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Blood Parasites of Mammals in New Zealand

Artefact resembling Hepatozoon cuniculi (Sangiorgi, 1914) — (Plate 1, Figs. 11–20)

Artefact resembling Hepatozoon cuniculi (Sangiorgi, 1914)
(Plate 1, Figs. 11–20)

Smears of the heart blood and organs of 21 of 48 specimens of the introduced rabbit, Oryctologus cuniculus (Linnaeus), mostly collected in the Wairarapa area during 1947–1949, were found to contain numerous examples of an organism which was at first taken to be a Hepatozoon. All the preparations were made within an hour or two of the death of the host, by an investigator primarily concerned with ectoparasites and intestinal protozoa and helminths. Peripheral-blood smears made from the vessels of the ear of rabbits kept alive in the laboratory after their capture proved uniformly negative for the organism, although heart-blood and organ smears made after the death of these same animals were heavily positive.

Patton (1908) recorded Leucocytozoon leporis as a new species of leucocytic haemogregarine from the Indian black-naped hare (Lepus nigricollis), but failed to publish a description. Sangiorgi (1914) described Leucocytogregarina cuniculi from the domestic rabbit in Italy. According to Porter (1919), who records Leucocytogregarina leporis (Patton) from South African rabbits, there is evidence that asexual multiplication of this parasite takes place in the lungs.

As there is considerable difficulty in obtaining Sangiorgi's account of Hepatozoon cuniculi today, and as no English translation has been published, an abbreviated translation is given below.

The cytoplasm of (Leucocytogregarina) = Hepatozoon cuniculi is finely and uniformly granular, and stains very light azure blue with Giemsa. The body of the parasite is elongate, slightly concave on one side and rounded at the extremities, which are not always perfectly symmetrical. Small masses of chromatic material are often present at the wider extremity. The nucleus, which is generally situated towards the narrower extremity, is of oval shape and contains chromatic masses interspersed with achromatic zones as does that of H. musculi. A fine and very regular clear space about the body corresponds with the capsule, and is very resistant to staining. The average measurements of the characteristic forms from splenic smears are 16.6μ to 18.2μ by 4.1μ to 4.9μ. Other, less common, forms reach the same average length but are more slender, being only 3.5μ in width. Still others are stout, measuring some 14.0μ by 6.6μ and having more rounded extremities and being more symmetrical than those first described. The nucleus of the stout form page 9 is compact and perfectly median (and appears from Sangiorgi's Fig. 2 to be of irregular shape). Smaller forms morphologically similar to that just described but measuring 12.5μ by 3.5μ are sometimes found in the cytoplasm of large mononuclear leucocytes. No multiplication stages have been found in preparations of the blood or organs, perhaps because the infection studied was an early one (Sangiorgi, 1914).

The organism in my material from Oryctolagus cuniculus (Plate 1, Figs. 12–20) corresponds so closely with that described by Sangiorgi as Hepatozoon cuniculi that it was at first identified with this species. It has granular cytoplasm resembling that of H. cuniculi, and assuming a light blue colour in suitably stained smears. A varying number of vacuoles may be present in the cytoplasm, which sometimes shows maculation (Plate 1, Fig. 17). The body of the common form resembles that of the equivalent form of H. cuniculi in being elongate with one side slightly concave (Plate 1, Figs. 13, 15–17), and in having rounded extremities, one of which is usually narrower than the other. The size range of this form is 15.0μ to 18.0μ by 4.0μ to 5.0μ, which compares closely with the 16.6μ to 18.2μ by 4.1μ to 4.9μ of H. cuniculi. In most cases the nucleus, which is situated either centrally or somewhat towards the narrower end of the body, stains densely and uniformly pink. It may be round (Plate 1, Figs. 13 and 16) or irregular (Plate 1, Figs. 14, 15, 17) in shape. Oval organisms are rarer, and these range in size from 10.8μ to 18.0μ by 4.9μ to 7.2μ. They compare well with the stout form of H. cuniculi, and usually have a dense and irregular nucleus not quite central in position (Plate 1, Figs. 12, 14, 19). One lightly stained example has a round nucleus stained light pink with deeper pink chromatic masses (Plate 1, Fig. 18). All forms so far described are surrounded by a capsule which, like that of H. cuniculi, takes up no stain and appears as a sharply defined halo surrounding the body. Some of the large oval examples (Plate 1, Fig. 19) have a faintly staining nucleus and numerous round extranuclear chromatic granules. These granules range in size from dot-like particles to bodies 1.2μ in diameter. They stain an intense blackish-red, and are indicative of pyknosis. A single intraleucocytic organism has been observed (Plate 1, Fig. 20). This has no capsule, and is within the cytoplasm of a large mononuclear leucocyte.

The dominant irregular shape of the nucleus and the usual uniformly dense staining reaction of this structure, coupled with the fact that with Giemsa the cytoplasm frequently assumes an intensely blue colour quite unlike that characteristically taken by the haemogregarines, raised some doubts in my mind concerning the systematic position of the organism from Oryctolagus cuniculus. These doubts were unexpectedly confirmed on perusal of an account of supposed blood parasites by Wenyon (1923), who actually described how the appearance of a haemogregarine infection may be produced by allowing a film of water containing large "bacilli" from the faeces of rabbits to dry on a slide, after which a blood film is prepared on the slide and stained with Leishman. The examples of vegetable cells figured by Wenyon (1923) from such an intentionally contaminated preparation (his Fig. 10) bear a striking resemblance to the organisms from my material from Oryctolagus cuniculus. Accordingly, smears were prepared from the faeces of rabbits which had proved positive for the Hepatozoon-like organism at autopsy. The smears, after staining with Giemsa, were found to contain very large numbers of cells (Plate 1, Figs. 12, 15, 16) similar in every respect to those from the blood and organs, which are thus identified as yeast-like vegetable cells of intestinal page 10 origin. The granules referred to above as indicative of pyknosis are probably volutin granules, which are often abundant in yeasts (Henrici, 1941).

Wenyon had probably not studied Sangiorgi's description of Hepatozoon cuniculi at the time of writing his paper. Otherwise he would surely have been impressed by the close resemblances between H. cuniculi and the organism from the alimentary tract of the rabbit, for he himself said of his experiment that it "will convince anyone of the difficulty there would be of distinguishing such organisms from Protozoa if they occurred unexpectedly in blood films. It is hardly necessary to emphasize the resemblance they have to some of the 'haemogregarines' described."

Smears of the blood and organs of freshly killed rabbits have been made under sterile conditions. As careful search has failed to reveal the presence of yeast-like cells in the peripheral blood of living specimens of Oryctolagus cuniculus, it thus seems likely that these cells invade the blood stream from the intestine soon after death. The intracellular form illustrated in Fig. 20 is explained as an organism which has been ingested by a large mononuclear leucocyte.

I consider the resemblances between the organism described as Hepatozoon cuniculi (Sangiorgi, 1914) and the yeast-like cells from Oryctolagus cuniculus to be strong enough to warrant "H. cuniculi" being regarded as an artefact of vegetable nature, unless definite evidence of its protozoan nature can be obtained.