TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1984) 78, CORRESPONDENCE 563

Kreutzer, R. D. & Christensen, H. A. (1980). Characteriza- tion of Leishmania spp. by isoenzyme electrophoresis. American Journal of Tropical Medicine and Hygiene. 29, 199-208.

Lanham, S. M., Grendon, J. M., Miles, M. A., Povoa, M. & Almeida de Souza, A. A. (1981). A comparison of electrophoretic methods for isoenzyme characterization of trypanosomatids I. Standard stocks of Typanosoma cruzi zymodemes from northeast Brazil. Transactions of the Royal Socieg of Tropical Medicine and Hygiene, 75, 742-750.

Mann, P. G. & Warhurst, D. C. (1983). Isolation of thermophilic Naegleria species from thermal springs associated with a case of primary amoebic meningo- encephalitis. Presented at the 3rd International Confer- ence on Small Free-living Amoebae Colorado State University.

Martinez, A. J. (1983). Free-living amoebae: pathogenic aspects. A review. Protozoological Abstracts, 7, 293-306.

Stevens, A. R., De Jonckheere, J. & Willaert, E. (1980). Naegleria lovaniensis new species: isolation and identifica- tion of six thermophilic strains of a new species found in association with Naegleria fowleri. International Journal for Parasitology, 10, 51-64.

according to the numbers of miracidia used for the infection.

It was shown that these organisms were viable by inoculating daughter sporocysts obtained into the cephalopodal region of laboratory-reared Biomphal- aria snails. These infections were successful.

The experiments are being reviewed in detail in order to establish a defined technique.

We acknowledge financial support from FINEP and CNPq, Brazil, and the World Health Organization.

LE~GENES H. PEREIRA TERESINHA E. VALADARES MARIA LUIZA DA CUNHA MARIA CANDIDA R. CORREA

Grupo Interdepartmental de Estudos sobre Esquistossomose (GIDE),

Universidade Federal de Minus Gerais C. Postal 2486 30000 Belo Horizonte, Brazil

Accepted for publication 16th March, 1984.

Recovery of young daughter sporocysts from snails infected with Schistosoma mansoni

We have developed in our laboratories a simple procedure for in vitro recovery of young daughter sporocysts of Schistosoma mansoni from experimen- tally infected Biomphaluria glabrata, as follows:

Experimental infection of B. glabrata was carried out using large numbers of miracidia (10 to 100) per snail, the ciliate larvae being recovered from livers of expe&mentally infected hamsters.

Followinp: the 10th dav of exnosure, snails were removed f&m their shells, the cephalopodal part of bodv separated and then cut into small pieces with scissors-This material was subsequently incubated in a snecial anuaratus with Hank’s balanced salt solution (thk solut& being prepared in the conventional way and then diluted to 30% in distilled water to reach the snail haemolymph osmolarity). The pH was settled to 7.2 and the temperature maintained at 28°C.

The apparatus: The apparatus consisted mainly of a glass tube with lateral legs, opened in its upper extremity, and with a wire mesh in the lower opening. This vessel was introduced into a small beaker with the wire mesh 0.5 cm above the bottom. (The receiver was similar to that described by BARBOSA et al., 1978.)

The snail fragments were transferred to the tube, over the wire mesh, the diluted Hank’s solution being introduced into the beaker, laterally to the vessel, until the medium reached the tissues enough to moisten them.

After 60 mi?,. the vessel was removed, and the medium contammg the larvae was centrifuged at 1,000 rpm for one min. The supernatant was dis- carded, the remaining 1.0 ml suspension transferred to a Petri dish and the daughter sporocysts counted under a dissecting microscope. The larvae moved characteristically. From each snail larvae were reco- vered in numbers ranging from 17 to over 1,000,

Reference Barbosa, M. A., Pellegrino, J., Coelho, I’. M. Z. &

Sampaio, I. B. M. (1978). Quantitative aspects of the migration and evolutive asynchronism of Schistosoma mansoni in mice. Revista do Institute de Medicina Tropical de SBo Paulo, 20, 121-132.

Accepted for publication 30th March, 1984.

isolation of Mycobacterium avium complex from an ovossum Didelbhis marsuDialis in Brazil

Opo&ums are wefi-known &ma1 reservoirs for such pathogens as Amphimerus pseudofelineus, the Arizona group enterobacteri?, the leptospires, Mesocestoides, Pasteurella multoczda, Rickettsia austra- lis, R. rickettsi, salmonellae, Schistosoma mansoni and Trichinella spiralis; they also serve as peri-domiciliary host of Typanosoma cruzi (see HUBBERT et al., 1975). Of all opossums, Didelphis marsupialis is the most common. This species is naturally infected with Francisella tularensis (see HUBBERT et al., 1975), Histoplasmu capsulatum (see TAYLOR & SHACKLETTE, 1962), Leishmania braziliensis guyanensis (see ARIAS et al., 1981), Parugonimus mexicanus (see MIYAZAKI & ISHII, 1968), Trypanosoma rungeli (see D’ALESSAN- DRO, 1976; MILES et al., 1983), T. rungeli-like trypanosomes (DEANE, 1958); it is also host to the flea Ctenocephalides felis, a vector of Rickettsia typhi (see ADAMS et al., 1970), and to ticks of the genus Omithodoros, vectors of Borrelia (see HUBBERT et al., 1975). Recently we recovered acid-fast bacilli belon- ging to the Mycobacterium avium complex from an apparently normal D. marsupialis caught in the central Amazon of Brazil.

A hamster with a slow-evolving, non-ulcerating nose lesion was referred to opur mycology laboratory by R. D. Naiff. 12 months previously, the hamster had been inoculated, as described by ARIAS et al. (1981), intradermally in the nose and hind feet with saline suspensions of pooled macerates of the liver and spleen of a common opossum. The hamster had been

564 TRANSACTIONS OF THE ROYAL SOCIETY or TROPICAL MEDICINE AND HYGIENE (1984) 78, CORRESPONDEKE

kept under observation for any probable cutaneous leishmaniasis. However, periodic microscopic ex- amination of Giemsa-stained impression smears of the nose lesion and biopsy material from the foot pads did not show any protozoan parasites. In our laboratory, similar tissue smears were processed with the Ziehl- Neelsen stain. Acids-fast bacilli were detected in the nose lesion. The hamster was killed. The spleen was found to be slightly enlarged and infected with acid-fast bacilli. On Lowenstein-Jensen medium, the nose, spleen and liver tissues yielded slow-growing, non-photochromogenic mycobacteria morphologic- ally suggestive of the avium complex. The isolate was identified by Dr. R. C. Good and Mrs. V. A. Silcox of the Centers for Disease Control, Atlanta, USA, as M. avium complex. We were later able to reproduce similar nasal and systemic infections in hamsters with the M. avium complex isolate.

M. avium complex cause avian and swine tubercu- losis and occasionally human tuberculosis. The two species that constitute the complex, M. avium and M. intracellulare. can onlv be differentiated bv virulence for chicks and by serotypes. Their known natural reservoirs are soil, birds and pigs (WOLINSKY, 1973). Our finding of natural mycobacterial infection in an Amazonian D. marsupialis not only registers the common opossum as an unusual host of the M. avium complex, but also confirms the susceptibility and the role as animal reservoir of D. marsupiazis to a variety of human pathogens.

It is noteworthy that our recovery of M. a&m complex from opossum was due largely to the natural susceptibility of the hamster to a common mycobacterial infection, a characteristic previously unrecognized but which can be of use in studies on experimental mycobacteriosis.

W. Y. MOK Institute National de Pesquisas

da Amaz&zia, Caixa Postal 478, 69000 Manaus, Amazonas, Brazil

References Adams, W. H,, Emmons, R. W. & Brooks, J. E. (1970).

The changmg ecology of murine (endemic) typhus in Southern California. American Journal of Tropical Medi- cine and Hygiene, 19, 311-318.

Arias, J. R., Naiff, R. D., Miles, M. A. & de Souza, A. A. (1981). The opossum, Didelphis marsupialis (Marsupialis: Didelphidae), as a reservoir host of Leishmania brazilien- sis guyanensis in the Amazon Basin of Brazil. Transactions of the Royal Society of Tropical Medicine and Hygiene, 75, 537-541.

D’Alessandro, A. (1976). Biology of Trypanosoma (Herpeto- soma) rangeli Tejara, 1920. In: Biology of the Kinetoplas- tidu. Vol. 1. W. H. R. Lumsden & D. A. Evans (Editors). London: Academic Press, pp. 328-403.

Deane, L. M. (1958). Encontro de tripanosomo do tipo rangeli em gambas de especie Didelphis marsupialis marsupialis, no estado do Para. Revista Brasileira de Malariologia e Doencas Tropicais, 10, 451-458.

Hubbert, W. T., McCulloch, W. F. & Schnurrenberger, P. R. (1975). Diseases Transmittedfrom Animals to Man. (6th edit.) Springfield, Illinois: Charles C. Thomas.

Miles, M. A., Arias, J. R., Valente, S. A. S., Naiff, R. D., de Souza, A. A., Povoa, M. M., Lima, J. A. N. & Cedillos, R. A. (1983). Vertebrate hosts and vectors of Trypanosoma rangeli in the Amazon Basin of Brazil. American Journal of Tropical Medicine and Hygiene, 32, 1251-1259.

Miyazaki, I. & Ishn, Y. (1968). Studies on the Mexican lung fluke, with special reference to a description of Parugoni- mus mexicanus sp. nov. (Trematoda: Troglotrematidae). ‘faoanese Yournal of Parasitoloev.17. 445-453.

Tailor, R. L. & Shacklette, M”: ‘H: (1962). Naturally acquired histoplasmosis in the mammals of the Panama Canal Zone. American Journal of Tropical Medicine, 11, 79h-799

I , - I _ , .

Wolinsky, E. (1973). Mycobacteria. In: Microbiology (2nd edit.) B. D. Davis, R. Dulbecco, H. N. Eisen, H. S. Ginsberg, W. B. Wood & M. McCarty (Editors). New York: Harper & Row, 843-870.

Accepted for publication 12th April, 1984.

Haem and haemoflagellates: A reply to J. R. Baker J. R. Baker (1984) ‘confidently await[s] enlighten-

ment from my biochemical colleagues’ as to how-if at all-haemoflagellates of some species grow axeni- tally in media in which the only blood component was serum. Issues are thereby posed: (i) has serum non-haems which allow the haem requirement to be bypassed?; (ii) if serum has such constituents, must the assumption that haem is an absolute growth-factor for the entire haemoflagellate line be extended to ‘haem or a functional equivalent’?

This puzzle is attended with interest for the biochemistry and phylogeny of the haemoflagellates, also for chemotherapy, for if man and other verte- brates synthesize haem-as all evidence indicates vertebrates do-and if haemoflagellates do wholly depend on host-synthesized haem, then the haem metabolism of haemoflagellates is an attractive target for antihaem chemotherapy. The weight of evidence is that the requirement for exogenous haem is indeed absolute. By supplying appropriate Fe-chelators, e.g. l-(2-pyridylazo)-2-naphthol, the haem requirement of the mosquito haemoflagellate Crithidiafasciculata may be lowered to one hundredth of the usual concentra- tion, implying that 99% of haem, as supplied in conventional defined media, serves as a rather non- specific source of Fe (SHAPIRO et al, 198 1). The issue thus comes down to which constituents of serum individually or collectively imitate the chelating agent (which had been tested in the presence of adequate Fe, Cu and MO). As long known, Cu is variously and intimately concerned with the utilization of Fe. A major constituent of serum is the Cu-protein cerulo- plasmin which apparently functions as a non-toxic reservoir or buffer for Cu (MATHER. 1982). Serum contains the Fe-transport protein transfer& which serves as a growth stimulant, perhaps even an absolute growth factor for various tissue cell lines (ROCKWELL et al., 1980). Serum contains a peptide which may be important for uptake of Fe and other trace transitional metal ions (PICKART & THALER, 1980). An additional mechanism may come into play to maintain haem’s status in vivo as a potent trace rather than gross growth factor: protection against its oxidative degradation by enzymatic and non- enzymatic reactions. Haem complexed to serum albumin is at least partlv protected against microso- ma1 haem oxygenases (J~O~HIDA & ~KUCHI, 1978). Further protection is provided by uric acid, a principal constituent of the excrement of mosquitoes