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Pathological and haematological responses of cats experimentally infected with Toxocara canis larvae
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Parasitology
PATHOLOGICAL AND HAEMATOLOGICAL RESPONSES OF CATS EXPERIMENTALLY INFECTED WITH TOXOCARA CANIS LARVAE J. C. *Department tDepartment
PARSONS,* D. D. BOWMAN? and R. B. GRIEVE*
of Pathology,
College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, U.S.A. of Microbiology, Immunology and Parasitology, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, U.S.A. (Received 23 August 1988; accepted 1 February 1989)
Abstract-PARSONS J. C., BOWMAN D. D. and GRIEVER. B. 1989. Pathological
and haematological responses of cats experimentally infected with Toxocara canis larvae. International Journal for Parasitology 19: 419488. Responses of eight adult cats to one or two infections with larvae of Toxocara canis were studied up to 39 days post infection (DPI). Clinically, all cats remained normal throughout the study. The major necropsy finding was multifocal, white to grey nodules mainly within the liver, lungs and kidneys; live larvae were found in liver nodules. Histologically, the nodules were eosinophilic granulomas. Granulomas containing a larval section were observed mainly within the liver. All infected cats had variably severe, eosinophilic arteritis and bronchiolitis and medial hypertrophy and hyperplasia of the pulmonary arteries. No inflammatory eye lesions were detected. Circulating eosinophil levels increased in all infected cats; peak values of 15,790 and 10,050 eosinophils pi- ’ were observed at 25 or 32 DPI in cats receiving a single or double infection, respectively. Bone marrow of all infected cats exhibited marked eosinophilic hyperplasia which did not correlate with the level of circulating eosinophilia. Thus, infection of cats by the larvae of T. canis causes disseminated eosinophilic and granulomatous disease with marked pulmonary artery and airway lesions. INDEX KEY WORDS: Toxocara canis; cats; histopathology; granuloma; larva migrans; eosinophils; eosinophilia; bone marrow; medial hypertrophy
HUMAN visceral larva migrans (VLM) is a syndrome resulting from prolonged migration of infective nematode larvae through the internal organs of the host. Overt VLM usually occurs in young children and presents as a clinical syndrome of chronic extreme eosinophilia, hepatosplenomegaly and some degree of pneumonitis (Beaver, Snyder, Carrera, Dent & Lafferty, 1952). In the original report of VLM by Beaver et al. (1952), liver biopsies from three affected children revealed extensive eosinophilic and granulomatous lesions, one of which contained an infective larva of the canine ascarid, Toxocara canis. Other ascaridoid larvae, including Toxocara cati and Baylisascaris procyonis, have been incriminated in additional human cases of VLM (reviewed by Beaver, 1966; Huff, Neafie, Binder, De Leon, Brown & Kazacos, 1984) but serological evidence suggests that T. canis remains the predominant worldwide cause of human VLM (Glickman & Schantz, 1981). An important sequela to larval toxocariasis in humans and dogs is ocular disease or ocular larva migrans (OLM) (Rubin & Saunders, 1965; Shields, 1984; Hughes, Dubielzig & Kazacos, 1987; Johnson, Kirkpatrick, Whiteley, Morton & Helper, in press). Both VLM and OLM have been well characterized in humans and laboratory animals, but little work has
granulomatous disease; visceral and hyperplasia; MHPA.
been done on larval toxocariasis in other vertebrate hosts, and it may be that this disease is often overlooked in these animals. Animal species other than mice, rabbits and guinea pigs, in which responses to systemic migration of T. canis larvae have been studied, include sheep (Hayat, Rehman & Ahmad, 1973), goats (Sinha, 1970), cattle (Fitzgerald & Mansfield, 1970; McGraw & Greenway, 1970), pigs (Ron&us, 1966; Zendulka, 1967), dogs (Magnusson, 1970; Hayden & van Kruiningen, 1973), monkeys (Beaver, 1962; Tomimura, Yokota & Takiguchi, 1976; Glickman & Summers, 1983), possums (Sweatman, 1962) and tortoises (Merdivenci & Sezen, 1965). Larval T. canis migration in several of these animal species including fowl has also been reviewed by Poynter (1966). However, the lesions of VLM in cats due to ascarid larval migration have not been extensively examined. Larvae of Toxocara cati have been recovered from the liver, lungs and musculature of experimentally infected cats (Sprent, 1956; Swerczek, Nielsen & Helmboldt, 1971), but only the pulmonary lesions were described (Swerczek, Nielsen & Helmboldt, 1970). In cats experimentally infected with T. canis, Bhowmick (1964) found multifocal, white to grey nodules within the kidneys, lung, liver and heart. We have recently described a case 419
J. C. PARSONS,D. D. BOWMANand R. B. GRIEVE
480
in which similar nodules were found at necropsy within the same range of organs from a domestic cat with naturally-occurring larval toxocariasis (Parsons, Bowman, Gillette & Grieve, 1988). Histologically, these nodules were found to be eosinophilic granulomas, some of which contained an infective T. canis larva. However, little could be determined about the temporal development of these lesions in this host. Thus, in order to further investigate the pathogenesis of feline VIM due to T. canis we have examined the haematological and pathological responses of cats infected on one or two occasions with embryonated eggs of T. canis. Additionally, we have examined the potential for larvae of T. canis to cause ocular larva migrans in the cat. MATERIALS
AND METHODS
Experimental animals. A group of seven neutered, adult, female, domestic shorthair (DSH) cats were housed in an American Association for the Accreditation of Laboratory Animal Care-approved facility for a period of at least one year. According to health care records, some cats had previously passed nematodes and may, therefore, not have been ascarid-naive. Cats 8 and 9 were intact, adult, male, specific-pathogen-free (SPF), DSH cats. The ancestors of these animals, obtained over 10 years previously, were caesarian-derived and colostrum-deprived and, therefore, were free of any feline ascarids (Swerczek et al., 1971). Prior to the commencement of the study, all cats were found to be helminth-free by use of a faecal flotation procedure. Experimental infection and sample collection. Eggs of Toxocara canis were collected and embryonated using a previously described method (Bowman, Mika-Grieve & Grieve, 1987a). All cats were anaesthetized by an intramuscular injection of ketamine hydrochloride (Ketaset; Bristol Laboratories, Syracuse, NY) prior to each sample collection and each egg inoculation. On Day 0 of the study all of the experimental cats were infected intragastrically, via a stomach tube, with 5000 embryonated T. canis eggs (i.e. approximately 1 epg body weight) in 10 ml of tap water. Fourteen days later, four of the cats were given a challenge dose of 5000 infective eggs. During the course of the study all cats were observed at least twice daily. Necropsies were performed on Days 18, 25, 32 and 39 post initial infection according to the schedule in Table 1. A control cat received an intragastric dose of 10 ml of water on each of Days 0 and 14 post infection and was necropsied on Day 32 post initial infection. Immediately prior to infection, challenge and necropsy, blood samples were collected by jugular venipuncture into EDTA-coated blood tubes (Vacutainer; Becton, Dickinson & Co., Rutherford, NJ). Whole blood smears were made and air dried prior to staining with Wright’s stain. On the day of necropsy all cats underwent a complete ophthalmoscopic examination. The cats were then killed by an intravenous overdose of barbiturate and immediately necropsied. Liver samples, each weighing approximately 0.1 g and containing a subcapsular lesion, were taken from Cats 1, 5, 6, 7 and 8, compressed between two microscope slides and examined for larvae. The entire gastrointestinal system was opened and examined for immature and mature helminths. All organs were examined for gross lesions and tissue samples were collected from the right caudal and left cranial lung lobes after fixation, and from the left and right ventricular free wall and
TABLE
I-INFECTIONANDNECROPSYSCHEDULEOFCATS
EXPERIMENTALLYINFECTEDONCEORTWlCEW,THEGGSOF
Toxocara canis Cat number
No. of eggs administered
FS FS FS FS FS M FS FS M
Day of necropsy
0 DPI
14 DPI
(DPI)
5000 5000 5ooo 5000 5000 5000 5000 5000 0
0 5000 0 5000 0 5000 0 5000 0
18 18 25 25 32 32 39 39 32
FS = Spayed female, M = male, DPI = days post initial infection.
interventricular septum of the heart, liver, kidneys, spleen, pancreas, adrenal glands, thyroid glands, thymus, mesenteric and tracheobronchial lymph nodes, small and large intestine, cerebellum, brain stem, cerebrum, sciatic nerve and femoral bone marrow. Muscle samples were collected from the diaphragm, tongue, masseter, biceps brachii and quadriceps femoris. In organs where gross lesions were present, representative examples were collected; all other tissues were sampled at random. All tissues except the lungs, eyes and bone marrow were immersion-fixed in 10% formalin. The lungs were fixed by an intratracheal infusion of 10% formalin and the eyes and bone marrow by immersion in Kamovsky’s fixative. Haematology and histopathology. Differential white cell counts were made using blood smears stained by Wright’s stain. Total whitecell counts (TWCC) and haematocrits were measured using a Coulterm counter (Coulter Electronics Inc., Hialeah, FL). Eyes were hemisccted around the pars plana and the anterior and posterior segments examined for gross lesions under a dissecting microscope. All tissue samples were paraffin-embedded and routinely processed for histopathology. Sections were cut at 5 m and stained with haematoxylin and eosin. Selected sections were also stained using Verhoff-Van Gieson. Alcian Blue-PAS, Luna’s eosinophil and Toluidine Blue stains. Bone marrow sections, cut at 3 m, were stained with Giemsa stain and the percentage of eosinophils in 500 nucleated cells was determined. T. canis larvae, when preserved within tissue sections, were identified on the basis of larval morphology and size (Nicholls, 1956). Larval viability was also assessed using morphological criteria. Representative tissue sections have been deposited with the U.S. National Parasite Collection (Beltsville, MD 20705). RESULTS
During the course of the study Cat 7 developed a mild cough and sinusitis; all other cats remained clinically normal. No antemortem ocular lesions were detected in any cats.
