Fatal toxoplasmosis in brown hares (Lepus europaeus): possible reasons of their high susceptibility to the infection

Veterinary Parasitology 93 (2000) 13–28

Fatal toxoplasmosis in brown hares (Lepus europaeus): possible reasons of their high susceptibility to the infection K. Sedlák a , I. Literák b,∗ , M. Faldyna c , M. Toman c , J. Benák b b

a State Veterinary Institute, S´ıdlištn´ı 24, 165 03 Prague, Czech Republic Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Palackého 1-3, 612 42 Brno, Czech Republic c Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic

Received 16 March 2000; received in revised form 14 June 2000; accepted 27 June 2000

Abstract Brown hares (Lepus europaeus) trapped in the countryside and domestic rabbits were experimentally infected with Toxoplasma gondii (K7 strain) oocysts. Hares (n = 12) were divided into groups of 4 and infected with 10, 103 and 105 oocysts. Rabbits (n = 12) were infected in the same way. The experimentally infected animals were monitored for 33 days after infection (p.i.). Most of the infected hares demonstrated behavioural changes, and all of them died between 8 and 19 days p.i. Three of the rabbits demonstrated only clinical changes related to the concurrent pasteurellosis. The typical pathological finding in the hares were haemorrhagic enteritis, enlargement and hyperaemia of mesenteric lymph nodes, splenomegaly and multiple miliary necrotic lesions in the parenchyma of the liver and other organs. Pathological changes in the rabbits were less pronounced than in the hares. In rabbit brains, tissue cysts of the T. gondii were found. The incidence of T. gondii antibodies both in the hares and the rabbits was first ascertained on day 7 p.i. On day 12 p.i., antibodies were already found in all the animals infected. Antibody titres in indirect fluorescence antibody test (IFAT) using the anti-rabbit conjugate were markedly higher in rabbits than in hares. In all hares, T. gondii was isolated post mortem from the liver, brain, spleen, kidney, lung, heart and skeletal muscles. Although T. gondii was also isolated in all rabbits, it was not always isolated in all their organs. In all hares, parasitemia was demonstrated on days 7 and 12 p.i. The percentage of rabbits with detected parasitemia was lower. In hares, a decrease in the numbers of leukocytes during the infection was observed. No such decrease was observed in the rabbits. The lymphocyte

∗ Corresponding author. Tel.: +420-5-4156-2307; fax: +420-5-7488-41 E-mail address: [email protected] (I. Liter´ak).

0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 3 2 5 - 3

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activity after the stimulation with non-specific mitogens showed significant differences between the hares and the rabbits even before the infection. After the infection, the hares infected with 103 and 105 doses and in rabbits infected with a 105 dose showed a decrease of lymphocyte activity. Rabbits infected with a 103 dose showed an increase of the lymphocyte activity. While in hares toxoplasmosis was an acute and fatal disease, the infection in rabbits had subclinical manifestations only and easily passed to a latent stage. The different courses of toxoplasmosis in the hare and the rabbit may be due to the differences in the natural sensitivity of the two species to the T. gondii infection or a negative impact of stress to the immune status of hares. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Toxoplasma gondii; Hare; Rabbit; Experimental infection

1. Introduction The brown hare (Lepus europaeus) is a common species of wild mammals in Europe, where they are extensively hunted and form a part of the local diet. They are supposed to be an exceptionally susceptible species to primary infection caused by Toxoplasma gondii, a protozoan parasite prevalent throughout the world. They demonstrate a high incidence of the acute fatal toxoplasmosis with a very low prevalence of latent, i.e. subclinical infections (Gustafsson and Uggla, 1994; Gustafsson, 1997). This observation is supported by a number of papers describing clinical toxoplasmosis in hares (Christiansen and Siim, 1951; Štˇerba et al., 1997; Gustafsson et al., 1988) and reports of a low seroprevalence and parasitological prevalence of T. gondii in their populations (Werner et al., 1973; Edelhofer et al., 1989). Some other studies, however, have reported a high seroprevalence and parasitological prevalence of toxoplasmosis in hares without any clinical signs of the disease or ˇ increased death rates (Levit et al., 1965; Eatár, 1972; Hejl´ıeˇ ek et al., 1997). The question of an increased sensitivity of hares to T. gondii infections might be answered by results of experimental infections. Gustafsson (1997) and Gustafsson et al. (1997a,b) set out to prove the hypothesis of an exceptionally high susceptibility of hares to T. gondii infection by experiments with mountain hares (Lepus timidus). They administered 50 oocysts of the Tg Swe F1 strain per orally to hares and rabbits. All the infected animals were sacrificed on 7 and 8 days post infection (p.i.). Using immunohistochemical techniques, they were able to demonstrated T. gondii in the organs of all the infected hares but in only a few of the rabbits. They found no difference between hares and rabbits as far as serological responses were concerned. In the lymphocyte transformation tests (LTT), no response to the T. gondii antigen was found in hares; rabbits, however, responded even before the experimental infection. The aim of the present study was to monitor the course of an experimental infection of brown hares with T. gondii oocysts and to analyse the pathogenesis of toxoplasmosis in hares. In a control group, a number of domestic rabbits were infected and examined in the same way. In rabbits, which together with the hares belong to the order Lagomorpha, clinical toxoplasmosis has been found only rarely (Dubey et al., 1992), although the seroprevalence and parasitological prevalence is very high in that species (Hejl´ıeˇ ek and Literák, 1994).

