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Acute fulminating babesiosis in hamsters infected with Babesia microti
hlernarionol Journal/iv Parasirologv, Vol. 26, No. 6. pp. 661-610. 1996 0 1996 Australian Society for Parasitology. Published by Elsevier Science Ltd Printed in Great Britam 002&7519/96 $15.00+0.00 SOO20-7519(96)00022-7 Copyright
Pergamon PII:
RESEARCH
NOTE
Acute Fulminating Babesiosis in Hamsters Infected with Babes&z microti HELIEH
S. OZ* and WALTER
T. HUGHES
St Jude Children’s Research Hospital, Memphis, TN 38105, U.S.A. (Received
18 September
1995;
accepted
I February
1996)
Abstract-Oz H. S. & Hughes W. T. 1996. Acute fulminating babesiosis in hamsters infected with Babesia microti. International Journal for Parusifofogy 26: 667470. In this study, Bubesia microti (ATCC30222) from mice was adapted to golden hamsters. The parasite was passaged to immmmsuppressed and then adapted to normal hamsters. When 30 normal hamsters were inoculated with this strain, parasitaemia increased to 74% of erytbrocytes by day 7 and 70% of the hamsters died. By day 12, parasitaemia extended to 90%, with 97% mortality. Hearts and kidneys from infected animals were enlarged. Histopatbology revealed acute myocarditis, hepatitis, pneumonitis, glomerulonepbritis and splenomegaly. Giemsa, Acridine Orange and Rhodamine staining of the parasite were compared. Scanning electron microscopy of blood from infected hamsters revealed from 1 to 5 intra-erytbrocytic parasites. Copyright 0 1996 Australian Society for Parasitology. Published by Elsevier Science Ltd. Key words:
Babesia
microti;
(ATCC30222);
acute
babesiosis;
Babesia microti, a common rodent parasite, is transmitted to humans by Zxodes tick bite or by blood transfusion. Human clinical cases caused by this strain have been reported from New England, Wisconsin and California in the United States pillar, While & Benach, 1991). The Gray strain of Babesia microti (ATCC30221) originally isolated from a human patient but adapted to hamsters is usually used as an experimental model. However, the disease is usually subacute in hamsters and peak parasitaemia occurs 2-3 weeks following inoculation although infections may prove fatal in about l-2 months (Weiss et al., 1993). The purpose of this study was to develop an animal model that mimicked human disease manifest by extensive parasitaemia, clinical disease and death. Male, IO-12-week-old golden hamsters, weighing about 170 g were obtained from Harlan SpragueDawley (Indianapolis, IN), and fed autoclavable Purina rodent chow and water ad libitum. They were inoculated i.p. with (ATCC30222) stock B. microti infected blood from mouse. This strain was originally *To
whom
correspondence
should
be addressed.
667
hamster;
parasitaemia.
isolated from a forest mouse Thamnomys surdaster in Kinshasa, Zaire in 1950, and was subsequently adapted to laboratory mice. Unlike the Gray strain (ATCC30221), this strain is not normally propagated in hamsters. Hamsters were therefore, immunosuppressed with one injection of 30mg Depomedrol followed by 2.5mglL of dexamethasone in daily drinking water. The hamsters were inoculated IP with B. microti (ATCC30222) and 10 days later, blood was collected and passaged into normal hamsters. Animals were anaesthetized with methofane and bled from the tail vein every 3 days until the end of the experiment. In order to transfer the infection, blood was collected from anaesthetized animals by cardiac puncture using a heparinized syringe. Blood smears were prepared and the percentage parasitaemia was determined by counting the number of infected erythrocytes per 500 red blood cells. After infections were established, hamsters were killed by CO, inhalation or they died as a consequence of babesiosis. Gross lesions were examined at post mortem and internal organs were removed, weighed and fixed in 10% formalin. Histological sections were prepared and stained with hematoxylin and eosin. Wet mount
H. S. Oz & W. T. Hughes
Fig.
1.
