EXPERIMENTAL
PARASITOLOGY
Brugia
38, 279-290
(1975)
pahangi: Susceptibility of Golden
and Macroscopic Hamsters
Pathology
B. MALONEI AND PAUL E. THOMPSON~
JOHN
Department
of Parasitology,
College of Veterinary Medicine, Athens, Georgh 30602
(Accepted
for publication
University
of Georgia,
March 25, 1975)
MALONE, J. B., AND THOMPSON, P. E. 1974. Brugia pahangi: Susceptibility and Parasitology 38, 279-290. macroscopic pathology of golden hamsters. Experimental Twenty male hamsters w-ere inoculated with 95 to 150 infective larvae of B. pahangi via the subcutaneous route. Worms recovered from 19 hamsters averaged 14% (O-32) from 11 hamsters killed at 105-195 days after infection and 16% (5-19) from 8 hamsters examined at 2345 days after infection. Approximately one-half of the worms recovered were from the lymphatic vessels of the testes, epididymis, and spermatic cord. A few were found in afferent or efferent vessels of regional lymph nodes. The remaining worms were from the heart and lungs. Low-level microfilaremias were observed in 10 of 12 hamsters held for over 100 days. The average prepatent period was 89 days (65-128). Worms were recovered for up to 3 weeks following inoculation of nine hamsters via ,the intraperitoneal route with 100-400 infective larvae of B. pahangi. Gross lymphatic pathologic lesions consisted of moderate to marked dilation of lymphatic vessels, enlargement of regional lymph nodes, and numerous lymphthrombi and emboli. Macroscopic changes were most consistent and severe in the lymphatic vessels of the testes, epididymis, and spermatic cord and were noted less frequently in the afferent or efferent vessels of various regional lymph nodes. Areas of reddish discoloration were observed frequently on the serosal surface of the lung in infected hamsters. INDEX DESCRIPTORS: Brugiu pahangi; golden hamsters; Mesocdcetus auratu.s; susceptibility; microfilaremia; pathology; lymphangitis; lymphadenitis; lymphangiectasis; genital filariasis; tropical eosinophilia.
INTR~DU~~N
Research on lymphatic-dwelling filariae has been hampered until recently by the lack of a small rodent host suitable for laboratory investigations. The demonstration that Mongolian jirds (Meriones un1 Present address: Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803. 2 Deceased, July 3, 1973.
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
guiculatus) can be infected readily with Brugia pahangi (Ash and Riley 1970a) or B. muluyi (Ash and Riley 1970b) provides such a host and has prompted efforts to develop the jird as a model for use in certain types of studies on filariasis. This has included investigations on the biology ( Foor 1974)) host-parasite relationship (Ash 1971; ElBihari and Ewert 1971; Gwadz and Chernin 1973; Ah et al. 1974), pathology (Ah and Thompson 1973), and
230
MALONE AND THOMPSON
chemotherapy of filarial infections (Wang and Saz 1974). The usefulness of the jird as an experimental host for lymphaticdwelling filariae has not been elucidated completely, however, and continued efforts to develop alternative rodent model systems are warranted. In particular, the use of hosts of lesser susceptibility may ultimately prove advantageous for certain studies on acquired immunity and pathogenesis relevant to various life-cycle stages of filariae. Early attempts to infect golden hamsters, hlesocricetus auratus, with B. pahangi met with limited success (Zaini et al. 1962; Edeson et al. 1962). Adult worms were found in the heart of one of four hamsters in both reports, but no circulating microfilariae were observed. More recently, Ash and Riley (1970a) recovered worms from the testes, heart and lungs, viscera, pelt, and carcass of male hamsters infected with B. pahungi. Subsequently, Harbut (1972, 1973) reported improved worm recoveries but low-level microfilaremias and concluded that the hamster was not promising as a reliable experimental host for studies on chemotherapy and other aspects of filariasis research. On the basis of the low microfilaremias found (Sucharit and MacDonald, 1972), hamsters have been placed in the category of inadequate hosts for B. pahangi infections along with cotton rats, Signzodon hispidis, white rats, and multimammate rats, Mastonzys nataknsis. The purposes of this study were to investigate the susceptibility and pathologic changes in hamsters infected with B. pahangi and to determine the usefulness of the B. pahangi-hamster system as a model for lymphatic-dwelling filariae. Our preliminary observations on B. pahangi infections in hamsters indicated that substantial adult worm recoveries could be obtained from lymphatic vessels and the heart and lungs and that pathologic changes were more severe and somewhat different in distribution in the hamster
than in the jird (Malone, Leininger, and 1974; Malone, Thompson Leininger, Thompson, and Chapman 1974). MATERIALS AND METHODS
Male golden hamsters 7-11 weeks of age (39-140 g) were used. The strain of Brugia pahangi was obtained from Dr. Paul C. Beaver (Tulane University School of Medicine, New Orleans) and maintained in the laboratory by passage in beagles. The intermediate host was a genetically selected strain of Aedes aegypti (Black eye strain) originally obtained from Dr. Ralph Barr (University of California, Los Angeles ) . Infective third-stage larvae were collected from mosquitoes by a previously described method (Ah and Thompson, 1973), suspended in Hank’s balanced salt solution (BSS), and injected subcutaneously in the inguinal region or intraperitoneally. Microfilariaemias were determined by examining 20 mm3 of blood taken from the orbital sinus at 7-15 day intervals beginning at 59-60 days after infection. Blood smears were stained with Giemsa’s stain, and total numbers of microfilariae were counted. Blood samples were taken also from the right ventricle at necropsy, and microfilariae were counted in a similar manner. Hamsters were euthanatized with ether. Necropsies and searches for filariae were done according to the dissecting and soaking techniques described by Ah and Thompson ( 1973). Visualization of lymph vessels and nodes was facilitated by subcutaneous injection of Evan’s blue ( 1% ) into the footpads and occasionally into other selected sites. A dissecting microscope was used to search major organs and major lymphatics in situ and after thorough teasing and soaking of tissues. After removal of all visible worms by dissection, selected tissues were taken for microscopic examination, fixed in 10% buffered formalin, embedded, sectioned, and
Brugiu pahangi IN HAMSTERS stained with hematoxylin and eosin or additional selected stains as needed (Luna 1968). These tissues were utilized for a histopathologic study reported elsewhere (Malone et al. 1975). Serial sections (8 u) were prepared (each 15th to 20th section) to enumerate worms included in the specimen that were not visible at the time of necropsy. Tissue sections were made so that three specimens were collected from each testicle: one specimen was taken through the anterior pole of the testicle and the head of the epididymis, one through the posterior pole and the tail of the epididymis, and one through the pampiniform plexus area. Lung was sectioned through the cardiac, diaphragmatic, and apical lobes. Central structures in the mediastinum were included. Other organs examined included the spleen, liver, pancreas, stomach, intestines, kidneys, seminal vesicles, urinary bladder, and scrotum. Lymphatic tissues collected included the subiliac, iliac, renal, popliteal, inguinal, and medistinal lymph nodes and associated lymphatic vessels and surrounding tissues. In addition, selected sections were taken of any other tissues having pathologic lesions at necropsy. Ten uninfected control hamsters were examined for both macroscopic and histopathologic lesions. Parasites were fixed in glacial acetic acid, placed in 70% ethanol containing 5% glycerin, and brought into pure glycerin by evaporation, Descriptions of B. puhangi (Schacher 1962) were consulted for identification of morphological characters of whole parasites and for worms found in tissue sections. RESULTS Parasitologic
findings
Subcutaneous infections. Worm recovery data were compiled on three separate groups of juvenile male hamsters following injection of 95-150 infective larvae into the inguinal region (Table I). The
231
percentage of worms rccovercd from eight animals necropsied 2345 days after inoculation was slightly higher than recoveries from 11 animals necropsied 105 days or more after inoculation ( 16 vs 14% ), The localization of worms differed in early and late infections. The heart and lungs and lymphatic vessels contained approximately equal numbers of worms in hamsters examined 39 or more days after inoculation, but only small numbers had reached the heart of three hamsters examined at 2324 days. Greater numbers of worms occurred in efferent or afferent vessels of regional lymph nodes in early infections than in later infections (Table II). Recovery percentages of worms from the lymphatic vessels of the testes, epididymis, and spermatic cord correspondingly increased with duration of infection. Low-level, evanescent microfilaremias, not exceeding 18 microfilariae in 20 mm3 of blood, were seen in 10 of 12 hamsters held for over 105 days (Table III). The average mean prepatent period was 90 days. One or more microfilariae were seen in three hamsters (Tl, T8, T9), which became negative in subsequent ocular blood samples and in heart blood taken at the time of necropsy. No adult worms were recovered from animal T9 despite relatively high microfilaria counts. In animal T8 mature microfilariae were present in the uterus of all four female worms recovered at necropsy at 187 days; three were from the heart and lungs, and one was from the genital lymphatics. A fifth female worm found in tissue sections of the testes parenchyma also contained microfilariae. Examination of worms from the third hamster (Tl) at 109 days revealed microfilariae in one worm from the heart and lungs and in one worm from the lymphatics. Three of the other four female worms recovered contained ova but no microfilariae. The remaining worm was small and did not contain recognizable ova or microfilariae. One hamster (T3)
2S%
MALONE
AND
THOMPSON
TABLE
1
Sunrbcr, Sex, and Distribution of Brugia pahangi Kccowrcd from Ziamslrw A jicr Subcutaneous Znjdion ruilh Znjwt ive Larvae Hamster No.~
Cl C2 Tl T2 T4 T-3 TG T7 T8 T9 TIO
Days to necrwp,y
105 106 109 110 141 146 747 181 187 188 195
Number
Itecovery
larvae injecte:!
(:a
97 9.5 110 103 110 103 110 109 110 110 106
Number,
sex, and location
Lymphatics
27.X i41.6 10.9 13, .i 7.:2 9.7 7.3 11.9 I:
PARASITOLOGY
Brugia
38, 279-290
(1975)
pahangi: Susceptibility of Golden
and Macroscopic Hamsters
Pathology
B. MALONEI AND PAUL E. THOMPSON~
JOHN
Department
of Parasitology,
College of Veterinary Medicine, Athens, Georgh 30602
(Accepted
for publication
University
of Georgia,
March 25, 1975)
MALONE, J. B., AND THOMPSON, P. E. 1974. Brugia pahangi: Susceptibility and Parasitology 38, 279-290. macroscopic pathology of golden hamsters. Experimental Twenty male hamsters w-ere inoculated with 95 to 150 infective larvae of B. pahangi via the subcutaneous route. Worms recovered from 19 hamsters averaged 14% (O-32) from 11 hamsters killed at 105-195 days after infection and 16% (5-19) from 8 hamsters examined at 2345 days after infection. Approximately one-half of the worms recovered were from the lymphatic vessels of the testes, epididymis, and spermatic cord. A few were found in afferent or efferent vessels of regional lymph nodes. The remaining worms were from the heart and lungs. Low-level microfilaremias were observed in 10 of 12 hamsters held for over 100 days. The average prepatent period was 89 days (65-128). Worms were recovered for up to 3 weeks following inoculation of nine hamsters via ,the intraperitoneal route with 100-400 infective larvae of B. pahangi. Gross lymphatic pathologic lesions consisted of moderate to marked dilation of lymphatic vessels, enlargement of regional lymph nodes, and numerous lymphthrombi and emboli. Macroscopic changes were most consistent and severe in the lymphatic vessels of the testes, epididymis, and spermatic cord and were noted less frequently in the afferent or efferent vessels of various regional lymph nodes. Areas of reddish discoloration were observed frequently on the serosal surface of the lung in infected hamsters. INDEX DESCRIPTORS: Brugiu pahangi; golden hamsters; Mesocdcetus auratu.s; susceptibility; microfilaremia; pathology; lymphangitis; lymphadenitis; lymphangiectasis; genital filariasis; tropical eosinophilia.
INTR~DU~~N
Research on lymphatic-dwelling filariae has been hampered until recently by the lack of a small rodent host suitable for laboratory investigations. The demonstration that Mongolian jirds (Meriones un1 Present address: Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803. 2 Deceased, July 3, 1973.
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
guiculatus) can be infected readily with Brugia pahangi (Ash and Riley 1970a) or B. muluyi (Ash and Riley 1970b) provides such a host and has prompted efforts to develop the jird as a model for use in certain types of studies on filariasis. This has included investigations on the biology ( Foor 1974)) host-parasite relationship (Ash 1971; ElBihari and Ewert 1971; Gwadz and Chernin 1973; Ah et al. 1974), pathology (Ah and Thompson 1973), and
230
MALONE AND THOMPSON
chemotherapy of filarial infections (Wang and Saz 1974). The usefulness of the jird as an experimental host for lymphaticdwelling filariae has not been elucidated completely, however, and continued efforts to develop alternative rodent model systems are warranted. In particular, the use of hosts of lesser susceptibility may ultimately prove advantageous for certain studies on acquired immunity and pathogenesis relevant to various life-cycle stages of filariae. Early attempts to infect golden hamsters, hlesocricetus auratus, with B. pahangi met with limited success (Zaini et al. 1962; Edeson et al. 1962). Adult worms were found in the heart of one of four hamsters in both reports, but no circulating microfilariae were observed. More recently, Ash and Riley (1970a) recovered worms from the testes, heart and lungs, viscera, pelt, and carcass of male hamsters infected with B. pahungi. Subsequently, Harbut (1972, 1973) reported improved worm recoveries but low-level microfilaremias and concluded that the hamster was not promising as a reliable experimental host for studies on chemotherapy and other aspects of filariasis research. On the basis of the low microfilaremias found (Sucharit and MacDonald, 1972), hamsters have been placed in the category of inadequate hosts for B. pahangi infections along with cotton rats, Signzodon hispidis, white rats, and multimammate rats, Mastonzys nataknsis. The purposes of this study were to investigate the susceptibility and pathologic changes in hamsters infected with B. pahangi and to determine the usefulness of the B. pahangi-hamster system as a model for lymphatic-dwelling filariae. Our preliminary observations on B. pahangi infections in hamsters indicated that substantial adult worm recoveries could be obtained from lymphatic vessels and the heart and lungs and that pathologic changes were more severe and somewhat different in distribution in the hamster
than in the jird (Malone, Leininger, and 1974; Malone, Thompson Leininger, Thompson, and Chapman 1974). MATERIALS AND METHODS
Male golden hamsters 7-11 weeks of age (39-140 g) were used. The strain of Brugia pahangi was obtained from Dr. Paul C. Beaver (Tulane University School of Medicine, New Orleans) and maintained in the laboratory by passage in beagles. The intermediate host was a genetically selected strain of Aedes aegypti (Black eye strain) originally obtained from Dr. Ralph Barr (University of California, Los Angeles ) . Infective third-stage larvae were collected from mosquitoes by a previously described method (Ah and Thompson, 1973), suspended in Hank’s balanced salt solution (BSS), and injected subcutaneously in the inguinal region or intraperitoneally. Microfilariaemias were determined by examining 20 mm3 of blood taken from the orbital sinus at 7-15 day intervals beginning at 59-60 days after infection. Blood smears were stained with Giemsa’s stain, and total numbers of microfilariae were counted. Blood samples were taken also from the right ventricle at necropsy, and microfilariae were counted in a similar manner. Hamsters were euthanatized with ether. Necropsies and searches for filariae were done according to the dissecting and soaking techniques described by Ah and Thompson ( 1973). Visualization of lymph vessels and nodes was facilitated by subcutaneous injection of Evan’s blue ( 1% ) into the footpads and occasionally into other selected sites. A dissecting microscope was used to search major organs and major lymphatics in situ and after thorough teasing and soaking of tissues. After removal of all visible worms by dissection, selected tissues were taken for microscopic examination, fixed in 10% buffered formalin, embedded, sectioned, and
Brugiu pahangi IN HAMSTERS stained with hematoxylin and eosin or additional selected stains as needed (Luna 1968). These tissues were utilized for a histopathologic study reported elsewhere (Malone et al. 1975). Serial sections (8 u) were prepared (each 15th to 20th section) to enumerate worms included in the specimen that were not visible at the time of necropsy. Tissue sections were made so that three specimens were collected from each testicle: one specimen was taken through the anterior pole of the testicle and the head of the epididymis, one through the posterior pole and the tail of the epididymis, and one through the pampiniform plexus area. Lung was sectioned through the cardiac, diaphragmatic, and apical lobes. Central structures in the mediastinum were included. Other organs examined included the spleen, liver, pancreas, stomach, intestines, kidneys, seminal vesicles, urinary bladder, and scrotum. Lymphatic tissues collected included the subiliac, iliac, renal, popliteal, inguinal, and medistinal lymph nodes and associated lymphatic vessels and surrounding tissues. In addition, selected sections were taken of any other tissues having pathologic lesions at necropsy. Ten uninfected control hamsters were examined for both macroscopic and histopathologic lesions. Parasites were fixed in glacial acetic acid, placed in 70% ethanol containing 5% glycerin, and brought into pure glycerin by evaporation, Descriptions of B. puhangi (Schacher 1962) were consulted for identification of morphological characters of whole parasites and for worms found in tissue sections. RESULTS Parasitologic
findings
Subcutaneous infections. Worm recovery data were compiled on three separate groups of juvenile male hamsters following injection of 95-150 infective larvae into the inguinal region (Table I). The
231
percentage of worms rccovercd from eight animals necropsied 2345 days after inoculation was slightly higher than recoveries from 11 animals necropsied 105 days or more after inoculation ( 16 vs 14% ), The localization of worms differed in early and late infections. The heart and lungs and lymphatic vessels contained approximately equal numbers of worms in hamsters examined 39 or more days after inoculation, but only small numbers had reached the heart of three hamsters examined at 2324 days. Greater numbers of worms occurred in efferent or afferent vessels of regional lymph nodes in early infections than in later infections (Table II). Recovery percentages of worms from the lymphatic vessels of the testes, epididymis, and spermatic cord correspondingly increased with duration of infection. Low-level, evanescent microfilaremias, not exceeding 18 microfilariae in 20 mm3 of blood, were seen in 10 of 12 hamsters held for over 105 days (Table III). The average mean prepatent period was 90 days. One or more microfilariae were seen in three hamsters (Tl, T8, T9), which became negative in subsequent ocular blood samples and in heart blood taken at the time of necropsy. No adult worms were recovered from animal T9 despite relatively high microfilaria counts. In animal T8 mature microfilariae were present in the uterus of all four female worms recovered at necropsy at 187 days; three were from the heart and lungs, and one was from the genital lymphatics. A fifth female worm found in tissue sections of the testes parenchyma also contained microfilariae. Examination of worms from the third hamster (Tl) at 109 days revealed microfilariae in one worm from the heart and lungs and in one worm from the lymphatics. Three of the other four female worms recovered contained ova but no microfilariae. The remaining worm was small and did not contain recognizable ova or microfilariae. One hamster (T3)
2S%
MALONE
AND
THOMPSON
TABLE
1
Sunrbcr, Sex, and Distribution of Brugia pahangi Kccowrcd from Ziamslrw A jicr Subcutaneous Znjdion ruilh Znjwt ive Larvae Hamster No.~
Cl C2 Tl T2 T4 T-3 TG T7 T8 T9 TIO
Days to necrwp,y
105 106 109 110 141 146 747 181 187 188 195
Number
Itecovery
larvae injecte:!
(:a
97 9.5 110 103 110 103 110 109 110 110 106
Number,
sex, and location
Lymphatics
27.X i41.6 10.9 13, .i 7.:2 9.7 7.3 11.9 I:
