protein concentration as low as 5 pg per ml. Previously, a correlation was found between virulence to hamster liver and in vitro toxicity of trophozoites of various strains. (Bos & VAN DE GRIEND, 1977). Now, also a correlation has been demonstrated between the CPEs of trophozoites and their soluble extracts. It may be concluded that E. histolytica has two different ways of killing host cells. One is a rapid process occurring at close contact; the other is slow, operating through soluble substances from disintegrating amoebae. The activity of these substances is neutralized by non-immune serum. Further study aims at elucidating the nature of this (these) toxin(s). I am, etc., H. J. Bos Leiden University, Medical Department of Parasitology, The Netherlands
Centre, Leiden,
References Bos, H. J. & van de Griend, R. J. (1977). Virulence and toxicity of axenic Entamoeba histolytica. Nature,
265,341-343.
Jarumilinta, R. & Kradolfer, F. (1964). The toxic effect of Entamoeba histolytica on leucocytes. Annals
of
tropical
Medicine
and
Hygiene,
58,
375381. Knight, R., Bird, R. G. & McCaul, T. F. (1975). Fine structural changes at Entamoeba histoiytica.rabbit kidnev cell (RK13) interface. Annals of Tropical
Me&cine
ad
Paraiitology,
64,299.
Lusbaugh, W. B., Kairalla, A. B., Cantey, J. R., Hofbauer, A. F., Pittman, J. C. & Pittman, F. E. (1977). Cytotoxicity of a cell-free extract of Entamoeba histolytica. on Amoebiasis. Mexico
Accepted
Diagnosis
for
publication
of
Abstracts of the 7th Seminar City, 3-5 November 1977.
4thJuly,
1978.
African trypanosomiasis in the bovine SIR-We feel that we must point out several errors in Dr. Lesley Griffin’s comments on trypanosomiasis diagnosis and epidemiology (GRIFFIN, 1978; MURRAY et al., 1977) in which she questioned the time-consuming nature of blood buffy coat examination by darkground microscopy as a diagnostic technique and whether it could be used under field conditions. In addition. she felt that trypanosome species identification was not important and suggested the need for a simple physiological test. In bovine trypanosomiasis, while a tentative diagnosis can be made by an experienced veterinary clinician, specific diagnosis must still depend on the detection of a trypanosome. However, in the bovine animal, parasitaemias are usually low and often transient. Thus the question of detection of low numbers of trypanosomes is paramount. In our experience, the buffy coat darkground technique is significantly more sensitive than other standard techniques currently in use (MURRAY et al., 1977). For this very reason the presence of trypanosomes can be detected more quickly by this technique than by other standard diagnostic tests. It is known that under certain circumstances the clinicopathological syndrome produced by Trypanosoma vivax is distinct from that of T. congolense (Losos & IKEDE, 1972), while the pathogenicity of T. brucei in bovine trypanosomiasis, where infection is often mixed, awaits evaluation. Thus, in order that the data being collected in an epidemiological study be meaningful it is important that the species of trypanosome be identified.
OF THE ROYAL
SOCIETY OP TROPICAL
MEDICINE
AND HYGIENE,
VOL.
73, No.
1, 1979
Correspondence To the Editor
Toxins of Entamoeba histolytica SIR-A long-standing controversy exists on the ability of Entamoeba histolytica to kill cells at a distance. JARUMILINTA & KRADOLFER (1964) demonstrated a contact-dependent lysis of mammalian white blood cells caused by the amoeba, but could not observe this phenomenon when using extracts of amoebic cultures. Others, e.g. KNIGHT, BIRD & MCCAUL (1975) described progressively appearing defects in monolayers of rabbit kidney cells during interaction with E. histolytica but failed to demonstrate damage caused by sonicated amoebae. During the 7th Seminar on Amoebiasis, held in Mexico Citv from 3rd to 5th November 1977. LUSBAUGH and co-workers from Charleston Medical University (South Carolina, USA) presented an interesting paper on the cytotoxicity of a cell-free extract of E. histolytica. They suggested that the addition of 5”/ foetal calf serum (FCS) inhibited the cytopathogenic effect (CPE) of amoebic sonicates. Using monolavers of babv hamster kidney cells, growing in Nunc microplates on L-15 medium, I found a clear CPE of amoebic sonicates, some of which were keot lvoohilized at 4°C for several years. Indeed, the preshnce of FCS largely inhibited cell damage. Cells were washed with L-15 medium without serum prior to incubation with antigens. CPE was measured after 20 hours and titrated. Six axenically cultivated strains of E. histolytica and E. invadens were tested, both the living amoebae and their sonicates. The sonicate of one strain produced marked CPE at a protein concentration as low as 5 pg per ml. Previously, a correlation was found between virulence to hamster liver and in vitro toxicity of trophozoites of various strains. (Bos & VAN DE GRIEND, 1977). Now, also a correlation has been demonstrated between the CPEs of trophozoites and their soluble extracts. It may be concluded that E. histolytica has two different ways of killing host cells. One is a rapid process occurring at close contact; the other is slow, operating through soluble substances from disintegrating amoebae. The activity of these substances is neutralized by non-immune serum. Further study aims at elucidating the nature of this (these) toxin(s). I am, etc., H. J. Bos Leiden University, Medical Department of Parasitology, The Netherlands
Centre, Leiden,
References Bos, H. J. & van de Griend, R. J. (1977). Virulence and toxicity of axenic Entamoeba histolytica. Nature,
265,341-343.
Jarumilinta, R. & Kradolfer, F. (1964). The toxic effect of Entamoeba histolytica on leucocytes. Annals
of
tropical
Medicine
and
Hygiene,
58,
375381. Knight, R., Bird, R. G. & McCaul, T. F. (1975). Fine structural changes at Entamoeba histoiytica.rabbit kidnev cell (RK13) interface. Annals of Tropical
Me&cine
ad
Paraiitology,
64,299.
Lusbaugh, W. B., Kairalla, A. B., Cantey, J. R., Hofbauer, A. F., Pittman, J. C. & Pittman, F. E. (1977). Cytotoxicity of a cell-free extract of Entamoeba histolytica. on Amoebiasis. Mexico
Accepted
Diagnosis
for
publication
of
Abstracts of the 7th Seminar City, 3-5 November 1977.
4thJuly,
1978.
African trypanosomiasis in the bovine SIR-We feel that we must point out several errors in Dr. Lesley Griffin’s comments on trypanosomiasis diagnosis and epidemiology (GRIFFIN, 1978; MURRAY et al., 1977) in which she questioned the time-consuming nature of blood buffy coat examination by darkground microscopy as a diagnostic technique and whether it could be used under field conditions. In addition. she felt that trypanosome species identification was not important and suggested the need for a simple physiological test. In bovine trypanosomiasis, while a tentative diagnosis can be made by an experienced veterinary clinician, specific diagnosis must still depend on the detection of a trypanosome. However, in the bovine animal, parasitaemias are usually low and often transient. Thus the question of detection of low numbers of trypanosomes is paramount. In our experience, the buffy coat darkground technique is significantly more sensitive than other standard techniques currently in use (MURRAY et al., 1977). For this very reason the presence of trypanosomes can be detected more quickly by this technique than by other standard diagnostic tests. It is known that under certain circumstances the clinicopathological syndrome produced by Trypanosoma vivax is distinct from that of T. congolense (Losos & IKEDE, 1972), while the pathogenicity of T. brucei in bovine trypanosomiasis, where infection is often mixed, awaits evaluation. Thus, in order that the data being collected in an epidemiological study be meaningful it is important that the species of trypanosome be identified.