Scanning
electron
micrographs of infected blood cells. Scale protruding parasites. (B) B. microti merozoites
were also made by mixing a drop of blood in, phosphate buffer saline (PBS) and then examining by phase-contrast microscopy. For Giemsa staining, thin blood smears were fixed in methanol and stained for 20 min in 0.5% Giemsa stock solution in 4% PBS. Additional blood smears were fixed in methanol and stained in 0.1% of fluorescent dye (Acridine Orange or Rhodamine B) diluted in Krebs Ringer-phosphate solution pH 7.4 and then examined 10 min later by fluorescent microscopy. For scanning electron microscopy blood samples were centrifuged at 1000 g for 10 min, the erythrocytes washed with PBS and then fixed in 2% glutaraldehyde in PBS with 4% sucrose. The cells were post-fixed in 1% osmium tetroxide, dehydrated, immersed in hexamethyldisilizane (Polysciences INC), sputter-coated with gold and examined on a JEOL 1200 EXII scanning electron microscope (Fig. 1). Efforts to infect normal hamsters by i.p. inoculaof mouse origin were tion of B. microti (ATCC30222) unsuccessful and no parasitaemia was detected. When golden hamsters were immunosuppressed with corticosteroids, 10% parasitaemia was observed 7 days after inoculation and had increased to about 90% after 10 days. All of the hamsters died as a result of infection. Parasites from immunosuppressed hamsters were transferred to 30 normal hamsters by i.p. inoculation of 1 x 10’ infected erythrocytes. The mean parasitaemia at days 34 after inoculation was
bars = 2pm. (A) Infected attached to a host cell.
erythrocytes
sho wing
8% and it increased to 74% by day 7 at which time 70% of the hamsters (21/30) had died. By day 12 the parasitemia in surviving animals extended to about 90% and all remaining animals became moribund and died as consequence of babesiosis. The hamsters were cachectic, and had lost about 50% of their body weight. Haematuria, pale mucous membranes and oedema of the head were evident 1 week after infection and these signs persisted until death. On post mortem, enlargement of the spleen and liver was noticeable whereas hearts and kidneys from infected animals had increased to 25% and 30% of the organ weight from control normal hamsters. Hydrothorax, acute pneumonitis (Fig. 2A), glomerulonephritis (Fig. 2B), hepatitis (Fig. 2C), splenomegaly (Fig. 2D) and myocarditis (Fig. 2E) were prominent features of babesiosis induced with this strain in hamsters as early as 1 week after infection. Parasites were visible in wet mounts examined by phase-contrast as halo structures inside infected red blood cells. This technique was proved to be a rapid and simple means to screen blood samples for Bubesiu before staining. Acridine Orange stained parasite nuclei bright yellow-green and the rest of the organism orange-red (Fig. 2F) whereas erythrocytes appeared ghost-like in outline. Staining with Rhodamine B was similar to that of A&dine Orange except that nucleus of the parasite stained red. Fluorescent dyes have previously been used to stain different blood parasites,
Research
note
Fig. 2. Histopathological changes in hamster tissues caused by B. microti (A-E). paraffin sections stained with H & E ( x 400 magnification). Scale bars= 10 pm. (A) Lung: congested, with diffuse infiltration of inflammatory cells, haemosiderin deposits in parenchyma (arrows) and acute pneumonitis. (B) Kidney: degeneration and necrosis of tubules, thickening of Bowman’s capsule, erythrocytes (arrows) and fibrin deposits in tubules. (C) Liver: hepatitis with infiltration of inflammatory cells around the portal vein, degeneration and necrosis of the hepatocytes. (D) Spleen: distortion of spleen structure, hyperplasia of reticuloendothelial cells and giant cells (arrow). (E) Heart: degeneration and necrosis of myocardial fibers (arrow), mild infiltration of inflammatory cells, acute myocarditis. (F) Blood smear stained with Acridine Orange examined by fluorescent microscopy ( x 1000 magnification). B. microti are visible inside the red blood cells as bright nuclei (arrows).
such as B.canis (Goldsmith & Rogers, 1977) and even Plasmodium sp. (Kazuyuli, 1983). We found the Acridine Orange stain to provide better visualization of B. microti than the Rhodamine stain. In addition, there were no significant differences in the percentage parasitaemia as determined by A&dine Orange and Giemsa staining. However Giemsa-stained slides were more stable and could be stored without degradation or bleaching for long periods. Although slides stained with fluorescent dyes could be restained. Scanning electron microscopy revealed infected
erythrocytes to be deformed and to contain from l-5 intra-erythrocytic bodies (Fig. 1A). Occasionally free organisms were observed attached to leucocytes (Fig. 1B). Free organisms were seen as well as phagocytosis of these organisms by macrophages or leucocytes in blood smears of infected hamsters or by means of transmission electron microscopy. We transferred B. microti (ATCC30222) from the laboratory mouse to the immunosuppressed hamster and then adapted it to normal hamsters. This strain showed high virulence as evidenced by acute clinical
670
H. S. Oz & W. T. Hughes
signs, 70% (mean) parasitaemia and death only 1 week after inocuIation. HistopathoIogicaI studies revealed acute pneumonitis, myocarditis, splenitis, hepatitis and glomerulonephritis. This strain adapted to hamsters mimicked the pattern of human babesiosis in immunocompromised patients. This model therefore provides greater opportunities for research on this important zoonosis. Acknowledgements-Grant
support: NIH (AI-20673,
21765); American Lebanese acknowledge Donna Davis, for their technical assistance.
Syrian Bonnie
CA-
Charities. The authors Greer and Joe Emmes
REFERENCES
Goldsmith J. M. & Rogers S. 1977. Preliminary report on the use of acridine orange 0 for the detection of Babesia canis in the blood. Central African Journal of Medicine 23: 35-36. Kazuyuli T. 1983. Staming of Plasmodium yoelii infected mouse erythrocytes with the fluorescent dye rhodamine 123. Journal of Protozoology 30: 707-710. Villar B. F., While D. J. & Benach J. L. 1991. Human babesiosis. Progress in Clinical Parasitology 2: 129-143. Weiss L. M., Wittner M., Wasserman S., 0.z H. S., Retsema J. & Tanowitz H. B. 1993. Efficacv of azithromvcin for treating Babe& microti infection & the hamster model. Journal of Infectious Diseases 168: 1289-1292.
Pergamon PII:
RESEARCH
NOTE
Acute Fulminating Babesiosis in Hamsters Infected with Babes&z microti HELIEH
S. OZ* and WALTER
T. HUGHES
St Jude Children’s Research Hospital, Memphis, TN 38105, U.S.A. (Received
18 September
1995;
accepted
I February
1996)
Abstract-Oz H. S. & Hughes W. T. 1996. Acute fulminating babesiosis in hamsters infected with Babesia microti. International Journal for Parusifofogy 26: 667470. In this study, Bubesia microti (ATCC30222) from mice was adapted to golden hamsters. The parasite was passaged to immmmsuppressed and then adapted to normal hamsters. When 30 normal hamsters were inoculated with this strain, parasitaemia increased to 74% of erytbrocytes by day 7 and 70% of the hamsters died. By day 12, parasitaemia extended to 90%, with 97% mortality. Hearts and kidneys from infected animals were enlarged. Histopatbology revealed acute myocarditis, hepatitis, pneumonitis, glomerulonepbritis and splenomegaly. Giemsa, Acridine Orange and Rhodamine staining of the parasite were compared. Scanning electron microscopy of blood from infected hamsters revealed from 1 to 5 intra-erytbrocytic parasites. Copyright 0 1996 Australian Society for Parasitology. Published by Elsevier Science Ltd. Key words:
Babesia
microti;
(ATCC30222);
acute
babesiosis;
Babesia microti, a common rodent parasite, is transmitted to humans by Zxodes tick bite or by blood transfusion. Human clinical cases caused by this strain have been reported from New England, Wisconsin and California in the United States pillar, While & Benach, 1991). The Gray strain of Babesia microti (ATCC30221) originally isolated from a human patient but adapted to hamsters is usually used as an experimental model. However, the disease is usually subacute in hamsters and peak parasitaemia occurs 2-3 weeks following inoculation although infections may prove fatal in about l-2 months (Weiss et al., 1993). The purpose of this study was to develop an animal model that mimicked human disease manifest by extensive parasitaemia, clinical disease and death. Male, IO-12-week-old golden hamsters, weighing about 170 g were obtained from Harlan SpragueDawley (Indianapolis, IN), and fed autoclavable Purina rodent chow and water ad libitum. They were inoculated i.p. with (ATCC30222) stock B. microti infected blood from mouse. This strain was originally *To
whom
correspondence
should
be addressed.
667
hamster;
parasitaemia.
isolated from a forest mouse Thamnomys surdaster in Kinshasa, Zaire in 1950, and was subsequently adapted to laboratory mice. Unlike the Gray strain (ATCC30221), this strain is not normally propagated in hamsters. Hamsters were therefore, immunosuppressed with one injection of 30mg Depomedrol followed by 2.5mglL of dexamethasone in daily drinking water. The hamsters were inoculated IP with B. microti (ATCC30222) and 10 days later, blood was collected and passaged into normal hamsters. Animals were anaesthetized with methofane and bled from the tail vein every 3 days until the end of the experiment. In order to transfer the infection, blood was collected from anaesthetized animals by cardiac puncture using a heparinized syringe. Blood smears were prepared and the percentage parasitaemia was determined by counting the number of infected erythrocytes per 500 red blood cells. After infections were established, hamsters were killed by CO, inhalation or they died as a consequence of babesiosis. Gross lesions were examined at post mortem and internal organs were removed, weighed and fixed in 10% formalin. Histological sections were prepared and stained with hematoxylin and eosin. Wet mount
H. S. Oz & W. T. Hughes
Fig.
1.
Scanning
electron
micrographs of infected blood cells. Scale protruding parasites. (B) B. microti merozoites
were also made by mixing a drop of blood in, phosphate buffer saline (PBS) and then examining by phase-contrast microscopy. For Giemsa staining, thin blood smears were fixed in methanol and stained for 20 min in 0.5% Giemsa stock solution in 4% PBS. Additional blood smears were fixed in methanol and stained in 0.1% of fluorescent dye (Acridine Orange or Rhodamine B) diluted in Krebs Ringer-phosphate solution pH 7.4 and then examined 10 min later by fluorescent microscopy. For scanning electron microscopy blood samples were centrifuged at 1000 g for 10 min, the erythrocytes washed with PBS and then fixed in 2% glutaraldehyde in PBS with 4% sucrose. The cells were post-fixed in 1% osmium tetroxide, dehydrated, immersed in hexamethyldisilizane (Polysciences INC), sputter-coated with gold and examined on a JEOL 1200 EXII scanning electron microscope (Fig. 1). Efforts to infect normal hamsters by i.p. inoculaof mouse origin were tion of B. microti (ATCC30222) unsuccessful and no parasitaemia was detected. When golden hamsters were immunosuppressed with corticosteroids, 10% parasitaemia was observed 7 days after inoculation and had increased to about 90% after 10 days. All of the hamsters died as a result of infection. Parasites from immunosuppressed hamsters were transferred to 30 normal hamsters by i.p. inoculation of 1 x 10’ infected erythrocytes. The mean parasitaemia at days 34 after inoculation was
bars = 2pm. (A) Infected attached to a host cell.