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2. Material and methods 2.1. Animals Brown hares were trapped in January 1999 in the south-east part of the Czech Republic. The trapped hares were taken to closed premises where they were kept in outdoor roofed boxes 2 m × 4 m × 0.8 m. The hares were fed meadow hay, complete feed mix for rabbits and oats ad libitum, and drinking water. After a 14-day acclimatisation, the hares were serologically examined and experimentally infected. The domestic rabbits (Hyla hybrid) were bought from a commercial large-scale animal husbandry facility at the age of 125–130 days and kept in outdoor boxes of the type used for growing rabbits. The rabbits were given the same food and water as the hares. Prior to the infection, the rabbits were serologically examined. Throughout the experiment, the hares and rabbits were exposed to central European winter weather with temperatures below the freezing point. Outbred toxoplasma-negative CD1 mice (Charles River — Anlab, Prague, Czech Republic) were used for passaging the T. gondii strain, infecting cat, producing T. gondii oocysts and for isolation experiments for the demonstration of T. gondii from hare and rabbit tissues following experimental infections. For the production of T. gondii oocysts, one serologically toxoplasma negative (ELISA, Immunocomb, Biogal Galed Labs, Israel) cat aged 3.5 months was infected. 2.2. T. gondii strain The K7 strain used was isolated in 1995 from a rabbit (Literák et al., 1998). Oocysts of that strain were obtained from the faeces of cats infected per os with a brain tissue suspension of the CD1 mice containing T. gondii tissue cysts (Dubey and Beattie, 1988). The oocysts were kept for 3–7 days in 2% sulphuric acid at room temperature to sporulate, and stored for 7 months at 4◦ C. Before inoculation, the oocyst suspension was neutralised in 3.3% sodium hydroxide. The number of oocysts was based on a visual count under the microscope. 2.3. Experimental design Hares and rabbits were infected perorally with oocyst suspension in phosphate buffered physiological saline (PBS) (7.2 pH). The volume inoculated was 0.1 ml. A total of 12 hares and 12 rabbits were divided into three groups of four animals each, and infected with a dose of 10, 103 and 105 oocysts. The health of the experimental animals was visually monitored on a daily basis. Immediately before the infection and on days 7, 12, 19, 26 and 33 p.i., blood samples from the vena auricularis were taken for serological and parasitemia examinations. The isolation assay and the polymerase chain reaction (PCR) were used to ascertain the presence of T. gondii in blood. Immediately before the infection and on days 7 and 12 p.i., blood samples for immunological examination were taken at the same time. The animals that survived the acute toxoplasmosis were sacrificed on day 33 p.i. All dead hares and

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rabbits were pathomorphologically examined (autopsy and histological examination), and their tissues were examined for the presence of T. gondii using the isolation assay and immunohistochemical examinations. 2.4. Serological examinations In the indirect fluorescent antibody test (IFAT), commercially available Sevatest Toxoplasma Antigen IFR (Sevac, Prague, Czech Republic) and anti-rabbit immunoglobulin SwAR/FITC (Sevac) were used. The sera were diluted in a two-fold series starting at 1:20 as the basic dilution. The titre ≥ 20 was considered positive. The latex agglutination test (LAT) was performed according to the manufacturer’s instructions (Pastorex Toxo, Sanofi Diagnostics Pasteur, France). A ten-fold dilution of the basic 1:10 titre was used, with the titre ≥ 10 being considered positive. 2.5. Isolation assay Tissues from individual hares and rabbits (brain, liver, spleen, heart, muscle, lung and kidney) were homogenised and suspended in 2 ml PBS. Two mice were injected intraperitoneally, each with 1 ml suspension from the organ of the animal tested. Blood samples were kept at 4◦ C. After coagulation, blood was homogenised and injected intraperitoneally in 1 ml doses to two mice. Five weeks later the mice were killed, bled and examined for T. gondii tissue cysts (brain squash preparations) and for T. gondii antibodies (IFAT). In this IFAT, anti-mouse immunoglobulin SwAM/FITC (Sevac Prague, Czech Republic) was used. Mouse sera were examined at the dilution of 1:20, with the titre of 20 being considered positive. 2.6. PCR Blood for PCR tests was taken to test tubes containing an anticoagulant (K3 EDTA solution). Each blood sample (500 ␮l) was mixed with an equal volume of 2% Dextran T 500 (Pharmacia Biotech AB, Uppsala, Sweden) in 0.85% physiological saline (Paugam et al., 1995). After 1 h of bench-top sedimentation, erythrocyte-free supernatant containing leukocytes and platelets was collected. After 10 min centrifugation at 2000 ×g, pellets were washed twice in PBS and kept frozen at −18◦ C. From the frozen cell pellets, DNA was extracted using the QIAamp Tissue Kit (Qiagen, Hilden, Germany). Toxoplasma DNA was amplified with primers based on the specific repeated TGR1E sequence (Cristina et al., 1991). The primer sequences were: forward — 50 GGA GAT GGT CGG GCG TAT TG 30 , reverse — 50 CAC CTG TGC CGC AAA TGA AA 30 . A total of 4 ␮l extracted DNA from each sample was amplified in a 40 ␮l reaction. The amplification reactions were induced by 20 ␮l of Taq PCR Master Mix Kit (Qiagen). The procedure used in setting the PCR cycles has been described elsewhere (Paugam et al., 1995). Results were evaluated by gel electrophoresis in a 2% agarose gel stained with ethidium bromide. The presence of the 194 base pair fragment indicated a positive result.