Necropsyjndings Gross lesions were found mainly within the liver, lungs and kidneys of the infected cats. In general,
Experimental larval toxocariasis in cats lesions were more numerous in cats receiving a dual infection and became progressively more numerous and larger with time. Marked hepatomegaly was found in Cats 6 and 8; splenomegaly could not be assessed as the barbiturate-based euthanasia agent caused marked dilation of this organ. No gross lesions were present in any organs, including eyes, of the control cat. Liver. Within the liver of all infected cats there were multifocal, often slightly raised, irregular, white to grey foci up to 2 mm in diameter. A living T. canis larva was found within squash preparations of hepatic lesions dissected from all cats thus examined. Lungs. All infected cats had either moderate or severe pneumonia and pleuritis. The pleural surface, in the early stages of infection or reinfection, was usually mottled with irregular, dark red and white areas, up to approximately 10 mm in diameter, which contained well-delineated, raised, white nodules, often with a haemorrhagic core. In the later stages of infection (Cats 5-Q focal areas of the visceral pleura were markedly thickened and were often lightly adherent to
481
kidney and pleura were present within the epicardium of Cat 8, the pancreas of Cats 1 and 3, and the mesenteric lymph node of Cat 3. All other organs of the infected cats appeared grossly normal. No ocular lesions were detected in hemisected eyes. No immature or mature helminths were found within the lumen of the gastrointestinal system of any cat. Cat 7 had moderate, suppurative rhinitis and sinusitis which was probably unrelated to T. canis infection. Histopathologicalfindings
The major histopathological findings in all infected cats were eosinophilic granulomas, within a range of visceral organs and somatic musculature, and medial hypertrophy and hyperplasia of the pulmonary arteries. Granulomas, which corresponded to the nodules seen grossly, were also found in sections of randomly-sampled tissues in which no gross lesions were detected. Morphologically-viable larvae were present in one or more tissues of six cats (Fig. 2a, b, d); most commonly these larvae were in the liver (Cats, 2, 3, 4, 6 and 8). All tissues from the control cat were histopathologically normal.
FIG. 1. Kidney from cat experimentally infected twice with eggs of Toxocara canis, Cat 8. a. Multiple subcapsular granulomas. Scale bar = 5 mm. b. Sectioned surface with three granulomas (arrows) extending towards the corticomedullary junction. Scale bar = 5 mm.
the parietal pleura. Nodular foci, similar to those on the pleura, were present within the lung parenchyma of all infected cats. Several of the larger airways of Cat 8 exuded pale yellow, mucoid material. Adjacent to these airways were firm, homogeneously white foci, up to 2 mm in diameter, with a fine, central, dark brown core. Kidneys. Multifocal, well-circumscribed, raised, white nodules up to 3 mm in diameter (Fig. la) were present on the capsular surface of one or both kidneys of all cats except Cat 5. The larger renal lesions were conical and extended for varying distances toward the corticomedullary junction (Fig. lb). Other organs. Nodular lesions similar to those in the
Liver. Generalized, mild or moderate, lymphoplasmacytic, portal hepatitis and mild periportal fatty change were present within the liver of each infected cat. The portal hepatitis was more severe and contained more eosinophils in areas directly involved in larval migration (Fig. 2a). In singlyinfected cats, early phase (Cats 1 and 3) lesions consisted of focal areas of acute hepatocellular necrosis surrounded by leucocyte debris, eosinophils and neutrophils. These lesions were scattered at random throughout the hepatic lobules. In Cat 5 these areas had become focally extensive granulomas with a necrotic core of eosinophilic leucocyte debris admixed
482
J. C. PARSONS, D. D. BOWMAN and R. B. GRIEVE
FIG 2. Development of disseminated eosinophilic granulomas in various tissues from cats infected once or twice with eggs of Toxocura canis. a. Liver, Cat 8. Area of moderate, eosinophilic, portal hepatitis with an adjacent T. canis larva in tangential section surrounded by an acute inflammatory response of eosinophils, neutrophils and necrotic hepatocytes, H&E. Scale bar = 100 m. b. Liver, Cat 5. Organizing granuloma containing eosinophilic cellular debris moulded around larva (arrows) which either died and disintegrated or migrated from the granuloma. Note giant cells and early fibroplasia at the periphery of the granuloma, H&E. Scale bar = 50 ,mn. c. Lung, Cat 2. Large eosinophilic granuloma surrounding a tangential section of a viable T. canislarva. Note mild medial hypertrophy and hyperplasia and sub-endothelial vacuolation in adjacent pulmonary artery, H&E. Scale bar = 75 ,um. d. ~#u~~~ce~~~~o~i~ muscle, Cat 5. Eosinophilic granuloma surrounding two cross-sections of a coiled, viable, T. canis larva. Note the prominent excretory columns within one larval section (arrow), H&E. Scale bar = 50 m.
with epithelioid cells and occasional foreign-body giant cells surrounded by a thin outer rim of mainly eosinophils with a few lymphocytes and plasma ceils (Fig. 2b). By 39 DPI (Cat 7) the granuiomas were more organized, but with little evidence of fibroplasia, and contained fewer eosinophils. Early phase lesions were also present in some liver sections from Cats 5 and 7. A similar range of lesions, including those of the early phase (Fig. Za), was present with the livers of the cats receiving a challenge infection. However, in Cat 2, large areas of hepatocellular necrosis with marked leucocyte infiltration, occasionally surrounding a larva, were present in addition to early phase lesions.
No macrophages or lymphocytes were observed in these lesions which were mainly subcapsular in location. This type of lesion was suggestive of an acute response to the challenge larvae. LLcngs.Within the lung parenchyma of all infected were focally extensive, eosinophil-rick cats granulomas (Fig. 2c) and mild to moderate pulmonary oedema. No difference in the nature and distribution of lesions was observed between the two sampled lung lobes. When a larva was present within an alveolar lumen, the accompanying inflammatory response occluded that lumen and those of severe adjoining alveoli (Fig. 2~). Pleural and subpleural granulomas,
Experimental larval toxocariasis in cats contiguous with extensive areas of eosinophilic pleuritis, were common in most of the cats. In the later
stages of infection (Cats 5-8) there was marked, focal, pleural fibrosis. Medial hypertrophy and hyperplasia of the pulmonary arteries and arterioles (MHPA) were also present in all infected cats (Figs. 2c, 3). This marked thickening of the tunica media was mainly due to proliferation of the medial myocytes, however, in some vessels individual smooth muscle cells were swollen with cytoplasmic vacuoles (Fig. 3d). MHPA was most severe in Cat 8 in which the greatly thickened and narrowed arteries corresponded to the white foci seen grossly adjacent to airways (Fig. 3~). Coexisting with MHPA in all infected cats was a variably severe, eosinophilic endarteritis, periarteritis, bronchiolitis and peribronchiolitis (Fig. 3b, c, d). Numerous mast cells were present within the vascular and airway
483
infiltrates which were most severe in animals receiving a challenge infection; severity increased with time following infection. In Cat 8, marked intimal proliferation, subendothelial vacuolation and endarteritis had greatly reduced or occluded the lumen of several vessels (Fig. 3~). Fragmentation of the internal elastic membrane had occurred in some of the more severely affected vessels of this cat. Normal pulmonary arteries were also present in sections from all infected cats. Perivascular oedema, independent of any arteritis, was observed only within the lungs of challenged cats. In Cat 2 the oedema was marked and contained a moderate infiltrate of eosinophils around most large arteries. This lesion became progressively more eosinophilic and less oedematous with time in Cats 4,6 and 8, respectively. No airway or pulmonary artery lesions were present in lung sections from the control cat (Fig. 3a).
FIG. 3. Eosinophilic endarteritis and medial hypertrophy and hyperplasia (MHPA) in the lungs ofcats experimentally infected once or twice with eggs of Toxocara canis. a. Cat 9. Normal pulmonary artery and bronchiole, H&E. Scale bar = 80 p. b. Cat 1. Mild eosinophilic periarteritis, endarteritis and bronchiolitis with moderate MHPA, H&E. Scale bar = 80 F. c. Cat 8. Severe eosinophilic endarteritis and MHPA which appears to have almost occluded the arterial lumen. Note the plug of eosinophils, mucus and sloughed epithelial cells within the adjacent bronchiole (arrow), H&E. Scale bar = 120 m. d. Cat 5. A pulmonary artery with severe eosinophilic periarteritis and endarteritis and marked cytoplasmic vacuolation of hypertrophied and hyperplastic medial myocytes, H&E. Scale bar = 80 m.
J. C. PARSONS,D. D. BOWMAN and R. B. GRIEVE
484
Kidneys. Nephritis was found in all infected cats except Cats 4 and 5. Large, eosinophilic granulomas incorporating glomeruli and renal tubules and extending towards the corticomedullary junction were present in Cats 6, 7 and 8. Hearf. Non-suppurative, eosinophilic, interstitial myocarditis without distinct granuloma formation was present in four cats (Cats 2,3,4 and 8). Skeletalmuscles. Five cats had granulomas in one or more skeletal muscles. The tongue (Cats 2, 7 and 8) and the quadriceps femoris (Cats 2, 5 and 8) (Fig. 2d) were most commonly affected. Lymph nodes. In Cats 2 and 3, the subcapsular sinus and medullary cords of the mesenteric lymph node (MLN) were focally distended by large numbers of eosinophils. A granuloma containing a viable larva surrounded by Splendore-Hoeppli material (see Johnson, 1976) was present within a germinal centre of the MLN of Cat 3.
Brain. Cats 4 and 6 had focal, granulomatous meningitis. Occasional eosinophils were present within the surface meninges and perivascular cuffs. Cat 6 had, in addition to meningitis, a mild, multifocal, granulomatous encephalitis mainly affecting the white matter of the brain stem and cerebrum. Larvae were not observed in any brain sections. Bone marrow. All infected cats had marked eosinophilic hyperplasia of the bone marrow (Table 2). The percentage of eosinophils in 500 nucleated bone marrow cells ranged from 6.4 in Cat 6 to 28.2 in Cat 3 (mean = 19.6). Other organs. Focal areas of eosinophilic and granulomatous infiltration were present within the pancreas of Cats 1 and 3 and within the submucosa of the colon of Cat 2 and of the small intestine of Cat 8. No additional lesions were present in the remaining tissue sections, including eyes, from the infected cats.
TABLE Z-HAEMATOLOGICAL RESPONSES OF CATSEXPERIMENTALLY EVFECTEDONCEORTWICEWITHEGGSOF Toxocara
canis
Days post initial infection
Cat number 0
14
18
lTwcc EOS PI-’ %EOSBM
9300 740 -
-
6100 850 6.6
TWCC EOS /X’ %EOSBM
I 1,800
TWCC EOS PI-’ %EOSBM
6800 680
TWCC EOS PI-’ %EOSBM
6200 500
TWCC EOS /J-’ %EOSBM
16,400 1640 -
TWCC EOS PI-’ %EOSBM
8300 420
TWCC EOS~I-’ %EOSBM
8900 530
TWCC EOS&’ %EOSBM
12,500 870
TWCC EOS pi-’ %EOSBM
15,200 3190 -
710 -
25
32
39
14,200 3410 22.6 32,900 15,790 28.2
-
14,800 6600 12.9
16,200 4050
-
-
16,100 3060 24.0
9100 1460
18,500 8330 6.4 15,300 1990 15.6 31,400 10,050 22.4
_ 8700 90
9200 90 -
-
-
8300 420 0.4
TWCC = Total white cell count, EOS ~1~ ’ = absolute number of circulating eosinophils ~1~ ‘, %EOSBM = percentage of eosinophils of 500 nucleated cells in bone marrow sections. *Normal
values (Jain, 1986): TWCC=
5.5-19.5.
x IO3 leucocytes
~1~‘; EOS pl -‘=&1500;
%EOSBM=
1.5-3.8%.
Experimental larval toxocariasis in cats Haematological responses
Prior to infection, the leucogram of each cat, except Cat 5, was within normal limits for adult cats (Table 2). On 0 DPI Cat 5 had an unexplained eosinophilia with an absolute eosinophil count of 1640 eosinophils ~1~’ and a normal TWCC. The mean eosinophil count of the eight test cats at 0 DPI was 760 ~1~‘. The haematocrit of all cats remained within normal limits throughout the study (data not shown). By 14 DPI three of the four cats sampled had a marked eosinophilia ranging from 1460 to 7390 eosinophils ~1~’ (mean=4020 eosinophils ~1~‘) and a normal TWCC. Each of the four cats that received a challenge infection had an eosinophilia at necropsy. The circulating eosinophil levels varied from 3410 ~1~’ in Cat 2 to 10,050 ~1~’ in Cat 8. Each of the cats that received a single infection, except Cat 1, had an eosinophiha at the time of necropsy. In the group of singly-infected cats, the peak level of eosinophilia (15,790 eosinophils ~1~‘) was seen at 25 DPI in Cat 3 and in the group of cats receiving a challenge infection the peak level of T. canis (10,050 eosinophils PI-‘) was seen in Cat 8 at 32 DPI. Occasional basophils, not exceeding 1% of the TWCC, were seen in blood smears during the course of infection in Cats 4,7 and 8. Two cats had a leucocytosis on the day of necropsy. Cat 3 had a leucocytosis with eosinophilia and Cat 8 had a neutrophilic leucocytosis. The control cat remained haematologically normal throughout the study. DISCUSSION The present study indicates that the experimental infection of adult cats with larvae of T. canis causes disseminated eosinophilic granulomatous disease which is similar to that described in visceral larva migrans of humans and other animals. The range of affected organs in the cats in this study was similar to those seen following experimental and natural infection of the cats with T. canis larvae (Bhowmick, 1964; Parsons et al., 1988) with the exception that heart lesions in this study were less common. Lesions due to larval migration within the pancreas and mesenteric lymph node have not been reported previously in this species. No clinical signs due to larval migration within major organs were detected in any of our study cats. Hence, with a lack of presenting signs, and the close association of cats and dogs, covert disease in cats due to T. canis may be more common than is presently suspected. The histopathology of the developing granuloma in larval toxocariasis has been described previously (Kayes & Oaks, 1978). A similar sequence of histological responses was observed in most tissues from the singly infected cats in this study. As in the study by Kayes & Oaks (1978) we found that both acute and chronic types of granuloma were often present in the same histological section. This response probably represented the migration from the more
485
chronic granuloma by the inciting larva which had then induced a subsequent reaction at another site. In general, challenge infection induced additional acute lesions without modifying the histological picture of either the acute or chronic response. However, within the lungs there was a strong association between challenge infection and the severity of eosinophilic arteritis, periarteritis, bronchiolitis and peribronchiolitis. In addition, perivascular oedema, independent of any arteritis present within the same lungfield, was seen only in challenged animals. All these lesions may represent an ongoing immediatetype hypersensitivity reaction to some larval product(s) present locally within the lungs in association with continued larval migration to this site (Weatherley & Hamilton, 1984). Alternatively, it may be a non-specific, hyperergic response of a lung, previously primed by T. canis larval migration, to an inhaled or ingested allergen (Kayes, 1986). Enumeration of larval numbers following artificial digestion of a range of tissue samples may give an estimate of larval dissemination within an infected animal; tissue digests of each cat were not performed in this study. However, the finding of larval granulomas in a wide range of randomly sampled organs without gross lesions suggests that, particularly in the case of the skeletal muscles, the level of dissemination of T. canis larvae following infection in cats is high. Many of these tissue granulomas contained a T. canis larva in section which helped to distinguish them from any pre-existing granulomas due to T. cati and from the non-effusive form of feline infectious peritonitis (Julian, 1985) and eosinophilic leukaemia (Simon & Holzworth, 1967); the gross lesions of the latter two conditions resemble those seen in the present study. Medial hypertrophy and hyperplasia of the pulmonary arteries (MHPA) were found in all of the infected cats of this study as well as in a natural case of VLM in a domestic cat due to T. canis described by Parsons et al. (1988). Prior infection by the feline lungworm, Aelurostrongylus abstrusus, was originally thought to be the main cause of MHPA in cats (Mackenzie, 1960; Hamilton, 1963). Subsequently, Swerczek (unpublished PhD thesis, University of Connecticut, 1969) and Swerczek et al. (1970) demonstrated that the lesion was associated with lung migration of any of several larval nematodes. MHPA was present in cats following infection by the larvae of T. canis, T. cati, Ascaris suum and A. abstrusus; however, it was not present following Toxascaris Zeonina infection in which larvae undergo little or no extraintestinal migration in cats (Sprent, 1959). Prior infection with A. abstrusus in the laboratory-reared cats used in this study is unlikely due to lack of exposure to the molluscan intermediate host. However, it is possible that previous infection of several cats with T. cati may have caused MHPA prior to the commencement of the study. Swerczek (1969, thesis cited above) and Swerczek et al. (1970)
486
J. C. PARSONS, D. D. BOWMAN and R. B. GRIEVE
previously observed mild MHPA, attributed to prior infection by T. cati, in conventionally-reared control cats but not in caesarian-derived, colostrum-deprived cats. Other non-parasitological causes of MHPA may also exist as the lesion has been reported within a colony of caesarian-derived, SPF cats (Rogers, Bishop & Rohovsky, 1971). However, as T. cati is largely transmitted by the transmammary route (Swerczek et al., 1971) colostrum deprivation is important in assuring ascarid-naive animals; it was not clear whether the cats in the colony of Rogers ef al. (1971) were colostrum-deprived. No immature or adult T. canis were found in the gastrointestinal system of the cats in the present study. Swerczek (1969, thesis cited above) was also unable to induce a patent infection of T. canis in cats following the administration of infective eggs. However, others have reported the recovery of adult T. canis from the intestine of cats following natural (reviewed by Sprent & Barrett, 1964; Rohde, 1962; Roth & Schneider, 1971) and experimental (Bhowmick, 1964) infection. Bhowmick (1964) recovered a total of eight adult worms from two young cats at Day 70 following a per OSinfection with 15,000-20,000 infective eggs and was also able to recover an additional 18 adult worms from four cats following intraperitoneal infection or the feeding of infected mice. Whether these intestinal infections in a non-definitive host were due to infection at a young host age or to large egg dosages is not clear. The prepatent period of T. canis in the canine definitive host is 30-35 days (Dubey, 1978); delaying the necropsy of Cats 7 and 8 in the present study until 70 DPI may have allowed patent infections to develop as observed in cats by Bhowmick (1964). Following larval T. cunis infection most animals develop a peak eosinophilia by 14-18 days which is then followed by a rapid decline to near baseline values (Sugane & Oshima, 1984a). However, peripheral eosinophilia can be induced even in the absence of infection by administration of excretory-secretory antigens of infective T. canis larvae (Sugane & Oshima, 1984b). Humans usually maintain elevated eosinophil counts for prolonged periods following even a single exposure to infective eggs (Smith 8~ Beaver, 1953; Chaudhuri & Saha, 1959). In the present study all infected cats except Cat 1 developed marked eosinophilia with peak eosinophil counts at 25 DPI and 39 DPI in a singly-infected cat and a doublyinfected cat, respectively. Challenge infection at 14 days after initial infection appeared to prolong the eosinophil peak associated with a single infection for a further 14 days. Accompanying this circulating eosinophilia was a corresponding elevation in the level of bone marrow eosinophils, although there was no direct correlation between the levels in the two compartments. Production of bone marrow eosinophils and the induction of peripheral eosinophilia in T. c&s-infected animals have been shown to be dependent, in part, on mast cell-derived factors (Nawa, Owhashi, Imai & Abe, 1987). Basophils were
found circulating in several of our infected cats with an accompanying eosinophilia. In addition, increased numbers of mast cells were found within selected tissue sections of our infected cats. Collectively, these observations may reflect morphological evidence of increased production of mast cell-derived eosinophilogenic factors. A spectrum of hypereosinophilic syndromes has been recorded in six cats presenting with enteritis (Hendrick, 1981). Although gastrointestinal signs were not noted in the present study, the eosinophil levels in both blood and bone marrow as well as the distribution and histological description of the lesions in the hypereosinophilic cats closely resembled those found in several of the cats in the present study. These similarities may warrant the future inclusion of feline larval T. canis infection within the spectrum of eosinophil disorders in cats. Ocular larva migrans due to T. canis may be a rare occurrence in the cat. Despite a thorough ophthalmological, microscopic and histopathologic examination of each eye, we were unable to document any ocular disease or the presence of intraocular larvae in the eight cats used in this study. Bhowmick (1964) was also unable to recover larvae from the eyes of nine cats obtained either 6 or 70-71 days post infection. Although the pathogenesis of OLM is at present unknown, the lack of ocular infection in these two studies may be a result of the small number of animals examined or the magnitude of infection. For example, when mice were infected at 100 times the dose used in this study, up to 86% of the eyes developed OLM by 34 days post challenge (Olson, 1976). In summary, in this study we have reproduced the lesions of naturally-occurring feline VLM by experimental infection of cats with eggs of T. canis. Most cats developed peripheral and bone marrow eosinophilia as well as eosinophilic and granulomatous lesions among a wide range of organs, not including the eye. Although not directly attributable to larval migration during this study, medial hypertrophy and hyperplasia of the pulmonary arteries (MHPA) were found in the lungs of all our infected cats. MHPA may represent an important feature of the response of the feline lung to larval migration in general. Finally, with the application of recentlyavailable, T. c&s-specific reagents (Bowman, MikaGrieve & Grieve, 1987b; Kennedy, Maizels, Meghji, Young, Qureshi & Smith, 1987) accurate serodiagnosis of this potentially common but clinically inapparent condition may be possible. Acknowledgements-This study was supported in part by National Institutes of Health Grant EY-05677. Durinrz the course of this study JCP received partial salary support from the Department of Pathobiological Sciences, University of Wisconsin-Madison. The authors would like to thank Dr R. R. Dubielzig for assistance with ocular histopathology, Dr K. M. Young for examining bone marrow sections and Dr A. J. Cooley for necropsy photography. Barbara Rose provided excellent technical assistance.
Experimental
larval toxocariasis
REFERENCES BEAVERP. C., SNYDER C. H., CARRERA G. M., DENT J. H. & LAFFERTYJ. W. 1952. Chronic eosinophilia due to visceral larva migrans. Pediatrics 9: 7-19. BEAVERP. C. 1962. Toxocariasis (visceral larva migrans) in relation to tropical eosinophilia. Bufletin de la Societe de Pathologie Exotique 55: 555-576. BEAVERP. C. 1966. Zoonoses, with particular reference to parasites of veterinary importance. In: Biology of Parasites (Edited by SOULSBY E. J. L.), pp. 215-227. Academic Press, New York. BHOWMICK D. K. 1964. Beitrage zu dem Problem der Wanderweg der Askaridenlarven (Ascaris lumbricoides Linne 1758 und Toxocara canis Werner 1782) im experimentellen und natilrlichen Wirt. Zeitschrtft fur Parasitenkunde 24: 121-168. BOWMAND. D., MIKA-GRIEVE M. & GRIEVE R. B. 1987a. Circulating excretory-secretory . antigen - levels and specific antibody responses in mice infected with Toxocara canis. American Journal of Tropical Medicine and Hvaiene 36: 75-82. BOWMAN D. D., M;KA&RIEVE M. & GRIEVE R. B. 1987b. Toxocara canis: monoclonal antibodies to larval excretory-secretory antigens that bind with genus and species specificity to the cuticular surface of infective larvae. Experimental Parasitology 64: 458465. CHAUDHURIR. N. & SAHA T. K. 1959. Tropical eosinophilia. Experiments with Toxocara canis. Lancet 2: 493-494. DUBEY J. P. 1978. Patent Toxocara canis infection in ascaridnaive dogs. Journal of Parasitology 64: 1021-1023. FITZGERALDP. R. & MANSFIELD M. E. 1970. Visceral larva migrans (Toxocara canis) in calves. American Journal of Veterinary Research 31: 561-565. GLICKMAN L. T. & SCHANTZ P. M. 1981. Epidemiology and pathogenesis of zoonotic toxocariasis. Epidemiologic Reviews 3: 230-250. GLICKMAN L. T. & SUMMERS B. A. 1983. Experimental Toxocara canis infection in cynomolgus macaques (Macaca fascicularis). American Journal of Veterinary Research 44: 2347-2354. HAMILTONJ. M. 1963. Aelurostrongylus abstrusus infestation of the cat. Veterinary Record 75: 417422. HAYAT C. S., REHMAN B. & AHMAD M. S. 1973. Migration of ascarids in sheep. Pakistan Journal of Zoology 5: 91-97. HAYDEN D. W. & KRUININGENH. J. VAN 1973. Eosinophilic gastroenteritis in German Shepherd dogs and its relationship to visceral larva migrans. Journal of the American Veterinary Medical Association 162: 379-384. HENDRICK M. 1981. A spectrum of hypereosinophilic syndromes exemplified by six cats with eosinophilic enteritis. Veterinary Pathologv 18: 188-200. HUFF D. S., NEAFIE.R. C., B~DER M. J., DE LEON G. A., BROWN L. W. & KAZACOS K. R. 1984. The first fatal Baylisascaris infection in humans: an infant with eosinophilic meningoencephalitis. Pediatric Pathology 2: 345-352. HUGHES P. L., DUBIELZIG R. R. SC KAZACOS K. R. 1987. Multifocal retinitis in New Zealand sheep dogs. Veterinary Pathology 24: 22-27. JAIN N. C. 1986. The cat: normal hematology with comments on response to disease. In: Schalm’s Veterinary Hematology, 4th Edn (Edited by JAIN N. C.), pp. 126139. Lea & Febiger, Philadelphia. JOHNSONB. W., KIRKPATRICK~. E., WHITELEYH. E., MORTON D. & HELPER L. C. (in press) Retinitis and intraocular larval migration in a group of Border Collies. Journalof the American Animal Hospital Association.
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JOHNSON F. B. 1976. Splendore-Hoeppli phenomenon. In: Pathology of Tropical and Extraordinary Diseases, Vol. 2 (Edited bv BINFORDC. H. & CONNOR D. H.). DD. 681-683. Armed Forces Institute of Pathology, Washington. JULIAN R. J. 1985. The peritoneum, retroperitoneum, and mesentery. In: Pathology of Domestic Animals, 3rd Edn, Vol. 2 (Edited by JUBB K. V. F., KENNEDYP. C. & PALMER N.), pp. 329-342. Academic Press, Orlando. KAYES S. G. & OAKS J. A. 1978. Development of the granulomatous response in murine toxocariasis. American Journal of Pathology 93: 277-294. KAYESS. G. 1986. Non-specific allergic granulomatosis in the lungs of mice infected with large but not small inocula of the canine ascarid, Toxocara canis. Clinical Immunology -. and Immunopathology 41: 55-65. KENNEDY M. W.. MAIZELS R. M.. MEGHJI M.. YOUNG L.. QURESHI F. & SMITH H. V. 1987. Species-specific and common epitopes on the secreted and surface antigens of Toxocara cati and Toxocara canis infective larvae. Parasite Immunology 9: 407420. MACKENZIE A. 1960. Pathological changes in lungworm infestation in two cats with special reference to changes in pulmonary arterial branches. Research in Veterinary Science 1: 255-259. MAGNUSSON G. 1970. Visceral larva migrans in dogs. Zeitschrift fir Versuchstierkunde 12: 4457. MCCRAW B. M. & GREENWAY 3. A. 1970. Ascaris suum infection in calves III. Pathology. Canadian Journal of Comparative Medicine 34: 247-255. MERDIVENCIA. & SEZENY. 1965. The resistance of tortoises, Testudo graeca L., against Toxocara canis infection. Zeitschrift fur Parasitenkunde 25: 387-392. NAWA Y., OWHASHI M., IMAI J. & ABE T. 1987. Eosinophil response in mast cell-deficient W/W” mice. International Archives of Allergy and Applied Immunology 83: 6-l 1. NICHOLLSR. L. 1956. The etiology of visceral larval migrans. I. Diagnostic morphology of infective second-stage Toxocara larvae. Journal of Parasitology 42: 349-362. OLSON L. J. 1976. Ocular toxocariasis in mice: distribution of larvae and lesions. International Journalfor Parasitology 6: 247-25 1. PARSONSJ. C., BOWMAND. D., GILLETTE D. M. & GRIEVE R. B. 1988. Disseminated granulomatous disease in a cat caused by larvae of Toxocara canis. Journal of Comparative Pathology 99: 343-346. POYNTER D. 1966. Some tissue reactions to nematode parasites of animals. Advances in Parasitology 4: 321-383. ROGERS W. A., BISHOP S. P. & ROHOVSKY M. W. 1971. Pulmonary artery medial hypertrophy and hyperplasia in conventional and specific-pathogen-free cats. American Journal of Veterinary Research 32: 767-774. ROHDE K. 1962. Helminthen aus Katzen und Hunden in Malaya; Bermerkungen zu ihrer epidemiologischen Bedeutung fur den Menschen. Zeitschrift fur Parasitenkunde 22: 237-244. RON&US 0. 1966. Studies on the aetiology and pathogenesis of white spots in the liver of pigs. Acta Veterinaria Scandinavica (Supplement 16) I: 1-I 12. ROTH B. & SCHNEIDER C. C. 1971. Untersuchungen zur Abhangigkeit des “weissen Blutbildes” bei Hauskatzen (Felis domestica) von Wurminfektion des Darmes. Berliner und Munchener Tierdrztliche Wochenschrtft 22: 43-37. RUBIN L. F. & SAUNDERS L. Z. 1965. Intraocular larva migrans in dogs. Pathologia Veterinaria 2: 566573. SHIELDSJ. A. 1984. Ocular toxocariasis. A review. Survey of Ophthalmology 28: 361-38 1,
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SIMON N. & HOLZWORTHJ. 1967. Eosinophilic leukemia in a cat. Cornell Veterinarian 57: 579-597. SINHA B. N. 1970. Some observations on the pathological lesions caused by migrating larvae of Toxocara canis in Spanish goats. Southwestern Veterinarian 23: 201-208. SMITH M. H. D. & BEAVER P. C. 1953. Persistence and distribution of Toxocara larvae in the tissues of children and mice. Pediatrics 12: 491497. SPREN~ J. F. A. 1956. The life history and development of Toxocara cari (Schrank 1788) in the domestic cat. Parasitology 46: 54-78. SPRENT J. F. A. 1959. The life history and development of Toxascaris leonina (von Linstow 1902) in the dog and cat. Parasitology 49: 33Cb-371. SPRENTJ. F. A. & BARREN M. G. 1964. Large roundworms of dogs and cats: differentiation of Toxocara canis and Toxascaris leonina. Australian Veterinary Journal 40: 166 171. SUGANE K. & OSH~MA T. 1984a. Interrelationship of eosinophilia and IgE antibody production to larval antigen in Toxocara canis infected mice. Parasite Immunology 6: 409420. SUGANEK. & OSHIMAT. 1984b. Induction of peripheral blood eosinophilia in mice by excretory and secretory antigen of
Toxocaracanislarvae. JournalofHelminthology58: 143-147. SWEATMANG. K. 1962. The potential role of the possum (Trichosurus vulpecula) as a transport host of Toxocara canis. New Zeal&d Veterinary Jo&al 10: 108-I 10. SWERCZEKT. W., NIELSEN S. W. & HELMBOLDTC. F. 1970. Ascariasis causing pulmonary arterial hyperplasia in cats. Research in Veterinary Science 11: 103-104. SWERCZEKT. W., NIELSEN S. W. & HELMBOLDTC. F. 1971. Transmammary passage of Toxocara cati in the cat. American Journal of Veterinary Research 32: 89-92. TOMIMURA T., YOKO~A M. & TAKIGUCHI H. 1976. Experimental visceral larva migrans in monkeys I. Clinical, hematological, biochemical and gross pathological observations on monkeys inoculated with embryonated eggs of the dog ascarid, Toxocara canis. Japanese Journal of Veterinary Science 38: 533-548. WEATHERLEYA. J. Kc HAMILTONJ. M. 1984. Possible role of histamine in the genesis of pulmonary arterial disease in cats infected with Toxocara cati. Veterinary Record 114: 347-349. ZENDULKAM. 1967. The differences between changes in pig livers caused by an experimental migration of larvae of Ascaris suum and Toxocara canis. Veterinarni Medicina 12: 551-556.
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PATHOLOGICAL AND HAEMATOLOGICAL RESPONSES OF CATS EXPERIMENTALLY INFECTED WITH TOXOCARA CANIS LARVAE J. C. *Department tDepartment
PARSONS,* D. D. BOWMAN? and R. B. GRIEVE*
of Pathology,
College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, U.S.A. of Microbiology, Immunology and Parasitology, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, U.S.A. (Received 23 August 1988; accepted 1 February 1989)
Abstract-PARSONS J. C., BOWMAN D. D. and GRIEVER. B. 1989. Pathological
and haematological responses of cats experimentally infected with Toxocara canis larvae. International Journal for Parasitology 19: 419488. Responses of eight adult cats to one or two infections with larvae of Toxocara canis were studied up to 39 days post infection (DPI). Clinically, all cats remained normal throughout the study. The major necropsy finding was multifocal, white to grey nodules mainly within the liver, lungs and kidneys; live larvae were found in liver nodules. Histologically, the nodules were eosinophilic granulomas. Granulomas containing a larval section were observed mainly within the liver. All infected cats had variably severe, eosinophilic arteritis and bronchiolitis and medial hypertrophy and hyperplasia of the pulmonary arteries. No inflammatory eye lesions were detected. Circulating eosinophil levels increased in all infected cats; peak values of 15,790 and 10,050 eosinophils pi- ’ were observed at 25 or 32 DPI in cats receiving a single or double infection, respectively. Bone marrow of all infected cats exhibited marked eosinophilic hyperplasia which did not correlate with the level of circulating eosinophilia. Thus, infection of cats by the larvae of T. canis causes disseminated eosinophilic and granulomatous disease with marked pulmonary artery and airway lesions. INDEX KEY WORDS: Toxocara canis; cats; histopathology; granuloma; larva migrans; eosinophils; eosinophilia; bone marrow; medial hypertrophy
HUMAN visceral larva migrans (VLM) is a syndrome resulting from prolonged migration of infective nematode larvae through the internal organs of the host. Overt VLM usually occurs in young children and presents as a clinical syndrome of chronic extreme eosinophilia, hepatosplenomegaly and some degree of pneumonitis (Beaver, Snyder, Carrera, Dent & Lafferty, 1952). In the original report of VLM by Beaver et al. (1952), liver biopsies from three affected children revealed extensive eosinophilic and granulomatous lesions, one of which contained an infective larva of the canine ascarid, Toxocara canis. Other ascaridoid larvae, including Toxocara cati and Baylisascaris procyonis, have been incriminated in additional human cases of VLM (reviewed by Beaver, 1966; Huff, Neafie, Binder, De Leon, Brown & Kazacos, 1984) but serological evidence suggests that T. canis remains the predominant worldwide cause of human VLM (Glickman & Schantz, 1981). An important sequela to larval toxocariasis in humans and dogs is ocular disease or ocular larva migrans (OLM) (Rubin & Saunders, 1965; Shields, 1984; Hughes, Dubielzig & Kazacos, 1987; Johnson, Kirkpatrick, Whiteley, Morton & Helper, in press). Both VLM and OLM have been well characterized in humans and laboratory animals, but little work has
granulomatous disease; visceral and hyperplasia; MHPA.
been done on larval toxocariasis in other vertebrate hosts, and it may be that this disease is often overlooked in these animals. Animal species other than mice, rabbits and guinea pigs, in which responses to systemic migration of T. canis larvae have been studied, include sheep (Hayat, Rehman & Ahmad, 1973), goats (Sinha, 1970), cattle (Fitzgerald & Mansfield, 1970; McGraw & Greenway, 1970), pigs (Ron&us, 1966; Zendulka, 1967), dogs (Magnusson, 1970; Hayden & van Kruiningen, 1973), monkeys (Beaver, 1962; Tomimura, Yokota & Takiguchi, 1976; Glickman & Summers, 1983), possums (Sweatman, 1962) and tortoises (Merdivenci & Sezen, 1965). Larval T. canis migration in several of these animal species including fowl has also been reviewed by Poynter (1966). However, the lesions of VLM in cats due to ascarid larval migration have not been extensively examined. Larvae of Toxocara cati have been recovered from the liver, lungs and musculature of experimentally infected cats (Sprent, 1956; Swerczek, Nielsen & Helmboldt, 1971), but only the pulmonary lesions were described (Swerczek, Nielsen & Helmboldt, 1970). In cats experimentally infected with T. canis, Bhowmick (1964) found multifocal, white to grey nodules within the kidneys, lung, liver and heart. We have recently described a case 419
J. C. PARSONS,D. D. BOWMANand R. B. GRIEVE
480
in which similar nodules were found at necropsy within the same range of organs from a domestic cat with naturally-occurring larval toxocariasis (Parsons, Bowman, Gillette & Grieve, 1988). Histologically, these nodules were found to be eosinophilic granulomas, some of which contained an infective T. canis larva. However, little could be determined about the temporal development of these lesions in this host. Thus, in order to further investigate the pathogenesis of feline VIM due to T. canis we have examined the haematological and pathological responses of cats infected on one or two occasions with embryonated eggs of T. canis. Additionally, we have examined the potential for larvae of T. canis to cause ocular larva migrans in the cat. MATERIALS
AND METHODS
Experimental animals. A group of seven neutered, adult, female, domestic shorthair (DSH) cats were housed in an American Association for the Accreditation of Laboratory Animal Care-approved facility for a period of at least one year. According to health care records, some cats had previously passed nematodes and may, therefore, not have been ascarid-naive. Cats 8 and 9 were intact, adult, male, specific-pathogen-free (SPF), DSH cats. The ancestors of these animals, obtained over 10 years previously, were caesarian-derived and colostrum-deprived and, therefore, were free of any feline ascarids (Swerczek et al., 1971). Prior to the commencement of the study, all cats were found to be helminth-free by use of a faecal flotation procedure. Experimental infection and sample collection. Eggs of Toxocara canis were collected and embryonated using a previously described method (Bowman, Mika-Grieve & Grieve, 1987a). All cats were anaesthetized by an intramuscular injection of ketamine hydrochloride (Ketaset; Bristol Laboratories, Syracuse, NY) prior to each sample collection and each egg inoculation. On Day 0 of the study all of the experimental cats were infected intragastrically, via a stomach tube, with 5000 embryonated T. canis eggs (i.e. approximately 1 epg body weight) in 10 ml of tap water. Fourteen days later, four of the cats were given a challenge dose of 5000 infective eggs. During the course of the study all cats were observed at least twice daily. Necropsies were performed on Days 18, 25, 32 and 39 post initial infection according to the schedule in Table 1. A control cat received an intragastric dose of 10 ml of water on each of Days 0 and 14 post infection and was necropsied on Day 32 post initial infection. Immediately prior to infection, challenge and necropsy, blood samples were collected by jugular venipuncture into EDTA-coated blood tubes (Vacutainer; Becton, Dickinson & Co., Rutherford, NJ). Whole blood smears were made and air dried prior to staining with Wright’s stain. On the day of necropsy all cats underwent a complete ophthalmoscopic examination. The cats were then killed by an intravenous overdose of barbiturate and immediately necropsied. Liver samples, each weighing approximately 0.1 g and containing a subcapsular lesion, were taken from Cats 1, 5, 6, 7 and 8, compressed between two microscope slides and examined for larvae. The entire gastrointestinal system was opened and examined for immature and mature helminths. All organs were examined for gross lesions and tissue samples were collected from the right caudal and left cranial lung lobes after fixation, and from the left and right ventricular free wall and
TABLE
I-INFECTIONANDNECROPSYSCHEDULEOFCATS
EXPERIMENTALLYINFECTEDONCEORTWlCEW,THEGGSOF
Toxocara canis Cat number
No. of eggs administered
FS FS FS FS FS M FS FS M
Day of necropsy
0 DPI
14 DPI
(DPI)
5000 5000 5ooo 5000 5000 5000 5000 5000 0
0 5000 0 5000 0 5000 0 5000 0
18 18 25 25 32 32 39 39 32
FS = Spayed female, M = male, DPI = days post initial infection.
interventricular septum of the heart, liver, kidneys, spleen, pancreas, adrenal glands, thyroid glands, thymus, mesenteric and tracheobronchial lymph nodes, small and large intestine, cerebellum, brain stem, cerebrum, sciatic nerve and femoral bone marrow. Muscle samples were collected from the diaphragm, tongue, masseter, biceps brachii and quadriceps femoris. In organs where gross lesions were present, representative examples were collected; all other tissues were sampled at random. All tissues except the lungs, eyes and bone marrow were immersion-fixed in 10% formalin. The lungs were fixed by an intratracheal infusion of 10% formalin and the eyes and bone marrow by immersion in Kamovsky’s fixative. Haematology and histopathology. Differential white cell counts were made using blood smears stained by Wright’s stain. Total whitecell counts (TWCC) and haematocrits were measured using a Coulterm counter (Coulter Electronics Inc., Hialeah, FL). Eyes were hemisccted around the pars plana and the anterior and posterior segments examined for gross lesions under a dissecting microscope. All tissue samples were paraffin-embedded and routinely processed for histopathology. Sections were cut at 5 m and stained with haematoxylin and eosin. Selected sections were also stained using Verhoff-Van Gieson. Alcian Blue-PAS, Luna’s eosinophil and Toluidine Blue stains. Bone marrow sections, cut at 3 m, were stained with Giemsa stain and the percentage of eosinophils in 500 nucleated cells was determined. T. canis larvae, when preserved within tissue sections, were identified on the basis of larval morphology and size (Nicholls, 1956). Larval viability was also assessed using morphological criteria. Representative tissue sections have been deposited with the U.S. National Parasite Collection (Beltsville, MD 20705). RESULTS
During the course of the study Cat 7 developed a mild cough and sinusitis; all other cats remained clinically normal. No antemortem ocular lesions were detected in any cats.
Necropsyjndings Gross lesions were found mainly within the liver, lungs and kidneys of the infected cats. In general,
Experimental larval toxocariasis in cats lesions were more numerous in cats receiving a dual infection and became progressively more numerous and larger with time. Marked hepatomegaly was found in Cats 6 and 8; splenomegaly could not be assessed as the barbiturate-based euthanasia agent caused marked dilation of this organ. No gross lesions were present in any organs, including eyes, of the control cat. Liver. Within the liver of all infected cats there were multifocal, often slightly raised, irregular, white to grey foci up to 2 mm in diameter. A living T. canis larva was found within squash preparations of hepatic lesions dissected from all cats thus examined. Lungs. All infected cats had either moderate or severe pneumonia and pleuritis. The pleural surface, in the early stages of infection or reinfection, was usually mottled with irregular, dark red and white areas, up to approximately 10 mm in diameter, which contained well-delineated, raised, white nodules, often with a haemorrhagic core. In the later stages of infection (Cats 5-Q focal areas of the visceral pleura were markedly thickened and were often lightly adherent to
481
kidney and pleura were present within the epicardium of Cat 8, the pancreas of Cats 1 and 3, and the mesenteric lymph node of Cat 3. All other organs of the infected cats appeared grossly normal. No ocular lesions were detected in hemisected eyes. No immature or mature helminths were found within the lumen of the gastrointestinal system of any cat. Cat 7 had moderate, suppurative rhinitis and sinusitis which was probably unrelated to T. canis infection. Histopathologicalfindings
The major histopathological findings in all infected cats were eosinophilic granulomas, within a range of visceral organs and somatic musculature, and medial hypertrophy and hyperplasia of the pulmonary arteries. Granulomas, which corresponded to the nodules seen grossly, were also found in sections of randomly-sampled tissues in which no gross lesions were detected. Morphologically-viable larvae were present in one or more tissues of six cats (Fig. 2a, b, d); most commonly these larvae were in the liver (Cats, 2, 3, 4, 6 and 8). All tissues from the control cat were histopathologically normal.
FIG. 1. Kidney from cat experimentally infected twice with eggs of Toxocara canis, Cat 8. a. Multiple subcapsular granulomas. Scale bar = 5 mm. b. Sectioned surface with three granulomas (arrows) extending towards the corticomedullary junction. Scale bar = 5 mm.
the parietal pleura. Nodular foci, similar to those on the pleura, were present within the lung parenchyma of all infected cats. Several of the larger airways of Cat 8 exuded pale yellow, mucoid material. Adjacent to these airways were firm, homogeneously white foci, up to 2 mm in diameter, with a fine, central, dark brown core. Kidneys. Multifocal, well-circumscribed, raised, white nodules up to 3 mm in diameter (Fig. la) were present on the capsular surface of one or both kidneys of all cats except Cat 5. The larger renal lesions were conical and extended for varying distances toward the corticomedullary junction (Fig. lb). Other organs. Nodular lesions similar to those in the
Liver. Generalized, mild or moderate, lymphoplasmacytic, portal hepatitis and mild periportal fatty change were present within the liver of each infected cat. The portal hepatitis was more severe and contained more eosinophils in areas directly involved in larval migration (Fig. 2a). In singlyinfected cats, early phase (Cats 1 and 3) lesions consisted of focal areas of acute hepatocellular necrosis surrounded by leucocyte debris, eosinophils and neutrophils. These lesions were scattered at random throughout the hepatic lobules. In Cat 5 these areas had become focally extensive granulomas with a necrotic core of eosinophilic leucocyte debris admixed
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J. C. PARSONS, D. D. BOWMAN and R. B. GRIEVE
FIG 2. Development of disseminated eosinophilic granulomas in various tissues from cats infected once or twice with eggs of Toxocura canis. a. Liver, Cat 8. Area of moderate, eosinophilic, portal hepatitis with an adjacent T. canis larva in tangential section surrounded by an acute inflammatory response of eosinophils, neutrophils and necrotic hepatocytes, H&E. Scale bar = 100 m. b. Liver, Cat 5. Organizing granuloma containing eosinophilic cellular debris moulded around larva (arrows) which either died and disintegrated or migrated from the granuloma. Note giant cells and early fibroplasia at the periphery of the granuloma, H&E. Scale bar = 50 ,mn. c. Lung, Cat 2. Large eosinophilic granuloma surrounding a tangential section of a viable T. canislarva. Note mild medial hypertrophy and hyperplasia and sub-endothelial vacuolation in adjacent pulmonary artery, H&E. Scale bar = 75 ,um. d. ~#u~~~ce~~~~o~i~ muscle, Cat 5. Eosinophilic granuloma surrounding two cross-sections of a coiled, viable, T. canis larva. Note the prominent excretory columns within one larval section (arrow), H&E. Scale bar = 50 m.
with epithelioid cells and occasional foreign-body giant cells surrounded by a thin outer rim of mainly eosinophils with a few lymphocytes and plasma ceils (Fig. 2b). By 39 DPI (Cat 7) the granuiomas were more organized, but with little evidence of fibroplasia, and contained fewer eosinophils. Early phase lesions were also present in some liver sections from Cats 5 and 7. A similar range of lesions, including those of the early phase (Fig. Za), was present with the livers of the cats receiving a challenge infection. However, in Cat 2, large areas of hepatocellular necrosis with marked leucocyte infiltration, occasionally surrounding a larva, were present in addition to early phase lesions.
No macrophages or lymphocytes were observed in these lesions which were mainly subcapsular in location. This type of lesion was suggestive of an acute response to the challenge larvae. LLcngs.Within the lung parenchyma of all infected were focally extensive, eosinophil-rick cats granulomas (Fig. 2c) and mild to moderate pulmonary oedema. No difference in the nature and distribution of lesions was observed between the two sampled lung lobes. When a larva was present within an alveolar lumen, the accompanying inflammatory response occluded that lumen and those of severe adjoining alveoli (Fig. 2~). Pleural and subpleural granulomas,
Experimental larval toxocariasis in cats contiguous with extensive areas of eosinophilic pleuritis, were common in most of the cats. In the later
stages of infection (Cats 5-8) there was marked, focal, pleural fibrosis. Medial hypertrophy and hyperplasia of the pulmonary arteries and arterioles (MHPA) were also present in all infected cats (Figs. 2c, 3). This marked thickening of the tunica media was mainly due to proliferation of the medial myocytes, however, in some vessels individual smooth muscle cells were swollen with cytoplasmic vacuoles (Fig. 3d). MHPA was most severe in Cat 8 in which the greatly thickened and narrowed arteries corresponded to the white foci seen grossly adjacent to airways (Fig. 3~). Coexisting with MHPA in all infected cats was a variably severe, eosinophilic endarteritis, periarteritis, bronchiolitis and peribronchiolitis (Fig. 3b, c, d). Numerous mast cells were present within the vascular and airway
483
infiltrates which were most severe in animals receiving a challenge infection; severity increased with time following infection. In Cat 8, marked intimal proliferation, subendothelial vacuolation and endarteritis had greatly reduced or occluded the lumen of several vessels (Fig. 3~). Fragmentation of the internal elastic membrane had occurred in some of the more severely affected vessels of this cat. Normal pulmonary arteries were also present in sections from all infected cats. Perivascular oedema, independent of any arteritis, was observed only within the lungs of challenged cats. In Cat 2 the oedema was marked and contained a moderate infiltrate of eosinophils around most large arteries. This lesion became progressively more eosinophilic and less oedematous with time in Cats 4,6 and 8, respectively. No airway or pulmonary artery lesions were present in lung sections from the control cat (Fig. 3a).
FIG. 3. Eosinophilic endarteritis and medial hypertrophy and hyperplasia (MHPA) in the lungs ofcats experimentally infected once or twice with eggs of Toxocara canis. a. Cat 9. Normal pulmonary artery and bronchiole, H&E. Scale bar = 80 p. b. Cat 1. Mild eosinophilic periarteritis, endarteritis and bronchiolitis with moderate MHPA, H&E. Scale bar = 80 F. c. Cat 8. Severe eosinophilic endarteritis and MHPA which appears to have almost occluded the arterial lumen. Note the plug of eosinophils, mucus and sloughed epithelial cells within the adjacent bronchiole (arrow), H&E. Scale bar = 120 m. d. Cat 5. A pulmonary artery with severe eosinophilic periarteritis and endarteritis and marked cytoplasmic vacuolation of hypertrophied and hyperplastic medial myocytes, H&E. Scale bar = 80 m.
J. C. PARSONS,D. D. BOWMAN and R. B. GRIEVE
484
Kidneys. Nephritis was found in all infected cats except Cats 4 and 5. Large, eosinophilic granulomas incorporating glomeruli and renal tubules and extending towards the corticomedullary junction were present in Cats 6, 7 and 8. Hearf. Non-suppurative, eosinophilic, interstitial myocarditis without distinct granuloma formation was present in four cats (Cats 2,3,4 and 8). Skeletalmuscles. Five cats had granulomas in one or more skeletal muscles. The tongue (Cats 2, 7 and 8) and the quadriceps femoris (Cats 2, 5 and 8) (Fig. 2d) were most commonly affected. Lymph nodes. In Cats 2 and 3, the subcapsular sinus and medullary cords of the mesenteric lymph node (MLN) were focally distended by large numbers of eosinophils. A granuloma containing a viable larva surrounded by Splendore-Hoeppli material (see Johnson, 1976) was present within a germinal centre of the MLN of Cat 3.
Brain. Cats 4 and 6 had focal, granulomatous meningitis. Occasional eosinophils were present within the surface meninges and perivascular cuffs. Cat 6 had, in addition to meningitis, a mild, multifocal, granulomatous encephalitis mainly affecting the white matter of the brain stem and cerebrum. Larvae were not observed in any brain sections. Bone marrow. All infected cats had marked eosinophilic hyperplasia of the bone marrow (Table 2). The percentage of eosinophils in 500 nucleated bone marrow cells ranged from 6.4 in Cat 6 to 28.2 in Cat 3 (mean = 19.6). Other organs. Focal areas of eosinophilic and granulomatous infiltration were present within the pancreas of Cats 1 and 3 and within the submucosa of the colon of Cat 2 and of the small intestine of Cat 8. No additional lesions were present in the remaining tissue sections, including eyes, from the infected cats.
TABLE Z-HAEMATOLOGICAL RESPONSES OF CATSEXPERIMENTALLY EVFECTEDONCEORTWICEWITHEGGSOF Toxocara
canis
Days post initial infection
Cat number 0
14
18
lTwcc EOS PI-’ %EOSBM
9300 740 -
-
6100 850 6.6
TWCC EOS /X’ %EOSBM
I 1,800
TWCC EOS PI-’ %EOSBM
6800 680
TWCC EOS PI-’ %EOSBM
6200 500
TWCC EOS /J-’ %EOSBM
16,400 1640 -
TWCC EOS PI-’ %EOSBM
8300 420
TWCC EOS~I-’ %EOSBM
8900 530
TWCC EOS&’ %EOSBM
12,500 870
TWCC EOS pi-’ %EOSBM
15,200 3190 -
710 -
25
32
39
14,200 3410 22.6 32,900 15,790 28.2
-
14,800 6600 12.9
16,200 4050
-
-
16,100 3060 24.0
9100 1460
18,500 8330 6.4 15,300 1990 15.6 31,400 10,050 22.4
_ 8700 90
9200 90 -
-
-
8300 420 0.4
TWCC = Total white cell count, EOS ~1~ ’ = absolute number of circulating eosinophils ~1~ ‘, %EOSBM = percentage of eosinophils of 500 nucleated cells in bone marrow sections. *Normal
values (Jain, 1986): TWCC=
5.5-19.5.
x IO3 leucocytes
~1~‘; EOS pl -‘=&1500;
%EOSBM=
1.5-3.8%.
Experimental larval toxocariasis in cats Haematological responses
Prior to infection, the leucogram of each cat, except Cat 5, was within normal limits for adult cats (Table 2). On 0 DPI Cat 5 had an unexplained eosinophilia with an absolute eosinophil count of 1640 eosinophils ~1~’ and a normal TWCC. The mean eosinophil count of the eight test cats at 0 DPI was 760 ~1~‘. The haematocrit of all cats remained within normal limits throughout the study (data not shown). By 14 DPI three of the four cats sampled had a marked eosinophilia ranging from 1460 to 7390 eosinophils ~1~’ (mean=4020 eosinophils ~1~‘) and a normal TWCC. Each of the four cats that received a challenge infection had an eosinophilia at necropsy. The circulating eosinophil levels varied from 3410 ~1~’ in Cat 2 to 10,050 ~1~’ in Cat 8. Each of the cats that received a single infection, except Cat 1, had an eosinophiha at the time of necropsy. In the group of singly-infected cats, the peak level of eosinophilia (15,790 eosinophils ~1~‘) was seen at 25 DPI in Cat 3 and in the group of cats receiving a challenge infection the peak level of T. canis (10,050 eosinophils PI-‘) was seen in Cat 8 at 32 DPI. Occasional basophils, not exceeding 1% of the TWCC, were seen in blood smears during the course of infection in Cats 4,7 and 8. Two cats had a leucocytosis on the day of necropsy. Cat 3 had a leucocytosis with eosinophilia and Cat 8 had a neutrophilic leucocytosis. The control cat remained haematologically normal throughout the study. DISCUSSION The present study indicates that the experimental infection of adult cats with larvae of T. canis causes disseminated eosinophilic granulomatous disease which is similar to that described in visceral larva migrans of humans and other animals. The range of affected organs in the cats in this study was similar to those seen following experimental and natural infection of the cats with T. canis larvae (Bhowmick, 1964; Parsons et al., 1988) with the exception that heart lesions in this study were less common. Lesions due to larval migration within the pancreas and mesenteric lymph node have not been reported previously in this species. No clinical signs due to larval migration within major organs were detected in any of our study cats. Hence, with a lack of presenting signs, and the close association of cats and dogs, covert disease in cats due to T. canis may be more common than is presently suspected. The histopathology of the developing granuloma in larval toxocariasis has been described previously (Kayes & Oaks, 1978). A similar sequence of histological responses was observed in most tissues from the singly infected cats in this study. As in the study by Kayes & Oaks (1978) we found that both acute and chronic types of granuloma were often present in the same histological section. This response probably represented the migration from the more
485
chronic granuloma by the inciting larva which had then induced a subsequent reaction at another site. In general, challenge infection induced additional acute lesions without modifying the histological picture of either the acute or chronic response. However, within the lungs there was a strong association between challenge infection and the severity of eosinophilic arteritis, periarteritis, bronchiolitis and peribronchiolitis. In addition, perivascular oedema, independent of any arteritis present within the same lungfield, was seen only in challenged animals. All these lesions may represent an ongoing immediatetype hypersensitivity reaction to some larval product(s) present locally within the lungs in association with continued larval migration to this site (Weatherley & Hamilton, 1984). Alternatively, it may be a non-specific, hyperergic response of a lung, previously primed by T. canis larval migration, to an inhaled or ingested allergen (Kayes, 1986). Enumeration of larval numbers following artificial digestion of a range of tissue samples may give an estimate of larval dissemination within an infected animal; tissue digests of each cat were not performed in this study. However, the finding of larval granulomas in a wide range of randomly sampled organs without gross lesions suggests that, particularly in the case of the skeletal muscles, the level of dissemination of T. canis larvae following infection in cats is high. Many of these tissue granulomas contained a T. canis larva in section which helped to distinguish them from any pre-existing granulomas due to T. cati and from the non-effusive form of feline infectious peritonitis (Julian, 1985) and eosinophilic leukaemia (Simon & Holzworth, 1967); the gross lesions of the latter two conditions resemble those seen in the present study. Medial hypertrophy and hyperplasia of the pulmonary arteries (MHPA) were found in all of the infected cats of this study as well as in a natural case of VLM in a domestic cat due to T. canis described by Parsons et al. (1988). Prior infection by the feline lungworm, Aelurostrongylus abstrusus, was originally thought to be the main cause of MHPA in cats (Mackenzie, 1960; Hamilton, 1963). Subsequently, Swerczek (unpublished PhD thesis, University of Connecticut, 1969) and Swerczek et al. (1970) demonstrated that the lesion was associated with lung migration of any of several larval nematodes. MHPA was present in cats following infection by the larvae of T. canis, T. cati, Ascaris suum and A. abstrusus; however, it was not present following Toxascaris Zeonina infection in which larvae undergo little or no extraintestinal migration in cats (Sprent, 1959). Prior infection with A. abstrusus in the laboratory-reared cats used in this study is unlikely due to lack of exposure to the molluscan intermediate host. However, it is possible that previous infection of several cats with T. cati may have caused MHPA prior to the commencement of the study. Swerczek (1969, thesis cited above) and Swerczek et al. (1970)
486
J. C. PARSONS, D. D. BOWMAN and R. B. GRIEVE
previously observed mild MHPA, attributed to prior infection by T. cati, in conventionally-reared control cats but not in caesarian-derived, colostrum-deprived cats. Other non-parasitological causes of MHPA may also exist as the lesion has been reported within a colony of caesarian-derived, SPF cats (Rogers, Bishop & Rohovsky, 1971). However, as T. cati is largely transmitted by the transmammary route (Swerczek et al., 1971) colostrum deprivation is important in assuring ascarid-naive animals; it was not clear whether the cats in the colony of Rogers ef al. (1971) were colostrum-deprived. No immature or adult T. canis were found in the gastrointestinal system of the cats in the present study. Swerczek (1969, thesis cited above) was also unable to induce a patent infection of T. canis in cats following the administration of infective eggs. However, others have reported the recovery of adult T. canis from the intestine of cats following natural (reviewed by Sprent & Barrett, 1964; Rohde, 1962; Roth & Schneider, 1971) and experimental (Bhowmick, 1964) infection. Bhowmick (1964) recovered a total of eight adult worms from two young cats at Day 70 following a per OSinfection with 15,000-20,000 infective eggs and was also able to recover an additional 18 adult worms from four cats following intraperitoneal infection or the feeding of infected mice. Whether these intestinal infections in a non-definitive host were due to infection at a young host age or to large egg dosages is not clear. The prepatent period of T. canis in the canine definitive host is 30-35 days (Dubey, 1978); delaying the necropsy of Cats 7 and 8 in the present study until 70 DPI may have allowed patent infections to develop as observed in cats by Bhowmick (1964). Following larval T. cunis infection most animals develop a peak eosinophilia by 14-18 days which is then followed by a rapid decline to near baseline values (Sugane & Oshima, 1984a). However, peripheral eosinophilia can be induced even in the absence of infection by administration of excretory-secretory antigens of infective T. canis larvae (Sugane & Oshima, 1984b). Humans usually maintain elevated eosinophil counts for prolonged periods following even a single exposure to infective eggs (Smith 8~ Beaver, 1953; Chaudhuri & Saha, 1959). In the present study all infected cats except Cat 1 developed marked eosinophilia with peak eosinophil counts at 25 DPI and 39 DPI in a singly-infected cat and a doublyinfected cat, respectively. Challenge infection at 14 days after initial infection appeared to prolong the eosinophil peak associated with a single infection for a further 14 days. Accompanying this circulating eosinophilia was a corresponding elevation in the level of bone marrow eosinophils, although there was no direct correlation between the levels in the two compartments. Production of bone marrow eosinophils and the induction of peripheral eosinophilia in T. c&s-infected animals have been shown to be dependent, in part, on mast cell-derived factors (Nawa, Owhashi, Imai & Abe, 1987). Basophils were
found circulating in several of our infected cats with an accompanying eosinophilia. In addition, increased numbers of mast cells were found within selected tissue sections of our infected cats. Collectively, these observations may reflect morphological evidence of increased production of mast cell-derived eosinophilogenic factors. A spectrum of hypereosinophilic syndromes has been recorded in six cats presenting with enteritis (Hendrick, 1981). Although gastrointestinal signs were not noted in the present study, the eosinophil levels in both blood and bone marrow as well as the distribution and histological description of the lesions in the hypereosinophilic cats closely resembled those found in several of the cats in the present study. These similarities may warrant the future inclusion of feline larval T. canis infection within the spectrum of eosinophil disorders in cats. Ocular larva migrans due to T. canis may be a rare occurrence in the cat. Despite a thorough ophthalmological, microscopic and histopathologic examination of each eye, we were unable to document any ocular disease or the presence of intraocular larvae in the eight cats used in this study. Bhowmick (1964) was also unable to recover larvae from the eyes of nine cats obtained either 6 or 70-71 days post infection. Although the pathogenesis of OLM is at present unknown, the lack of ocular infection in these two studies may be a result of the small number of animals examined or the magnitude of infection. For example, when mice were infected at 100 times the dose used in this study, up to 86% of the eyes developed OLM by 34 days post challenge (Olson, 1976). In summary, in this study we have reproduced the lesions of naturally-occurring feline VLM by experimental infection of cats with eggs of T. canis. Most cats developed peripheral and bone marrow eosinophilia as well as eosinophilic and granulomatous lesions among a wide range of organs, not including the eye. Although not directly attributable to larval migration during this study, medial hypertrophy and hyperplasia of the pulmonary arteries (MHPA) were found in the lungs of all our infected cats. MHPA may represent an important feature of the response of the feline lung to larval migration in general. Finally, with the application of recentlyavailable, T. c&s-specific reagents (Bowman, MikaGrieve & Grieve, 1987b; Kennedy, Maizels, Meghji, Young, Qureshi & Smith, 1987) accurate serodiagnosis of this potentially common but clinically inapparent condition may be possible. Acknowledgements-This study was supported in part by National Institutes of Health Grant EY-05677. Durinrz the course of this study JCP received partial salary support from the Department of Pathobiological Sciences, University of Wisconsin-Madison. The authors would like to thank Dr R. R. Dubielzig for assistance with ocular histopathology, Dr K. M. Young for examining bone marrow sections and Dr A. J. Cooley for necropsy photography. Barbara Rose provided excellent technical assistance.
Experimental
larval toxocariasis
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