- Email: [email protected]
Comparison of the inflammatory responses of mice infected with American and Australian Trichinella pseudospiralis or Trichinella spiralis
ELSEVIER
International Journal for Parasitology 28 (1998) 343-348
RESEARCH NOTE
Comparison of the inflammatory responses of mice infected with American and Australian Trichinda pseudospiralis or Trichinella
spiralis
K. Alford,” D.L. Obendorf,b T.M. Fredeking,’ G.L. Stewart”*
E. Haehling” and
“Center for Parasitology, Box 19498, The University of Texas at Arlington, Arlington, TX 76019, U.S.A. ‘Animal Health Laboratory, Mt Pleasant Laboratories, P.O. Box 46, Kings Meadows, Tasmania 7249, Australitr ‘Antibody Systems, Inc., 1901 Norwood Drive, Hurst. TX 76OJ4, U.S.A.
Received 16 June 1997; accepted 14 October 1997
Abstract
This study wasdesignedto determineif the Tasmaniandevil isolateof Trichineflu pseudospirulis suppresseci inflammation asdoesthe original isolate.White adult worm numberswere similar in al.1groups,lower enteritis occurredin devil isolate-infectedmicecomparedwith miceinfectedwith the original isolateof T. pseudospiru&QT with Tricknelfu spirnlis. Diaphragm muscleinflammationwasgreater in T. spiraks-infectedthan in mice infectedwith either isolate of T. pseudospiralis or concurrently infected with T. spiralis and either of the isolatesof T. pseudospiralis. Granukxna inflammationwaslower in miceinfectedwith either isolateof T. pseudospiralis comparedwith uninfectedor 7: spiralisinfected mice. The devil isolate down-regulatedinflammation more profoundly during the intestinal phasethan the musclephasecomparedto the original isolate, differenceswhich may be related to the biology of their natural hosts. ((2 1998Australian Society for Parasitology.Publishedby ElsevierScienceLtd. Key words:
Trichinella
spiralis;
Trichinella
pseudospiralis;
Myeloperoxidase: Enteritis; Myositis; String granuloma; Tasmanian devil:
Mouse
Endoparasites of vertebrates display an arsenal of immunoevasive strategies. Members of the genus Trichinella establish a long-term and intimate relationship with their vertebrate host [l] and the host responds to infection with a vigorous inflammatory reaction against all stages in the life-cycle of this parasite. While the encapsulating species of this genus (e.g., Trichinella spiralis) induce the host
*Corresponding author. Tel: (817) 272-2423;Fax:(817) 2722855; e-mail: [email protected]
to form a collagenous capsule around the muscIe larva to protect it from inflammation, the muscle larvae of Trichinella pseudospiralis, which are not enclosed within a protective capsule, down-regulate host inflammatory capacity during the muscle and enteric phases of infection [2]. This parasite was originally recovered from a raccoon (Pxoc):on iotor; [3]), and was occasionally isolated from sever4 different species of carnivorous wiid birds [4]. The recent discovery of a sylvatic cycle for T. pseudospiralis in Tasmanian dasyurids is the first observation of a consistent natural focus for this parasite
SOO20-7519198 $19.00+0.00 $2 1998 Australian Society for Parasitology. Published by ElsevimerScience Ltd. Printed in Great Britain PII: s002075199700184-7
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and has demonstrated that it routinely infects an ancient group of marsupials [5], showing a high prevalence of infection among Tasmanian devils (Surcophifus havrisiz) and a lower rate of infection among other dasyurids. Here we have determined that this new isolate of unencapsulated Trichinelhz evades immunological destruction by host inflammatory response in a manner similar to that of the original isolate of T, pseudospiralis. Female S-lo-week-old ICR Swiss albino mice (Harlan SpragueDawley, Houston, TX, U.S.A.) were used in all experiments. Animals were maintained on a 12-h light-dark cycle and provided with food and water ad &turn. Trichinella spiralis has been maintained in ICR Swiss outbred mice since 1944 when it was isolated from a pig at Drake University in Iowa, U.S.A. Trichinella pseudospiralis (original raccoon isolate) was obtained in 1978 from Dr Dickson Despommier of Columbia University, New York, U.S.A. and has since been maintained in ICR Swiss outbred mice. The methods used for isolation of muscle larvae of all species of parasite and infection of hosts have been described previously [6]. The Tasmanian devil isolate of T. pseudospirulis was obtained from Tasmanian devil muscle tissue provided by one of the authors (Dr David Obendorf) and was recovered by methods used for isolation of the original isolate of T. pseudospiralis [6]. The Tasmanian devil isolate was passaged through ICR Swiss outbred mice six times before being used in the experiments described here. The pepsin-HCl digestion method described by Stewart et al. [6] was used in recovery of muscle larvae on day 40 p.i. for determining muscle larva burdens in the diaphragm muscles of infected mice. In mixed infections, the numbers of encapsulated and unencapsulated muscle larvae were determined among 200 larvae examined on a compound microscope (40 x ) in each of three freshly isolated diaphragm muscles sandwiched between two glass microscope slides. The percentage of encapsulated and unencapsulated worms was used to determine the number of larvae isolated from the diaphragm muscle which were T. spiralis or T. pseudospiralis. In infection with a single species of parasite, mice were given 500 infective larvae of either isolate of T. pseudospiralis or 250 infective larvae of T. spiralis per OS. Concurrently infected
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animals received 250 infective T. spiralis larvae and 500 infective larvae of the devil isolate or original isolate of T. pseudospiralis. For determination of total number of adult worms, mice were given 250 infective larvae orally, and on days 6, 9, 11 and 14 p.i. the small bowel was removed from each of at least four mice, split lengthwise and incubated at 37°C in 25 ml of PBS for 1 h. Following incubation, adult worms were counted at 10 x on a stereoscopic microscope. Inflammation was measured in diaphragm muscle, small intestine and around cotton string implants by determining myeloperoxidase activity (a marker enzyme for granulocytes) following the recovery and homogenisation of diaphragm muscle [7] or mucosa from the entire small intestine [8], or following removal and homogenisation of the entire granuloma surrounding cotton string implants [9]. Myeloperoxidase activity was assayed by a modification [lo] of the method of Maehly and Chance [ 111. Protein determinations were by the spectrophotometric Coomassie blue binding technique of Sedmak and Grossberg [12]. Enteritis was measured on days 6, 9, 11 and 14 p.i. in the small bowel of each of four or five mice for each experimental group. Diaphragm inflammation was assessed in each of four mice from each group on day 25 p.i. The total number of larvae of each species was determined for diaphragm muscles from each of five or six mice for each species of parasite in both single and concurrent infections. Because the numbers of muscle larvae becoming established differed among each of the three species of parasite tested, total mean myeloperoxidase activity (mg protein)-‘min-’ (diaphragm inflammation) was divided by the mean number of larvae recovered from this muscle to calculate specific myeloperoxidase units per larva. To measure the systemic, non-specific suppression accompanying infection with each species of parasite under study, a l-cm section of sterile 5-ply cotton string was implanted subcutaneously (s.c.) as described by Stewart et al. [9] in each of five uninfected mice and in five mice from each of the three infection groups on day 10 p.i. All strings and associated granulation tissue were removed 5 days following implantation and inflammatory response was measured in terms of myeloperoxidase activity by the methods outlined above. Data were analysed by analysis of vari-
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Fig. 1. Enteritis ((pm01 H,Or decomposed) min’(mg protein))‘) in the mucosa of the small bowel recovered from mice infected for 9, 11 or 14 days with TrichineNu spiralis (Ts), the original isolate of Trichinellupseudospiralis (Tp) or the devil isolate of T. p.wudospirnlis (TTp). Standard errors are shown above each bar.
ante, followed by multiple range analysis. Differences between means were considered significant at P < 0.05. Inflammation in the small bowel of mice infected with either of the three parasites tested was undetectable on day 6 p.i. (data not shown). On days 9, 11 and 14 pi. inflammation in the small bowel of mice infected with the devil isolate of T. pseudospiralis was significantly reduced compared with that seen in mice infected with either the original isolate of T. pseudospiralis or T. spiralis (Fig. 1). Similar numbers of adult worms were present in the small intestines of mice in all three groups on days 6 and 9 pi. (Table 1). By day 11 pi. similar numbers Table 1 Mean number of adult worms recovered on days 6, 9, 11 and 14 pi. from the small intestines of mice infected with either Trichinella spiralis, the original isolate of T. pseudospiralis or the devil isolate of Trichinella pseudospiralis Day post-infection
Ts” (S.E.)
Tp” (SE.)
TTp” (SE.)
Day 6 Day 9 Day 1I Day 14
105 (23)b 59 (3l)b 49 (13)d 10 (8)d
132 (35)b 98 (25)b 15 (6)“* 4 (6)’
141 (22) 65 (28) 38 (6)* 1 (I.2)d
“Ts = T. spiralis; Tp = T. pseudospiralis; of T. pseudospiralis. bn=5;
TTp = the devil isolate
“n= 10; *n=4.
*Differed significantly from the other two groups (P
of adult worms were recovered from mice infected with the devil isolate or with T. spiraiis, while the number of adult worms present in the bowel of mice infected with the original isolate of T. pseudospiraiis was significantly less than in the former two groups, By day 14 p.i., le,ss than 10 worms were found in the intestines of mice from any of the three infection groups. Inflammation in the diaphragm muscles of mice expressed as specific myeloperoxidase units per larva (Fig. 2) was dramatically reduced in muscle infected with the original isolate compared to that seen in muscle infected with the devil isolate. Compared to mice infected with T. spiralis alone (Fig. 2). diaphragm inflammation in mice infected concurrently with the devil isolate of T. pseudospiralis and T. spiraiis or the original isolate of T. pseudospiralisand T. spiralis was similar in the former group and reduced by 83% in the latter group. Mean total, muscle larvae of both species in concurrent infection, and mean total muscle larvae of T. pseudospirah and T. spiralis recovered from diaphragms of mice concurrently infected with 2: spiralis and the original isolate of T. pseudsopiralis were significantly greater than those recovered from mice infected with the devil isolate of 7’. pseuduspiralis and T. spiralis (Table 2). Inflammation measured in the granuloma forming around se. implanted cotton string was reduced in mice infected with the devil isolate of T. pseudospiralis by 51% and that of mice infected with the original
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800
700
1 T
EXPEWMENTK INFECTION
GAOUP
Fig. 2. Myositis presented as myeloperoxidase activity per larva ((nmol HZO, decomposed)(mg protein))‘min-‘larva-‘) in homogenates of diaphragm muscle recovered from mice infected with Trichinella spiralis (Ts), original isolate of Trichinella pseudospiralis (Tp), the devil isolate of T. pseudospiralis (TTp), or infected concurrently with T. spiralis and the original isolate of T. pseudospiralis (Tsf Tp), or with T. spiralis and the devil isolate of T. pseudospiralis (Ts+TTp). Standard errors are shown above each bar (n = 4).
Table 2 Mean total number of larvae per diaphragm muscle from mice concurrently infected with Trichinella spiralis and the original isolate of T. pseudospiralis or T. spiralis and the devil isolate of T. pseudospiralis
Ts+Tp Mean total muscle larvae of both species recovered Mean total muscle larvae of T. spiralis recovered Mean total muscle larvae of T. pseudospiralis
(S.E.pb Ts+TTp
1742 (307)
623 (143)*
1036 (182)
346 (98)*
706 (125)
277 (52)*
(S.E.)+
recovered
“Ts + Tp = T. spiralis and the original isolate of T. pseudospiralis; Ts +TTp = T. spiralis and the devil isolate of T. pseudospiralis. %=5.
*Differed significantly from Ts + Tp (P < 0.05).
isolate by 43% of that seen in mice infected with T. spiralis (Fig. 3). Based on differences between uninfected mice and infected mice in total myeloperoxidase activity around implanted string and the total number of muscle larvae recovered from
GROilP
Fig. 3. Granuloma inflammation, shown as myeloperoxidase activity ((pm01 H,Oz decomposed)min-‘(mg protein)-‘), around cotton string implanted S.C.in uninfected mice (UI), mice infected with Trichinellu spiralis (Ts), with the original isolate of Trichinella pseudospiralis (Tp), or with the devil isolate of T. pseudospiralis (TTp). Standard errors are shown above each bar (n=5).
mice (mean total larvae per mouse: 13 410 + 1926, IZ = 5 for devil isolate; 20 275 ) 2248, n = 5 for original isolate) it was possible to assessthe relationship between reduction in granuloma myeloperoxidase activity seen in infected mice and the number of muscle larvae present in the host. Accordingly, in mice infected with the original isolate of T. pseudospiralis, string granuloma inflammation was reduced below that seen in uninfected mice by 938 f 102 myeloperoxidase units for every 1000 larvae present in the host. A similar reduction was seen in mice infected with the devil isolate of T. pseudospirah (1006 ) 95 myeloperoxidase units per 1000 larvae). Suppression of host inflammatory response during the intestinal and muscle phases of infection with the original isolate of T. pseudospirulis is essential for the survival of adult worms and the unencapsulated muscle larvae, respectively [ 131. Suppression of enteritis in mice infected with the devil isolate of T. pseudospiralis was much greater than that seen in infection with the original isolate of the parasite. Identification of the molecule(s) and mechanism(s) by which the devil isolate suppresses inflammation in the enteric compartment would be of considerable value and interest in characterising the arsenal of immunoevasive strategies employed
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by this parasite and in evaluating this substance for application in the treatment of enteric autoinflammatory disorders. Although inflammation per larva in diaphragm muscles of mice infected with the devil isolate was less than half that seen in mice infected with T. spiralis, it was four times greater than inflammation per larva in muscles from mice infected with the original isolate of T. pseudospiralis. Significantly fewer muscle larvae in hosts infected with the devil isolate could reflect a lack of acclimation in this parasite to the murine host. On the other hand, this effect may be a function of the inverse relationship between levels of muscle inflammation and establishment of muscle larvae [7. 131. High levels of host plasma corticosterone are seen during infection with the original isolate of T. pseudospiralis and are the basis for reduced myositis seen in animals infected with this parasite [9, 131. If a similar mechanism is employed by the devil isolate to suppress myositis, it may be applied with greater caution since in Tasmania this parasite infects predominantly animals that feed on carrion, exposing them to a wide variety of pathogens that could easily kill an immunocompromised host. Compared with the original isolate, the devil isolate was less capable of down-regulating inflammatory reaction in host muscle. This is clearly seen in mice infected concurrently with the devil isolate of T. pseudospiralis and T. spiralis, where it was apparent that the devil isolate was unable to reduce the development of myositis in response to T. spiralis. Infection with T. spiralis has never been reported for wild or domestic animals in Australia [Sj. Therefore, it is unlikely that the devil isolate of T. pseudospiralis has had an opportunity to adapt to sharing a host with T. spiralis. Interspecific competition in the muscles or gut of the host between the devil isolate and T. spiralis or the reduced ability of the devil isolate to suppress myositis may be the basis for reductions in muscle larvae of both species of parasite in concurrently infected mice [7, 131. The devil isolate failed to reduce T. spiralis-induced infected animals. myositis in concurrently However, inflammation around cotton string implants in mice infected with the devil isolate was reduced to a level similar to that seen in mice infected with the original isolate of T. pseudospiraiis. Myositis in diaphragm muscles from mice
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.347
infected with the devil isolate is four times greater than that seen in mice infected with the original isolate of T. pseudospiralis and, as in infections with T. spiralis [7], might recruit inflammatory cetls into infected muscle and away from cotton string implants, accounting for a portion of the reduction seen in inflammation to implants in mice infected with the devil isolate of T. pseudospiralis. The original isolate of T. pseudospiralis occupies a wide geographic range [14] in which it has most commonly been recovered from carnivorous birds [4]. The migratory habits of this group of hosts and their ability to cross great distances unimpeded by natural barriers that would block quadrupeds is probably the basis for the wide geographic distribution of the original isolate of T. pseudospiralis. Since the tolerances for flight are narrower than those for mobility on land, it would be in the best interests of a parasite that primarily infects birds to avoid induction of high levels of myositis [7’j. This may be the basis for the more potent systemic anti-inflammatory effects seen during infection with the original isolate of this parasite. Acknowledgements--The authors acknowledge financial support of this project provided by Antibody Systems Inc., Bedford, TX. U.S.A.
References
Ill Despommier DD. Biology. In: Campbell WC. editor. inella
Trich-
and trichinosis. New York: Plenum Press. 1983;75 -
154.
PI Stewart CL. Biological and immunological characterisncs
of Trichinelia pseudospiralis. Parasitol Today 1989:5:344349. isolated from wild [31 Garkavi BL. Species of Trichinella animals. Veterinariya 1972; l&90-91 [41 Wheeldon EB, Dick TA, Shulz TA. First report of Trichinella spiralis var. pseudospiralis in North America. J Parasitol 1983;69:781-782. f51Obendorf DL, Handlinger JH, Mason RM, Clarke KP. Trichinella pseudospiralis infection in Tasmanian wildlife. Austral Vet J 1990:67:10X-110. PI Stewart CL, Kennedy RR, Larsen E. Alterations in the longevity and fecundity of adult TrichbzeUa pseudospiralis related to method of isolation of infective larvae .I Parasitol 1990;76:297-30 1. 171 Stewart CL, Wood B, Boley RB. Modulation of host response by Trichineila pseudospiralis. Parasite Immunol 1985;7:223--233.
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[8] Stewart GL, Kramar GW, Kramar M, Charniga L. The effects of cortisone on fecundity, number and distribution of adult Trichinella spiralis and on trichinous enteritis in the host. J Parasitol 1982;68:909-915, (91 Stewart GL, Niederkorn JY, Kennedy RR, Mayhew E. Effect of acute versus chronic Trichinella pseudospiralis infections on systemic cell-mediated immunity. Int J Parasitol 1991;21:935940. [lo] Smith JW, Castro GA. Relation of peroxidase activity in gut mucosa to inflammation. Am J Physiol 1978;234:R72R79. [ll] Maehly AC, Chance B. The assay of catalases and peroxidases. In: Glick D, editor. Methods of biochemical analysis. New York: Interscience, 1954;357408.
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[12] Sedmak JJ, Grossberg SF. A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G-250. Anal Biochem 1977;79:544-552. [13] Stewart GL, