Veterinary Parasitology, 23 (1987) 169--178 169 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

C U L T I V A T I O N OF B E S N O I T I A B E S N O I T I AND E V A L U A T I O N OF S U S C E P T I B I L I T Y OF L A B O R A T O R Y A N I M A L S TO C U L T U R E D P A R A S I T E S

VARDA SHKAP, E. PIPANO and C. GREENBLATT ~

Department of Parasitology, Kimron Veterinary Institute, Bet Dagan (Israel)

1Department of Protozoology, Hadassah Medical School, Hebrew University, Jerusalem (Israel)

(Accepted for publication 19 March 1986)

ABSTRACT

Shkap, V., Pipano, E. and Greenblatt, C., 1987. Cultivation of Besnoitia besnoiti and evaluation of susceptibility of laboratory animals to cultured parasites. Vet. Parasitol., 23: 169--178.

Cultivation of Besnoitia besnoiti on five mammalian cell types showed that Vero (green monkey kidney), L929 (mouse fibroblasts) and BEK (bovine embryo kidney) cells were highly susceptible and were almost destroyed after 5 days of incubation, while MDBK (Madin--Darby bovine kidney) and BL (Theileria annulata-infected bovine lymphoid) cells were less affected.

Intraperitoneal infection with culture-derived endozoites was fatal for gerbils (Me- riones tristrami) and sand rats (Psammomys obesus). Rabbits showed fever, conjunctivitis and orchitis, but survived the infection. Mice, rats, guinea pigs, hamsters and Microtus guentheri did not show signs of illness, but developed specific antibodies to B. besnoiti.

B. besnoiti subcultivated for 2 years in BL cells remained virulent for gerbils.

INTRODUCTION

Besnoit ia besnoit i (Marotel , 1912) is a tissue cys t - forming p r o t o z o o n (Fayer , 1980; Tadros and Laarman , 1981) tha t infects catt le and has con- siderable e c o n o m i c impor t ance in cer tain regions o f Africa and Asia. Since large-scale exper iments on cat t le are cost ly , l abora to ry animal models and cul t ivat ion in vitro are used in studies on B. besnoiti . The parasite has been cul t ivated o n pr imary cell cul tures (bovine and ovine k idney , ovine t h y r o i d cells), bu t there are obvious advantages in using established cell lines. Cultiva- t ion on Hela and BHK cells, however , was slow and p roceeded for no more than six passages (Neuman , 1974) and only the Ve to cell line was satis- f a c to ry for con t i nuous p ropaga t ion (Bigalke et al., 1974) .

Gerbils, rabbits and hamsters were r epor ted to be susceptible to B. besnoit i (Pols, 1960; Bigalke, 1968; N e u m a n and Nobel , 1981) , bu t discrepancies

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exist in the literature concerning the susceptibility of mice and guinea pigs (Pols, 1960; Neuman, 1965; Peteshev et al., 1974; Kaggwa et al., 1979; Neuman and Nobel, 1981).

The purpose of this report was to study the multiplication rate of B. besnoiti in five cell lines, to evaluate the influence of long-term cultivation on the virulence of the parasite and to assess the response of common laboratory animals to an Israeli isolate of B. besnoiti.

MATERIALS AND METHODS

Source o f parasites

Subcutaneous tissues from a naturally infected bull were minced with scissors, washed 3--4 times in phosphate buffered saline (PBS) containing penicillin (200 u ml-1), streptomycin (100 mg ml-l), neomycin (100 mg ml-1), kanamycin (100 mg ml-l). The tissue pieces were digested in 0.5% trypsin solution by stirring at 4°C overnight. Non-digested particles were allowed to settle and the supernatant centrifuged at 400 X g for 10 min. The pellet containing free cystozoites was washed twice with PBS by centri- fugation and after resuspension inoculated intraperitoneally (i.p.) into gerbils.

Within 6--8 days terminally ill gerbils were killed by ether anaesthesia. The abdomen was opened aseptically and the peritoneal cavity rinsed with PBS containing 100 units of heparine ml- 1. Peritoneal washings from several infected gerbils were pooled and rinsed in PBS by centrifugation at 400 X g for 10 min. The pelleted parasites were resuspended in culture medium, counted in a Thomas chamber and used for infecting cell cultures. In ad- dition one cell line (BL, see below) was inoculated with cystozoites obtained directly from cysts after trypsin dispension of infected bull tissues.

Cells and culture media

Four established cell lines were used: mouse fibroblasts (L92~), Madin- Darby adult bovine kidney cells (MDBK), green monkey kidney cells (Vero) and bovine lymphoblastoid cells infected with Theileria annulata schizonts (BL). In addition, primary bovine embryo kidney cells {BEK) were ob- tained by trypsinization of kidney tissues from a 2-month-old bovine em- bryo.

Monolayers of cultures were dispersed with a 0.25% trypsin solution sup- plemented with 0.02% EDTA. Dispersed cells were counted and their num- ber adjusted with culture medium to 4 X l0 s cells ml -t. One ml of cell suspension was introduced into Leighton tubes containing coverslips.

The medium for all cultures except BL consisted of Eagle's Minimum Essential Medium supplemented with 10% calf serum and 0.3% tryptose phosphate broth solution. BL cells were grown in the same medium sup-

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plemented with 20% calf serum and wi thout t ryptose phosphate. Penicillin (100 U ml-1), s treptomicine (100 mg m1-1) and mycostat in (50 U ml -~) were added to all media before use. Cultures were incubated at 37°C.

Infection of cells and evaluation of rate of infection

About 1 h after seeding the Leighton tube with cells, 4 × l 0 s B. besnoiti parasites per tube were introduced (1 parasite per cell). Coverslips removed after 3, 6, 12, 24, 48, 72, 96 and 120 h were rinsed in PBS, air<lried, fixed in methanol for 10 min and stained with Giemsa. Twelve coverslips were examined for each cell type and each t ime interval. The number of para- sites found in 200 host cells at tached to the coverslips was counted on each preparation and the percentage of infected cells was determined.

Infection of animals

Rabbits, hamsters, guinea pigs, rats, white mice, inbred mice (CsH, Black/- 57 and BALB/C), gerbils (Meriones tristrami), sand rats (Psammomys o- besus) and Microtus guentheri were inoculated i.p. with B. besnoiti. Each animal received 107 endozoites derived from Vero cultures. All animals were observed for 30 days except for rabbits, which were observed for 3 months. To determine whether death occurred as a result of B. besnoiti infection, about 50% of terminally ill gerbils and sand rats were killed and the washings of the peritoneal cavity examined microscopically for zoites. Rectal temperature was measured in rabbits every day for a month, blood smears were prepared during the febrile period, stained with Giemsa and examined for parasites.

To determine the LDs0 in gerbils, seven groups of 12 animals each were inoculated i.p. with 10-fold dilutions of cultured endozoites ranging from 107 to 10 organisms m1-1. An identical set of animals was inoculated with endozoites separated from the culture cells on a cellulose column as des- cribed by Bodner et al. (1972) for T. gondii derived from mouse peritoneal fluid.

In another exper iment a group of 30 white mice was inoculated i.p. with 107 Besnoitia endozoites. Two or three mice were sacrificed daily f rom Day 1 to 6, then every 2 days up to Day 21, and three mice were sacrificed on Day 30. The peritoneal washings were examined microscopical- ly for Besnoitia and the brains were fixed in formalin for histological ex- amination. Paraffin-embedded sections of fixed tissues were cut at 5pm and stained with hematoxyl in and eosin.

Prolonged cultivation

Two Besnoitia-infected BL lines were subcultivated in 7 5 ~ m 2 plastic vessels (Falcon, USA). In the first line, the Besnoitia-infected monolayer

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was passaged regularly twice a week. In the second line Besnoitia-infected cells were mixed with the same number of fresh Besnoitia-intact BL cells at each passage in parallel with the first line.

Serology

All animals were bled before inoculation and those surviving were also bled a month later. B. besnoiti antigen for use in the indirect immuno- fluorescence ant ibody test (IFAT) was prepared from cultured endozoites as described by Goldman and Pipano (1983). Commercial specific fluores- cent antiglobulin conjugates were used (Bio-Yeda, Israel).

RESULTS

Cultivation o f B. besnoiti in cell cultures

The percentage of cells infected during 120 h of cultivation in various cell types is shown in Fig. 1.