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2.7. Number and activity of leucocytes Blood for immulogical examinations was taken to test tubes containing heparin. Total leukocyte counts were determined using the Digicell 500 cell counter (Contraves AG, Switzerland). The lymphocyte activity was tested in the whole blood using the LTT. Blood samples were diluted at the 1:10 ratio with the RPMI 1640 medium (Sevapharma, Prague, Czech Republic) supplemented with 5% precolostral calf serum and antibiotics. The 200 ␮l of diluted blood was pipetted into microtitre plate wells in triplicates. Then 20 ␮l of mitogens PHA (Phytohaemagglutinin) (Murex Diagnostics Ltd., Dartford, UK) (40 ␮g/ml), Con A (Concanavalin A) (Pharmacia Biotech AB, Uppsala, Sweden) (10 ␮g/ml), PWM (pokeweed mitogen) (Sigma Chemicals Co., St. Louis, USA) (10 ␮g/ml) were added to each well. The microplates were incubated at 37◦ C for 3 days (non-specific LTT) or 6 days (specific LTT) with 3 H-thymidine (1 ␮Ci/well) labelling during the last 20 h. The incorporation of 3 H-thymidine was measured using a liquid scintillation counter (Packard Tricarb CA 600, Canberra-Packard, USA). The results were expressed in terms of stimulation indices calculated as the ratio between the activity of the stimulated and the non-stimulated cells. 2.8. Pathomorphology and immunohistochemistry All the hares and rabbits that died or were sacrificed were autopsied in a standard manner. Specimens for histological examination were fixed at 10% neutral buffered formaldehyde solution and processed using a standard paraffin technique. Tissue sections were 5 ␮ thick and stained with HE (hematoxilin-eosin). For immunohistochemical examinations, the avidin-biotin peroxidase complex on Superfrost Plus glass plates (VWR Scientzific, West Chester, USA) was used. The specimens were deparaffinized in xylene (2 × 1 min) and hydrated with ethanol at gradually decreasing concentrations (3 × 1 min, 1 min and 1 min with 100, 95 and 70% ethanol, respectively). Endogenous peroxidase was blocked by 0.3% hydrogen peroxide in methanol (30 min at room temperature). To demask the antigen, citrate buffer in water bath (40 min at 95–99◦ C, 20 min cooling) was used. Specimens were blocked with normal rabbit serum (20 min, room temperature) and incubated with Rabbit-anti-T. gondii (DAKO, Carpinteria, USA) primary antiserum for 90 min in a wet chamber. They were then washed three times in PBS and the Goat anti Rabbit biotinylated antiserum (Vector Lab., Burlingame, USA) was applied at 1:500 titre for 30 min in a wet chamber. After washing (3 × PBS), the specimens were treated with Strept-Avidin (Zymed, San Francisco, USA) for 30 min at the room temperature. Specimens were then washed again (2 × PBS), treated with DAB Substrate Kit chromogen (Vector Lab., Burlingame, USA), washed in water and HE stained. 2.9. Statistical analysis All statistics were calculated with MS-Excel 6.0® . Statistical differences between groups were estimated with the Student’s t-test.

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3. Results 3.1. Changes in behaviour and mortality No changes in the behaviour of four hares infected with a dose of 10 oocysts were observed. The hares died on days 12, 15, 15 and 19 p.i., respectively. Two hares infected with 103 oocysts spent most of their time sitting in the corner of their box starting from days 9 and 15 p.i., respectively. They also exhibited a loss of shyness and their fur was ruffled. These two hares died on days 12 and 16 p.i., respectively. Another two hares infected with the dose of 103 oocysts demonstrated no behavioural changes and died on day 15 p.i. In two hares infected with 105 oocysts, a loss of shyness, reduced food intake and lassitude were observed from days 3 and 4 p.i., respectively. The hares mostly sat in the corner of their box and died on day 8 p.i. In another two hares infected with the dose of 105 oocysts, no changes in behaviour were observed. These hares died on day 9 p.i. In three rabbits (doses 10, 103 and 105 ), nasal discharge was observed during the experiment and the rabbits lost weight. Two of these rabbits (doses 103 and 105 ) died on days 9 and 19 p.i., respectively. Microbiological and histological examinations confirmed the diagnosis of pasteurellosis in all the three rabbits, in which, at the same time, two pathogens — Pasteurella multocida and T. gondii — were found. Other rabbits infected with doses of 10 (n = 3) and 103 (n = 3) showed no behavioural changes. None of these rabbits died. None of the rabbits infected with a dose of 105 (n = 3) died and only a reduced food intake was observed among them between day 5 and 12 p.i. 3.2. Pathomorphological findings At the time of death, the nutritional status of the hares was good. In the hares infected with 10 and 103 oocysts, the post mortem examination showed an oedema, hyperaemia and minor haemorrhages of the jejunal and iliac walls. Two hares exhibited a pronounced haemorrhagic enteritis in the terminal section of the jejunum and the ileum. The mesenteric lymph nodes were markedly enlarged, oedematous and hyperaemic. Enlarged spleens were found in six hares. In several hares, a slight steatosis of the liver with focal hyperaemia was present. Two hares had multiple small liver necroses. In hares infected with a dose of 105 oocysts, changes were more prominent than in hares infected with lower doses. All four animals exhibited changes in the digestive tract, which included an oedema, hyperaemia and small haemorrhages of the walls of the duodenum, a larger part of the jejunum and haemorrhagic enteritis in the terminal section of the jejunum and the ileum. The mesenteric lymph nodes were markedly enlarged, oedematous and hyperaemic. The spleen was enlarged, with rounded edges and tightened capsule, and, when cut, its pulp was colliquative. The liver demonstrated numerous small necrotic foci. Pulmonary and pericardial coherences and renal granulomas were found in one hare in which a concurrent Klebsiella pneumoniae infection was also diagnosed. The autopsy showed that 9 out of a total of 12 experimentally infected rabbits were in good health and their organs showed no macroscopic changes. The remaining three rabbits were characterised by ill health (cachexia), pneumonia, and enlarged and oedematous mesenteric lymph nodes. Moreover, one of these three rabbits also suffered from the miliary liver

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necrosis. In all of the three rabbits, microbiological examinations confirmed the concurrent P. multocida infection. In two hares and one rabbit that died having been infected with a dose of 105 oocysts, a fairly large amount of peritoneal exsudate was found. Isolation assays showed the presence of T. gondii in the exsudate in one hare. Histological examinations of all the hares showed pathological changes, mainly in the small intestine, liver and mesenteric lymph nodes. The lesions ascertained in small intestines included the necrosis of the epithelial cells and their desquamation into the intestinal lumen, hyperaemia and small haemorrhages in the lamina propria mucosea. The submucosa contained a mixed inflammatory infiltrate. In the epithelial lining, T. gondii tachyzoises were found. The livers contained irregular coagulation necrosis foci. Mesenteric lymph nodes and spleen contained coagulation necrosis foci with a slight peripheral inflammatory reaction and a slight depletion of follicular lymphocytes. Changes in the heart and skeletal muscles included small necrotic foci. Necrotic foci in the diaphragm were more extensive, with slight mineralisation in some cases. In the hare with a positive cultivation of K. pneumoniae, focal granulomatous pleuropneumonia and focal granulomatous interstitial nephritis were found. Milder histological changes were ascertained in hares infected with 10 or 103 oocysts. In the intestines, only oedema and haemorrhages in the lamina propria mucosae and the submucosa with a mild desquamation of the epithelium were found. Tachyzoises were found only rarely in the intestines. Skeletal muscle, myocardium and diaphragm necrosis was not a uniform finding in all the hares and, when found, it affected individual fibres only. Histopathological changes in rabbits were less pronounced than in hares. Changes in the liver were limited to small lymphocytic nodes and a mild lymphocytic infiltration in the portal fields. An interstitial pneumonia was found in four rabbits. The few necrotic foci found were small. Brain lesions consisted of mild perivascular infiltrations with infrequent glial nodes and the T. gondii tissue cysts found in both white and grey matter. 3.3. Antibodies to T. gondii and T. gondii in organs and blood Prior to the infection, no antibodies were found in the hares. Seven days after the infection, only the IFAT was able to demonstrate antibodies in 2 out of the 12 hares. Twelve days after the infection, antibodies were demonstrated in all the infected hares by both the IFAT and the LAT (Table 1). Antibodies were found (IFAT, titre 40) in one of the rabbits tested before the infection. After the infection, antibodies (IFAT) in rabbits were first demonstrated on day 7 p.i., and on day 19 p.i., antibodies were demonstrated in all the rabbits. Between days 19 and 33 p.i., antibody titres showed an increase in nine rabbits and remained the same in one rabbit (Table 2). By the LAT, antibodies were demonstrated later and at lower titres (Table 3). All the infected hares died between day 8 and 19 p.i. In all cases, the isolation assay in hares infected with doses of 10 oocysts (n = 4), 103 oocysts (n = 4) and 105 oocysts (n = 3) demonstrated the presence of T. gondii in the liver, brain, spleen, kidneys, lung, heart and the skeletal muscles. T. gondii was isolated from organs of all the infected rabbits: from the liver in 27% of cases (3 positive/11 tested), from the brain in 100% of cases (8/8), from the spleen in 90%

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Table 1 Results of serological examinations of brown hares experimentally infected with T. gondii oocystsa Hare no.

Dose of oocysts

IFAT titre 7 dpi

LAT titre 7 dpi

IFAT titre 12 dpi

LAT titre 12 dpi

1 2 3 4 5 6 7 8 9 10 11 12

10 10 10 10 103 103 103 103 105 105 105 105

40 – – – – – 20 – – – – –

– – – – – – – – – – – –

80 160 nt 320 160 320 nt 640 nt nt nt nt

100 10 nt 100 100 10 nt 100 nt nt nt nt

a

nt: not tested, dpi: days post infection.

of cases (10/11), from the kidneys in the 72% of cases (8/11), from the lung in the 100% of cases (11/11), from the heart in the 92% of cases (11/12) and from the skeletal muscles in the 82% of cases (9/11) (Table 4). Parasitemia was demonstrated in all experimentally infected hares on days 7 and 12 p.i. The sensitivity of the biological experiment was 100% (n = 19), PCR sensitivity was 79% (n = 19). The number of rabbits with parasitemia after experimental infection was lower that that of hares. On day 7 p.i., parasitemia was demonstrated in 83% of the rabbits (10 positive/12 tested), on day 12 p.i. only 27% (3/11), on day 19 it was 20% (2/10), on day 26 it was 40% (4/10) and on day 33 it was 20% (2/10) of rabbits. The results of the biological test and the PCR were the same in 79.2% of cases (n = 53), in six cases (11.3%) the results Table 2 Antibodies to T. gondii in rabbits experimentally infected with T. gondii oocysts (indirect fluorescence antibody test)a Rabbit no.

Dose of oocysts

Titre 0 dpi

Titre 7 dpi

Titre 12 dpi

Titre 19 dpi

Titre 26 dpi

Titre 33 dpi

1 2 3 4 5 6 7 8 9 10 11 12

10 10 10 10 103 103 103 103 105 105 105 105

– – – – – – – 40 – – – –

– – – – – – – 40 20 – 40 80

80 160 – 20 10240 nt 5120 10240 5120 5120 40960 20480

640 1240 2480 81020 10240 nt 10240 40960 20480 10240 nt 10240

10240 10240 20480 81020 20480 nt 20480 81020 40960 40960 nt 81020

40960 81020 40960 81020 81020 nt 81020 324080 81020 40960 nt 81020

a

nt: not tested, dpi: days post infection.

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Table 3 Antibodies to T. gondii in rabbits experimentally infected with T. gondii oocysts (latex agglutination test)a Rabbit no.

Dose of oocysts

Titre 0 dpi

Titre 7 dpi

Titre 12 dpi

Titre 19 dpi

Titre 26 dpi

Titre 33 dpi

1 2 3 4 5 6 7 8 9 10 11 12

10 10 10 10 103 103 103 103 105 105 105 105

– – – – – – – – – – – –

– – – – – – – – – – – –

100 100 – – 100 nt 100 100 100 100 100 100

10 10 – 10 100 nt 100 1000 100 100 nt 1000

100 100 – 10 1000 nt 1000 1000 1000 1000 nt 1000

100 100 100 100 1000 nt 1000 1000 1000 1000 nt 1000

a

nt: not tested, dpi: days post infection.

of the biological experiment were positive while the results of the PCR were negative; in five cases (9.4%) the results of the biological experiment were negative and the PCR results were positive. Imunohistochemical examinations regularly demonstrated the T. gondii antigen in a majority of tissues and organs in hares, while positive results in rabbits were far less numerous (Tables 4 and 5).

Table 4 Presence of T. gondii in organs of rabbits infected with T. gondii oocystsa Rabbit Dose of Liver Brain Spleen Kidney Lung Heart Skeletal no. oocysts muscle 1 2 3 4 5 6d 7 8 9 10 11e 12

10 10 10 10 103 103 103 103 105 105 105 105 a

–b /−c +/− −/− −/− +/+ nt/+ −/− −/− −/+ −/− +/+ −/−

+/− +/− +/− +/+ +/− nt/+ +/+ +/+ +/+ nt/+ nt/+ nt/+

+/+ +/+ −/− +/+ +/− nt/+ +/− +/+ +/+ +/+ +/+ +/+

−/+ +/+ +/− +/+ +/+ nt/− +/+ −/+ −/− +/+ +/+ +/+

+/+ +/+ +/− +/+ +/+ nt/+ +/+ +/+ +/+ +/+ +/+ +/+

+/+ −/+ +/+ +/+ +/+ +/+ +/− +/+ +/+ +/+ +/+ +/+

Examination on day 33 p.i. Result of the isolation. c Result of the immunohistochemical examination. d Died on day 9 p.i. (toxoplasmosis and pasteurellosis). e Died on day 19 p.i. (toxoplasmosis and pasteurellosis). b

−/+ +/+ −/− +/− +/+ nt/− +/+ +/+ +/+ +/+ +/+ +/+

Diaph- Small Mesenteric Gonads ragm intestine lymphnodes nt/− nt/− nt/− nt/− nt/+ nt/− nt/− nt/− nt/− nt/+ nt/+ nt/−

nt/+ nt/− nt/− nt/− nt/− nt/+ nt/− nt/+ nt/+ nt/− nt/+ nt/−

nt/+ nt/+ nt/+ nt/+ nt/+ nt/+ nt/+ nt/− nt/+ nt/+ nt/+ nt/+

nt/− nt/− nt/− nt/− nt/− nt/− nt/− nt/− nt/− nt/− nt/− nt/−

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Table 5 Immunohistochemical assay of T. gondii in organs of died hares infected with T. gondii oocystsa Hare Dose of no. oocysts

Died Liver Brain Spleen Kidney Lung Heart Skeletal Diaph- Small Mesenteric Gonads (dpi) muscle ragm intestine lymphnodes

1 2 3 4 5 6 7 8 9 10 11 12

19 15 15 12 16 15 15 12 9 9 8 8

10 10 10 10 103 103 103 103 105 105 105 105 a

+ + + + + + + + + + + +

+ + − + + + + + + + + +

+ + + + + + + + + + + +

− − + + − − − + + + + −

− + − − + + − + + + + +

+ − + + − + − − + + + +

− + + − − + + − − + + +

+ + + − + nt + nt + + + +

+ + + + + + + + + + + +

+ + + + + + + + + + + +

− − − − + − − + − − − +

nt: not tested, dpi: days post infection.

3.4. Number and activity of leucocytes Before the infection, the total leukocytes count in hares was 5.8 ± 1.4 × 109 /l. The total leukocyte count in rabbits before infection was 11.1 ± 5.0 × 109 /l. There was a significant difference in leukocyte counts before infection between hares and rabbits (P < 0.01). In the course of the disease, the experimentally infected hares showed a marked decrease in the number of leukocytes (Table 6). None of the experimentally infected rabbits showed any significant change in the number of leukocytes during the infection. Before experimental infection, stimulation indices were tested in both rabbits and hares. Stimulation indices after PHA stimulation were 71.1 ± 34.7 and 19.9 ± 18.7 in rabbits and in hares, respectively. After ConA stimulation, they were 113.0 ± 58.6 and 12.0 ± 12.1 in rabbits and in hares, respectively, and after PWM stimulation, 4.8 ± 2.6 and 19.9 ± 14.7 in rabbits and in hares, respectively. The differences between rabbits and hares were significant (p < 0.01) (Fig. 1). In experimentally infected hares, the stimulation index in the non-specific LTT dropped significantly during the infection in the hares infected with a dose of 103 oocysts when

Table 6 Total leukocyte count in the blood of hares experimentally infected with T. gondii oocysts (×109 /l) (mean ± S.D.) Dose of oocyst 10 oocysts (n) 103 oocysts (n) 105 oocysts (n) a

0 dpi

7 dpi

12 dpi

4.7 ± 0.9 (4) 6.3 ± 1.0 (4) 7.0 ± 1.5 (4)

2.5 ± 0.6∗∗

1.4 ± 0.4∗∗ (4) 2.0 ± 0.6∗∗ (3) nta

(4) 3.7 ± 1.0∗ (4) 2.8 ± 1.1∗ (4)

nt: not tested. Significant changes (p < 0.01) against collection at day 0. ∗∗ Significant changes (p < 0.05) against collection at day 0. ∗

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Fig. 1. Comparison of LTT results in the peripheral blood of rabbits and hares immediately prior to infection, **: significant differences (p < 0.01) between hares and rabbits.

tested with the PHA and the PWM, and in the hares infected with a dose of 105 oocysts tested with the ConA (Table 7). In experimentally infected rabbits, a significant decrease in the stimulation index in the non-specific LTT was observed in rabbits infected with a dose of 105 oocysts tested with the PHA (Table 8).

4. Discussion Among hares, toxoplasmosis is often an acute fatal disease. Clinical symptoms were observed at infection doses of only 10 T. gondii oocysts of the K7 strain, which, moreover, shows only a low virulence in mice. None of 10, 0/10, 0/10, 2/10 and 10/10 CD1 mice died when infected orally with 10, 102 , 103 , 104 and 105 oocysts of K7 strain, respectively (Sedlák and Literák, unpublished observations). Experimentally infected hares died between days 8 and 19 p.i. At a high infection dose, clinical symptoms were manifested as early as Table 7 Results of non-specific LTT in the peripheral blood of hares infected with T. gondii oocysts (mean ± S.D.) Dose of oocysts

Day after infection

n

Unstimulated (CPM)a

PHA (SI)b

ConA (SI)b

PWM (SI)b

10 10 10 103 103 103 105 105

0 7 12 0 7 12 0 7

4 4 4 4 4 3 4 4

112 ± 75 80 ± 23 85 ± 26 76 ± 20 69 ± 20 64 ± 7 72 ± 11 72 ± 18

24.2 ± 20.7 4.3 ± 3.5 1.4 ± 0.6 18.7 ± 6.7 6.0 ± 5.9 1.3 ± 0.4∗ 11.4 ± 9.1 1.3 ± 0.2

15.8 ± 19.5 7.1 ± 6.9 2.6 ± 1.8 12.5 ± 12.6 2.5 ± 1.0 2.5 ± 1.7 11.5 ± 6.2 1.5 ± 0.2*

16.8 ± 18.0 5.5 ± 1.4 1.2 ± 0.1 22.9 ± 3.5 11.7 ± 5.1 5.0 ± 4.4∗∗ 25.7 ± 19.3 3.1 ± 0.7

a

Counts per minute. Stimulation index. ∗ Significant changes (p < 0.05) against collection at day 0. ∗∗ Significant changes (p < 0.01) against collection at day 0. b

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Table 8 Results of non-specific LTT in the peripheral blood of rabbits infected with T. gondii oocysts (mean ± S.D.) Dose of oocysts

Day after infection

n

Unstimulated (CPM)a

PHA (SI)b

ConA (SI)b

PWM (SI)b

10 10 10 103 103 103 105 105 105

0 7 12 0 7 12 0 7 12

2 2 2 4 4 3 4 4 4

193 ± 160 268 ± 242 110 ± 25 159 ± 79 101 ± 43 68 ± 8 113 ± 52 104 ± 42 113 ± 22

74.9 ± 64.8 39.6 ± 26.9 17.0 ± 6.8 45.8 ± 28.0 19.9 ± 29.7 22.7 ± 17.6 83.4 ± 24.0 9.6 ± 12.8∗ 5.2 ± 4.0∗∗

84.8 ± 33.0 73.9 ± 75.4 41.5 ± 30.4 83.4 ± 29.0 43.7 ± 43.0 47.9 ± 18.9 128.3 ± 85.9 18.3 ± 16.9 15.0 ± 12.8

3.6 ± 2.6 1.9 ± 1.3 3.0 ± 0.9 4.5 ± 2.6 2.9 ± 0.8 3.9 ± 1.1 5.1 ± 3.7 3.6 ± 4.3 1.3 ± 0.5

a

Counts per minute. Stimulation index. ∗ Significant changes (p < 0.05) against collection at day 0. ∗∗ Significant changes (p < 0.01) against collection at day 0. b

day 3 p.i. Symptoms of toxoplasmosis induced by the experimental infection corresponded with those described in hares with a spontaneous T. gondii infection (Hülphers et al., 1947; Christiansen and Siim, 1951; Englert and Karle, 1956; Walzl, 1959). In experimentally infected rabbits, no clinical symptoms of toxoplasmosis were observed. In the three rabbits with the nasal discharge and a loss of weight, two of which died during the experiment, pasteurellosis was diagnosed. Clinical manifestations of toxoplasmosis including mortality were much more pronounced in hares than in rabbits, which proves that hares are the more susceptible to toxoplasmosis of the two. In infected hares, standard pathomorphological findings included haemorrhagic enteritis, enlargement and hyperaemia of mesenteric nodes, splenomegaly and numerous miliary necrotic foci in the liver parenchyma. In histopathology, the predominant finding was necrosis of the small intestine, liver and mesenteric lymph nodes, with immunohistochemically demonstrated presence of T. gondii tachyzoises. The changes observed were identical with the findings in experimentally infected hares described elsewhere (Gustafsson et al., 1997a), and consistent with the results of previous studies into spontaneous toxoplasmosis in hares (Hülphers et al., 1947; Štˇerba et al., 1997). The much less severe pathomorphological findings in rabbits are also consistent with previous findings in experimental toxoplasmosis in rabbits (Gustafsson et al., 1997a). In hares, parasitemia in experimental infections appeared in all cases and persisted until the death of the animal. No differences were found among groups of hares with different infection doses. In rabbits, parasitemia was detected irregularly throughout the entire monitoring period, i.e. from day 7 to day 33 p.i. The largest and the smallest numbers of parasitemia were, however, recorded on days 7 and 33 p.i., respectively. We believe that the decreasing ratio of parasitemic rabbits was due to the specific immune response they acquired and to the effect of that response on the conversion of tachyzoises into bradyzoises. After an experimental infection, antibodies to T. gondii were found in both hares and rabbits. The IFAT was able to detect them at an earlier stage and at higher titres than the LAT. Some hares infected with 105 oocysts died before T. gondii antibodies were demonstrated in them. The higher titres of T. gondii antibodies in rabbits than in hares detected by the IFAT

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might mean that the response of rabbits’ humoral component of immunity was stronger and that may partly explain the fact that rabbits enjoy better protection against toxoplasmosis infection. The higher titres of rabbits might have, however, been also due to the fact that we used a homologous (anti-rabbit) conjugate for the IFAT (heterologous conjugate was used in hares). Anti-rabbit conjugate is successfully used in the IFAT serodiagnostics of toxoplasmosis in hares (Edelhofer et al., 1989; Gustafsson and Uggla, 1994; Gustafsson et al., 1997b), but no comparison of IFAT results in hares between heterologous anti-rabbit and homologous anti-hare conjugates has been made to date. The differences in titres ascertained by the LAT were not so big, which may have been partly due to its lower sensitivity compared with the IFAT. The host organism responds to the T. gondii infection by activating its immune system, the aim of which is to limit the replication of tachyzoises before the onset of T-cell mediated immunity, and to provide for an adequate T-cell response (Denkers and Gazzinelli, 1998). It is a complicated process in which macrophages start producing cytokins, particularly interferon-gama, and reactive nitrogen intermediates. An uncontrolled production of anti-inflammatory cytokins may, on the other hand, lead to severe immunopathological changes and may even be a threat to life. For that reason it is essential that their production be accurately regulated by a feedback system. The same feedback regulatory system, however, causes the suppression of mitogen-stimulated LTT in the acute phase of the T. gondii infection (Chan et al., 1986). No suppression has been observed in the chronic stage of the infection (Luft et al., 1984). A reduced ability of lymphocytes to respond to mitogenic stimuli (immunosuppression) was also observed in the present study. We believe that infections caused by higher doses of T. gondii oocyst stimulate a more intensive immunological response and thus also a more intensive suppression. The mechanisms responsible for this phenomenon in laboratory mice and men included cytokins IL-10, IL-4, transforming growth factor-beta (Oswald et al., 1992; Candolfi et al., 1995; Roberts et al., 1996) and nitrogen oxide (Hayashi et al., 1996). The question which cytokins may be responsible for the onset of the immunosuppression observed in hares and rabbits during acute toxoplasmosis still remains to be answered. We suggest that the different courses of acute toxoplasmosis in hares and in rabbits after experimental infection may have three possible explanations. The first is that hares as a species are naturally more susceptible to toxoplasmosis than rabbits. This hypothesis was also formulated by Gustafsson et al. (1997a,b), who also tried to confirm it in an experimental infection study involving mountain hares. We, however, do not believe that the results of her study allow any final conclusions to be drawn because the hares and rabbits in her study were already sacrificed on day 7 p.i., and the higher susceptibility of hares was deduced from the fact that they did not respond in the lymphocyte activity test following a specific antigen stimulation. The rabbits in that test nevertheless showed response even before infection, which would most probably point to an earlier infection and were not suitable as controls. The second alternative explanation of the higher susceptibility of hares to experimental infection is the effect of stress on their immunity response. There is no doubt that the hares are stressed because they were trapped, kept in a confined space and handled when being infected and when biological samples were being taken. In that case, experimentally infected hares would have to be treated as immunocompromised subjects. The course of infection in animals with suppressed immune response is more dramatic, which has been documented

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in studies into the relationships between immunosuppression and the development of encephalopathy caused by an uncontrolled multiplication of parasites (e.g. Gazzinelli et al., 1993). It seems that the influence of stress and the ensuing immunosuppression play the decisive role in the pathogenesis of toxoplasmosis in hares. Rubarth (1948) noticed that the character of some of histological findings in the liver of hares with the diagnosis of acute lethal toxoplasmosis corresponded to that of a chronic infection without any direct relationship to necrotic foci. That led him to a conclusion that it was a case of a ‘fairly old infection, possibly contracted in summer’. The lack of food and dietetic disturbances, exposure to cold, concurrent infections and unnatural living conditions in which trapped hares are kept at concentration sites and during transportation are considered as the main cause of the exacerbation of the latent toxoplasmosis in hares (Englert and Karle, 1956; Møller, 1962). The third possible explanation is the cumulative effect of immunosuppression induced by toxoplasmosis and by stress. With regard to the higher susceptibility of hares, the different ability of non-specific mitogens to stimulate lymphocytes before infection might be interpreted as supportive of both the biological and the stress principle theories. The immune system of hares as a biological species may respond differently than that of rabbits, although it is not really expected in species that are so close to each other in evolutionary terms. The repeated TGR1E sequence has been used in the PCR for the detection of T. gondii in people with toxoplasmosis (Franzen et al., 1997), and in mice experimentally infected with T. gondii (Paugam et al., 1995). Compared with the more frequently used B1 sequence, the TGR1E offers the benefits of a higher sensitivity (Paugam et al., 1995). Hitt and Filice (1992) used the PCR (B1 sequence) to detect toxoplasma DNA in the blood of rabbits subcutaneously infected with tachyzoises between days 6 and 14 p.i. When the ‘mouse inoculation’ was used, parasitemia was detected even on day 32 p.i. Mouse inoculation was much more sensitive than the PCR with the B1 sequence. In our study, the sensitivity of the PCR and the TGR1E sequence was the same as that of the biological experiment with mice.

Acknowledgements This study was funded by Grants nos. 524/98/0111 (Grant Agency of the Czech Republic), 1121/98 and 161 700 001 (Ministry of Education, Youth and Sport of the Czech republic), MZEM 03/9801 (Ministry of Agriculture of the Czech Republic), and 1/VFU/98 (Veterinary and Pharmaceutical University, Brno, Czech Republic). We are grateful to F. Vitula for his technical cooperation. References Candolfi, E., Hunter, C.A., Remington, J.S., 1995. Roles of interferon and other cytokines in suppression of the spleen cell proliferative response to concanavalin A and toxoplasma antigen during acute toxoplasmosis. Infect. Immunol. 63, 751–756. Chan, J., Siegel, J.P., Luft, B.J., 1986. Demonstration of T-cell dysfunction during acute toxoplasma infection. Cell. Immunol. 98, 422–433.

K. Sedl´ak et al. / Veterinary Parasitology 93 (2000) 13–28

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Christiansen, M., Siim, J.C., 1951. Toxoplasmosis in hares in Denmark. Serological identity of human and hare strains of Toxoplasma. Lancet 1, 1201–1203. Cristina, N., Liaud, M.F., Santoro, F., Oury, B., Ambroise-Thomas, P., 1991. A family of repeated DNA sequence in Toxoplasma gondii: cloning, sequence analysis, and use in strain characterization. Exp. Parasitol. 73, 73–81. Denkers, E.Y., Gazzinelli, R.T., 1998. Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection. Clin. Microbiol. Rev. 11, 569–588. Dubey, J.P., Beattie, C.P., 1988. Toxoplasmosis of Animals and Man. CRC Press, Boca Raton, Florida, 220 pp. Dubey, J.P., Brown, C.A., Carpenter, J.L., Moore III, J.J., 1992. Fatal toxoplasmosis in domestic rabbits in the USA. Vet. Parasitol. 44, 305–309. ˇ Eatár, G., 1972. Studies on toxoplasmosis as regards its natural focality in Slovakia. Folia Parasitol. 19, 253–256. Edelhofer, R., Heppe-Winger, E.M., Hassl, A., Aspöck, H., 1989. Toxoplasma-Infektionen bei jagdbaren Wildtieren in Ostösterreich. Mitteilungen der Österraischische Gesselschaft für Tropenmedizinische Parasitologie 11, 119–123. Englert, H.K., Karle, L., 1956. Endemische Toxoplasmose bei Feldhasen im Schwarzwald. Deut. Tieräztl. Woch. 63, 163–168. Franzen, C., Altfeld, M., Hegeneger, P., Hartmann, P., Arendt, G., Jablonowski, H., Rockstroh, J., Diehl, V., Salzberger, B., Fatkenheuer, G., 1997. Limited value of PCR for detection of Toxoplasma gondii in blood from human immunodeficiency virus-infected patients. J. Clin. Med. 35, 2639–2641. Gazzinelli, R.T., Eltoum, I., Wynn, T.A., Sher, A., 1993. Acute cerebral toxoplasmosis is induced by in vivo neutralization of TNF-␣ and correlates with down-regulated expression of inducible nitric oxide synthase and other markers of macrophage activation. J. Immunol. 151, 3672–3681. Gustafsson, K., 1997. Studies of toxoplasmosis in hares and capercaillie. Doctor’s Dissertation, Uppsala. Gustafsson, K., Uggla, A., 1994. Serologic survey for Toxoplasma gondii infection in the brown hare (Lepus europaeus P.) in Sweden. J. Wildl. Dis. 30, 201–204. Gustafsson, K., Uggla, A., Svensson, T., Sjöland, L., 1988. Detection of Toxoplasma gondii in liver tissue sections from brown hares (Lepus europaeus P.) and mountain hare (Lepus timidus L.) using peroxidase anti-peroxidase technique as a complement to conventional histopatology. J. Vet. Med. B 30, 402–407. Gustafsson, K., Uggla, A., Järplid, B., 1997a. Toxoplasma gondii infection in the mountain hare (Lepus timidus) and domestic rabbit (Oryctolagus cuniculus). I. Pathology J. Comp. Path. 117, 351–360. Gustafsson, K., Wattrng, E., Fossum, C., Heegaard, P., Lind, P., Uggla, A., 1997b. Toxoplasma gondii infection in the mountain hare (Lepus timidus) and domestic rabit (Oryctolagus cuniculus): II. Early immune reactions. J. Comp. Pathol. 117, 361–369. Hayashi, S., Chan, C.C., Gazzinelli, R., Roberge, F.G., 1996. Contribution of nitric oxide to the host parasite equilibrium in toxoplasmosis. J. Immunol. 154, 1476–1481. Hejl´ıeˇ ek, K., Literák, I., 1994. Prevalence of toxoplasmosis in rabbits in south Bohemia. Acta Vet. Brno 63, 145–150. Hejl´ıeˇ ek, K., Literák, I., Nezval, J., 1997. Toxoplasmosis in wild mammals from the Czech Republic. J. Wildl. Dis. 33, 480–485. Hitt, J.A., Filice, G.A., 1992. Detection of Toxoplasma gondii parasitemia by gene amplification, cell culture, and mouse inoculation. J. Clin. Microbiol. 30, 3181–3184. Hülphers, G., Lillengen, K., Rubarth, S., 1947. Toxoplasmos hos hare och tjäder. Sven. Veterinärtidskrift 52, 295–332. Levit, A.V., Vustina, U.D., Gubenko, P.N., Fadeev, V.A., 1965. Toksoplazmoz zajceobraznych. In: Galuzo, I.G. (Ed.), Toksoplazmoz zhivotnych. Nauka, Alma-Ata, pp. 224–237. Literák, I., Rychl´ık, I., Svobodová, V., Posp´ıšil, Z., 1998. Restriction fragment length polymorphism and virulence of Czech Toxoplasma gondii strains. Int. J. Parasitol. 28, 1367–1374. Luft, B.J., Kansas, G., Engleman, E.G., Remington, J.S., 1984. Functional and quantitative alteration in T lymphocyte subpopulation in acute toxoplasmosis. J. Inf. Dis. 150, 761–767. Møller, T., 1962. Three casuistic reports of toxoplasmosis in Zoo-animals (Macropus bennetti, Marmota marmota, Lepus timidus). Nord. Vet.-Med. 14, 233–243. Oswald, I.P., Gazzinelli, R.T., Sher, A., James, S.L., 1992. IL-10 synergizes with IL-4 and transforming growth factor-beta to inhibit macrophage cytotoxic activity. J. Immunol. 148, 3578–3582. Paugam, A., Dupouy-Camet, J., Sumuyen, M.H., Romand, S., Lamoril, J., Derouin, F., 1995. Detection of Toxoplasma gondii parasitemia by polymerase chain reaction in perorally infected mice. Parasite 2, 181–184.

28

K. Sedl´ak et al. / Veterinary Parasitology 93 (2000) 13–28

Roberts, C.W., Ferguson, D.J.P., Jebbari, H., Satoskar, A., Bluethmann, H., Alexander, J., 1996. Different roles for interleukin-4 during course of Toxoplasma gondii infection. Infect. Immunol. 64, 897–904. Rubarth, S., 1948. Discussion. Acta Path. Microbiol. Scand. 25, 235. Štˇerba, F., Literák, I., Brandstätter, L., 1997. Toxoplasmosis in brown hare (Lepus europaeus) in the Czech Republic. Eur. J. Vet. Pathol. 3, 91–93. Walzl, H., 1959. Ein Beitrag zur pathologischen Histologie der Toxoplasmose. Wien. Tierärztl. Monat. 46, 544– 546. Werner, H., Aspöck, H., Janitschke, K., 1973. Serologische Untersuchungen über die Verrbreitung von Toxoplasma gondii unter Wildtieren (Mammalia) in Ostösterreich. Zbl. Bakt., Abt. I. Orig. A 224, 257–263.