平成3年6月15日

第19巻第2号
内

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The

容

説
fundamental

responses

andnatureofChagasdisease：aprovided

by

immme

idiotypes−Daniel原

view

G．ColleyandMichaelJ．Howard

175−190著

ナイジェリアにおけるオンコセルカ症の疫学（英文）・・Bertram

E．B．Nwoke，塩飽邦憲，高橋弘

191−201

ブラジル，レシフェ市内小学生の好酸球増多症，高IgE血症の病因（英文）
一浜田

篤郎，渡辺

直煕，ノ』・林

仁，奥沢

英一，野崎

智義，

Ivete Barbosa，建野正毅，小林昭夫

203−208

Lo∫sh耀n毎ρσn佛卿廊promastigote型原虫から精製した皮内反応用抗原の効果判
定とその特性（英文）………一………・……古谷正人，野中 薫雄，E．A．L．Gomez，橋口 義久

209−217

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日本熱帯医学会雑誌

発行所 日本熱帯医学会

第19巻

編集者 板

第2号

平成3年6月15日
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Jpn. J. Trop Med Hyg Vol 19 No 2 1991, pp. 175‑190

175

Review

THE FUNDAMENTAL NATURE OF CHAGAS' DISEASE=
A VIEW PROVIDED BY IMMUNE RESPONSESAND IDIOTYPESDANIEL G. COLLEY AND MICHAEL J. HOWARD
Received November I 1990/Accepted December 2 1990

Abstract: Chagas' disease is caused by the protozoan Tmpanosoma cruzi which
infects 10‑15 million people in endemic areas throughout Latin America and is naturallytransmitted by insect vectors of the family Reduviidae. Infection can also occur bycongenital passage, oral ingestion, Iaboratory accident, and in organ transplants and bloodtransfusiohs. There are 3 Iife‑cycle forms of T. cruzi. Epimastigotes multiply in the
midgut of the insect vector, differentiate in the hind‑gut into infectious trypomastigotes,
and are excreted with the feces or urine after a blood meal. They enter the body by

mucous membranes or abraded skin, enter mammalian host cells, escape thephagolysosomal vacuole, and differentiate into amastigotes. Intracellular amastigotes
multiply in host cells, redifferentiate to trypomastigotes, and are released upon cell
rupture.
Acute Chagas' disease can be asymptomatic, or a mild to severe illness. Morbidity canbe localized and/or systemic and is usually accompanied by general immunosuppression
with blood and tissue parasitemia. It can be fatal. Usually, symptoms and parasitemia
decrease within months, followed by a life‑long, chronic infection with little morbidity andfew apparent parasites. Serologically‑positive, chronic asymptomatic patients are termed
indeterminate (D . Morbidity develops in 20‑30% of chronic patients after 10‑30 years andoften involves myocardiopathy (Cardiac disease; O , ranging from minor electrocardiogra‑
phic changes to sudden death by heart failure. Severe "digestive" megasyndromes can also
develop. Chagasic myocardial inflammatory infiltrates are associated with cardiac muscle
and neuron destruction, followed by progressive fibfotic replacement. Asymptomatic I‑
patients who die of unrelated causes have identical (but less intense) Iesions.

Responses against T. cruzi antigens, their immunoregulation, and responses against
related idiotypes ･(Ids) correlate with the presence of the different clinical forms of theinfection. Although there are numerous findings of autoimmune lymphocyte and antibody
reactivities in chagasic patients, a causal relationship is always difficult to prove.

Chagasic patients' peripheral blood mononuclear cells (PBMO respond to T. cruzi
epimastigQ]teiantigens ( PD with̲Irarying vig:or*̲̲̲̲Almost all low ̲responders are C‑
patients, and their respohs6s are usually augmented by removal of adherent macrophage
suppressor cells or the addition of indomethacin. Chronic I‑patients are medium or highresponders, and exhibit little adherent cell‑mediated immunoregulation. Westem blotting

Veterans Affairs Medical Center, and: the Departments of Microbiology and Immunology, andMedicine, Vanderbilt University School of Medicine, Nashville, Tennessee, U.S.A.

176

studies indicate patients' Ab and cell‑mediated responses to EPI show differences between

C‑ and I‑patients. PBMC from I‑patients responded more often to high molecular weightcomponents (lO0‑150 kD), while both I‑ and C‑patients responded well to moieties
between 28‑32 and 48‑57 kD. All chagasic patients' sera had Abs and PBMC responses toT. cruzi GP57/51 antigen. "Chronic Chagas' patients have peripheral blood anti‑Id T cells that respond to anti‑
EPI Abs purified from patients' sera. Some patients' PBMC anti‑Id responses to anti‑EPI

lds from C‑patients (Id‑O are inhibited by chloroquine (Group 1), but some are notinhibited by chloroquine, anti‑HLA Class 11 antigens, or sodium azide (Group 2). Most
patients in Group I are asymptomatic, but all Group 2 patients have severe disease. Direct(non‑processed; non‑MHC‑presented) stimulation of anti‑Id T cells from C‑patients by

lds expressed on anti‑EPI Abs from C‑patients could be immunopathogenic. Anti‑Id
specific rabbit sera detect Id differences in the anti‑EPI Abs from pooled or individual C

vs. I‑cases. Competitive ELISA assays and Westem blot analyses of Abs show that Ids on
I‑patients' anti‑EPI Abs are primary structure expressions, while Ids on C‑patients' anti‑

EPI Abs are defined by intact Ab molecules.
The chronic, endemic nature of Chagas' disease allows maternal/neonatal Id inter‑actions that might influence later immune response and immunoregulatory abilities ofchildren born of infected mothers. Such interactions occur because cord blood mono‑
nuclear cells from chagasic mothers' children respond to Ids on anti‑EPI Abs. A hypothe‑
sis based on Id‑induced pathology and immunoregulation will be described that could
account for certain aspects of the immunology and pathology of Chagas' disease.

History and prevalence
Chagas' disease (South American Trypanosomiasis) is an endemic zoonosis produced by
Tmpanosoma cruzi which is found presently only in the Americas. The disease is named forthe 29 year old Brazilian physician, Carlos Chagas, who in 1909 identified and described theparasite in the hind‑gut of insects from the family Reduviidae. Dr. Chagas later observed its

pathogenicity in mammals and located and described many of the domestic (dogs, cats, goats,etc.) and wild (armadillo, opossum, etc.) reservoirs. He went on to demonstrate this infection

in humans and described its acute and chronic stages. Recent epidemiologic studies in SouthAmerica suggest there may be 10 to 15 million people serologically positive for T. cruzi(Brener, 1982; Chagas, 1988) . Reports on the incidence of infection crudely estimate it to beover 800,000 new cases each year with an annual mortality of around 60,000 (Dias, 1987) .
Like many parasitic infections, Chagas' disease is most common in, but not confined to,people occupying the lower socioeconomic classes. Contact with the vector and transmission

have been correlated most strongly to poor housing construction and inadequate vector
control (Marsden, 1984) . These factors play a major role in transmission of Chagas' disease

as the vector and trypanosome are much more widely distributed than is human infection(Grogl et al., 1984; Beard et al., 1988; Yaeger, 1988).

Transmission in endemic areas is largely vector‑related and thus limited to theAmericas, but recent reports have shown that congenital passage (Azogue et al., 1985),
laboratory accidents (Brener, 1987), organ transplants and blood transfusions (Kirchhoff,1989) play a considerable role in transmission in endemic countries and may become a serious
problem for non‑endemic countries as well. Two recent reports of transfusion related T.

cruzi infection in North American hospitals have stressed the danger which T. cruzi

177

contaminated blood could pose for non‑endemic countries (Grant ,et al.. 1989; Nickerson etal., 1989). These cases involved two patients undergoing chemotheraceutic treatments forleukemias. Each of these patients had received platelets from Latin American immigrants.
These blood products were screened for conventional infectious contaminants and deemed fit
for use, but there are no protocols in the United States of America or Canada which consider

T. cruzi contamination. Screening of donors within endemic areas of Latin America is
becoming more common and there are highly sensitive and specific serological assays
available (complement fixation, indirect immunofluorescence and ELISA) which should help
decrease the risk of transfusion or transplant related infection. Presently, Brazil reports 10,

OOO to 20,000 transfusion related T. cruzi infections a year (Brener and Camargo, 1982) , and
5‑51% of the blood units recently tested in Bolivia were serologically positive (Carrasco etal., 1990). With the recent increase in immigration of people of Japanese heritage from
Brazil to Japan, transfusion related Chagas' disease may become something that the Japanese
medical community will need to consider in the future.

Biology of Trypanosoma cruziTr)panosoma cruzi has a complex life cycle involving both vertebrate and invertebrate
hosts (Pereira, 1990) . Under natural conditions, the trypanosome is transmitted to its
definitive mammalian host via the feces or urine of an infected reduviid bug of the genera
Triatominae. Rhodnius, or Panstrongylus (Ghauri, 1973) . T.hose strictly hematophagous
reduviids which are the most efficient vectors of human infections defecate shortly after
taking a blood meal. The feces and urine contain the infectious trypomastigote stage whichdevelops from the non‑infectious, asexually dividing epimastigote form found in the hind‑gutof the vector. Trypomastigotes cannot penetrate intact epidermis but enter through breaks
in the skin or through intact mucous membranes. Therefore, infection is not due to a direct
inoculation by the insect vector, but rather indirectly through the victim wiping and scratch‑

ing at the bite and subsequently contaminating the wound, their eyes or mouth. Evidence ofprimary infection is sometimes seen at the site of entry as localized inflammatory reaction
resulting in an indurated nodule at the bite (chagoma) or by unilateral periorbital swellingand conjunctivitis (Romana's sign) .
The non‑dividing blood stage trypomastigote form has the well described ability to avoid
complement lysis (Joiner et al., 1988; Rimoldi et al.. 1988) and phagocyte killing (Osuna etal., 1986; Reed, 1988; Reed et al.. 1989) and is capable of receptor mediated entry of a wide
variety of host cell types (Velge et al., 1988; Davis and Kuhn, 1990) . Once inside the cell thetrypomastigote produces a recently described perforin‑like molecule, analogous to C9 of thecomplement cascade, and passes from the parasitoferous vacuole into the cytoplasm of thecell (Andrews et al., 1990) . The trypomastigote differentiates into the amastigote form in thecytoplasm. Amastigotes multiply asexually and develop into trypomastigotes whileliberating antigens into the host cell cytoplasm (Andrews et al.. 1988). These antigens
theoretically would be expressed in a Class I restricted manner on the surface of infected
cells. Whether acute and chronic lesions are related to host cell lysis by either immune
cytotoxicity or passive rupture due to parasite load is not clear. The release of parasites
results in trypomastigotes and amastigotes in the circulation. These organisms are capableof disseminating and parasitizing diverse tissues throughout the host or can be taken up bya feeding reduviid to complete the cycle (Andrews et al., 1987) .

178

The intracellular, dividing amastigote stage is readily identified in histologic sections ofcardiac, intestinal and cerebral tissues from infected individuals early in the course of
infection and is localized predominantly to neuronal Or conductive fibers within the paren‑chyma of these organs (Andrade et al.. 1978, 1984; Said et al.. 1985). Sequestration of theparasite in these intracellular sites confers a significant survival advantage to T. cruzi. The

intracytoplasmic residence of these organisms means that effective sterilizing chemotherapy
regimens would need to achieve trypanocidal concentrations of drugs inside host cells. This
is not thought to occur with the current drugs of choice (nifurtimox and benzinidazole) andposes a major challenge in the area of drug development and chemotherapy (Filardi andBrener, 1987; McCabe, 1988) . The intracellular location of the parasite also allows it tolargely avoid the immune response. The host's early immune responses induce a resolutionof blood parasitemia but they are unable to clear the persistent, intracellular infection. It is

commonly assumed that intact host cells with resident intracellular amastigotes do not elicit

inflammatory responses early in infection. The intense focal inflammation typical of acuteinfection is believed to occur only after the rupture of parasitized cells which exposes the hostto concentrations of parasite antigen. However, during chronic infection parasites are scarcein blood and are usually not found within the observed diffuse infiammatory responses in hosttissue. This has lead to the widely proposed, but still controversial, theory of an autoimmune

basis for Chagas' disease (Takle and Hudson, 1989; Hudson and Hindmarsh, 1985) .
Clinical aspects of infection
Chagas' disease, Iike many chronic endemic parasitic infections, demonstrates a spec‑
trum of clinical presentations. The initial infection can go completely unnoticed or result in

lethal cardiac infarctions. Chronic patients range in presentation from asymptomatic but
serologically‑positive to severe debilitation and death. This heterogeneity is no doubt due toa multitude of parasite and host factors but recent reports have begun to link specific hostimmune responses during infection to the various clinical stages (Gazzinelli et al.. 1988a, b;
Reis et al.. 1989; Gazzinelli et al., 1990; Colley et al., 1990) . Interestingly, although the acutephase of'primary infection does occur in endemic human populations it is rarely seen byhealth care workers. Oftentimes, acute cases of parasitic diseases in non‑endemic people
present as severe infections in contrast to those of endemic peoples (von Lichtenberg, 1987;
Nash et al., 1982; Ottesen, 1984). These findings suggest that there are many individuals
within endemic populations who are somehow able to better modulate the severity of diseasein the acute, and possibly chronic, phases of infection with parasites.

When acute chagasic infection is clinically apparent it is usually defined by a marked
parasitemia with symptoms ranging from mild fatigue and fever to severe myocarditis anddeath (5‑lO% of acute patients) (Nogueira, 1986) . Diagnosis is made by demonstrating
parasites in stained blood smears or live parasite isolation (xenodiagnosis or hemoculture)and is occasionally aided by description of a chagoma or Romana's sign. Diagnosis of acute
Chagas' disease by serological testing is generally of little use possibly due to the rapid andsevere immunosuppression seen in these patients (Brener, 1980; Kuhn, 1981; Beltz et al.,1989) .

Because the acute phase is not often recognized, data on cellular and humoral events
during acute Chagas' disease are generally not available. A few reports discuss the humoral
response progression during .acute accidental infection of laboratory workers from sero‑

179

negativity through to serocdnversion and frank parasitemia (Brener, 1984, 1987; Hofflin et al.,1987). But little is known about cellular reactivities during this phase of human infection.
The acute phase of experimental' Chagas disease has been widely studied (Teixeira et al..

1975; Tarleton and Scott, 1987). Acute murine infection with T. cruzi demonstrates atremendous polyclonal stimulation involving both B and T cells. Nearly half of all spleencells appear to be blast cells undergoing mitosis. This response is truly polyclonal: all Vh‑gene families studied (covering more than 95% of the repertoire of the entire locus) are
stimulated (Minoprio et al.. 1989). The majority of these activated splenic and lymph nodeplaque forming cells are not specific for the parasite (Minoprio et al.. 1988). Ther6 are noreports in the literature concerning the existence or the extent of this polyclonal activationin humans but its presence has been proposed to play a role in the marked immunosuppression
and possible autoimmunity seen in human trypanosomiasis (Petry and Eisen, 1989). Similar
panlymphocytic, polyclonal proliferation has been reported in viral infections, bacterial(leprosy) infections, and Leishmania infections (Petry and Eisen, 1989). All of these organ‑
isms develop in macrophages. During the acute phase of infection T. cruzi also largely infects

macrophages (McCabe et al., 1984; Villalta and Kierszenbaum, 1984). Because of thisunifying observation, several researchers have proposed roles for cytokines produced by theinfected macrophages as being responsible for the immunological disturbances seen in T.
cruzi infections (Kierszenbaum and Wirth, 1987; Reed, 1988; Wirth and Kierszenbaum, 1988;
Tarleton 1988; Reed et al., 1989).
The chronic phase of the disease usually. presents 20‑30 years after initial infection andoften without history of acute disease (Nogtieira, 1986) . Intracellular parasites are believed

to maintain a lifelong tissue infection but trypomastigotes are typically scarce or absent in

the blood as parasitemia is tightly controlled by the host's immune response. This becomesvery apparent in those chronic patients who, for reasons unrelated to their Chagas' disease,receive immunosuppressive therapy and subsequently develop a new "acute" infection com‑plete with patent parasitemia (Brener, 1980; Kierszenbaum et al.. 1983; Hudson and Britten,1985) . In the chronic phase, demonstration of 1)arasitemia is generally very difiicult becauseof the low numbers seen in the peripheral circulation. Simple blood smears are completely
useless for diagnostic purposes. This requires very sensitive techniques which rely on a longmultiplicative phase either in uninfected reduviid bugs allowed to feed on patients' blood(xenodiagnosis) or in vitro culture (hemoculture) of parasites from patient blood directly.
For this reason serology is usually the method of choice for positive diagnosis of chronic
Chagas' disease (Brener, 1982) . New approaches using molecular biological techniques suchas the polymerase chain reaction (PCR) for highly sensitive and specific detection of organ‑
isms are now being explored (Moser et al.. 1989; Sturm et al.. 1989) .
Most chronic patients are asymptomatic and typically present with positive serology andsub‑patent parasitemia. Between 60% and 80% of people infected with T. cruzi fall into thiscategory and are termed "indeterminate" (Brener and Camargo, 1982) . By definition, thesepeople do not suffer from disease related morbidity and mortality, but this does not mean thatthey are free from pathology caused by their infections. A series of autopsies performed on"sudden death" cases in an endemic area indicate varying degrees of sub‑patent chagasic
cardiac pathology are present in indeterminate individuals (Brener and Camargo, 1982;
Pereira‑Barretto et al., 1986). This has lead to the hypothesis that the clinical forms of
Chagas' disease represent a progression rather than distinct and unrelated disease states.

180

Why certain infected individuals progress more rapidly to morbidity while others regulate theinfection their entire lives without clinically apparent disease is not known.

The minority of chronic patients (20‑30%) demonstrate a more severe chronic course(Brener and Camargo, 1982) . Patients with the "cardiac" clinical form demonstrate severe
lesions to the conducting and muscular structures of the heart which commonly lead to adilative cardiomyopathy and congestive heart failure. Lesions of the conducting system of
the heart may lead to right bundle branch block and life threatening arrhythmias. This isevident in electrocardiographic changes, and rich inflammatory infiltrates can be demon‑
strated in post‑mortem sections of the heart‑ often in the absence of demonstrable parasites
or parasite antigens (Andrade et al., 1978). The clinical finding of chagasic cardiopathy
include recurrent palpitations and ECG changes combined with cardiac insufficiency andheart enlargement. The electrocardiographic changes commonly involve right bundle branch
block with occasional complete atrio‑ventricular block. Fibrosis and chronic inflammation
are prominent features of the histology of chronic chagasic cardiac pathology (Ribeiro dos
Santos and Rossi, 1985; Carrasco‑Guerra et al.. 1987; Brener and Krettli, 1990). It is thiscardiopathology which contributes to Chagas' disease being among the leading cause of deathin adults in some endemic areas.
A further subgroup of chronic patients with severe Chagas' disease present with aperi‑
stalsis and dilation of the esophagus, Iarge bowel or both. These patients have "megadisease"or the "digestive" clinical form. Histology of lesions typically shows mononuclear infiltrates

in the myenteric plexus of the gut wall with demyelination and sclerosis of surrounding
conductive fibers. The loss of autonomic control and subsequent dilatation are believed dueto the inflammatory destruction of the ganglionic plexus and nerve fibers. However, direct
destruction by intracellular parasitism no longer observed at the site cannot be ruled out(Hudson and Hindmarsh, 1985; Hudson and Britten, 1985). A small minority of patientsdemonstrate both cardiac and digestive involvement.

Lesions and their possible immune etiology
The etiology of chronic chagasic pathology has been examined in a number of experi‑mental animal models and in human infection. In 1974, Cossio and co‑workers (Cossio et al..
1974) reported a series of experiments concerning antibodies reacting with endocardium,
vascular structures, and interstitium of striated muscle (EVI antibodies) present in the seraof patients with chronic chagasic cardiopathy. This was one of the first findings whichsuggested a mechanism for the l sions to heart and digestive tract tissues and implicated an

autoimmune etiology. These EVI antibodies reacted with human, mouse, bovine and guinea
pig heart tissue from non‑infected subjects. In these studies, similar cross‑reactive antibodies

were found in over 90% of patients with chronic chagasic cardiopathology. Later findingsthat these antibodies could be absorbed by epimastigotes and by laminin lent further support
to this theory (Szarfman et al.. 1982; Brener et al., 1983; Gazzinelli et al.. 1988d) . However,these results have been difiicult to reproduce, and it is now known that EVI‑autoantibodies
are also found in the sera of over 40% of indeterminate patients and in patients with otherparasitic infections (malaria, Ieishmaniasis, African trypanosomiasis and Tmpanosoma
rangeli) (Khoury et al., 1983; Avila et al., 1984; Kierszenbaum, 1986; Avila et al., 1987;
Towbin et al., 1987). These findings have fueled debate over the significance of these
antibodies in the pathogenesis of T. cruzi (Kierszenbaum and Hudson, 1985).

181

Many mouse and rat monoclonal antibodies exist which are reported to cross‑react with
T. cruzi and host tissue. Some of these antibodies and the parasite and host epitopes
recognized have been defined in detail. For example, Eisen and colleagues have described twoanti‑T. cruzi monoclonals, VESP 6.2 and VESP 8.2, which were shown by indirect
immunofiuorescence to recoguize different glycolipids on the surface of fixed, CL‑strain
trypomastigotes and also epitopes on cerebellar neurons in culture and frozen sections ofheart, digestive tract and peripheral nervous tissues (Petry and Eisen, 1989) . Another mousemonoclonal antibody (CE5) raised by Wood and colleagues against membranes of rat dorsal
root ganglia also recognizes epitopes on the surface of fixed Y‑strain epimastigotes (Woodet al., 1982). Western blots using CE5 demonstrate a complex banding pattern of proteins(50‑100 kD) in both epimastigote and amastigote antigen preparations which may reflect a
common amino acid sequence in the distinct proteins of these two life cycle stages. CE5 also
recognizes several bands on Western blots of preparations from rat heart, intestine and brain
but not from tissues known not to be affected in Chagas' disease. Snary and his co‑workers
have used monoclonal antibodies derived from immunized and infected mice to demonstratecross‑reactivity between epitopes on the parasite surface and those found on mouse central
and peripheral neurons and glia (Snary et al., 1983) . A11 of the monoclonal antibodies
discussed were shown to have no activity against the culture medium or fetal bovine serum
used to raise the parasites, making it unlikely that these cross‑reactivities are due to antigen

scavenging by T. cruzi.
The existence of cross‑reactive clones such as those described above is not unexpectedin situations of intense lymphocyte polyclonal activation. This has been shown in, and iscorrelated with, pathogenesis in a number of autoimmune diseases (Klinman and Steinberg,
1987) . If Chagas' disease does have an autoimmune component, its etiology may be related

to polyclonal stimulation of lymphocytes such as that obserVed in experimental T. cruzi
infection of mice. It is interesting to note that some systemic autoimmune disease in bothhumans and mice are characterized by hypergammaglobulinemia and abnormally high levelsof serum autoantibodies, two things known to occur in acute human and experimental
Chagas' disease (Minoprio et al.. 1989). How these observations correlate with pathology
and what is responsible for the breakdown of normal control of humoral autoreactivity inthese diseases are not clear at present.

Cell transfer studies by Laguens and colleagues gave the first evidence suggesting a T
cell‑mediated autoimmune etiology in experimental Chagas' disease (Laguens et al.. 1981) .
These studies used "parasite‑free" non‑adherent spleen cells isolated from infected mice tocreate lesions in non‑infected syngeneic recipient mice only 4 days after cell transfer. In
similar studies, some recipient animals later died of T. cruzi infections so that parasite

generated pathology could not be ruled out. Recently, Hontebeyrie‑Joskowicz and co‑workers have used CD4 + T cell clones to create truly parasite free pathology in an MHC‑
restricted manner in healthy mice. These CD4 + clones were shown to have specificity forboth epimastigote antigen and syngeneic murine sciatic nerve antigen giving strong support

for a cell‑mediated autoimmune mechanism in chagasic cardiopathy (Hontebeyrie‑
Joskowicz et al., 1987; Ben Younes‑Chennoufi et al.. 1988) .

The above observations have lead to several theories on the development of thepathogenesis associated with T. cruzi infection. The most widely tested hypothesis involves

generation of autoreactivity due to infection. The mechanisms involved in the development

182

of this auto‑reactivity are unknown but several models are commonly addressed in theliterature. A few of these include: 1. Antigen mimicry; 2. Polyclonal blastogenesis of both B

and T cells, stimulating a B cell repertoire already naturally expressing a large number ofanti‑self reactivities; 3. Development of autologous responses to the idiotypes or the isotypesof the original antigen specific clones. How these possibilities might occur and how theywould relate to specific lesion development in chronic infection with T. cruzi is only beginningto be examined.

The spectrum of immunologic responses
The clinical spectrum observed in patients infected with T. cruzi allows speculation
regarding differential host immunoregulatory mechanisms that may contribute to thesedifferences in morbidity. The remainder of this article will summarize findings concerning

responses against T. cruzi antigens, and their immunoregulation, and responses against
idiotypes (Ids) associated with these anti‑ T. cruzi responses. It will present, analyze andhypothesize about immunologic profiles which correlate with the pr sentation of different
clinical forms of the infection.

In Chagas' disease, peripheral blood mononuclear cellS (PBMO proliferate to soluble(Morato et al., 1986) or nitrocellulose‑fixed (Gazzinelli et al.. 1990

parasite antigen. If thein vitro responses to soluble epimastigote antigens ( .FD are subdivided into levels of
responsiveness and analyzed, almost all of the low responders are .clinically classifie as"cardiac" (O patients (Gazzinelli et al., 1988a; Colley et al., 1990; Morato, M.J.F. et al..

submitted for publication) . The responses of these patients are also augmented more,
relative to those of "indeterminate" (D patients, by the removal of adherent cells (Morato,
M.J.F. et al., submitted for publication) . PBMC from chronic I patiepts usually respond wellto EPI and these responses and are not apparently regulated by adherent cells. These data,
coupled with the observations that C patient PBMC responses are often partly augmented by
the addition of indomethacin (Morato, M.J.F. et al., submitted for publication) , suggests arole for prostaglandin‑mediated adherent macrophage suppression in the development or
expression of clinical disease. It is possible that strong cellular responses against some of theparasite components in EPI are essential to hold the parasite in check, thus decreasing thechance of parasite‑induced related morbidity.
There have been a number of studies examining humoral responses of patients infectedwith T. cruzi (Kuhn, 1981; Snary, 1985; Nogueira, 1986) . Recent research has comparedpatients' antibody responses by Western immunoblotting against separated EPI in parallelwith their cell‑mediated responses by T cell‑Western blotting (Gazzinelli et al.. 1988a, 1990) .

These experiments have shown some differences between C and I patients. PBMC from I
patients responded more often to high molecular weight EPI components (lO0‑150 kD) whileboth C and I patients cells responded to molecules in the 28‑32 kD and 48‑57 kD ranges. But
while there was an apparent difference in the recognition by T cells of separated antigens,none of the specific antigen bands were recognized preferentially by sera of patients with one

clinical form vs. another. These studies further describe a glycoprotein component of EPI
which migrates diffusely between 57 and 51 kD (GP57/51) (Scharfstein et al., 1986; Murta etal., 1988) . GP57/51 antigen is recognized by all patients' sera and cells, and a given patient's

PBMC responses to this purified antigen correlate with their responsiveness to crude EPI. It
appears that a major portion of the cellular reactivity against crude EPI is due to responses

183

specific for GP57/51. The biological sign,ificance of GP57/51 and its potential use as adiagnostic antigen are the focus of ongoing studies.
The role of Id/anti‑Id interactions in the regulation and maintenance of the immune
response was first proposed by Jerne in 1974 (Jerne, 1974). It has been demonstrated sincethat time that repeated, Iong‑term exposure to antigens can lead to the development of
dominant cross‑reactive Ids (CRD , and that these CRI and anti‑Ids against them may play
multiple roles in the responses to immunizations or during infections (Bona, 1987; Kearney,
1989; Cerny and Hiernaux, 1990). Id/anti‑Id interactions have been demonstrated to be
integral to the ability of the host to regulate some responses to immunologic challenges andhave also been implicated in the pathogenesis of certain autoimmune diseases. We havebegun to explore the possibility that Id/anti‑Id systems, which occur in Chagas' disease, arerelated to the clinical differences that occur in this infection.

In studies which parallel findings in human schistosomiasis mansoni (Colley et al.. 1989) ,chagasic patients' PBMCS proliferate upon exposure to anti‑EPI immunoafflnity‑purified
antibodies (anti‑EPD from patients' serum (Gazzinelli et al.. 1988b, c; Colley et al., 1990) . Asin schistosomiasis, PBMC from patients who have never been exposed to T. cruzi are notstimulated by these antigen specific antibodies. Controls for these experiments demonstratethat neither normal lg or immunoafflnity‑purified antibodies to antigens of other non‑relatedparasites stimulate PBMC of chagasic patients to proliferate.
Gazzinelli and co‑workers (Gazzinelli et al.. 1988b) observed that anti‑EPI purified from

sera of chronic chagasic patients with cardiac disease (Id‑O were generally more stimula‑
tory for PBMC (or T Iymphocytes) than anti‑EPI Id isolated from sera of indeterminate
patients (Id‑D . This was seen regardless of the clinical form of the donor of the responding
cells. This was the first dem,onstration of anti‑Id T Iymphocytes in Chagas' disease, and thecorrelation with clinical disease suggested a possible role for Id/anti‑Id responses in thegeneration of chronic chagasic cardiopathy.
This Id stimulation of auto‑anti‑Id T cells from Chagas' patients was observed to require
the presence of an adherent cell population. Recently, a difference in the requirements forchronic chagasic T cell‑recognition and response to Id‑C and Id‑1 became evident while
examining this role of adherent cells in antigen processing (Colley et al.. 1990; Gazzinelli, R.

T. et al., submitted for publication) . As expected, antigen (EPD stimulated responses of all

patients are completely ablated in the presence of 25 pM chloroquine. This level of chloro‑

quine is known to block lysosomal enzymatic degradation by raising the pH of lysosomes andthus blocking processing and presentation of antigen to T cells (Unanue, 1984) . Chloroquinealso inhibited Id‑1 stimulation of PBMC from either C or I patients, but strikingly, when Id‑
C preparations were used as the stimulus, two different response patterns were observed in
the presence of chloroquine (Colley et al., 1990; Gazzinelli, R.T. et al., ubmitted for publica‑tion) . The PBMC responses of one group of patients (Group 1) resembled the responses toeither Id‑1 or EPI. Their anti‑Id‑C responses were markedly inhibited by chloroquine(responses were decreased by greater than 40% ) . In contrast, a second group of patients(Group 2) had anti‑Id‑C responses which were not significantly inhibited or were augmented
in the presence of chloroquine. Most Group I patients were I, and surprisingly, all 14 Group
2 patients had severe Chagas' disease‑related morbidity. This is a striking clinical correla‑tion. It is possible that the direct (non‑processed; non‑MHC‑presented) stimulation ofanti‑Id T cells seen with cells and Ids from C‑patients could be related to the immunopatho‑

184

genesis of the'disease.
Recent experiments in progress (Reis et al.. 1989) have shown that anti‑Id specific rabbit

sera can detect Id differences in the anti‑EPI Abs from pooled or individual sera from C vs.
I patients (i.e. Id‑C vs. Id‑D. Preliminary competitive ELISA assays and Western immuno‑
blot analy es of these Ids confirm that the Ids on Id‑1 are associated with the primarystructure of lg heavy and light chains, while the Ids on Id‑C are defined by conformation,requiring the presence of intact, non‑denatured immunoglobulin molecules. These data arecompletely compatible with those results from the in vitro anti‑Id T cell proliferation studies

where processing and presentation was required for Id‑1 stimulation, but not Id‑C stimula‑tioh, and delineated the different clinical forms.

Presently, it is conjectural how differential processing requirements for anti‑Id T cell

responses among patients with different clinical forms of Chagas' disease is related topathogenesis.̲ It is at least possible that the lesions could result from repeated, unregulated

direct stimulation of anti‑Id T cells, and thus be a unique form of autoimmunity based on an
ld etiology. Further dissection of the response at both the cellular and Id levels may clarify

why T. cruzi infection in one patient stabilizes and remains asymptomatic, while in anotherit leads to severe morbidity.

Maternal fetal effects of idiotypes in Chagas' disease

In areas endemic fof Chagas' disease individuals become infected early in life andmaintain life‑10ng, chronic infections. In areas of high prevalence, this pattern predicts thatmany women of child bearing age are actively infected at the time of their pregnancy. Thissituation could create an opportunity fo'r the mothers' infection, or their immunologicalresponses to that infection, to greatly influence the developing repertoire and future reactivityof her child. An active infection at the time of pregnancy would lead to placental or milktransfer of the mothers' high titer lgG antibodies (bearing their Ids) and circulating T. cruzi

antigens to the developing child. The impact of these Ids or antigens on the future develop‑

ment of pathology when that child becomes infected with T. cruzi is currently only specula‑tive. This may, however, explain the observation in many parasitic infections that people
from non‑endemic areas (i.e., who would be unlikely to be born of infected mothers) areinuch more likely to suffer severe disease than are residents of the endemic area. People bornof infected mothers may be primed in utero to elicit regulatory or non‑pathogenic Id/anti‑Id

responses by specific maternal Ids or anti‑Ids on antibodies developed during their chronic
infections (Bona, 1987; Kearney, 1989; Cerny and Hiernaux, 1990).
Specific anti‑Id priming in utero (possibly caused by antigen, Id or anti‑Id) has been

demonstrated in humans by Eloi‑Santos and her colleagues in studies of the responsivenessof cord blood mononuclear cells (CBMO from neonates born of mothers with Chagas'
disease (Eloi‑Santos et al.. 1989). CBMC from neonates born of mothers infected with T.
cruzi responded to soluble antigen from epimastigotes but not from unrelated antigens (Eloi‑

Santos et al.. 1988) . Their studies also showed that anti‑EPI antibodies, but not anti‑
schistosome egg antigen antibodies (anti‑SEA) or normal human lg, stimulated these CBMC.
Furthermore, anti‑EPI Ids did not stimulate CBMC from neonates of uninfected or Schis‑tosoma mansoni‑infected mothers. These data strongly support the hypothesis that in utero
sensitization of anti‑Id T‑cells occurs. We further hypothesize that such powerful influenceson the^ developing repertoire of the neonate play an important role in determining the185

responses and regulatory mechanisms expressed by most children in endemic areas when they
are subsequently exposed to the same chronic infection.

This work was supported by grants from the Financidadora de Estudos e Projetos(FlNEP) , the Conselho Nacional de Pesquisas (CNPq) , the Department of Veteran Affairs
and the NlH (AI 26505 and T32‑GM‑07347: Medical Scientist Training Program Grant) .

REFERENCES
1 ) Andrade, Z.A., Andrade, S.G., Oliveira, G.B. and Alonso, D.R. (1978): Histopathology of the
conducting tissue of the heart in Chagas' myocarditis, Am. Heart J., g5, 316‑3242 ) Andrade, Z.A., Andrade, S.G. and Sadigursky, M. (1984): Damage and healing in the conduct‑
ing tissue of the heart (an experimental study in dogs infected with Tmpanosoma cruzi), J.
Pathol., 143, 93‑lO13 ) Andrews, N.W., Hong, K.S., Robbins, E.S. and Nussenzweig, V. (1987) : Stage‑specific surface

antigens expressed during the morphogenesis of vertebrate forms of Tmpanosoma cruzi, Exp.
Parasitol., 64, 474‑484

4 ) Andrews, N.W., Robbins, E.S., Ley, V., Ho,ng, K.S. and Nussenzweig, V. (1988): Develop‑
mentally regulated, phospholipase C‑mediated release of the major surface glycoprotein of
amastigotes of Tmpanosoma cruzi. J. Exp. Med., 167, 300‑3145 ) Andrews, N.W., Abrams, C.K., Slatin, S.L. and Grifflths, G. (1990) : A T. cruzi‑secreted protein

immunologically related to the complement component C9: evidence for membrane pore‑
forming activity at low pH, Cell, 61, 1277‑1287
6 ) Avila, J.L., Rojas, M. and Rieber, M. (1984.) : Antibodies to laminin in American cutaneousleishmaniasis, Infect. Immun., 43, 402‑406
7 ) Avila, J.L., R,ojas, M., Velazquez Avila, G. and Rieber, M. (1987): Antibodies to laminin in

Tmpanosoma rangeli‑infected subjects, Parasitol. Res., 73, 178‑179

8 ) Azogue, E., La Fuente, C. and Darras, C. (1985): Congenital Chagas' disease in Bolivia:
epidemiological aspects and pathological findings, Trans. R. Soc. Trop. Med. Hyg., 79, 176‑180
9 ) Beard, C.B., Young, D.G., Butler, J.F. and Evans, D.A. (1988): First isolation of Tmpanosoma

cruzi from a wild‑caught Triatoma sanguisuga (LeConte) (Hemiptera: Triatominae) in
Florida, U.S.A., J. Parasitol., 74, 343‑344

10) Beltz, L.A., Kierszenbaum, F. and Sztein, M.B. (1989): Selective suppressive effects ofTmpanosoma cruzi on activated human lymphocytes. Infect. Immun., 57, 2301‑2305
11) Ben Younes Chennoufi, A., Said, G., Eisen, H., Durand, A. and Hontebeyrie Joskowicz, M.
(1988) : Cellular immunity to Tmpanosoma cruzi is mediated by helper T cells (CD4 + ) , Trans.

R. Soc. Trop. Med. Hyg., 82, 84‑89

12) Bona, C.A. (1987): Idiotypes and Lmphocytes (New York: Academic Press, Inc.)
13) Brener, Z. (1980): Immunity to Tmpanosoma cruzi. Adv. Parasitol., 18, 247‑292
14) Brener, Z. and Camargo, E.P. (1982): Perspectives of vaccination in Chagas' disease. hePontificiae Academiae Scientiarum Scripta Varia. C. Chagas, ed. (Rome: Pontificia Academia
Scientiarum), pp. 145‑168
15) Brener, Z. (1982): Recent developments in the field of Chagas' disease, Bull. W.H.Q., 60,
463‑473
16) Brener, Z., Ramirez, L.E., Krettli, A.U. and Cancado, J.R. (1983): EVI antibodies in patientswith Chagas' disease: relationship with anti‑ Tr)panosoma cruzi immunoglobulins and effects ofspecific treatment, Mem. Inst. Oswaldo. Cruz., 78, 437‑442

17) Brener, Z. (1984): Laboratory aquired Chagas' disease: an endemic disease among par‑

186

asitologists? in Genes and Antigens; Alaboratory Manual (Rio de Janeiro, Brazil: FIOCRUZ)18) Brener. Z. (1987): Laboratory‑acquired,Chagas' disease: comment [letter] , Trans. R. Soc.
Trop. Med. Hyg., 81, 527
19) Brener, Z. nd Krettli. A.U., (1990): Immunology of Chagas' disease. In Modern Parasite
Biology: Cellular, Immunological and Mo,lecular Aspects. D.J. Wyler, ed. (New York: W.H.
Fireeman and Company) , p.p,. '247‑261 '20) darrasco Guerra: H.A., Palacios Pru. E., Dagert de Scorza, C.. Molina, C., Inglessis. G. andMendoza, R.V. (1987): Clinical, histochemical, and ultrastructural correlation in septal
endomyocardial biopsies from chronic chagasic patients: 'detection of early myocardial dam‑age. Am. Heart J., 113, 716‑724
21) Catrasco, R., 'Migtiez, H., Camacho. C., Echalar, L., Re 0110. S., Ampuero, T. and Dedet, J.P.

(1990): PreValence of Tmpanosoma cruzi infection in blood banks of seven departments ofBolivia, M m. Inst. Oswaldo. Cruz., 85, 69‑73
22) Cerhy, J, and Hiernaux. J. (1990) : Idiotypic Network and Disease (Washington, D.C.: Am. Soc.
Microbiol.)23) C･hagas. C'. (1988): A short chronicle of the discovery of Chagas' disease, PACE., 11, 1108‑1113
24) Colley, D.G., Montesan6, M.A., Eloi Santos, S.M., Powell, M.R., Parra, J.C., Goes, A., Doughty,

B.L.. Correa Oliveira, R. Rocha, R.S. and Gazzinelli, G. (1989): Idiotype networks in Schis‑tosomiasis. In New Strategies in Parasitology. P.W.J. McAdam, ed. (London: Churchill
Livingston) ; pp. 179‑190
25) Colley. D.G., Gazzinelli. R.T., Brener, Z., Monte ano, M.A., Eloi Santos, S.M., Correa Oliveira,R., Parra, J.C., Lima, M.S. and Gazzinelli, G. (1990) : Idiotypes and anti‑idiotypes in chronicparasitic endemic diseases. In Idiotype Networks in Biology and Medicine. A.D.M.E.
Osterhaus, and F.G.C.M. UytdeHaag, eds. (Amsterdam: Elsevier Science Publishers B.V.) , pp.

219‑224 ･ , = '26) Cossio, P.M., Diez. C., Szarfman, A., Kreutzer, E.. Candido, B. and Arana, R.M. (1974):
Chagasic cardibphthy. Demonstration of a serum gammaglobulin factor which reacts withendocardium and vascular structures, Circ., 49, 13‑2127) Davis, C.D. and' Kuhn. R.E. (1990): Selective binding of Tmpanosoma cruzi to host cell
membrane polypeptides, Infect. Immun., 58, 1‑628) Dia , J.C. (1987):" Cbntrol of Chagas' dis ase in Brazil, Parasit. Today, 3, 336‑341

29) Eloi Santos, S.M:, Galvao, L.M., Gazzinelli, R.T., Colley, D.G.. Gazzinelli, G. and Correa
Oliveira, R. (1988) : In utero idiotype and antigen sensitization in Tmpanosoma cruzi infectedpatients, Mem. Inst. Oswaldo. Cruz., 83 (Suppl. 1), 155 (Abstract)30) Eloi Santos, S.M., Novato Silva, E., Maselli, V.M.. Gazzinelli, G., Colley, D.G. and Correa
Oliveira, R. (1989): Idiotypic sensitization in utero of children born to mothers with schis‑tosoiniasis or Chagas' disease, J. Clin. Invest., 84, 1028‑l031
31) Filardi. L.S. and Brener, Z. (1987): Susceptibility and natural resistance of Tmpanosoma cruzi
strains to drugs used clinically in Chagas' disease, Trans. R. Soc. Trop. Med. Hyg., 81, 755‑759

32) Gazzinelli. R.T.. Leme, V.M., Cancado, J.R.. Gazzinelli, G. and Scharfstein. J. (1988a):ldentification of Tmpanosoma cruzi antigens recognized by T cells and immune sera fromcha asic patients. Mem. Inst. Oswaldo. Cruz., 83 (Suppl. 1), 272‑279
33) Gazzinelli. R.T., Morato, M.J., Nunes, R.M., Cancado. J.R., Brener, Z. and Gazzinelli, G.
(1988b): Idiotype stimulation of T Iymphocytes from' Tmpanosoma cruzi‑infected patients, J.

Immunol., 140, 3167‑3172
34) Gazzinelli, R.T., Parra, J.F., Correa Oliveira, R., Cancado, J.R., Rocha, R.S., Gazzinelli, G. andColley, D.G. (1988c): Idiotypic/anti‑idiotypic interactions in schistosomiasis and Chagas'
disease, Am. J. Trop. Med. Hyg., 39, 288‑29435) Gazzinelli, R.T., Galvao, L.M., Cardoso, J.E.. Cancado, J.R., Krettli, A.U., Brener, Z. and187

Gazzinelli, G. (1988d) : Anti‑Ttypanosoma cruzi and anti‑laminin antibodies in chagasic
patients after specific treatment, J. Clin. Microbiol., 26, 1795‑1800

36) Gazzinelli, R.T., Leme, V.M.. Cancado, J.R., Gazzinelli, G. and Scharfstein, J. (1990):
ldentification and partial characterization of Tmpanosoma cruzi antigensrecognized by T cells
and immune sera from. patients with Chagas' disease, Infect. Immun., 58, 1437‑1444

37) Ghauri, M.S.K. (1973) : Hemiptera (Bugs) . In Insects and Other Arthropods of Medical
Importance. K.G.V. Smith, ed. (London: British Museum Trustees) , pp. 373‑38538) Grant, I.H., Gold. J.W.. Wittner, M., Tanowitz, H:B., Nathan, C., Mayer, K., Reich, L., Wollner,

N., Steinherz. L., Ghavimi, F. and et al. (1989) : Transfusion‑associated acute Chagas' diseaseacquired in the United States, Ann. Intern. Med., 111, 849‑851
39) Grogl, M., Kuhn, R.E., Davis, D.S. and Green, G.E. (1984): Antibodies to Tmpanosoma cruzi incoyotes in Texas, J. Parasitol., 70, 189‑191
40) Hofflin, J.M., Sadler, R.H.. Araujo, F.G., Page. W.E. and Remington. J.S. (1987): Laboratory‑

acquired Chagas' disease, Trans. R. Soc. Trop. Med. Hyg., 81, 437‑440
41) Hontebeyrie Joskowicz, M., Said, G., Milon. G., Marchal, G. and Eisen, H. (1987): L3T4+ T
cells able to mediate parasite‑specific delayed‑type hypersensitivity play a role in the patho‑

logy of experimental Chagas' disease, Eur. J. Immunol., 17, 1027‑1033
42) Hudson, L. and Britten, V. (1985): Immune response to South American trypanosomiasis andits relationship to Chagas' disease, Br. Med. Bull., 41, 175‑18043) Hudson, L. and Hindmarsh, P.J. (1985): The relationship between autoimmunity and Chagas'
disease: causal or coincidental?, Curr. Top. Microbiol. Immunol., 117, 167‑177

44) Jerne, N.K. (1974): Towards a network theory of immune response, Ann. Immunol., 125,373‑38945) Joiner, K.A., daSilva, W.D., Rimoldi, M.T., Hammer. C.H., Sher, A. and Kipnis, T.L. (1988) :Biochemical characterization of a factor produced by trypomastigotes of Tr)panosoma cruzithat accelerates the decay of complement C3 convertases, J. Biol. Chem., 263, I1327‑11335
46) Kearney, J.F. (1989): Idibtypic networks. In Fundamental Immunology. W.E. Paul, ed. (NewYork: Raven Press) , pp. 663‑676

47) Khoury, E.L.. Diez, C., Cossio, P.M. and Arana, R.M. (1983): Heterophil nature .of EVI
antibody in Tmpanosoma Cruzi infection, Clin. Immunol. Immunopathol.. 27. 283‑288 ･‑48) Kierszenbaum, F., Gottlieb, C.A. and Budzko. D.B. (1983): Exacerbation of Tmpanosoma cruzi

infection in mice treated with the immunoregulatory agent cyclosporin A, Tropenmed.
Parasitol., 34, 4‑649) Kierszenbaum, F. and Hudson, L. (1985): Autoimmunity‑ in Chagas' disease: cause or symp‑
tom?, Parasit. Today, 1, 4‑950) Kierszenbaum, F. (1986): Autoimmunity in Chagas' disease. J. Parasitol., 72, 201‑211
51) Kierszenbaum, F. and Wirth. J.J. (1987): Activity of recombinant tumor necrosis factor on
Touoplasma gondii and Tmpanosoma cruzi [letter] , J. Immunol., 138, 33052) Kirchhoff, L.V. (1989): Is Tmpanosoma cruzi a new threat to our blood supply?, Ann. Intern.
Med., 111, 773‑775
53) Klinman, D.M. and Steinberg, A.D. (1987) : Systemic autoimmune disease arises from poly‑clonal B cell activation, J. Exp. Med., 165, 1755‑1760
54) Kuhn, R. (1981): Immunology of Tmpanosoma cruzi infection. In Parasitic Diseases, Vol. 1,

The Immunology. J.M. Mansfield, ed. (New York: Marcel Dekker), pp. 137‑166
55) Laguens, R.P., Cabeza‑Mekert, P.M.; Chambo, J.G. and Gelpi, R. (1981): Chronic Chagas'
disease in the mouse. II. Transfer of the heart disease by means of immunocompetent cells,
Medicina (Buenos Aires) , 41, 40‑4256) Marsden, P.D. (1984) : Selective primary health care: strategies for control of disease in thedeveloping world. XVI. Chagas' disease, Rev. Infect. Dis., 6, 855‑865

18857) McCabe, R.E., Remington. J.S. and Araujo, F.G. (1984): Mechanisms of invasion and replica‑tion of the. intracellular stage in Tmpanosoma cruzi. Infect. Immun., 46, 372‑376
58) McCabe. R.E. (1988): Primaquine is lethal for intracellular but not extracellular Tmpanosoma
cruzi. J. Parasitol., 74, 748‑753

59) Minoprio, P., Burlen, O., Pereira, P.. Guilbert, B.. Andrade, L., Hontebeyrie Joskowicz, M. andCoutinho, A. (1988): Most B cells in zicute Tr)panosoma cruzi infection lack parasite
specificity. Scand. J. Immunol., 28, 553‑561

60) Minoprio. P., Itohara, S., Heusser. C., Tonegawa, S. and Coutinho. A. (1989): Immunobiologyof murine T. cruzi infection: the predominance of parasite‑nonspecific responses and theactivation of TCRI T cells, Immunol. Rev., I12, 183‑207
61) Morato, M.J.. Brener. Z.. Cancado, J.R., Nunes, R. M., Chiari, E. and Gazzinelli, G. (1986) :Cellular immune responses of chagasic patients to antigens derived from different
Tmpanosoma cruzi strains and clones, Am. J. Trop. Med. Hyg., 35, 505‑511
62) Moser. D.R,, Kirchhoff, L.V. and Donelson, J.E. (1989): Detection of Tmpanosoma cruzi by
DNA amplification using the polymerase chain reaction, J. Clin. Microbiol., 27, 1477‑1482
63) Murta, A.C., Leme, V.C., Milani, S.R., Travassos, L.R. and Scharfstein, J. (1988): Glycoprotein

GP57/51 of Tmpanosoma cruzi: structural and conformational epitopes defined with mono‑clonal antibodies, Mem. Inst. Oswaldo. Cruz., 83, (Suppl. 1), 419‑422

64) Nash, T.E., Cheever, A.W., Ottesen, E.A. and Cook, J.A. (1982): Schistosome infections inhumans: perspectives and recent findings. Ann. Intem. Med., 97, 740‑75465) Nickerson, P., Orr, P., Schroeder. M.L., Sekla, L. and Johnston, J.B. (1989): Transfusion‑associated Tmpanosoma cruzi infection in a non‑endemic area, Ann. Intern. Med., 111, 851‑853
66) Nogueira, N. (1986): American Trypanosomiasis: Antigen and hostparasite interactions. In

Parasite Antigens‑ Towards New Stategies for Vaccines. T.W. Pearson, ed. (New York:
Merkel Dekker, Inc.) , pp. 91‑11067) Osuna, A., Gamarro, F., Castanys, S. and Ruiz Perez, L.M. (1986): Inhibition of lysosomal
fusion by Tmpanosoma cruzi in peritoneal macrophages, Int. J. Parasitol., 16, 629‑632
68) Ottesen, E.A. (1984) : Immunological aspects of lymphatic filariasis and onchocerciasis in man,Trans. R. Soc. Trop. Med. Hyg., 78, 9‑1869) Pereira Barretto, A.C., Mady, C., Arteaga Fernandez, E., Stolf, N., Lopes, E.A.. Higuchi, M.L.,
Bellotti, G. and Pileggi, F. (1986) : Right ventricular endomyocardial biopsy in chronic Chagas'

disease, Am. Heart J., 111, 307‑312

70) Pereira, M.E. (1990): Cell biology of Tmpanosoma cruzi. In Modern Parasite Biology:
Cellular. Immunological, and Molecular Aspects. D.J. Wyler, ed. (New York: W.H. Freeman
and Company) , pp. 64‑78
71) Petry. K. and Eisen, H. (1989) : Chagas' disease: a model for the study of autoimmune diseases,

Parasit. Today, 5, 111‑116

72) Reed, S.G. (1988): In vivo administration of recombinant IFN‑gamma induces macrophageactivation, and prevents acute disease, immune suppression, and death in experimental
Tmpanosoma cruzi infections. J. Immunol., 140, 4342‑4347
73) Reed, S.G.. Pihl, D.L. and Grabstein. K.H. (1989): Immune deficiency in chronic Tmpanosoma
cruzi' infection. Recombinant IL‑1 restores Th function for antibody production. J. Immunol.,142, 2067‑2071
74) Reis, D.d'A., Gazzinelli, R.T.. Cancado, J.R., Colley. D.G.. Brener, Z. and Gazzinelli, G. (1989):

ldiotypic differences on immunoaffinity‑purified anti‑epimastigote antibodies from pooled orindividual cardiac or indeterminate chagasic patients. Mem. Inst. Oswaldo. Cruz., 84 (Suppl. 2) ,87 (Abstract)75) Ribeiro dos Santos, R. and Rossi, M.A. (1985): Immunopathologia. In Cardiopatia Chagasica.
R.R. Cancado, and J.M. Chuster, eds. (Belo Horizonte, Brazil.: Fundacao Carlos Chagas de

189

Pesquisas Medicas) , pp. 371‑380
76) Rimoldi, M.T., Sher. A., Heiny, S., Lituchy, A., Hammer, C.H. and J̲oiner. K. (1988): Develop‑

mentally regu,lated expression by Tmpanosoma cruzi of molecules that accelerate the decay ofcomplement C3 convertases, Proc. Natl. Acad: Sci. U.S.A., 85, 193‑1'97
77) Said, G., Joskowicz, M., Barreira. A.A. and Eisen, H. (1985): Neuropathy associated withexperimental Chagas' disease, Ann. Neurol., 18, 676‑683
78) Scharfstein. J., Schechter, M., Senna, M., Peralta, J.M.. Mendonca Previato, L. and Miles, M.

A. (1986): Tmpanosoma cruzi: characterization and isolation of a 57/51,000 m.w. surface
glycoprotein (GP57/51) expressed by epimastigotes and bloodstream trypomastigotes, J.
Immunol., 137, 1336‑1341
79) Snary, D., Flint, J.E., Wood, J.N., Scott, M.T., Chapman, M.D., Dodd, J., Jessell, T.M. and Miles,

M.A. (1983): A monoclonal antibody with specificity for Tmpanosoma cruzi, central andperipheral neurones and glia, Clin. Exp. Immunol., 54, 617‑624
80) Snary, D. (1985): The cell surface of Tmpanosoma cruzi, Curr. Top. Microbiol. Immunol., I17,

75‑9281) Sturm, N.R. Degrave, W., Morel, C. and Simpson, L. (1989): Sensitive detection andschizodeme classification of Tmpanosoma cruzi cells by amplification of kinetoplast minicircle

DNA sequences: use in diagnosis of Chagas' disease, Mol. Biochem. Parasitol., 33, 205‑214
82) Szarfman, A., Terranova, V.P.. Rennard, S.1., Fordart, J.M., Lima, M.F. and Scheinman, J.1.
(1982): Antibodies to Laminin in Chagas' disease. J. Exp. Med., 155, 1161
83) Takle, G.B. and Hudson, L. (1989): Autoimmunity and Chagas' disease, Curr. Top. Microbiol.
Immunol., 145, 79‑92
84) Tarleton, R.L. (1988) : Tumour necrosis factor (cachectin) production during experimental
Chagas' disease, Clin. Exp. Immunol., 73, 186‑190
85) Tarleton, R.L. and Scott, D.W. (1987) : Initial induction of immunity, followed by suppressionof responses to parasite antigents during Tmpanosoma cruzi infection of mice, Parasite.
Immunol., 9, 579‑589
86) Teixeira, A.R., Teixeira, M.L. and Santos‑Buch. C.A. (1975): The immunology of experimen‑
tal Chagas' disease. IV. Production of lesions in rabbits similar to those of chronic Chagas'
disease in man, Am. J. Pathol., 80, 163‑180
87) Towbin, H., Rosenfelder, G., Wieslander, J.. Avila, J.L.. Rojas, M., Szarfman, A., Esser, K.,
Nowack, H. and Timpl, R. (1987) : Circulating antibodies to mouse laminin in Chagas' disease,

American cutaneous leishmaniasis, and normal individuals recognize terminal galactosyl(alpha 1‑3)‑galactose epitopes, J. Exp. Med., 166, 419‑432
88) Unanue, E.R. (1984): Antigen‑presenting function of the macrophage, Ann. Rev. Immunol., 2,

395‑42889) Velge, P., Ouaissi, M.A., Cornette, J., Afchain, D. and Capron, A. (1988): Identification andisolation of Tmpanosoma cruzi trypomastigote collagen‑binding proteins: possible role incell‑parasite interaction, Parasitology, 97, 255‑268
90) Villalta. F. and Kierszenbaum, F. (1984): Role of infiammatory cells in Chagas' disease. II.

Interactions of mouse macrophages and human monocytes with intracellular forms of
Tmpanosoma cruzi: uptake and mechanism of destruction, J. Immunol., 133, 3338‑3343
9D von Lichtenberg. F. (1987): Consequences of infections with schistosomes. In The Biology of

Schistosomes from Genes to Latrines. D. Rollinson, and A.J.G. Simpson, eds. (London:
Academic Press. Inc.) , pp. 185‑23292) Wirth, J.J. and Kierszenbaum, F. (1988): Recombinant tumor necrosis factor enhances
macrophage destruction of Tmpanosoma cruzi in the presence of bacterial endotoxin, J.
Immunol., 141, 286‑28893) Wood. J.N., Hudson, L.. Jessel, T.M. and Yamamoto, M. (1982) : A monoclonal antibody

190
de且ning

antigenic

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Jpn. J. Trop. Med. Hyg., Vol. 19 No. 2 1991, pp. 191‑201

191

NIGERIAN ONCHOCERCIASIS=EPIDEMIOLOGICAL PERSPECTIVE
BERTRAM E. B. NWOKEl, KUNINORI SHIWAKU2
AND HIROSHI TAKAHASH13
Received August 23 1990/Accepted February 20 1991

Abstract: Onchocerciasis is a widespread filarial disease in Nigeria that produces
grave socio‑economic effects. The great majority of the communities are mesoendemic
for onchocerciasis while only few are hyperendemic especially in the savanna zone. Sex‑
related infection depends on the degree of endemicity while age‑infection increases
gradually with advancing age. Visible and palpable nodules are more abundant aroundthe pelvic region. The nodules are countered more in the rainforest zone even whenmicrofilarial density (MFD) is moderate, while the nodules are less numerous in thesavanna form with high MFD. The microfilariae in concert with host's immune response
precipitate various skin lesions. The resulting pruritus, scratching and itching are
generalized in the rainforest and localized in the savanna. In the eye, various ocularlesions are associated with the death of microfilariae. There is high incidence of eye lesionin the savanna zone than in the rainforest zone, especially the anterior lesions. The
epidemiological picture presented by onchocerciasis in Nigeria is the summation of acomplex array of contributing factors, both intrinsic to the microfilariae and resulting
from the host‑immune response, bioclimatic factors and vector species complex.

INTRODUCTION
Human onchocerciasis is a chronic parasitic disease caused by Onchocerca volvulus. In

Nigeria the main vectors are the Simulium damnosum Theobald, 1903 complex of which
there are at least 26 species (Dunbar, 1976). The distribution of these cytospecies is to alarger extent related to the two main climatic regions in West Africa: rainforest and savannal Department of Zoology, Faculty of Natural Science, University of Jos, Jos, Plateau State,
N igeria

2 Department of Parasitology. Aichi Medical University, Yazako, Nagakute‑cho. Aichi 480‑11,

Japan
3 The Japan Cooperation Agency, University of Jos, Jos, Plateau State, Nigeria
This study was supported by University of Jos, Nigeria and Japan International Cooperation
Agency (JICA) Medical Research Project (JUJIP No. 16)
Present address:
1 Medical Entomology & Parasitology Unit. School of Biological Science. Imo State University,

Okigwe, Nigeria
2 Department of Environmental Medicine, Shimane Medical University, Izumo, Shimane 693,
Ja pan3 Hayamiya 1‑40‑ll, Nerima‑ku, Tokyo 176. Japan

192(Townson and Meredith, 1979) . It is not important to note the absolute demarcation because
there are cases of savanna‑type microconditions within the rainforest zone and pockets ofrainforest environment within the savanna zone especially along river banks. The six main
vector cytospecies in West Africa have been grouped according to phytogeographical condi‑
tions as follows: savanna species (S. sirbaum and S. damnosum s.s.) , rainforest species in big

rivers (S. santipauli and S. soubrense), rainforest species in small rivers (S. yahense and S.

squamosum) and in rainforest pockets within the savanna zone (S. squamosum) .
Onchocerciasis was first reported in Nigeria by Parsons (1908) . It seems that onchocer‑

ciasis produces grave socio‑economic effects and much human suffering to about 750,000
Nigerians (Dipeolu and Gemade, 1983) . Yet, the geographical distribution as well as its
complications are inadequately coordinated in the country. We have therefore used available
literature to make for a better understanding of epidemiology of Nigerian onchocerciasis.
Some believe that the South American disease has been imported from Africa throughthe slave trade, because only negro offsprings whose ancestors have been forced to workaround the coastal region in the 16th to 18th centuries are infected and the clinical features

is likely to be of African type. On the other hand, the disease in Guatemala and South Mexico
are apparently different from the South American regions: clinical manifestations are charac‑
teristic and the parasite is transmitted by a different species of blackfiy unlike to those ofSouth America.
Although there are marked differences in the clinical manifestations and the vector‑
parasite complexes between the Guatemalan and West African disease (Duke, 1974; De Leonand Duke, 1966) , no distinct morphological differences in the parasites from the two regions

are ever recognized by Franz (1980). Results on the chromosomes of Nigerian andGuatemalan O. volvulus (Hirai et al.. 1987) showed that both parasites had basically thesame chromosomal construct (2n= 8, xy type) which corresponded to the findings of Bazanez
et al. (1983) in Venezuela. Also the nuclei number of 88.0 distributed in the CS‑NR portionof the microfilariae of Nigerian O. volvulus (Mimori et al., 1986) suggests that there is no
siguificant taxonomical difference in O. volvulus from Nigeria and Guatemala; 87.8 (Tada etal.. 1981) and 81.7 nuclei number from Amazonas State, Venezuela (Yarzabal et al., 1983).
Even though results so far indicate that the Nigerian O. volvulus is biologically quite close to

the Guatemalan species, polymorphism of O. volvulus by iso‑enzYme technique and gene
analysis are needed to strongly back up the argument. '='PREVALENCE AND DISTRIBUTION
One of the satisfactory methods of studying the epidemiological aspects of onchocerciasis

is by the direct demonstrating of O. volvulus microfilariae in skin biopsies. About 750,000
Nigerians are infected by human onchocerciasis (Dipeolu and Gemade, 1983). This disease
is wide spread in Nigeria (Crosskey, 1954; Bradley, 1976; Edungbola et al., 1983; Nwoke, 1986;

Nwoke et al.. 1989) . These studies have shown that according to the recommendation of the

WHO (1966) , few areas in Nigeria must be regarded as hypoendemic, many mesoendemic
while few others as the middle Hawal area, Jarawa District on the Jos Plateau are hyperen‑
demic areas (Fig. 1) .