erythrocytes
sho wing
8% and it increased to 74% by day 7 at which time 70% of the hamsters (21/30) had died. By day 12 the parasitemia in surviving animals extended to about 90% and all remaining animals became moribund and died as consequence of babesiosis. The hamsters were cachectic, and had lost about 50% of their body weight. Haematuria, pale mucous membranes and oedema of the head were evident 1 week after infection and these signs persisted until death. On post mortem, enlargement of the spleen and liver was noticeable whereas hearts and kidneys from infected animals had increased to 25% and 30% of the organ weight from control normal hamsters. Hydrothorax, acute pneumonitis (Fig. 2A), glomerulonephritis (Fig. 2B), hepatitis (Fig. 2C), splenomegaly (Fig. 2D) and myocarditis (Fig. 2E) were prominent features of babesiosis induced with this strain in hamsters as early as 1 week after infection. Parasites were visible in wet mounts examined by phase-contrast as halo structures inside infected red blood cells. This technique was proved to be a rapid and simple means to screen blood samples for Bubesiu before staining. Acridine Orange stained parasite nuclei bright yellow-green and the rest of the organism orange-red (Fig. 2F) whereas erythrocytes appeared ghost-like in outline. Staining with Rhodamine B was similar to that of A&dine Orange except that nucleus of the parasite stained red. Fluorescent dyes have previously been used to stain different blood parasites,
Research
note
Fig. 2. Histopathological changes in hamster tissues caused by B. microti (A-E). paraffin sections stained with H & E ( x 400 magnification). Scale bars= 10 pm. (A) Lung: congested, with diffuse infiltration of inflammatory cells, haemosiderin deposits in parenchyma (arrows) and acute pneumonitis. (B) Kidney: degeneration and necrosis of tubules, thickening of Bowman’s capsule, erythrocytes (arrows) and fibrin deposits in tubules. (C) Liver: hepatitis with infiltration of inflammatory cells around the portal vein, degeneration and necrosis of the hepatocytes. (D) Spleen: distortion of spleen structure, hyperplasia of reticuloendothelial cells and giant cells (arrow). (E) Heart: degeneration and necrosis of myocardial fibers (arrow), mild infiltration of inflammatory cells, acute myocarditis. (F) Blood smear stained with Acridine Orange examined by fluorescent microscopy ( x 1000 magnification). B. microti are visible inside the red blood cells as bright nuclei (arrows).
such as B.canis (Goldsmith & Rogers, 1977) and even Plasmodium sp. (Kazuyuli, 1983). We found the Acridine Orange stain to provide better visualization of B. microti than the Rhodamine stain. In addition, there were no significant differences in the percentage parasitaemia as determined by A&dine Orange and Giemsa staining. However Giemsa-stained slides were more stable and could be stored without degradation or bleaching for long periods. Although slides stained with fluorescent dyes could be restained. Scanning electron microscopy revealed infected
erythrocytes to be deformed and to contain from l-5 intra-erythrocytic bodies (Fig. 1A). Occasionally free organisms were observed attached to leucocytes (Fig. 1B). Free organisms were seen as well as phagocytosis of these organisms by macrophages or leucocytes in blood smears of infected hamsters or by means of transmission electron microscopy. We transferred B. microti (ATCC30222) from the laboratory mouse to the immunosuppressed hamster and then adapted it to normal hamsters. This strain showed high virulence as evidenced by acute clinical
670
H. S. Oz & W. T. Hughes
signs, 70% (mean) parasitaemia and death only 1 week after inocuIation. HistopathoIogicaI studies revealed acute pneumonitis, myocarditis, splenitis, hepatitis and glomerulonephritis. This strain adapted to hamsters mimicked the pattern of human babesiosis in immunocompromised patients. This model therefore provides greater opportunities for research on this important zoonosis. Acknowledgements-Grant
support: NIH (AI-20673,
21765); American Lebanese acknowledge Donna Davis, for their technical assistance.
Syrian Bonnie
CA-
Charities. The authors Greer and Joe Emmes
REFERENCES
Goldsmith J. M. & Rogers S. 1977. Preliminary report on the use of acridine orange 0 for the detection of Babesia canis in the blood. Central African Journal of Medicine 23: 35-36. Kazuyuli T. 1983. Staming of Plasmodium yoelii infected mouse erythrocytes with the fluorescent dye rhodamine 123. Journal of Protozoology 30: 707-710. Villar B. F., While D. J. & Benach J. L. 1991. Human babesiosis. Progress in Clinical Parasitology 2: 129-143. Weiss L. M., Wittner M., Wasserman S., 0.z H. S., Retsema J. & Tanowitz H. B. 1993. Efficacv of azithromvcin for treating Babe& microti infection & the hamster model. Journal of Infectious Diseases 168: 1289-1292.