Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 104, Num. 2, 2009

Memórias do Instituto Oswaldo Cruz, Vol. 104, No. 2, March, 2009, pp.

A new protozoan parasite of rabbit found in histological lesions similar to human Kala-Azar⁺

Preliminary description made by Alfonso Splendore MD

Chair Bacteriological Lab at Portuguese Hospital, S Paulo, Brazil

Code Number : oc09024

Supplementary Data

A new rabbit disease that histologically resembles the human protozoan disease Kala-Azar has recently appeared in my laboratory, in the same cage where a previously reported mixoma outbreak had occurred. This new disease, which affected half a dozen rabbits, did not show distinctive clinical characteristics. All of the animals but one (showing paralysis of posterior limbs, two days before death) died because of wasting disease without specific organ involvement. No parasite or relevant modifications in blood smears were evident.

Upon necropsy, characteristic lesions were found in internal organs, mainly in spleen, liver, lung, lymph node, and large intestine. Hypertrophic spleen samples displayed a general marbleized pseudo-tubercular aspect, with a red-brown surface containing many small grey-white scattered lesions of different sizes. Gray spots were found in a hypertrophic liver, in lung, and in lymph nodes. Less frequently, ulcerations were found in the large intestine, and the dorsal skin was blistered in one animal. Peritoneal exudative material, sometimes bloody, was always found.

Optical microscopy revealed the presence of special cells in these internal organs and in bone-marrow, and sometimes in the kidneys.

These cells were particularly visible under Giemsa staining and could be found both inside or outside the cells in smears.

These cells were observed both in isolation and in groups; however, in histological preparations they occurred mostly in groups. They were always in parenchyma cells, never in red blood cells and rarely in white blood cells.

In fresh wet preparations, the cytoplasm displayed hyaline while the nucleus was granular. Neither flagella nor active movement were ever observed. Morphologically, these cells were kidney-shaped, with one of the extremities enlarged and rounded. Only some of them were oval-shaped as a consequence of a duplicative activity. Irregular or circular cells were infrequently observed.

In addition to these cells, regular and irregular circular cysts were found. Several kidney-shaped cells or many sparse granular lumps were randomly distributed in the cytoplasm of the cysts.

The dimensions of the kidney-shaped or oval cells was 5-8 μm in the length and 2.5-4 μm at the greatest width. Cyst size varied between 8 and 40 μm.

Giemsa staining revealed a pale blue cytoplasm surrounding chromatin lumps inside a red nucleus, and between these, a light colourless halo could be seen.

Rosette-like chromatin structures or, in the case of cells in the active mitotic phase, irregularly distributed lumps of chromatin granules, filled the maximum diameter of the nucleus as well as the area around that structure.

In several oval cells, the chromatin was observed to be distributed in two identical distinct blocks and the full body appeared as an assemblage of two kidney-shaped splitting cells. In many cases, well-defined and distinct kidney-shaped cells could be detected in pairs. This aspect seemed to be a characteristic two-by-two junction, which is a feature typical of parasitic cells.

Extra-nuclear chromatin that would suggest a defined blepharoplast was never detected, although a small, red coloured amount of chromatin (perhaps a rudimentary blepharoplast?) was occasionally observed at one of the extremities.

Besides these characteristic cells, Giemsa staining sometimes revealed additional small free cells. They were round or amoeboid, as large as one red blood cell or slightly smaller. They were slightly darker blue, had less condensed cytoplasm and a red violet lump of chromatin located at the periphery, which showed a width of almost one third of the overall cell surface. Some cells displayed barely observable chromatin granules in the middle of the cell or at the border. Others seemed chromatin-free. In a liver-derived histological preparation, small bulbshaped cells were detected, showing long and thin extremities similar to flagella. These also displayed light blue cytoplasm without red chromatin. Furthermore, a nucleus-like dark blue dense body could be seen in the middle of the cell. These lumps had a diameter that was three or four times that of a red blood cell (RBC), had a maximum width that was two thirds the diameter of a RBC, and the hair-shaped extremities were each as long as the full body.

When oval or kidney-shaped cells were undetectable after cyst staining, large amounts of red chromatin lumps could be seen. These were sometimes polygonal, usually randomly dispersed in the pale blue cytoplasm, and resembled schizogony of several sporozoa.

Unquestionably, the elements described above seemed to represent several development phases of a protozoan parasite whose life cycle, at least in part, oc-those of the human Kala-Azar. However, this new parasite of rabbit was as different from the Leishmania genus as any other so far described.

Prowazek, to whom I sent two spleen preparations, wrote me his competent opinion that this seems to be within a new Protozoa group that is similar to one of the mammal gregarine described by Balfour and others.

I do not believe we can make a definitive classification of this protozoan before its vital cycle has been fully recognized, an objective that I hope to achieve by personally carrying out further research.

On Rogers (citrate serum) and on McNeal (blooded agar), cultivation produced negative results. Transmission by subcutaneous injection or by feeding healthy rabbits and other animal species was unfruitful, as was inoculation of biological material from dead infected rabbits transmitted by fleas.

A more detailed report of my experiments, including illustrative material, will be the object of another paper.

⁺ Kindly translated by Wilma Buffolano from the original paper in Italian published in Rev Soc Sci S Paulo 3: 109-112, 1909.

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