The prevalence of Nigerian onchocerciasis shows significant variations of villages withthose villages near the breeding sites of vectors having the highest infection rates (Onwuliri

193
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Figure I Endemicity of onchocerciasis (river blindness) in Nigeria(After several authors) .

et al., 1978) . The many workers (Edungbola et al., 1983; Somorin, 1983; Nwoke, 1986) haveshown that the onchocerciasis infection rate increases gradually with advancing age in
Nigeria. This infection pattern bears witness to the chronic and cyptic nature of humanonchocerciasis infections (Buck, 1974) . In Nigeria, children make frequent visits to the fociof infection to swim, collect water for domestic use, hunt and fish. These activities together

with farming are maintained throughout life in these rural communities. As a result thecontact with infected Simulium are permanently maintained and therefore the age‑related
prevalence and intensity shows a gradual increase.
Sex‑related onchocercal infection in Nigeria varies depending on the degree of en‑
demicity. In hyperendemic villages male subjects have slightly higher but insignificant
infection rate than females. This is because inhabitants in such hyperendemic villages live
in homes very close to the foci of infection. This results that even when there are sex
differential in occupation, both sexes are more or less equally exposed to the same numberof infective vectors (Bradley, 1976; Edungbola et al.. 1983) . On the other hand, in hypo‑ andmeso‑endemic villages and living far away from foci of infection male subjects show
significantly higher intensity and prevalence rate than females. This is because the surround‑ings of such villages are not easily accessible to vector flies and therefore occupational
exposure tend to play more prominent role (Crosskey, 1954). In occupational groups, field

workers, the nomadic or migrant fulani tribesmen are more affected by onchocerciasis(Onwuliri et al.. 1987).

194

Photo. I Onchocercal nodule in the right thorax of a patient at Mai‑
jemu, Jos Plateau.

THE ADULT PARASrrES AND NoDULES
The adult parasites of O. volvulus are characteristically contained in fibrous subcutane‑

ous nodules (Phato. 1) . However, the great frequency of individuals with microfilariae of O.
volvulus demonstrable by skin biopsies, but without visible or palpable nodules indicates that

adult worms may be lying free, unencapsulated in the subcutis (Nnochiri, 1964; Nwoke et al..
1988) ; or may be small and deeply located (Leichsenrjng et al.. 1990) .

In Nigeria (Dipeolu and Gemade, 1983; Nwake et al.. Ig87) and most other Africancountries (Browne, Ig61; Leichsenring et al., 1990) the great majority of detectable nodules

occur in the pelvic region. This is in contrast to which obtains in Guatemala where as manyas 70% of the nodules are found in the head region (Lagraulet et al.. 1964). Yemen
onchacerciasis rarely present with onchocercomata (Somorin, 1983) .
There exi8t two form of nodules in West Africa. In the rainforest form, the disease ischaracterized by abundant nodules and moderate microfilariae density (MFD) while in thesavanna form the nodules are les8 numerous with characteristic high MFD in the skin
(Onwuliri et al.. 1987; Nwoke, 1986) .

THE MrcROFrLAR:rAE
The microfilariae are distributed widely in the dermis and the eyes. Most investigators
believe that the distribution of microfilariae in the skin is correlated with the onchocercal

nodules. In Nigeria and West Africa visible and palpable nodules are mainly in the lower
parts of the body and the hjghest density of microfilariae is recorded in pelvic region

195(Kershaw et al., 1954; Edungbola, 1982; Nwoke et al.. 1989). In East Africa, Nelson (1958)

noted that the microfilariae are mostly concentrated around the buttocks and upper thigh,whereas the skin below the knee is less affected. On the other hand, Guatemalan onchocer‑
ciasis is characterized by high MFD in the upper part of the body which corresponds to theanatomical distribution of nodules (De Leon and Duke, 1966) .
In most onchocerciasis‑endemic countries including Nigeria, the MFD has shown varying
concentrations in the skin depending on the time of the day or month of the year, the strainof parasite or environmental conditions. In the Haute‑Volta, the microfilariae were muchhigher in the skin at 16 hr than at 10 hr (Lartique, 1967) while in Cameroon Duke et al. (1967)

reported an elevation in the MFD during the afternoon in both the rainforest and Sudan‑savanna forms which they noted correlated: well with the curves for temperature/saturation
deficiency and for the biting density of S.

d'amnosum vectors. However, they noted that thefiuctuation in MFD were too small to affect the results of diagnosis. Also Picq and Jardel(1973) in Haute‑Volta reported insignific nt changes in the skin MFD. In Guatemala, Tada

and Figuerroa (1974) observed that thdaily variations of MFD were not consistent.

Hashiguchi et al. (1983) in Guatemala observed that the pattern of microfilarial intake by S.

onchraceum did not correlate well with the diurnal pattern of MFD. Recently Nwoke (1988)
in the savanna part of Nigeria observed insignificant variations in the seasonal, daily anddiurnal MFD. However, direct sun‑shine on the skin of patients working in the field and low
temperatures (8‑15'O had a significant reducing effect on MFD in Nigeria (Nwoke, 1986) .

ONCHODERMATITIS (SKlN LESIONS)The subcutaneous microfilariae, in concert with the host's immune response precipitate

various dermatological lesions such as pruritus with papular rash, depigmentation andatrophy. The most common dermatological manifestations of onchocerciasis in Nigeria are
pruritus, scratching and itching (Edungbola et al., 1983) which may be generalizes in therainforest type, Iocalized in the savanna or American type and segmental in the Yemen type
(Somorin, 1983) . Pruritus may be accompanied by a nonspecific rash which may be macular,
papular or pustular. In Nigeria, pruritus occurs in all ages but is significantly higher amongolder age groups. After some period of scratching lesions develop dry, scaly and in elastic
skin with folds resulting in lichenification. It is often impossible distinguish the skin changes

from those seen vitamin A deficiency patients. Rodger (1962) suggested that the parasite
competes for vitamin A in the skin or that vitamin A deficiency predisposes the skin toheavier infections. Sowda, the Yemen type of onchocerciasis characterized by dermatic
hyperpigmented (black) segmented limb associated with enlarged' inguinal lymphatic glandhas been observed in the rainforest of Nigeria (Somorin, 1983) .
The lichenified pruritus areas also may be associated with pigmentary changes (hyper‑or hypopigmentation) resulting in a striking dermatologic feature commonly called leopard
skin (Photo. 2). In Nigeria, this clinical manifestation of onchocerciasis is associated withlong‑standing onchodermatitis. Leopard skin affects the skin and anatomical sites such as
the enis, buttock, thigh, scrotum, inguinal fold and axillae (Connor and Palmieri, 1985;
Nwoke et al.. 19'89) which are not readily exposed to biting blackflies. Connor and Palmieri(1985) revealed progression of leopard skin: increasing number of microfilariae accumulates
and degenerates in the upper dermis which consequently provokes acute inflammation at first,

196

Photo. 2 Leopard skin on the shin in a patient at Maijemu, JoS
Plateau, characterized .by focal depigmentation.

around degenerating microfilariae, then chronic. There is melanin drop into the upper dermis

where it is phagocytased and eventually carried to regional lymph nodes. Nigerian onchocer‑
ciasis is characterized by relatively high prevalence rate of leopard skin in the savanna zone
than in the rainforest (Edungbola et ed,, 1983) , This has been attributed to differences in thepathogenicity and antigenic peculiarities of the savanna and rainforest strains of the para‑sites or to differences in the host immunological responses (Anderson and Fuglsang, 1977;
Anderson et al., 1974) .

ONCHOCERCAL LYMPH ADENIt{SAntigens released from microfilariae lead to the deposition of immune. complex in tissues,

which in turn causes inflammation and fibrosis and eventually obstructive lymphadenitis.
Lymphadenitis may sometimes lead to hanging groin and elephantiasis (Buck, 1974; Gibson
and Connor, 1978) . Adenolymphocele is a feature of onchocerciasis in Zaire, Central Africa
Republic. Chad, Cameroon and Nigeria and perhaps other African countries. These pendu‑

197

Photo. 3 Enlarged scrotum in a patient at Maiiemu, Jos Plateau.

10us sacs are observed in male adults and often contain both femoral and ing"uinal lymph
glands. Somorin (1983) in Nigeria and Connor and Palmieri (1985) in Africa describedfemale patients af onchocerciasis with fullness in the femoralnd inguinal regions revealing

underlying uni‑ or bilateral lymphadenopathy. No difference has been recorded between theprevalence of onchocercal lymphadenitis in the savanna and rainforest zones of Nigeria. ,In
Nigeria, Nwoke (1986, 1989) observed that these dreadful malformations due to onchocercal
lymphadenitis cause a lot of socio‑cultural discomfcrt to patients (Photo, 3). In affectedpatients with the pendulous genital sacs, sexual life is greatly affected.

OCULAR LESIONS
Ocular complicatians are the most serious clinicai manifestations of onchocerciasis. Lossof vision can manifest either as anterior or p08terior lesions. In Nigeria, Dipeolu and Gemade(1983) estimated that 30,000‑40.000 people have ocular complications of onchocerciasis. The
pathologic picture in ocular onchocerciasis is the summation of a complex array of cantribut‑

198

ing factors, both intrinsic of microfilariae and resulting from the immune response andbioclimatic factors. The release of large amount of microfilariae antigens have more serious

consequences in the eye and skin than in the organs with a more efi:cient antigen‑cleaningsystem, such as the spleen and liver (Mackenzie et al.. 1985).

Ocular onchocerciasis was found to be more severe in the savanna region than in therainforest of Nigeria (Budden, 1963; Anderson and Fuglsang, 1974) . Duke (1976, 1981) notedthat the microfilariae of the savanna strain were more invasive and more pathogenic on thecornea than those of the rainforest strain. In addition, the infiuence of climatic factors cannot

be ruled out completely. Sclerozing keratitis having a much higher prevalence in the savanna
than in the rainforest (Duke and Anderson, 1972) caused the invasion of microfilariae on thecornea. Microfilariae trend to involve that parts of the cornea which is exposed to light(WHO, 1973). However, it is possible to observe a comparatively low blindness rate inonchocerciasis hyperendemic communities in the savanna zone of Nigeria. According to
Nwoke (1986) and Nwoke et al. (1987) this low blindness rate may be attributed to thehabitual migration of disabled people in the savanna zone to urban centers tQ beg for alms,

consequently reducing the actual number of blind people in the rural communities.
Ocular lesions of onchocerciasis are much more prevalent among productive age groupin Nigeria (Budden, 1963; Dipeolu and Gemade, 1983; Onwuliri et al.. 1987). The pattern ofsocio‑economic liability that has emerged due to onchocerciasis blindness in the settle nents
has been damaging (Bradley, 1976; Nwoke et al.. 1988; Nwoke, 1989) . Onchocerciasis in

Nigeria has been instrumental to abandonment of some river valleys especially in thesavanna that are agriculturally fertile (Bradley, 1976) .

REFERENCES
1 ) Anderson, J. and Fuglsang, H. (1974) : Studies on onchocerciasis in the United Cameroon
Republic. II. Comparison of onchocerciasis in rain forest and Sudan‑savanna, Trans. Roy. Soc.

Trop. Med. Hyg., 68, 209‑222
2 ) Anderson, J., Fuglsang, H., Hamilton, P.J.S. and Marshal T.F.De C. (1974): Studies on
onchocerciasis in the United Cameroon Republic. I. Comparison of population with andwithout O. volvulus. Trans. Roy. Soc. Trop. Med. Hyg., 68, 190‑208
3 ) Anderson, J. and Fuglsang, H. (1977) : Ocular onchocerciasis, Trop. Diseases Bull., 74, 257‑272

4 ) Bazanez, M.G., Botto, C. and Yarzabal, L. (1983): Characteristica de undeme de O. volvulus

s.1. del alto Orinoco (Terr,itorio Federal Amazonas, Venezuela). Estudio citogenetico
preliminar. Las filariasis humans en el Territorio Federal Amazonas, In Pricet Amazonas (ed)Caracas Publ. Cient., 2, 79‑82
5 ) Bradley, A.K. (1976): Effects of onchocerciasis on settlement in the Middle Hawal Valley;

Nigeria, Trans. Roy. Soc. Trop. Med. Hyg., 70, 225‑229
6 ) Browne, S.G. (1961): Localization of onchocercomata, Trans. Roy. Soc. Trop. Med. Hyg., 55,

258‑2627 ) Buck, A.A. (1974): Onchocerciasis. Symptomatology, pathology, diagnosis, Geneva, Wld.
Hlth. Org.

8 ) Budden, F.H. (1963): Comparative study of ocular onchocerciasis in Sudan‑savanna andrainforest, Trans. Roy. Soc. Trop. Med. Hyg., 57, 64‑70
9 ) Connor, D.H. and Palmieri, J.R. (1985) : Blackfly bites, onchocerciasis, and leopard skin, Trans.

Roy. Soc. Trop. Med. Hyg., 79, 415‑417
10) Crosskey, R.W. (1954): Onchocerciasis in the Galma Valley area, Northern Nigeria, W. Afr.

199
Med. J., 3, 75‑79

11) De Leon, R.J. and Duke, B.O.L. (1966): Experimental studies on the transmissio, n of
Guatemalan and West African strains of Onchocerca volvulus by Simulium ochraceum. S.
metallicum and S. callidum. Trans. Roy. Soc. Trop. Med. Hyg., 60, 735‑752
12) Dipeolu, 0.0. and Gemade, E.1.1. (1983) : Onchocerciasis in the Benue State of Nigeria. IV. The
prevalence of the disease among the population in Manor. Int. J. Zoonosis, 10, 85‑95
13) Duke, B.O.L. (1974): Research and control of onchocerciasis in the Western Hemisphere, Pan.

American Health Organization, Washington D.C., 298, 25‑29
14) Duke, B.O.L. (1976): Strains of Onchocerca volvulus and their pathogenicity. Tropenmed.
Parasitol., 27, 21‑22

15) Duke, B.O.L. (1981): Geographical aspects of onchocerciasis, Ann. Soc. Belge. Med. Trop., 61,

179‑186
16) Duke. B.O.L. and Anderson. J. (1972): A comparison of lesions produced in the cornea of
rabbit eye of microfilariae of forest and savanna strains of O. volvulus from Cameroon. I. The
clinical pictures. Tropenmed. Parasitol., 23, 354‑368
17) Duke, B.O.L., Sheffel, D.P., Guyon, J. and Moore, J.P. (1967): The concentration of Onchocerca

volvulus microfilariae in skin snips taken over twenty‑four hours, Ann. Trop. Med. Parasitol.,
61, 206‑219

18) Dunbar, R.W. (1976): The East African situation and a review of S. damnosum complex asa whole. WHO/VBC/SC
19) Edungbola, L.D. (1982): Prevalence of onchocerciasis in Ife‑Ife District (ifelodun) , Kwara
State, Nigeria. Trop. Geog. Med., 34, 231‑239
20) Edungbola, L.D., Oni, G.A. and Aiyedun, B.A. (1983): Babana Parasitic Diseases Project: I.

The study area and a preliminary assessment of onchocercal endemicity based on the preva‑
lence of "leopard skin", Trans. Roy. Soc. Trop. Med. Hyg., 77, 303‑309
21) Franz, M. (1980): Electron microscope study of the cuticle of male and female O. volvulus
from various geographical areas, Tropenmed. Parasitol., 31, 149‑16422) Gibson, D.W. and Connor, D.H. (1978) : Onchocercal lymphadenitis: Clinicopathologic studyof 34 patients. Trans. Roy. Soc. Trop. Med. Hyg., 72, 137‑154
23) Hashiguchi, Y., Kawabata, M.. Takaoka, M. and Flores, O.C. (1983): Microfilarial density in
Guatemalan onchocerciasis patient's skin with special reference to the hourly intake by
Simulium ochraceum. Japan. J. Trop. Med. Hyg., 11, 25‑33
24) Hirai, H., Tada. I.. Takahashi, H., Nwoke. B.E.B. and Ufomadu, G.O. (1987): Chromosomesof Onchocerca volvulus (Spirurida: Onchocercidae) . A comparative study between Nigeria andGuatemala. J. Helminthol., 61, 43‑46
25) Kershaw, W.E.. Duke. B.O.L. and Budden, F.H. (1954): Distribution of microfilariae of O.
volvulus in the human skin. Br. Med. J., 2, 724‑729
26) Largraulet, J.. Monjusiau, A.G.M. and Durand, B. (1964): Etude des localisations des nodles
dan I'onchocercose; Ieurs relations avec le lieu de piqure des simulies, Med. Trop., 24, 566‑572
27) Lartique. J.J. (1967) : Variations du nombre de microfilaires d'Onchocerca volvulus contenues

dan des biopsies cutanees pratiquees a'differentes heures de la journee, Bull. Wld. Hlth. Org.,
36, 491‑494 .

28) Leichsenring, M., Troger, J., Nelle M., Buttner, D.W., Darge, K. and Doering‑Schwerdtfeger E.

(1990): Ultrasonographical investigations of onchocerciasis in Liberia, Am. J. Trop. Med.
Hyg., 43, 380‑38529) Mackenzie. C.D.. William. J.F.. Sisley. B.M.. Steward. M.W. and O'Day, J. (1985) : Variations

in host responses and the pathogenesis of human onchocerciasis, Rev. Inf. Dis., 7, 802‑80830) Mimori, T., Tada, I., Shiwaku, K., Ufomadu, G.O. and Nwoke, B.E.B. (1986) : A biometricstudy of Onchocerca volvulus microfilariae from Nigeria using the nuclear counting method, Z.

200
Parasitenkd., 72, 835‑836
31) Nelson. G.S. (1958) : Onchocerci.asis in the West Nile District of Uganda, Trans. Roy. Soc.

Trop. Med. Hyg., 52, 368‑37632) Nnochiri. E. (1964) : Observations on onchocercal lesions seen in autopsy specimens in Western
Nigeria, Ann. Trop. Med. Parasitol., 58, 89‑93
33) Nwoke. B.E.B. (1986): Studies on the field epidemiology of human onchocerciasis on the Jos
Plateau, Nigeria. Ph. D. Thesis University of Jos, Nigeria, pp. 343
34) Nwoke, B.E.B. (1988) : Studies on the field epidemiology of human onchocefciasis on the Jos
P1.ateau, Nigeria VII. The effects of climatic factors on the diurnal biting behaviors of

Simulium damnosum Theobald (Diptera: Simuliidae) , Insect Sci. Applic., 9, 323‑32835) Nwoke,'B.E.B. (1989): The socio‑economic aspects of human onchocerciasis in Africa: present
appraisal, J. Hyg. Epidemiol. Microbiol. Immunol., 33, 19‑2836) Nwoke, B.E.B., Onwuliri; C.O.E., Iwuala, M.O.E., Ufomadu, G.O., Takahashi, H., Tada, I. andShiwaku, K. (1987): Studies on the field epidemiology of human onchocerciasis on the Jos
Plateau, Nigeria. IV. Clinical manifestation, socio‑economic' importance and local disease
perception and treatment. Proc. Nigeria‑Japan Joint Int. Conf. Trace Metals, Diarrhea, Med.
Entomol: Epidemiol. Studies, pp. 217‑221, Jos, Nigeria37) Nwoke. B.E.B.. Onwuliri, C.O.E., Iwuala, M.O.E., Takahashi, H., Tada. I. and Ufomadu, G.O.
(1988) : Studies on the field epidemiology of human onchocerciasis on the Jos Plateau, Nigeria.

V. The efficacy of nodulectomy as a treatment procedure, Trop. Geogr. Med., in press.
38) Nwoke, B.E.B.. Onwuliri, C.O.E.. Iwuala, M.O.E., Ufomadu, G.O., Shiwaku, K., Tada; I. andTakahashi, H. (1989): Endemic onchocerciasis on the Jarawa Valley area of Jos Plateau,
Nigeria, Japan. J. Trop. Med. Hyg., 17, 205‑211
39) Onwuliri, C.O.E., Nwoke, B.E.B., Lawal. I.A. and lwuala, M.O.E. (1987): Onchocerciasis in
Plateau State of Nigeria. II. The prevalence among residents around the Assob River Area.
Ann. Trop* Med. Parasitol., 81, 49‑52
40) Parsons, A.C. (1908) : Filaria volvulus Leuckart, its distribution, structure and pathologicaleffects, Pa.rasitology, 1, 359‑368

41) Picq, J.J. and Jardel, J.P. (1973) : Une methode d'evaluation des densites microfilariennes
d' Onchocerca volvulus Leuckart 1883. Variations des densites microfilarienne au cours des24 heures. WHO Monograph Document WH0/0NCH0/73, 103, 2542) Rodger, F.C. (1962): A review of recent advances in the scientific knowledge of thesymptomatology, pathology and pathogenesis of onchocercal infections, Bull. Wld. Hlth. Org.,
27, 429‑44843) Somorin, A.O. (1983): Onchocerciasis, Int. J. Dermatol., 22, 182‑188
44) Tada, I. and Figuerroa, F.H. (1974): The density of O. volvulus microfilariae in the skin atdifferent times of the day in Guatemala, Jpn. J. Parasitol., 23, 220‑22545) Tada. I., Mimori, T., Sakaguchi, Y., Kusano, M., Hashiguchi, Y. and Recinos, M.C. (1981): The

use of aceto‑orecein‑stained squash preparations for enumeration of nuclei in microfilariae ofvarious filarial parasites, Am. J. Trop. Med. Hyg., 30, 593‑597

46) Townson. H. and Meredith, S.E.O. (1979): Identification of Simuliidae in relation to theonchocerciasis, pp. 145‑174, In Problems in the Identification of Parasites and Their Vectors,

Tayler. A.E.R. and Muller, R. ed., Symp. Br. Soc. Parasitol., Vol. 17. Blackwell Scientific

Publication, Oxford
47) WHO (1966): Onchocerciasis, Wld. Hlth. Org. Tech. Rep., No. 335
48) WHO study group (1973) : The prevention of blindness. Wld. Hlth. Org. Tech. Rep., No. 518
49) Yarzabal, L., Potralanda, I., Arango, M.. Lobol, L. and Botto, C. (1983): Acid phosphatasepatterns in microfilariae of Onchocerca volvulus s.1. from the Upper Orinoco Basin, Venezuela,

Tropenmed. Parasitol., 34, 109‑112

201

ナイジェリアにおけるオンコセルカ症の疫学
Bertram E．B．Nwoke1・塩飽

邦憲2・高橋弘3

オンコセルカ症はナイジェリアに広く流行しており，社会経済的に多大の悪影響を及ぼしている。ナイジェリアにおけるオンコセルカ症の分布，および合併症についての知見は不十分である

ため，これまでに報告された研究を整理し，ナイジェリアにおけるオンコセルカ症の記述疫学的な記載を行った。ナイジェリアの大部分の地区は，WHOの基準にしたがうとオンコセルカ症の中等度の流行地であるが，サバンナ地帯に高度流行地が点在する。地区でのオンコセルカ症の感染率における性差は，流行の程度に依存し，低流行地で性差が著しく，男性で高率である。また，感染率は加齢とともに増加する。視認および触知可能な皮下腫瘤は，腰部に多く存在する。熱帯雨林地帯ではミクロフィラリア密度が少ないにも関わらず，皮下腫瘤を多く認めるが，サバンナ

地帯ではミクロフィラリア密度が多いにも関わらず，皮下腫瘤は少ない。ミクロフィラリアは宿主の免疫反応と相まって様々な皮膚病変を引き起こす。皮膚掻痒症の発現は熱帯雨林では一般的であるが，サバンナ地帯では一部に留まる。様々な眼科病変は，死亡したミクロフィラリアによって引き起こされることが明らかになってきた。眼，特に前房の病変は，熱帯雨林地帯よりもサバンナ地帯に多い。ナイジェリアにおけるオンコセルカ症は，ミクロフィラリア自身の性質および宿主の免疫反応，生物気候学的因子，伝播するブユの亜種の多要因によって複雑な疫学像を呈している。

1 ジョス大学理学部
2 愛知医科大学寄生虫学教室
3 国際協力事業団・ジョス大学医学部
現住所
1 イモ大学生物学部
2 島根医科大学環境保健医学教室
3 東京都練馬区早宮1−40−11
この研究は，日本一ナイジェリア医学協力事業によった（JUJIP−No．16）。

Jpn. J Trop Med Hyg Vol 19 No 2 1991, pp. 203‑208

203

THE ETIOLOGICAL FACTOR FOR EOSlNOPHILIAAND HYPERGLOBULlNEMIA E IN BRAZILIANSCHOOL CHILDRENATSUO HAMADA*, NAOHIRO WATANABE*, MASASHI KOBAYASH12EIICHI OKUSAWA=, TOMOYOSHI NoZAK13, IVETE BARBOSA',SEIKI TATENO' AND AKIO KOBAYASHI*
Received March 5 1991/Accepted April 10 1991

Abstract: The etiology of peripheral blood eosinophilia and hyperglobulinemia E,
which are commonly found among residents in tropical area, were studied with two groupsof Brazilian children from different primary schools. These two groups of children had
different infection rates with soil‑transmitted helminthiases, but similar positive rates ofantibody against a common allergic antigen. The prevalence rate of children witheosinophilia or hyperglobulinemia E was significantly higher in a group with higher
infection rate with soil‑transmitted helminthiases than in another group with lower
infection rate. It was suggested that eosinophilia and hyperglobulinemia E in this area
were mainly caused by soil‑transmitted helminthiases.

INTRODUCTION
Peripheral blood eosinophilia and hyperglobulinemia E (hyperlgE) are common labora‑
tory findings among residents in tropical areas (Johansson et al.. 1968; Mahmoud, 1984). It
has been empirically supposed that these abnormal findings are caused by helininth infections,which are major health problem in tropical areas. However, the comparisons in the numberof eosinophils and serum lgE Ievel between helminth infected and uninfected subjects in one
community usually failed to show prominent differences (Bruce‑Tagoe et al.. 1977; Kaplan
et al.. 1980) .

Allergic diseases are another etiological factor for eosinophilia and hyperlgE especiallyin residents in temperate areas. It has been demonstrated that the morbidity of allergic
diseases was not so low even in tropical residents (Lynch et al.. 1984). Therefore theparticipation of allergic diseases on eosinophilia and hyperlgE in tropical residents also

1 Department of Parasitology, Jikei University School of Medicine, Japan.
2 Department of Parasitology, School of Medicine, Chiba University, Japan.
3 Department of Parasitology, School of Medicine, Keio University, Japan.

4 Laboratorio de Imunopatologia Prof. Keizo Asami, Universidade Federal de Pernambuco,
Brazil.

This study was supported in part by the Japan International Cooperation Agency, and grant‑in‑aid
for scientific research (No. 0277027) from the Ministry of Education, Science and Culture of Japan.

Address reprint request to Atsuo Hamada, M.D. Department of Parasitology, Jikei University
School of Medicine, 3‑25‑8 Nishishinbashi, Minato‑ku, Tokyo 105, Japan.

204should be considered.
To know the etiolQgy of eosinophilia and hyperlgE in tropical residents, we examined thenumber ofbsinophils and sefum lgE Ievel in tivo grotips o,f Brazilian children living in a city,

in relation to soil‑transmitted helminthiases and allergic diseases.

METHODS
Participan ts

Recife, Iocated in the northern part of Brazil, is a port city with a population of one

million. Two primary school in Recife were chosen for testing. One in Varzea (VAR)
located in the suburbs, where sanitary condition is poor. The other in Madalena (MAD) isin the center of the city with good sanitation. The stool and peripheral blood were collectedfrom 92 school children in VAR and 18 school children in MAD (age range from 6 to 12 years
old) .

Stool

Fecal samples were collected and examined for helminth eggs by modified Kato‑Katz

method.
Blood
The number of eosinophils was microscopically counted with Hinkelman's staining. The
presence of eosinophils more than 500lmm3 was considered as eosinophilia. Serum lgE Ievel

were measured by using Phadezym lgE PRIST kits (Pharmacia fine chemicals) and wereexpressed as international units per ml (IU/ml) . HyperlgE was defined as serum lgE Ievel
more than 500 IU/ml. The level of anti‑house dust lgE antibody was determined by Radio
Allergo Sorbent Test (Pharmacia fine chemicals) . Scores higher than 2 were considered as
positive.
Statistical analysis

Data were analysed by the application of x2 test or student's t test.

RESULTS
Stool examination
Results of the stool examination are shown in Table 1. Fifty eight (63% ) of 92 children
Table 1 Results of stool examinations among school children in Recife,
BrazilNo. (% ) children positive for

School in VAR& T.t.

helminth eggs

A.1.

T.t.

5s (63%)*

19

20

19

1 (6%)

o

l

o

A.1.

(N = 92)

School in MAD(N = 18)

A.1.: Ascaris lumbricoides. T.t.: Trichuris trichiuraThis percentage was significantly higher than that of school in MAD(p < 0.001) .

205

Table 2 Number of eosinophils and serum I gE

levelschool children in Recife, Brazil

No. eosinophils*

among primary

School in VAR

School in MAD(N = 92)(N = 18)l , 053PS

317 5180( Imm3 )No.(%) children
with eosinophilat

Serum lgE Ievel*

66 (72%)3 (17%)

1 , 202525

114< O . OOl

175

(IU/ml )No.(%) children
with hyperlgE$*t$

5 (28%)

67 (73%)< O . OO1

Number indicate mean :SEM.
Number of eosinphils more than 500/mm3.
Serum lgE Ievel more than 500 IU/ml.
P value calculated frorTl the percentages.

in VAR were found to be･ positive for hel‑minth eggs (Ascaris lumbricoides alone, 19;
Trichuris trichiura alone, 20; Both, 19). Of
the 18 children in MAD, only one (6% ) was
positive for helminth eggs. The prevalence
rate of soil‑transmitted helminthiases weresignificantly higher in VAR children than in
MAD children (P<0.001) .
Anti‑house dust lgE
As a marker of allergic diseases, anti‑
house dust lgE antibody was measured in 32

children in VAR and 18 children in MAD.

Only 3 (9%) and 2 (11%) students were

6
e

E

=
oe
o
UJ

e

4

oo

lbe
2 ee
,ee

e
e e

e

ee

o

e

ee eeeo)eaPepositive in VAR and MAD children, respec‑tively. Therefore, with respect to the preva‑

e

,,

O
O

lence of anti‑house dust lgE antibody, no
significant difference was observed between
these two groups.
Number of eosinophils and lgE Ievel

2

4

Eosinophils(1000/mm 3 )Prominent increase of the mean numberof eosinophils and serum lgE Ievel were ob‑served in the children of VAR (eosinophils:

Figure 1

Correlation of the number of

1,053: :80/mm3, IgE: 1,202ill4 IU/ml)

eosinophils and serum lgE Ievel in(Table 2). The percentage of children withprimary school children in VAR.
The number of eosinophils showedeosinophilia (eosinophils>500/mm3) was
72%, and children with hyperlgE (IgE > 500
IU/ml) was 73%. Significant correlation wasa significant correlation with serumlgE Ievel (r=0.24314, p<0.05) .

206

demonstrated between the number of eosinophis and lgE Ievel (p< 0.05, Figure 1) . Therefore,

it is supposed t h, a.t frequently observed eosinophilia and hyperlgE in VAR children werecaused by the same etiology, such as soil‑transmitted helminthiases.

On the other hands, in MAD children, the mean number of eosinophils and serum lgElevel were nearly normal values (eosinophils: 317 51 mm3, IgE: 525

175 IU/ml) (Table 2) .

The percentage of children with eosinophilia was 17% and, children with hyperlgE was 28%.

When percentages of children with eosinophilia were compared between VAR and MAD
children, significantly higher percentage was observed in VAR than in MAD (P<0.001).
Similarly, the percentage of children with hyperlgE was significantly higher in VAR than in

MAD (P<0.001) .
DISCUSSION
Eosinophilia and hyperlgE has been known to occur in association with helminth infec‑
tions or allergic diseases (Bennich and Johansson, 1971; Gleich and Adolphson, 1986) . Many

previous studies demonstrated that eosinophilia and hyperlgE were common in communities
with high prevalence of helminth infections (Johansson et al., 1968; Mahmoud, 1984).
However, it has been also reported that the number of eosinophils and serum lgE Ievel were
not significantly different between helminth infections positive and negative subjects within
a community (Bruce‑Tagoe et al.. 1975; Kaplan et al.. 1980).

In the present study, eosinophilia and hyperlgE were frequently found in VAR children

compared with MAD children. The difference between these two communities was also
apparent in regard to the rate of helminthic infections. These results strongly suggest thateosinophilia and hyperlgE in VAR children were caused by soil‑transmitted helminthiases.
These two schools were located in the same city, and race and life style of the children
was almost the same. The recent epidemics of schistosomiasis and filariasis were not foundin this city (Dr. Ivete Barbosa, personal communication) , suggesting that helminth infections

other than soil‑transmitted may be excluded from the etiology for eosinophilia and hyperlgE.
It has been reported that the incidence of allergic diseases in tropical area is identical tothose in temperate area, especially in urban district (Lynch et al.. 1984) . Anti‑house dust lgEantibody was chosen as a marker of allergic diseases in this study, since house dust antigen
is the most popular allergen in tropical residents (Lynch et al.. 1984) . The prevalence rates

of anti‑house dust lgE antibodies were almost the same in children of both VAR and MAD,suggesting that allergic diseases were not involved in the major etiologies for eosinophilia

and hyperlgE in VAR children.
The number of eosinophils and serum lgE Ievel in VAR children were significantly
correlated. Therefore, it is likely that infected helminths concurrently caused eosinophilia

and hyperlgE. Helminthic infections preferentially induce one of the subsets of helper T

lymphocytes (TH2), which produce both interleukin‑4 (IL‑4) and interleukin‑5 (IL‑5)(Mosmann and Coffman, 1989) . These lymphokines have the function to elicit coincidently
hyperlgE and eosinophilia, namely IL‑4 accelerates B Iymphocytes to secrete lgE (Stavnezer
et al.. 1988) , and IL‑5 promote proliferation of eosinophils in bone marrow (Coffman et al..

1989). It is also possible that lgE antibody, produced by stimulations with helminths,
subsequently gives signals to mast cells to release eosinophil chemotactic factor which cause
eosinophilia.

207

Additional investigation into the precise mechanisms for eosinophilia and hyperlgE inthese patients with soil‑transmitted helminthiases is needed.

ACKNOWLEDGEMENT
The authors wish to thank Ms. Iran Duarte and Mr. Amaro Cosme for their technicalassistance.

REFEREN CESl ) Bennich, H. and Johansson, S.G.O. (197D: Structure and function of human immunoglobulin
E, Adv. Immunol., 13, 1‑55
2 ) Bruce‑Tagoe, A:A., Belcher, D.W., Wurapa, F.K., Turkson, P., Nicholas, D.D. and Ofosu‑
Amaah, S. (1977): Hematological values in a rural Ghanaian population, Trop. geogr. Med.,29, 237‑244

3 ) Coffman, R.L., Seymour, B.W.P., Hudak, S., Jackson, J. and Rennick, D. (1989): Antibody tointerleukin‑5 inhibits helminth‑induced eosinophilia in mice, Science, 245, 308‑310
4 ) Gleich, G.L. and Adolphson, C.R. (1986) : The eosinophilic leukocyte: Structure and function,

Adv. Immunol., 39, 177‑253
5 ) Johansson, S.G.O., Mellbin, T. and Vahlquist, B. (1968): Immunoglobulin levels in Ethiopian
preschool children with special reference to high concentrations of immunoglobulin E (IgND) ,Lancet, 1, 1118‑1121

6 ) Kaplan, J.E., Larrick, J.W. and Yost, J.A. (1980) : Hyperimmunoglobulinemia E ip the
Waorani, an isolated amerindian populatipon, Am. J. Trop. Med. Hyg., 29, 1012‑1017
7 ) Lynch, N.R., Medouze, L., DiPrisco‑Fuenmayor, M.C., Verde, O., Lopez, R.1. and Malave, C.
(1984): Incidence of atopic disease in a tropical environment: Partial independence fromintestinal helminthiasis, J. Allergy Clin. Immunol., 73, 229‑233

8 ) Mahmoud, A.A.F. (1984): Tropical and Geographic Medicine, 70‑75 McGraw‑Hill, New York9 ) Mosmann, T.R. and Coffman, R.L. (1989): THI and TH2 cells: different patterns of lympho‑
kine secretion lead to different functional properties, Annu. Rev. ImmunQl. 7, 145‑173
10) Stavenzer, J.. Radcliffe. G., Lin, U.C., Nietupski, J., Berggren, L., Sitia, R. and Severinson, E.

(1988): Immunoglobulin heavy‑chain switching may be directed by prior induction of tran‑
scripts from constant‑region genes, Proc. Natl. Acad. Sci. USA, 85, 7704‑7708

208

ブラジル，レシフェ市内小学生の好酸球増多症，高IgE血症の病因
浜田 篤郎1・渡辺 直煕1・小林
野崎 智義3・Ive￠Barbosa4・建野

仁2・奥沢 英一3
正毅4・小林昭夫1・

熱帯地住民に高率にみられる好酸球増多症および高IgE血症の病因を究明するため，ブラジル，レシフェ市内の2つの小学校生徒を対象に，末梢血好酸球数，血清総IgE値を測定した。また，蠕虫感染の指標として糞便内蠕虫卵検査，アトピー性疾患の指標として血清抗ハウスダスト

IgE抗体価の測定を施行した。2校の生徒間で，抗ハウスダストIgE抗体陽性率には差がなかったが，糞便内蠕虫卵陽性率には著明な差を認めた。検出された虫卵は，全て土壌伝播蠕虫卵であった。好酸球増多症（好酸球数500／mm3以上），高IgE血症（lgE値5001U／ml以上）を呈する生徒
は，糞便内蠕虫卵陽性率が高い小学校において，有意に高率に認められた。

以上から，この地方における小学生の好酸球増多症および高IgE血症の病因として，土壌伝播
蠕虫感染が考えられた。

東京慈恵会医科大学寄生虫学教室
千葉大学医学部寄生虫学教室
慶応大学医学部寄生虫学教室
浅見敬三免疫病理センター

Jpn. J. Trop. Med. Hyg., Vol. 19 No. 2 1991, pp. 209‑217

209

EVALUATION AND CHARACTERIZATION OF

PARTIALLY PURIFIED SKIN TEST ANTIGENSPREPARED FROM LEISHMANIA PANAMENSISPROMASTIGOTESMASATO FURUYAl, SHIGEO NoNAKA2, EDUARDO A. GOMEZ L.3
AND YOSHIHISA HASHIGUCHI'
Received April 5 1991/Accepted May 2 1991

Abstract: The present study was designed to evaluate skin test preparations preparedfrom Leishmania panamensis promastigotes in 30 active cutaneous leishmaniasis patients.
The crude antigen preparation (CA) used was l0,000 X g supernatant of the parasites‑
homogenate. The soluble extract was further resolved into 4 preparations (FA‑1 to ‑4)
with the aid of a Sephacryl S‑200 gel filtration. There was no significant difference in thepositive ratio and the average induration size between CA (10 pg protein/test) andMontenegro's antigen (MA; 5 x 106 parasites/test). The rea tivity of the delayed‑typehypersensitivity to 10 pg dose of CA was shown with much the same intensity in the 25 pg
dose of CA. In FAS (lO pg protein dose, except for 7.5 pg in FA‑4) , the positive ratio was

as follows: 90.0% in FA‑1, 77.8% in FA‑2, 75.0% in FA‑3 and 37.5% in FA‑4. The positive
ratio and the intensity of skin test response in FA‑4 were remarkably low in comparison
with those in CA, or MA. Significant difference was found in the intensity of response
between FA‑3 and CA or MA. Based on these results, therefore, we concluded that 10 pg
protein dose of CA of L. panamensis and same dose of the fractionated preparations,
FA‑1 and ‑2, were very suitable for the diagnosis of cutaneous leishmaniasis in endemicareas of the New World. Furthermore, it was estimated that at least some or all of the
5 proteins, approximately 66, 55, 45, 28, and 26 kD, were related to a specific delayed‑typehypersensitivity in cutaneous leishmaniasis of the New World.

INTRODUCTION
The intradermal skin test is widely used for a presumptive diagnosis of visceral andcutaneous leishmaniasis in endemic areas of the Central and South America. Although theantigen commonly used is a suspension of whole parasites in phenolized saline (Buss, 1929) ,1 Institute for Laboratory Animals, Kochi Medical School, Nankoku, Kochi 783. Japan
2 Department of Dermatology. School of Medicine, Nagasaki University, Nagasaki, Nagasaki
852, Japan
3 Departamento de Medicina Tropical, Facultad de Medicina, Universidad Catolica Santiago de
Guayaquil, Guayaquil, Ecuador
4 Department of Parasitology, Kochi Medical School, Nankoku, Kochi 783, Japan
This study was supported by the Grand‑in‑Aid for Overseas Scientific Research of the
Ministry of Education, Science and Culture, Japan (Nos. 62043055 and 63041096) .

210

a little information on other preparations prepared from promastigote‑extracts have been
also reported (Furtado and Pellegrino, 1956; La Placa et al., 1975; Shaw and Lainson, 1975;
Reed et al.. 1986) . Among them, Reed et al. (t 986) have reported that a crude soluble extract

prepared from ruptured Leishmania chagasi promastigotes was highly sensitive and specificfor the diagnosis of American visceral leishmaniasis. We have also demonstrated that asimilarly prepared soluble extract obtained from L. braziliensis complex was very useful forthe screening of cutaneous leishmaniasis in endemic areas of Ecuador (Furuya et al.. 1989) .
In order to gain a better information on a standardization of skin test preparation and theantigen dose, we designed preliminary examinations to evaluate skin test preparations
prepared from L, panamensis promastigotes. The present paper reveals that the soluble
promastigotes extract and fractionated prepara ions of the soluble extract are highly sensi‑
tive for the diagnosis of active cutaneous leishpraniasis, and a characterization of protein

components of the preparations will be discussed in this paper.

MATERIALS AND METHODSSkin test preparations

Crude antigen preparation (CA): L. panamensis (MHOM/PA/71/LS94) obtained fromDr. P. Desjeux, PDP, WHO (formerly Instituto

oliviano de Biologia de Altura, Bolivia) was

cultured with the medium described by Pan (1984). After washing of parasites with abalanced salt solution, the harvested promastigotes were ruptured by a freeze‑thawing
procedure and centrifuged at 10,000 X g for 30 min at 4'C (Furuya et al.. 1989) . The super‑
natant was filtered through 0.45 p sterile filter and lyophilized as CA.

Fractionated antigen preparation (FA): The above mentioned soluble extract was
further resolved into 4 preparations, designated FA‑1, ‑2, ‑3, and ‑4, with the aid of aSephacryl S‑200 (Pharmasia, Uppsala, Sweden) column being in equilibrium with 0.02 M
phosphate buffered saline (pH 7.2) . The solution of each peaks was condenced by ultrafilter(MW cut‑out 10,000; Advantex Co., Japan) , dialyzed against PBS, and then centrifuged by
10,000 Xg for 30 min at 4'C. After filtration, these FAS Were adjusted 100 pg protein
concentration per ml, except for 75 pg in FA‑4.
Montenegro's antigen preparation (MA): 5 x 107 whole promastigotes per ml in sterile
saline containing 0.5% phenol was used (Bray, 1985) .
Skin test

Skin test was made on 30 patients with active cutaneous leishmanial lesions. The
preparations were injected intradermally in 0.1 ml in flexor surface of the forearm. The skintest area was observed for erythema and induration at 48 hrs. Induration size of more than
5 mm (mean value of length and "breadth) at the injection site was considered a positive
reaction.

Analysis of compone,ets of CA and FAS
CA and FAS Were solubilized with 60 mM Tris‑HCI buffer (pH 6.8) containing 2% SDS,
10% glycerol, and 5% 2‑mercaptoethanol. Samples were analyzed by SDS‑PAGE as de‑scribed by Laemmli (1970). After electrophoresis gel was stained by 0.25% Coomassie
brilliant blue R250.

211

RESULTS
Skin test

Intradermal skin test using MA, CA, and FAS Was carried out against 30 patients. The
results were shown iri Tables I and 2. Most of the patients had one or two active cutaneous
lesions infected at 3 to 8 months ago. Parasites were confirmed from the lesions of 29 patientsby smear specimens or culture method. Ten strains of Leishmania isolated from thesepatients were characterized by performing zymodeme, serodeme and schizodeme analysis(McMahon‑Pratt et al.. 1982; Lopes et al.. 1984) : 9 strains were identified as L. panamensis.

and one strain was L. braziliensis (Table 1). The detailed characterization of these strains

will be reported elsewhere.

Intensity of induration size of skin test using CA and MA was shown in Table 2. The CA

at 25 pg protein dose gave positive response in all 14 individuals, but, a superabundant
response was also recognized in 2 subjects (Table 2) . The 10 pg protein dose of CA and MA
gave 87.5% (14/16) and 93.8% (15/16) positivity, respectively. No response against CA andMA was observed in one patient. This patient had numerous lesions on the upper side of hisright arm and had been treated with a corticosteroid for 3 months. There was no significant
difference in the positive ratio and the average induration size between 25 and 10 pg dose of
CA. Moreover, the reactivity of the delayed‑type hypersensitivity (DTH) to 10 pg dose of

CA was shown with much the same intensity in the MA.
As shown in Fig. 1, the soluble promastigotes extract was separated seven to eight peaks

by Sephacryl S‑200 gel filtration. An evaluation of skin test using FAS Was made on 10

Table I Intradermal skin test responses to Montenegro's antigen (MA) , crude antigen (CA) ,and fractionated antigens (FA) prepafed from Leishmania panamensis in 10 individ‑
uals with active cutaneous leishmanial lesions

ParasiteMonths. Induration size (mm)No. Sex* Age after CA FA‑1 FA‑2 FA‑3 FA‑4 Smeart Speciest

infection MA

2
3
4

5
6
7
8
9

10

M
M
M

28

3

9

8

6

7

6

41

4

16

20

25

19

8

5

23

3

12

10

16

18

16

9

F

25

3

10

11

20

2

2

2

M
M
M
M
M
M

19

3

6

4

5

5

5

22

4

lO

11

4

2

3

2

16

7

3

4

16

17

10

21

4

20

12

7

7

7

6

38

12

12

15

22

N.D.

N .D.

N.D.

40

?

13

18

lO

10

N.D.

N.D.

21

Positive ratio

lOO . O 90 . O 90 . O 77 . 8 75 . O 37 . 5

Average induration

12.4
12.6 12.5 9.6 6.8 4.4(4 . 1) (4 . 9) (7 . 7) (6 . 6) (4 . 3) (2 . 3)

size (!ISD)* M, male; F, femalet + , positive; ‑ , negativet L.p., Leishmania panamensis: L.b., Leishmania braziliensis‑

+
+
+
+
+

L.p.

+
+
+
+

L,p.

L. p.

L.b.
L. p.

L.p.

L.p,
L.p,

212

Table 2 Frequency distribution of induration size of lei‑
shmanial skin test using crude antigen (CA) andMontenegro's antigen (MA) in 30 patients withactive cutaneous leishmanial lesions

MA

CA

IndurationlO

25

size (mm)5 x 106(parasites)(pg protem)

<5

2

5 ‑lO

3

4

11‑15
16‑20
21‑25

6

7

6

6

3

3

1

2

> 26

2*

Averageinduration

20 . l12 . 7

13 . 3:!:7. 2

13 .

1+5. 8size* Induration size of 2 examinees was 38 x 27 mm and 80 X
40 mm, ,respectively.

patients. The positive ratio against each FAS Was as follows: 90.0% (9/10) in FA‑1, 77.8%(7/9) in FA‑2, 75.0% (6/8) in FA‑3, and 37.5% (3/8) in FA=4. Among 8 subjects received
all of the FAs, 3 patients reacted to all of the FAs, 2 patients reacted to FA‑1, ‑2 and ‑3, and1 patient only reacted to FA‑1. In the last patient (No. 4), however, 6, 13, and 6 mm size oferythema was observed in the injection site of FA‑2, ‑3, and ‑4, respectively. On the other

hand, one patient (No. 6), showing positive reaction against MA and CA, did not reactagainst all of the FAs. There was significant difference in po itive ratio of skin test response

between FA‑4 and CA or MA (p<0.001). Furthermore, significant difference was found in
the average indurat, ion size between FA‑3 and CA or MA (p<0.025) .

SDS‑PAGE profiles of CA and FAS
Lane I in Fig. 2 shows the Coomassie brilliant blue R250‑ tained profile of the* PA, andlanes 2 to 5 show the pattern of FAs. About 35 bands were recognized in lane I (CA*) . Sevenbands were weakly stained in lane 2 (FA‑1) , 25 bands were recognized in lane 3 (FA‑2) , 18

bands were observed in lane 4 (FA‑3), and 9 bands in lane 5 (FA‑4).̲ Among these bands,
4 bands migrating in the region approximately 66, 55, 45, and 26 kilodalton (kD) werecommon to all of the FAs. SDS‑PAGE proved that most of protein components of the soluble
promastigotes extract of L. panamensis was recovered in FA‑2 area by Sephacryl S‑200 gel
filtration.

DISCUSSION
Until recently, no exact information has been available on species or subspecies level
characterization of the genus Leishmania of cutaneous leishmaniasis in this country. Threestrains of Leishmania isolated from active cutaneous leishmaniasis patients have been firstcharacterized as L. panamensis by isoenzyme electrophoresis and monoclonal antibodies

213

A 2 50 nm
FA ‑ 3

o.5

o.3

T

FAf 2

FA7‑1

.,.. ' :

FA‑4
o,

30

60

90

1 20

Frac'ion number
Figure 1 Ef uent pattern of saluble protein extract of L. pan:amelcsis by
Sephacryl S‑200 gel filtration. Each of the shadded area of figure
was collected and concentrated by ultrafilter as a FA preparation ofskin test, column, 2.5x60 cm; buffer, O.02 M phosphate bufferedsaline (pH 7.2); sample, 2.5 ml of soluble protein extract of L.
panamensis; flow rate, 18 ml/hr; fraction, 3 ml/tube.

kD .. 1 2 3 4 5
2 O 5 i,‑;* ,1 i 6 ･*;,,*" .....,'{･{:'t!'*:9 7.4 )･i#

e6

･*

45 ･*

34.7 *
2 9 F
2 4 ･*:'{

1 8,4 {{

i 4,3
Figure 2 SDS‑PAGE profile of soluble extract preparations prepared from L.

panameltsis‑promastigotes. Lanes: l, crude antigen preparation(CA; 45 ;eg protein) ; 2, fractionated antigen preparation FA‑1 (i4,4g protein) ; 3, FA2 (48 fseg protein) ; 4, FA‑3 (28 ,tg protein) ; 5,

FA‑4 (17 ; g protein) . The electrophoresis was done at 6.5 mA for

12 hrs. The positions of molecular size are indicated. kD,
kilodaltons. 12% gel.

214

(Mimori et al., 1989). Of the 26 strains newly isolated in this country, 23 were identified as

L. braziliensis complex (12 L. panamensis, 7 L. guyanensis and 4 L. braziliensis) by enzyme
electrophoresis (Armijos et al.. 1990) . In the present studies, therefore, we used L. panamen‑

sis promastigotes for preparing of the present skin test preparations.
Although a standerdization of antigen concentration in intradermal skin test for visceraland cutaneous leishmaniasis had not yet been done, 50 to 25 pg protein dose is used normally(Kerdel‑Vegas, 1982; Bray, 1985; Reed et al.. 1986). In the present study, it was definitely

shown that 10 pg dose of CA was able to stand comparison with a relatively large numberof promastigotes of MA in detecting DTH in patients with active cutaneous lesions. There
was no siguificant difference in the positive ratio and the average induration size between
25 pg and 10 pg antigen dose of CA. In the test with 25 pg protein dose of CA, superabundant

intradermal response was observed in a few active and some cured cutaneous leishmaniasis
individuals (data not shown) . From these results, 10 pg protein dose of soluble promastigotes

extracts (CA) of L. panamensis will be suitable for the diagnosis using intradermal skin test

against cutaneous leishmaniasis in the New World.
For a characterization of skin test antigens, it has been recently reported that two definedglycoconjugates purified from L. amazonensis was able to induce a specific DTH response toinfected susceptible and resistant mice strains, and that one of the glycoconjugates was adegradation product of a 17 kD antigen present in promastigotes and amastigotes (Rodrigues
et al., 1986a, b). Partially purified antigens containing 94 to 64 kD proteins, derived from L.

infantum or L. major promastigotes and isolated under reducing conditions with SDS‑PAGE,
were also able to induce specific DTH reactions in mice (Frommel et al.. 1988) . In thepresent studies using partially purified preparations, no significant difference was observed in

the positive ratio and the average induration size between FA‑1 and ‑2 and CA or MA. By
SDS‑PAGE analysis of CA and FAs, 5 bands migrating the region approximately 66, 55, 45,
28 and 26 kD were common to the both FAs. From these results, it is assumed that at least
some or all of these 5 antigens of L. panamensis may be related to a specific DTH response
in active cutaneous leishmaniasis patients infected with L. braziliensis complex.

Cross‑reactivity at the skin test level between different leishmanial species has been
demonstrated in humans and experimental animals (Manson‑Bahr, 1961; Adler and Gunders,
1964; Bryceson et al.. 1970) , although the reactions to heterologous organisms appeared to beof lesser magnitude (Weissberger et al.. 1973; Neal and Miles, 1976). Recently, Reed et al.

(1986) also reported that a crude soluble promastigotes extract prepared from a heterologous
parasite, L. amazonensis, was clearly less effective than a crude extract prepared from ahomologous parasite, L. chagasi, in detecting DTH in cured American visceral leishmaniasis
patients. In the present examinations, it was found that the present preparation was highly
sensitive in the intradermal skin test against active cutaneous leishmaniasis patients sufferingfrom heterologous organisms, L. braziliensis. There was no appreciable difference in theintensity of responses in patients caused by homologous and heterologous organismus. The
results suggest that the present CA and FAS contain some common antigens, may be highly
antigenic components, to L. braziliensis. It was concluded that the soluble extracts of L.
panamensis would be very useful for diaguosis of active or cured cutaneous leishmaniasis
caused by L. braziliensis complex in the endemic areas of the New World.

21 5

ACKNOWLEDGEMENTS
We express our heartfelt thanks to Dr. F. Parra Gil, of Instituto Nacional de Hygiene y

Medicina Tropical (INHMT) , Guayaquil, Ecuador, and also express our appreciation to Dr.

R.B. Tesh, Department of Epidemiology and Public Health, School of Medicine, Yale
University, Dr. G. Grimaldi, Jr., Department of Immu ology, Instituto Oswardocruz, and Dr.

R. D. Kreutzer, Biology Department, Youngstown State University, for identification of
species of 10 isolated strains. We are much indebted to all the members of the Departamento

de Parasitologia, INHMT.

REFERENCES
1 ) Adler, S. and Gunders, A.E. (1964): Immunity to Leishmania mexicana following spontaneousrecovery from oriental sore, Trans.' R. Soc. Trop. Med. Hyg., 58, 274‑2772 ) Armijos, R.X.. Chico, M.E., Cruz, M.E.. Guderian, R.H., Kreutzer, R.D.. Berman, J.D., Rogers,
M.D. and Grogl, M. (1990): Human cutaneous leishmaniasis in Ecuador: Identification ofparasites by enzyme electrophoresis. Am. J. Trop. Med. Hyg., 42, 424‑428
3 ) Bray, R.S. (1985): Immunodiagnosis of leishmaniasis. In: Leishmaniasis (Edited by Chang,

K‑P. and Bray. R.S.), 177‑182. Amsterdam‑New York‑Oxford, Elsevier
4 ) Bryceson, A.D.M.. Bray, R.S., Wolstencroft, R.A. and Dumonde, D.C. (1970): Immunity incutaneous leishl raniasis of the guinea‑pig. Clin. Exp. Immunol., 7, 301‑341
5 ) Buss, G. (1929): Untersuchungen mit Leishmania Vakzine, Arch. F. Schiffs. N. Trop. Hyg., 33,

65‑726 ) Frommel, D., Ogunkolade, B.W., Vouldoukis, I. and Monjour, L. (1988): Vaccine‑induced
immunity against cutaneous leishmaniasis in BALB/C mice, Infect. Immun., 56, 843‑848
7 ) Furtado. T.A. and Pellegrino, J. (1956) : Intradermal test in American leishmaniasis with apolysaccharide fraction isolated from Leishmania brasiliensis. J. Invest. Dermatol., 27, 53‑59

8 ) Furuya, M., Mimori, T., Gomez. E.A.L., Coronel, V.V., Kawabata, M. and Hashiguchi, Y.
(1989) : Epidemiological survey of leishmaniasis using skin test and ELISA in Ecuador, Japan.

J. Trop. Med. Hyg., 17, 331‑338
9 ) Kerdel‑Vegas, F. (1982) : American leishmaniasis, Int. J. Dermatol., 21, 291‑303
10) La Placa, M., Pampiglione. S., Borgatti. M. and Zerbini, M. (1975): Complement fixation andintradermal skin test with partially purified "proteinic" and "polysaccharidic" antigens from

Leishmania donovani. Trans. R. Soc. Trop. Med. Hyg., 69, 396‑398
11) Laemmli, U.K. (1970) : Cleavage o,f structural proteins during the assembly of the head ofbacteriophage T4, Nature, 227, 680‑685
12) Lopes, U.G., Momen, H., Grimaldi. G. Jr., Marzochi, M.C., Pacheco, R.S. and Morel. C,M.
(1984) : Schizodeme and zymodeme characterization of Leishmania in the investigation of fociof visceral and cutaneous leishmaniasis, J. Parasitol., 70, 89‑98

13) Manson‑Bahr. P.E.C. (1961): Immunity in Kala‑azar, Trans. R. Soc. Trop. Med. Hyg., 5,
550‑555
14) McMahon‑Pratt, D., Bennet, E. and David, J.R. (1982): Monoclonal antibodies that distinguishsubspecies of Leishmania braziliensis. J. Immunol., 129, 926‑927
15) Mimori, T.. Grimaldi, G. Jr.. Kreutzer, R.D., Gomez, E.A.L., McMahon‑Pratt, D., Tesh, R.B.

and Hashiguchi, Y. (1989): Identification of Leishmania isolated from humans and wild
animals in Ecuador, using isoenzyme electrophoresis and monoclonal antibodies, Am. J. Trop.

Med. Hyg., 40, 156‑160

16) Neal, R.A. and Miles, R.A. (1976): The Montenegro reaction in guinea‑pig infected by

216

Leishmania enriettii and the effect of antigens prepared from various Leishmania isolates, J.

Trop. Med. Hyg., 79, 32‑37
17) Pan, A.A. (1984): Leishmania mexicana: Serial cultivation of intracellular stages in a cell‑freemedium, Exp. Parasitol., 58, 72‑80
18) Reed, S.G., Badaro, R., Masur, H., Carvalho, E.M., Lorenco, R., Lisboa, A., Teixeira, R.,
Johnson, W.D. Jr. and Jones, T.C. (1986) : Selection of a skin test antigen for American visceralleishmaniasis, Am. J. Trop. Med. Hyg., 35, 79‑85
19) Rodrigues, M.M., Xavier, M.T., Previato, L.M. and Barcinski, M.A. (1986a): Characterizationof cellular immune response to chemically defined glycoconjugates from Leishmania mexicana
subsp. amazonensis. Infect. Immun., 51, 80‑86

20) Rodrigues, M.M., Xavier, M.T., Previato, L.M. and Barcinski, M.A. (1986b): Novel 17‑
kilodalton Leishmania antigen revealed by immunochemical studies of a purified glycoprotein
fraction recognized by murine T Iymphocytes, Infect. Immun., 56, 1766‑1770
2D Shaw, J.J. and Lainson, R. (1975): Leishmaniasis in Brazil: X. Some observations on intrader‑mal reactions to different trypanosomatid antigens of patients suffering from cutaneous andmucocutaneous leishmaniasis, Trans. R. Soc. Trop. Med. Hyg., 69, 323‑335
22) Weissberger, H., Spira, D.T. and Zuckerman, A. (1973): Delayed hypersensitivity to various
leishmania antigens in guinea‑pigs infected with Leishmania enriettii. J. Protozool., 20, 534‑535

217

L召ゑsh㎜痂αρ伽σ卿伽sゑspromastigote型原虫から精製した

皮内反応用抗原の効果判定とその特性
古谷正人1・野中薫雄2・E．A．L．Gomez3・橋口

義久4

五傭h脚痂α加％吻8％s∫s promastigote型原虫から作成した種々皮内反応用抗原の効果を30人の皮膚型リーシュマニア症患者で判定した。原虫ホモジネートの10，000×g遠心上清を粗抗原（CA）とし，更にSephacryl

S−200ゲルを用いて4画分（FA−1からFA−4）を得た。CA（10μgタンパク量／テスト）による皮内反応の陽性率，および硬結径はMontenegro抗原（MA；5x106
原虫／テスト）でのそれらと比較して優位差がなかった。更に，10μgタンパク量のCAによって誘発される遅延型皮内反応の反応の強さは，同抗原液を25μgタンパク量で用いた場合と同等であった。分画抗原（FA−4は7．5μgタンパク量，他は10μgタンパク量）での皮内反応陽性率はFA−1が90．0％，FA−2が77．8％，FA−3が75．0％，FA−4が37．5％であった。これら4抗原のうち，FA−4は陽性率および反応の強さの両面で，又FA−3は反応の強さの点でCAやMAでのそれらと比較して著しく劣っていることが判明した。以上の結果から，L，勿n佛朋廊原虫から作成した皮内反応用抗原のうち，10μgタンパク量のCAおよび同タンパク量のFA−1，FA−2分画抗原が新大陸での皮膚型リーシュマニア症の診断に適していることが結論づけられた。更に，これらの抗原液を構成しているタンパク質のうち，少なくとも66，55，45，28，26kDタンパクの全て，又は一部が新大陸での皮膚リーシュマニア症の遅延型皮内反応惹起に関与している可能性が示唆された。

高知医科大学医学部附属動物実験施設
長崎大学医学部皮膚科学教室
Departamento

deMedicina

Guayaquil，Ecuador
4 高知医科大学寄生虫学教室

Tropica1，Facultad

deMedicina，Universidad Catolica Santiago deISSN 0304‑2]46

JAPANESE JOURNAL OF
TROPICAL MEDIClNE AND HYGIENE
Vol. 19 No. 2

June 1991

CONTENTS
Review
Colley, D. G. and Howard, M. J.

The Fundamental Nature of Chagas' Disease:a View Provided by Immune Responses and Idiotypes """ ' 175‑190
Original article

Nwoke, B. E. B., Shiwaku, K. and Takahashi, H.

Nigerian Onchocerciasis: Epidemiological Perspective """ ' 191‑201
Hamada, A., Watanabe, N., Kobayashi, M., Okusawa, E., Nozaki, T., Barbosa I
Tateno, S. and Kobayashi, A.
The Etiological Factor for Eosinophilia and Hyperglobulinemia E
in Brazilian School Children" " " " " " """ """ " " " " "" " " """ ": . . . . . ... . . . . . . 203‑208

Furuya, M., Nonaka, S., Gomez L., E. A. and Hashiguchi, Y.
Evaluation and Characterization of Partially Purified Skin Test Antigens

Prepared from Leishmania panamensis Promastigotes" "" " "" """ " "" " " " "' 209‑217

Jpn. J. Trop.

Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDIClNE