PARASITOLOGICAL ASPECTS OF SCHZSTOSOMA HAEMATOBZUM (IRAN) INFECTION IN THE AMERICAN OPOSSUM (DZDELPHIS MA RSUPZALZS L.)” ROBERT E. KUNTZ, BETTY JUNE MYERS, JERRY A. MOORE and TAO-CHENG HUANG Parasitology,
Southwest
Foundation
for Research
and Education,
San Antonio,
Texas
78284,
U.S.A.
(Received 13 August 1973) Abstract-Kuwz
R. E., MYERS B. J., MOOREJ. A. and HUANC T. C. 1975. Parasitological aspects of Schistosoma haematohium (Iran) infection in the American opossum (Didelphis marsupialis L.). Internntional Journal/or Parasitology 5: 21-26. Parasitological studies on 25 American opossums (Didelphis mursupialis) exposed to 1000 cercariae each of S. haemutobium (Iran) have indicated a
host-parasite situation and general parasitological conditions which show this marsupial to be a satisfactory host for experimental schistosomiasis. The return of schistosomes in relation to cercarial exposure was not excessively high, but there were egg deposits in the major visceral organs. Varied macroscopic pathology due to egg deposits in different organs was demonstrated as well as a potential for urinary bladder involvement, including tumor formation. Even though the precise nature of tumor pathology is not yet known, availability of the host, a good host-parasite compatability, and a low death rate in infected hosts favor this mammal as a model for experimental schistosomiasis haematobia. INDEX KEY WORDS: Schistosomu haematobium; parasitological aspects of infection; tumor formation.
INTRODUCTION
Didelphis marsupialis;
American
opossum:
disease more or less parallel those observed in schistosomiasis in man, there is a distinct need for small mammals in which different biological parameters may be evaluated. Parasitological studies of S. haematobium infection in different species of animals have been somewhat limited (Gear, Davis & Pitchford, 1966; Kuntz & Malakatis, 1955a, b; Kuntz, 1961 ; Wright & Bennett, 1967a, 6). However, representatives of some of the major categories of American mammals have been investigated for their biomedical potentials in this laboratory (unpubl. data). The American opossum (Didelphis marsupialis), included in preliminary studies, showed an apparent unusual potential for bladder pathology 18-53 weeks post exposure to S. haematobium (Iran) (Kuntz, Myers & Cheever, I97 I a). Follow up research has focused on the overall host-parasitological aspects of S. haematobium infection in the opossum and constitutes the body of the present report.
THE SCHISTOSOMIASES have been recognized
as major diseases for several decades. Continuing surveillance and an analysis of epidemiological situations in endemic areas strongly suggest even greater spread of these diseases in the future. A general understanding of the basic biology of schistosomiasis has accrued as a result of extensive researches at the laboratory level, the majority of which have been concerned with Schistosoma mansoni and S. japonicum infections, since these parasites can be handled with relative efficiency under experimental conditions. Studies with S. haematobium have been somewhat limited due to difficulties encountered in maintenance of the cycle outside the endemic areas. Furthermore, this schistosome is characterized by a much narrower spectrum of infectivity for lower mammals, thus restricting to a certain extent, use of some commonly available animals as definitive hosts. Even though the nonhuman primates have shown great potential as subjects in which conditions of
MATERIALS
AND METHODS
Opossums (Didelphis marsupialis) captured near San Antonio, Texas were exposed to S. haemutobium (Iran) propagated in Bulks truncatus rohlj..i (Clessin) from West Africa. Cercariae pooled from a number of Bulks were counted on coverslips, then the coverslips with the cercariae suspended in several drops of water, were applied to the cleansed abdominal skin of anesthetized (phencyclidine plus pentabarbitol) hosts for 30 min. Approximately 40 young or young adult hosts were
* This investigation was supported by the United StatesJapan Cooperative Medical Science Program, administered by the National Institute of Allergy and Infectious Diseases, under grant 5 R AI-08207 and by grant I R 22 CA-13208, National Cancer Institute, National Institutes of Health, Department of Health, Education and Welfare. 21
22
RCIBERTE. KUNTZ cl ul.
I.J.P. VOL. 5.
1975
exposed to different numbers of cercariae, but the present study is directed primarily to a group of 25 opossums exposed to 1000 cercariae each. Opossums held in individual metal cages adapted well to confinement and to a balanced diet consisting of dry Purina Dog Chow. Excreta were collected from screen covered trays under cages and examined at irregular intervals. Since no schistosome eggs were found in urine and stool samples examined by sedimentation (Kuntz et al., 1971~). there were no egg count reports. At sacrifice, or upon death, viscera were removed from the body and examined separately as quickly as feasible. The venous system was perfused when necessary to enhance parasite recovery. Fresh crushes were made from samples of tissue taken at random from different organs to determine relative distribution, viability, and abundance of eggs. Most tissues were refrigerated until time of digestion for eggs. In some instances, however, the urogenital system was fixed (10% buffered formalin) in toto for preliminary examinations and photography. Refrigerated tissues were digested in 2.5% potassium hydroxide solution for 12-24 h at 40’C and fixed organs for 16-20 h at 56°C (Cheever, 1968). RESUL,TS Schistosome recovery and distribution in the different major organs for 25 opossums exposed to 1000 cercariae each of S. haemarobium (Iran) held for 12-53 weeks are given in Table I. Total body egg counts, distribution of eggs (percentage), and eggs per worm pair (EWP) for hosts examined by KOH digest technic are presented in Table 2, and the regional distribution of worms in the venous system and in different organs, based upon the total recovery of parasites from 2.5 hosts, is indicated in Fig. I. Even though there was a broad range of parasite returns (2.2-26.5 per cent), there is no apparent significant correlation with duration of infection. In an additional five hosts exposed to 100-400 cercariae each at time of weaning, and sacrificed at 14-l 8 weeks post-exposure, recoveries ranged from 2 to approximately 40 per cent. Parasites were scattered widely through the hosts, with slightly more than half residing within the liver. A few worms were found in the walls of the stomach and in the lungs, but a much greater number of paired parasites resided in the lieno-splenic vein, especially in the pancreas. Most of the schistosomes in the superior mesenteric venous circulation occurred in the smaller, secondary and tertiary branches of the main vessel rather than in the walls of the small intestine. Only a few occurred in the upper part of the large intestine but IO.9 per cent of all parasites resided in the walls or tissues of the terminal section of the large intestine. Paired S. haematobiunz occurred in veins associated with the urinary bladder and ureters in only two hosts, but accounted for approximately 5 per cent of the total worm count. Two of the 25 opossums exposed to 1000 parasites succumbed in the course of the study. Even though cercariae were pooled from a number
FIG;. I. Parasite (percentage) location based upon total number of parasites obtained at autopsy of 25 opossums 12-53 weeks post infection. of snails to enhance the opportunity for infections with a balanced sex ratio of schistosomes, 24 hosts harbored unmated males. Excess females occurred in two hosts. In five hosts exposed to cercariae shed from more than 100 snails, the unmated males ranged from 46 to 169, whereas in another six hosts in which cercarial suspensions consisted of parasites from only eight snails, the unmated males numbered from 6 to 97. Excess females and the majority of single males were removed from the portal veins and especially the liver. Most hosts in the present study were sacrificed or examined considerabLy after the time for schistosome maturation. Consequently, there was ample opportunity for egg deposition by the parasites in different organs of the body. There were broad ranges in the total body counts and in the numbers of eggs per worm pair (EWP), as well as in the number of eggs per gram (EPG) of tissue. There was a general tendency toward an increase in the numbers of eggs as duration of infection lengthened. Exceptions occurred, however. As an example, one host (O-58) at 53 weeks had a total of only 19,401 eggs total body count, in comparison to another host (O-21) in
S~~~s~oso~a~ioemutobium infection in Didelphis ma~~upialis
I.J.P. VOL.5. 1975
RECOVERIESFROM AMERICAN OPOSSUMS (Didelpkis marsupialb)
TABLE I-WORM
Duration (weeks)
0 21 0 65
2 Y
0 18
J
--
Major organ systems Total recovery Pancreas-. Spleen* Liver S. ht. L. ht. Genital Urinary? Actual Lungs --_I.-Prs d d Prs d” a” Prs ii 0” Prs (f S Prs d 8 Prs $ 3 Prs $ $3 Prs 6 6 Percentage
I2 14
0
0
0
0
18
0 0 00
019
Southwest
Foundation
for Research
and Education,
San Antonio,
Texas
78284,
U.S.A.
(Received 13 August 1973) Abstract-Kuwz
R. E., MYERS B. J., MOOREJ. A. and HUANC T. C. 1975. Parasitological aspects of Schistosoma haematohium (Iran) infection in the American opossum (Didelphis marsupialis L.). Internntional Journal/or Parasitology 5: 21-26. Parasitological studies on 25 American opossums (Didelphis mursupialis) exposed to 1000 cercariae each of S. haemutobium (Iran) have indicated a
host-parasite situation and general parasitological conditions which show this marsupial to be a satisfactory host for experimental schistosomiasis. The return of schistosomes in relation to cercarial exposure was not excessively high, but there were egg deposits in the major visceral organs. Varied macroscopic pathology due to egg deposits in different organs was demonstrated as well as a potential for urinary bladder involvement, including tumor formation. Even though the precise nature of tumor pathology is not yet known, availability of the host, a good host-parasite compatability, and a low death rate in infected hosts favor this mammal as a model for experimental schistosomiasis haematobia. INDEX KEY WORDS: Schistosomu haematobium; parasitological aspects of infection; tumor formation.
INTRODUCTION
Didelphis marsupialis;
American
opossum:
disease more or less parallel those observed in schistosomiasis in man, there is a distinct need for small mammals in which different biological parameters may be evaluated. Parasitological studies of S. haematobium infection in different species of animals have been somewhat limited (Gear, Davis & Pitchford, 1966; Kuntz & Malakatis, 1955a, b; Kuntz, 1961 ; Wright & Bennett, 1967a, 6). However, representatives of some of the major categories of American mammals have been investigated for their biomedical potentials in this laboratory (unpubl. data). The American opossum (Didelphis marsupialis), included in preliminary studies, showed an apparent unusual potential for bladder pathology 18-53 weeks post exposure to S. haematobium (Iran) (Kuntz, Myers & Cheever, I97 I a). Follow up research has focused on the overall host-parasitological aspects of S. haematobium infection in the opossum and constitutes the body of the present report.
THE SCHISTOSOMIASES have been recognized
as major diseases for several decades. Continuing surveillance and an analysis of epidemiological situations in endemic areas strongly suggest even greater spread of these diseases in the future. A general understanding of the basic biology of schistosomiasis has accrued as a result of extensive researches at the laboratory level, the majority of which have been concerned with Schistosoma mansoni and S. japonicum infections, since these parasites can be handled with relative efficiency under experimental conditions. Studies with S. haematobium have been somewhat limited due to difficulties encountered in maintenance of the cycle outside the endemic areas. Furthermore, this schistosome is characterized by a much narrower spectrum of infectivity for lower mammals, thus restricting to a certain extent, use of some commonly available animals as definitive hosts. Even though the nonhuman primates have shown great potential as subjects in which conditions of
MATERIALS
AND METHODS
Opossums (Didelphis marsupialis) captured near San Antonio, Texas were exposed to S. haemutobium (Iran) propagated in Bulks truncatus rohlj..i (Clessin) from West Africa. Cercariae pooled from a number of Bulks were counted on coverslips, then the coverslips with the cercariae suspended in several drops of water, were applied to the cleansed abdominal skin of anesthetized (phencyclidine plus pentabarbitol) hosts for 30 min. Approximately 40 young or young adult hosts were
* This investigation was supported by the United StatesJapan Cooperative Medical Science Program, administered by the National Institute of Allergy and Infectious Diseases, under grant 5 R AI-08207 and by grant I R 22 CA-13208, National Cancer Institute, National Institutes of Health, Department of Health, Education and Welfare. 21
22
RCIBERTE. KUNTZ cl ul.
I.J.P. VOL. 5.
1975
exposed to different numbers of cercariae, but the present study is directed primarily to a group of 25 opossums exposed to 1000 cercariae each. Opossums held in individual metal cages adapted well to confinement and to a balanced diet consisting of dry Purina Dog Chow. Excreta were collected from screen covered trays under cages and examined at irregular intervals. Since no schistosome eggs were found in urine and stool samples examined by sedimentation (Kuntz et al., 1971~). there were no egg count reports. At sacrifice, or upon death, viscera were removed from the body and examined separately as quickly as feasible. The venous system was perfused when necessary to enhance parasite recovery. Fresh crushes were made from samples of tissue taken at random from different organs to determine relative distribution, viability, and abundance of eggs. Most tissues were refrigerated until time of digestion for eggs. In some instances, however, the urogenital system was fixed (10% buffered formalin) in toto for preliminary examinations and photography. Refrigerated tissues were digested in 2.5% potassium hydroxide solution for 12-24 h at 40’C and fixed organs for 16-20 h at 56°C (Cheever, 1968). RESUL,TS Schistosome recovery and distribution in the different major organs for 25 opossums exposed to 1000 cercariae each of S. haemarobium (Iran) held for 12-53 weeks are given in Table I. Total body egg counts, distribution of eggs (percentage), and eggs per worm pair (EWP) for hosts examined by KOH digest technic are presented in Table 2, and the regional distribution of worms in the venous system and in different organs, based upon the total recovery of parasites from 2.5 hosts, is indicated in Fig. I. Even though there was a broad range of parasite returns (2.2-26.5 per cent), there is no apparent significant correlation with duration of infection. In an additional five hosts exposed to 100-400 cercariae each at time of weaning, and sacrificed at 14-l 8 weeks post-exposure, recoveries ranged from 2 to approximately 40 per cent. Parasites were scattered widely through the hosts, with slightly more than half residing within the liver. A few worms were found in the walls of the stomach and in the lungs, but a much greater number of paired parasites resided in the lieno-splenic vein, especially in the pancreas. Most of the schistosomes in the superior mesenteric venous circulation occurred in the smaller, secondary and tertiary branches of the main vessel rather than in the walls of the small intestine. Only a few occurred in the upper part of the large intestine but IO.9 per cent of all parasites resided in the walls or tissues of the terminal section of the large intestine. Paired S. haematobiunz occurred in veins associated with the urinary bladder and ureters in only two hosts, but accounted for approximately 5 per cent of the total worm count. Two of the 25 opossums exposed to 1000 parasites succumbed in the course of the study. Even though cercariae were pooled from a number
FIG;. I. Parasite (percentage) location based upon total number of parasites obtained at autopsy of 25 opossums 12-53 weeks post infection. of snails to enhance the opportunity for infections with a balanced sex ratio of schistosomes, 24 hosts harbored unmated males. Excess females occurred in two hosts. In five hosts exposed to cercariae shed from more than 100 snails, the unmated males ranged from 46 to 169, whereas in another six hosts in which cercarial suspensions consisted of parasites from only eight snails, the unmated males numbered from 6 to 97. Excess females and the majority of single males were removed from the portal veins and especially the liver. Most hosts in the present study were sacrificed or examined considerabLy after the time for schistosome maturation. Consequently, there was ample opportunity for egg deposition by the parasites in different organs of the body. There were broad ranges in the total body counts and in the numbers of eggs per worm pair (EWP), as well as in the number of eggs per gram (EPG) of tissue. There was a general tendency toward an increase in the numbers of eggs as duration of infection lengthened. Exceptions occurred, however. As an example, one host (O-58) at 53 weeks had a total of only 19,401 eggs total body count, in comparison to another host (O-21) in
S~~~s~oso~a~ioemutobium infection in Didelphis ma~~upialis
I.J.P. VOL.5. 1975
RECOVERIESFROM AMERICAN OPOSSUMS (Didelpkis marsupialb)
TABLE I-WORM
Duration (weeks)
0 21 0 65
2 Y
0 18
J
--
Major organ systems Total recovery Pancreas-. Spleen* Liver S. ht. L. ht. Genital Urinary? Actual Lungs --_I.-Prs d d Prs d” a” Prs ii 0” Prs (f S Prs d 8 Prs $ 3 Prs $ $3 Prs 6 6 Percentage
I2 14
0
0
0
0
18
0 0 00
019