BEK cells appeared to be the most susceptible to B. besnoiti; ~ 30% of the cells were infected after 3 h of incubation. At that t ime only 7% of MDBK and about 20% of Veto, L~2~ and BL cells had become infected. At all cultivation times tested BEK, L~29 and Vero cells showed higher rates of infection than did MDBK and BL cells. Infected monolayers of Vero, L~29 and BEK cells were almost completely destroyed, but no sig-

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Fig. 1. In fec t ion rates of B. besnoi t i in various cell cultures.

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Figs. 2--7 Observation on Besnoitia besnoiti cultured in various types of cells. Photo- graphs made using Zeis$ microscope, magnification × 1000. (2) B. besnoiti organized in a typical rosette formation in L9:9 cells at 24 h of incubation. (3) Three vacuoles, containing up to 15 parasites in BL cells. 72 h of incubation. (4) Large aggregates of parasites in BEK cells. 72 h of incubation. (5) Destructed heavily infected Veto cells. Liberation of free parasites into intracellular spaces. 96 h of incubation. (6) Oval-shaped B. besnoiti in L9~9 cells, 48 h incubation. (7) Crescentic B. besnoiti parasites in MDBK cells, 96 h of incubation.

n i f icant changes were observed in MDBK and BL mono laye r s dur ing in- cuba t ion for 5 days.

Rose t t e f o r m a t i o n was seen in all cell types after 24 h o f incuba t ion (Fig. 2). In BL cells 1--3 separated vacuoles conta in ing up to 10 parasites each were seen after 42- -72 h (Fig. 3). At tha t t ime large aggregates o f intracellular organisms were fo rmed in the cy top lasm of Vero, L929 and BEK cells (Fig. 4). Dur ing the nex t 2 days the number o f heavily infected cells increased, releasing free endozo i tes into the cul ture m e d i u m u p o n dis integrat ion (Fig. 5).

In L929 and BL cells B. be sno i t i endozoi tes became shor t and oval-shaped

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with enlarged intensively stained nuclei (Fig. 6), while crescentic forms prevailed in BEK, MDBK and Vero cultures during the whole incubation period (Figs. 5, 7).

As shown in Table I long-term cultivation of B. besnoiti in Theileria-in- fected bovine lymphocytes did not alter the virulence of this parasite as evaluated by i.p. inoculation into gerbils.

TABLE I

Mortality in gerbils after i.p. inoculation with 2 x 106 Besnoitia besnoiti parasites grown in BL cells

Parasites No. of passages Days No. gerbils subcultured (up to) in culture died/inoculated

Without 50 140 14/15 addition of 105 360 14/15 intact cells By addition 100 300 15/17 of intact 225 680 15/20 cells

Sensitivity o f laboratory animals to B. besnoiti

Results of inoculation of B. besnoiti culture-derived endozoites are shown in Table II. Gerbils and sand rats appeared normal for 4--6 days post-inoculation, then diminished activity, apathy, anorexia, cahexia and t remor occurred. All animals died within 6--8 days. Fibrinous peritonitis wi thout exudate was observed in dying animals and the peritoneal washings contained many B. besnoiti endozoites. Rabbits showed fever (41.0 and 41.8°C) commencing 2--3 days after inoculation and persisting for 8--10 days. During the febrile period conjunctivitis and swelling of the testes were observed; blood smears were negative for parasites.

In white mice, a few endozoites (2--3 per 10 microscopic fields) were detected in peritoneal washings from Day 1 to Day 5 post-inoculation, but from Days 6 to 30 after inoculation no parasites could be detected. Cysts were not found in histological preparation of brains of inoculated mice. As shown in Table II, inbred mice (C3H, Black/57 and BALB/C) as well as 12 white mice that were inoculated with B. besnoiti survived for 30 days.

Rabbits, mice, rats, guinea pigs and hamsters were negative for antibodies against B. besnoiti prior to inoculation, but developed titers mostly ranging from 1:32 to 1:64 when examined 30 days after inoculation (Table II).

The LDs0 of B. besnoiti endozoites obtained from dispersed Veto-infected cells for gerbils was 103.6 as calculated by the Reed and Munch method (1938). A similar mortal i ty rate LDso, 104, was observed in groups of gerbils

1 7 5

which received column separated cell-free endozoites (Table III). Six weeks after primary inoculation surviving gerbils were challenged i.p. with 10 7 culture derived B. besnoiti; all animals previously inoculated with 10 or-

TABLE II

Response of l abora tory animals to i.p. inocula t ion wi th 107 B. besnoiti grown in cell cul ture

Animals tes ted Reac t ion

Died / inocula ted Reciprocal min-max t i ters in I F A T b

Gerbils 6/6 n.d. a Sand rats 6/6 n.d. Rabbi ts 0/6 256-1024 White rats 0/8 32-64 Guinea pigs 0/5 32-64 Hamsters 0/5 32-64 White mice 0/12 16-64 C3H mice 0/6 n.d. Black/57 mice 0/6 n.d. BALB/C mice 0/6 n.d. Microtus 0/6 n.d.

an.d. = no t done. b I F A T = indirect f luorescent an t ibody test.

TABLE III

Morta l i ty of gerbils fol lowing i.p. inocula t ion of various number s of B. besnoiti f rom cell cul ture

No. of Endozo i t e s and cul ture cells Cell-free endozo i t e s parasites inocula ted Pr imary a Challenge b Pr imary a Challenge b

inocula t ion inocula t ion

107 100 c - - 92 c 0 106 84 50 c (2) d 83.3 0 10 S 84 0 (2) 50 50 c 104 46 28.5 (7) 50 66.6 103 50 33.3 (6) 34 42.8 102 34 37.5 (8) 42 85.7 10 0 100 (12) 0 100 10 ~ 100 (12) I00

(1) d (2) (6) (6) (7) (7)

(12) (12)

aTwelve gerbils were inocula ted for each concen t r a t i on o f parasites. bChal lenged wi th 107 endozo i t e s per dose. c% = mor ta l i ty . d N u m b e r o f animals challenged in parentheses .

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ganisms and all controls died within 6--8 days, while 15%--50% of the remaining gerbils survived reinfection.

DISCUSSION

In the present s tudy B. besnoiti penetrated and multiplied in cells of various origins -- monkey (Vero), mice (L929) and bovine (MDBK, BEK, BL), showing a lack of species specificity as previously observed by Bigalke (1962), Bigalke et al. (1974) and Neuman (1974). The multiplication rate of B. besnoiti was similar in epithelial (Vero, BEK) and fibroblast (L929) cells, although according to Akinshina and Doby (1969) another species of Besnoitia, B. ]ellisoni grew more rapidly in fibroblast-like cells. The MDBK cells proved to be a less favorable substrate for multiplication of B. besnoiti and, as shown by Fayer et al. (1969), these cells provided a poor support also for growth of B. jellisoni. The presence of 2--3 discrete vacuoles containing B. besnoiti in BL cells is probably the result of multi- ple infection by single parasites followed by multiplication and formation of independent germinative centers.

The results of our laboratory animal infection experiments confirm the susceptibility of gerbils to B. besnoiti as previously reported by Neuman (1962), and demonstrate that another rodent species Psarnmomys obesus, is also highly susceptible. These animals might be considered as suitable laboratory models for studies on acute experimental besnoitiosis. The slight difference between the LDs0 of cell-free and unpurified endozoites for gerbils is believed to be due to small numbers of intracellular organisms that were overlooked during the counting. From the results obtained here it appears that in some animals doses of parasites varying from 102 to 106 induced asymptomatic infection followed by protect ion against lethal challenge. In other animals the same dosage was not infective and the re- cipients remained completely susceptible to challenge.

Rabbits developed the typical clinical signs described by Pols (1960) and Bigalke {1968); we did not see the endozoites in the blood smears as they did.

In the present study, results from guinea pigs, hamsters and mice are in agreement with those reported by Pols (1960), Bigalke (19G8), Kaggwa (1977) and Kaggwa et al. (1979). However, according to Neuman and Nobel (1981), guinea pigs and hamsters developed nervous symptoms and died following i.p. inoculation of endozoites, while in mice large num- bers of endozoites were found in peritoneal exudate.

Long term cultivation of B. besnoiti in bovine lymphoid cells did not influence the virulence of this parasite for gerbils. Negative results were also obtained with the similar parasite, T. gondii, upon prolonged in vitro cultivation (Cook and Jacobs, 1958; Shulova, 1960; Bickford and Burn- stein, 1966).

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ACKNOWLEDGEMENT

T h e a u t h o r s t h a n k P ro fe s so r Dr . T . N o b e l , Head of t h e P a t h o l o g y De- p a r t m e n t o f t h e K i m r o n V e t e r i n a r y I n s t i t u t e fo r t h e h i s t o p a t h o l o g i c a l e x a m i n a t i o n s .

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