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Leishmaniasis in Brazil: IV. The fox, Cerdocyon thous (l ) as a reservoir of Leishmania donovani in Para state, Brazil
741 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.
Vol. 63.
No. 6.
1969.
LEISHMANIASIS IN BRAZIL: IV. THE FOX, CERDOCYON THOU$ (L) AS A RESERVOIR OF LEISHMANIA DONOVANI IN PARA STATE, BRAZIL R. LAINSON, J. J. SHAW AND Z. C. LINS Instituto Evandro Chagas-FSESP, Beldm, Pard, Brazil
Introduction The epidemiology of kala-azar in Brazil has been discussed in such detail by other workers (DEANE,1956;-DEANE & DEANE,1962) that it will suffice here to make but the briefest summary of these authors' studies. Visceral leishmaniasis has been found principally in the North-eastern and Upper Eastern regions of this country, in particular in the State of Cear~. The endemic areas in general have a tropical climate, the major foci of infection being associated with the semi-arid regions which often experience long droughts. Though the disease is widespread in both the plains and plateaus, it is in the foothills and valleys between the hills that kala-azar is most prevalent. The vegetation is relatively sparse, composed of low trees with shrubby undergrowth. Lutzomyia longipalpis is generally regarded as the vector, its distribution and incidence coinciding exactly with that of visceral leishmaniasis. While the North-east remains the real "home" of kala-azar in Brazil, it is fininteresting fact that most of the earliest reported cases came from Parfi State, in the Lower Amazon Region (CHAGAS,et al., 1937, 1938), and that the first major epidemiological studies were made in the Instituto Evandro Chagas by these workers. Here, however, we have a very different terrain to consider--that of the high tropical rain-forest--and in this region kala-azar remains but a sporadic disease of man. As pointed out by DEANE & DEANE (1962) "It was unfortunate that Chagas and co-workers made their studies in a region (the Amazon) which, as we presently realize, was far from typical The importance of the problem went unrecognized and the idea widely spread that the disease was rare in Brazil". A commission to study the epidemiology of kala-azar in the State of Par~ was set up between the years 1936-1940, under the able leadership of Evandro Chagas. Appropriately enough the team included Bel6m-born Leonidas Deane who, with his future wife Maria yon Paumgartten, was later to make outstanding contributions to our knowledge on the epidemiolog~, of the disease in Cear& The studies were principally on the Abaetetuba and Mojfi Districts, some 60 kilometres from Bel6m, and where 8 human cases were described. The commission concluded that the disease was associated only with the drier "terra firme" forests and never with the wetter "varzea". In such areas infections were also found in 7 dogs These studies were conducted under the auspices of the Wellcome Trust, London, the World Health Organization, Geneva and the Instituto Evandro Chagas da Fundag~o Servigo Especial de Safde Pflblica of Brazil. We are indebted to Dr. Alfonso Wisniewski, Director of the Instituto de Pesquisa Experimental Agropecugria do Norte (IPEAN), Bel6m, Parfi, Brazil, who kindly allowed us to obtain foxes from the Institute grounds.
742
FOX RESERVOIR OF L. D O N O V A N 1 I N BRAZIL
and one cat but exhaustive attempts to find infected wild animals were unsuccessful (CHAGAS, et ai., 1937, 1938). The conviction has nevertheless remained that, unlike,kala-azar of the drier Northeast regions, the disease in the forested North is principally one of wild animals (DEANE, 1956). The low incidence in man and dogs supports this view; thus, the total cases reported in the whole of Parfi between the years 1934-1966 was only 30 (COSTA, 1966). The failure to find infected wild animals in Parfi doubtless stimulated interest in Cearfi, where kala-azar is so much more common, and DEANE and DEANE (1954) finally incriminated the fox, Lycalopex vetulus, as an important reservoir. These authors were careful to stress, however, that the situation regarding kala-azar in the North-east is very different from that in Patti, for in the former regions man and dogs are so commonly infected that they can really be regarded as major hosts and major sources of infection. It was also noted that the disease in Lycalopex is particularly severe and that this animal is probably "not an ancient indigenous reservoir such as the one searched for by Chagas, but that it is, on the contrary, a rather recent host of L. donovani". Recently, on the outskirts of Bel6m, Parfi, increasing numbers of foxes have reawakened our interest in reservoirs of L. donovani in this region and we have commenced a search for infections in another member of the Canidae, Cerdocyon thous (L) (Fig. 5). This fox would appear to form an ideal reservoir of infection for man and his domestic animals, for at night it leaves its home in the neighbouring forest and is to be found wandering in search of food, close to human dwelling-places. To date we have examined only 6 animals, for the only effective way of obtaining foxes is to shoot them, at night, We have been fortunate enough, however, to isolate L. donovani from the second fox examined. Materials and methods Five of the foxes, including the infected one, were shot in open, cultivated land in the grounds of the Instituto de Pesqnisa Experimental Agropecufiria do Norte (IPEAN), Bel6m, (Fig. 1) and the sixth from the Utinga area nearby. They were taken as soon as possible to the laboratory and placed in the 4°C. refrigerator prior to examination the next day. All the foxes appeared in excellent condition and presented no evidence of cutaneous lesions or gross pathology of the viscera. Portions of liver and spleen were removed from each animal, triturated together in physiological saline and inoculated into 2 hamsters by the intraperitoneal route. In addition, one of us (Z.C.L.) separately inoculated hamsters with triturated kidney from 4 of the foxes in an attempt to isolate Leptospira. Impression smears of liver, spleen, kidney and lung were stained by the usual Giemsa method and portions of these organs were fixed in Carnoy's fluid for histology. Results One month later, one of the hamsters inoculated with kidney suspension from the second fox died and its companion was killed. Examination of smears of liver, spleen, lung and kidney of both the hamsters showed large numbers of Leishmania (Fig. 6). The parasite was successfully passaged into further hamsters in which it is now maintained and from which it has been isolated in N N N culture. To date, no infections have been demonstrated in the remaining 5 foxes, but subinoculations of further hamsters have yet to be completed.
R. LAINSON~ J. J. SHAW AND Z. C. LINS
743
FIG. 1. Open pasture-land of the Instituto de Pesquisa Experimental Agro-pecugtria do Norte (IPEAN), Beldm, Parfi, where the infected fox was shot. Bordering forest in background. FIGS. 2-4. Sections of kidney from the infected fox. Low-power views showing (2 and 3) confluent foci of round-cell infiltration of interstitial tissue with resulting dissociation of tubules ; and (4) large atrophic area of medulla with ensuing fibrosis (haematoxylin and eosin). FIG. 5. Cerdocyon thous (L) Young male. FIG. 6. Spleen smear from hamster inoculated one month previously with kidney suspension of fox figured above: intra-cellular groups of L. donovani. (Giemsa, × 1400).
744
FOX RESERVOIR OF L. D O N O V A N I I N BRAZIL
Strangely enough, the hamsters inoculated with liver and spleen from the infected fox have also remained negative and this prompted us to make a thorough histological study of the tissues of this fox. No parasites were found in impression smears of the viscera and sections of the foxes' liver, spleen and lung appeared normal. In contrast, sections of the kidney showed huge areas of inflammatory reaction and fibrosis consistent with visceral leishmaniasis (Figs. 2-4). Undoubted parasites have remained difficult to demonstrate in the kidney sections and were clearly scanty in number.
Discussion Obviously, many more animals must be examined before we can say much about the importance of the fox, Cerdocyon thous, in the epidemiology of "sylvatic kala-azar" in North Brazil. The present finding does support the view, however, that the source of human and canine infection lies in the wild forest animals. Two points of interest may be raised. Firstly, the infected fox appeared in perfect health and might indeed be regarded as the "ancient indigenous reservoir" discussed by DEANE and DEANE (1962), unlike its counterpart Lycalopex in Cear~. Secondly, it is strange that the parasite could only be isolated from the kidney. This raises interesting conjectures as to the viscera one should routinely examine in attempts to isolate the parasite from wild animals (normally only liver and spleen). The screening of animals for L. donovani by the examination of smears of the viscera alone is clearly inadequate, for we were unable to find parasites in dab-smears of the kidney which nevertheless produced infection in the inoculated hamsters. The great value of this laboratory animal as a tool in the isolation of Leishmania is once more accentuated. Finally, similar examinations of the viscera of other wild animals in the same area are needed. It is interesting to speculate, for example, on the possible role of opossums as reservoirs of L. donovani, especially Didelphis which is even more closely associated with man than are foxes.
Summary
Leishmania donovani was isolated from 1 of 6 foxes (Cerdocyon thous L.) from the outskirts of Bel6m, Par~, Brazil. Isolation was by the intraperitoneal inoculation of hamsters with triturated kidney tissue, these animals showing a generalized visceral infection one month later. Histologically the kidney of the infected fox showed considerable pathological changes, but otherwise the animal appeared perfectly healthy. Kala-azar is only a sporadic disease of man and dogs in the forested regions of the North of Brazil and is probably a Zoonosis among the wild animals. Sumfirio
Leishmania donovani foi encontrada em uma entre seis rap6sas (Cerdocyon thous) provenientes das redondezas de Bel6m. Isolamento foi obtido por inocula~o intraperitoneal em hamster corn triturado de rim, vindo os referidos animals a morrer um m~s depois com infec~ho visceral generalizada. Cortes histol6gicos do rim da rap6sa infectada mostraram consider~iveis manifestaq6es patol6gicas, embora o animal apresentasse apar~ncia perfeitamente saud~ivel.
R. LAINSON, ~. J. SHAW AND Z. C. LINS
745
Calazar 6 doenqa esporfidica em homen e c~es nas regimes florestais do Norte do Brasil e constitfii, mui provhvelmente, uma zoonose entre animais silvestres. REFERENCES CHAGAS, E., CUNHA, A. M., CASTRO, G. O., FERR~IRA, L. C., & ROMA~A, C., (1937). Meres Inst. Oswaldo Cruz, 32, 321. ,- - , FERREIRA, L. C., DEANE, L., DEANE, G., GUIMARAES, F. N., PAUMGARTTEN~ M. J. & SA, B. (1938). Ibid., 33, 89. COSTA, O. R. (1966). Revta Serv. esp. Sagtde pf~bl., Rio de ft., 12, 91. DWANE, L. M. (1956). "Leishmaniose visceral no Brasil. Estudos s6bre reservat6rios e transmissores realizados no Estado do Cearfi". Thesis. Fac. Med. Univ., S. Paulo, Brasil. Ed. Servi~o Nacional de Educaoo Saniuiria, Rio de Janeiro, Brasil. & DEANE, M. P. (1954). 0 Hospital, Rio deffaneiro, 45, 419. &~ (1962). Revta Inst. IVied. trop., S. Paulo, 4, 198.
Vol. 63.
No. 6.
1969.
LEISHMANIASIS IN BRAZIL: IV. THE FOX, CERDOCYON THOU$ (L) AS A RESERVOIR OF LEISHMANIA DONOVANI IN PARA STATE, BRAZIL R. LAINSON, J. J. SHAW AND Z. C. LINS Instituto Evandro Chagas-FSESP, Beldm, Pard, Brazil
Introduction The epidemiology of kala-azar in Brazil has been discussed in such detail by other workers (DEANE,1956;-DEANE & DEANE,1962) that it will suffice here to make but the briefest summary of these authors' studies. Visceral leishmaniasis has been found principally in the North-eastern and Upper Eastern regions of this country, in particular in the State of Cear~. The endemic areas in general have a tropical climate, the major foci of infection being associated with the semi-arid regions which often experience long droughts. Though the disease is widespread in both the plains and plateaus, it is in the foothills and valleys between the hills that kala-azar is most prevalent. The vegetation is relatively sparse, composed of low trees with shrubby undergrowth. Lutzomyia longipalpis is generally regarded as the vector, its distribution and incidence coinciding exactly with that of visceral leishmaniasis. While the North-east remains the real "home" of kala-azar in Brazil, it is fininteresting fact that most of the earliest reported cases came from Parfi State, in the Lower Amazon Region (CHAGAS,et al., 1937, 1938), and that the first major epidemiological studies were made in the Instituto Evandro Chagas by these workers. Here, however, we have a very different terrain to consider--that of the high tropical rain-forest--and in this region kala-azar remains but a sporadic disease of man. As pointed out by DEANE & DEANE (1962) "It was unfortunate that Chagas and co-workers made their studies in a region (the Amazon) which, as we presently realize, was far from typical The importance of the problem went unrecognized and the idea widely spread that the disease was rare in Brazil". A commission to study the epidemiology of kala-azar in the State of Par~ was set up between the years 1936-1940, under the able leadership of Evandro Chagas. Appropriately enough the team included Bel6m-born Leonidas Deane who, with his future wife Maria yon Paumgartten, was later to make outstanding contributions to our knowledge on the epidemiolog~, of the disease in Cear& The studies were principally on the Abaetetuba and Mojfi Districts, some 60 kilometres from Bel6m, and where 8 human cases were described. The commission concluded that the disease was associated only with the drier "terra firme" forests and never with the wetter "varzea". In such areas infections were also found in 7 dogs These studies were conducted under the auspices of the Wellcome Trust, London, the World Health Organization, Geneva and the Instituto Evandro Chagas da Fundag~o Servigo Especial de Safde Pflblica of Brazil. We are indebted to Dr. Alfonso Wisniewski, Director of the Instituto de Pesquisa Experimental Agropecugria do Norte (IPEAN), Bel6m, Parfi, Brazil, who kindly allowed us to obtain foxes from the Institute grounds.
742
FOX RESERVOIR OF L. D O N O V A N 1 I N BRAZIL
and one cat but exhaustive attempts to find infected wild animals were unsuccessful (CHAGAS, et ai., 1937, 1938). The conviction has nevertheless remained that, unlike,kala-azar of the drier Northeast regions, the disease in the forested North is principally one of wild animals (DEANE, 1956). The low incidence in man and dogs supports this view; thus, the total cases reported in the whole of Parfi between the years 1934-1966 was only 30 (COSTA, 1966). The failure to find infected wild animals in Parfi doubtless stimulated interest in Cearfi, where kala-azar is so much more common, and DEANE and DEANE (1954) finally incriminated the fox, Lycalopex vetulus, as an important reservoir. These authors were careful to stress, however, that the situation regarding kala-azar in the North-east is very different from that in Patti, for in the former regions man and dogs are so commonly infected that they can really be regarded as major hosts and major sources of infection. It was also noted that the disease in Lycalopex is particularly severe and that this animal is probably "not an ancient indigenous reservoir such as the one searched for by Chagas, but that it is, on the contrary, a rather recent host of L. donovani". Recently, on the outskirts of Bel6m, Parfi, increasing numbers of foxes have reawakened our interest in reservoirs of L. donovani in this region and we have commenced a search for infections in another member of the Canidae, Cerdocyon thous (L) (Fig. 5). This fox would appear to form an ideal reservoir of infection for man and his domestic animals, for at night it leaves its home in the neighbouring forest and is to be found wandering in search of food, close to human dwelling-places. To date we have examined only 6 animals, for the only effective way of obtaining foxes is to shoot them, at night, We have been fortunate enough, however, to isolate L. donovani from the second fox examined. Materials and methods Five of the foxes, including the infected one, were shot in open, cultivated land in the grounds of the Instituto de Pesqnisa Experimental Agropecufiria do Norte (IPEAN), Bel6m, (Fig. 1) and the sixth from the Utinga area nearby. They were taken as soon as possible to the laboratory and placed in the 4°C. refrigerator prior to examination the next day. All the foxes appeared in excellent condition and presented no evidence of cutaneous lesions or gross pathology of the viscera. Portions of liver and spleen were removed from each animal, triturated together in physiological saline and inoculated into 2 hamsters by the intraperitoneal route. In addition, one of us (Z.C.L.) separately inoculated hamsters with triturated kidney from 4 of the foxes in an attempt to isolate Leptospira. Impression smears of liver, spleen, kidney and lung were stained by the usual Giemsa method and portions of these organs were fixed in Carnoy's fluid for histology. Results One month later, one of the hamsters inoculated with kidney suspension from the second fox died and its companion was killed. Examination of smears of liver, spleen, lung and kidney of both the hamsters showed large numbers of Leishmania (Fig. 6). The parasite was successfully passaged into further hamsters in which it is now maintained and from which it has been isolated in N N N culture. To date, no infections have been demonstrated in the remaining 5 foxes, but subinoculations of further hamsters have yet to be completed.
R. LAINSON~ J. J. SHAW AND Z. C. LINS
743
FIG. 1. Open pasture-land of the Instituto de Pesquisa Experimental Agro-pecugtria do Norte (IPEAN), Beldm, Parfi, where the infected fox was shot. Bordering forest in background. FIGS. 2-4. Sections of kidney from the infected fox. Low-power views showing (2 and 3) confluent foci of round-cell infiltration of interstitial tissue with resulting dissociation of tubules ; and (4) large atrophic area of medulla with ensuing fibrosis (haematoxylin and eosin). FIG. 5. Cerdocyon thous (L) Young male. FIG. 6. Spleen smear from hamster inoculated one month previously with kidney suspension of fox figured above: intra-cellular groups of L. donovani. (Giemsa, × 1400).
744
FOX RESERVOIR OF L. D O N O V A N I I N BRAZIL
Strangely enough, the hamsters inoculated with liver and spleen from the infected fox have also remained negative and this prompted us to make a thorough histological study of the tissues of this fox. No parasites were found in impression smears of the viscera and sections of the foxes' liver, spleen and lung appeared normal. In contrast, sections of the kidney showed huge areas of inflammatory reaction and fibrosis consistent with visceral leishmaniasis (Figs. 2-4). Undoubted parasites have remained difficult to demonstrate in the kidney sections and were clearly scanty in number.
Discussion Obviously, many more animals must be examined before we can say much about the importance of the fox, Cerdocyon thous, in the epidemiology of "sylvatic kala-azar" in North Brazil. The present finding does support the view, however, that the source of human and canine infection lies in the wild forest animals. Two points of interest may be raised. Firstly, the infected fox appeared in perfect health and might indeed be regarded as the "ancient indigenous reservoir" discussed by DEANE and DEANE (1962), unlike its counterpart Lycalopex in Cear~. Secondly, it is strange that the parasite could only be isolated from the kidney. This raises interesting conjectures as to the viscera one should routinely examine in attempts to isolate the parasite from wild animals (normally only liver and spleen). The screening of animals for L. donovani by the examination of smears of the viscera alone is clearly inadequate, for we were unable to find parasites in dab-smears of the kidney which nevertheless produced infection in the inoculated hamsters. The great value of this laboratory animal as a tool in the isolation of Leishmania is once more accentuated. Finally, similar examinations of the viscera of other wild animals in the same area are needed. It is interesting to speculate, for example, on the possible role of opossums as reservoirs of L. donovani, especially Didelphis which is even more closely associated with man than are foxes.
Summary
Leishmania donovani was isolated from 1 of 6 foxes (Cerdocyon thous L.) from the outskirts of Bel6m, Par~, Brazil. Isolation was by the intraperitoneal inoculation of hamsters with triturated kidney tissue, these animals showing a generalized visceral infection one month later. Histologically the kidney of the infected fox showed considerable pathological changes, but otherwise the animal appeared perfectly healthy. Kala-azar is only a sporadic disease of man and dogs in the forested regions of the North of Brazil and is probably a Zoonosis among the wild animals. Sumfirio
Leishmania donovani foi encontrada em uma entre seis rap6sas (Cerdocyon thous) provenientes das redondezas de Bel6m. Isolamento foi obtido por inocula~o intraperitoneal em hamster corn triturado de rim, vindo os referidos animals a morrer um m~s depois com infec~ho visceral generalizada. Cortes histol6gicos do rim da rap6sa infectada mostraram consider~iveis manifestaq6es patol6gicas, embora o animal apresentasse apar~ncia perfeitamente saud~ivel.
R. LAINSON, ~. J. SHAW AND Z. C. LINS
745
Calazar 6 doenqa esporfidica em homen e c~es nas regimes florestais do Norte do Brasil e constitfii, mui provhvelmente, uma zoonose entre animais silvestres. REFERENCES CHAGAS, E., CUNHA, A. M., CASTRO, G. O., FERR~IRA, L. C., & ROMA~A, C., (1937). Meres Inst. Oswaldo Cruz, 32, 321. ,- - , FERREIRA, L. C., DEANE, L., DEANE, G., GUIMARAES, F. N., PAUMGARTTEN~ M. J. & SA, B. (1938). Ibid., 33, 89. COSTA, O. R. (1966). Revta Serv. esp. Sagtde pf~bl., Rio de ft., 12, 91. DWANE, L. M. (1956). "Leishmaniose visceral no Brasil. Estudos s6bre reservat6rios e transmissores realizados no Estado do Cearfi". Thesis. Fac. Med. Univ., S. Paulo, Brasil. Ed. Servi~o Nacional de Educaoo Saniuiria, Rio de Janeiro, Brasil. & DEANE, M. P. (1954). 0 Hospital, Rio deffaneiro, 45, 419. &~ (1962). Revta Inst. IVied. trop., S. Paulo, 4, 198.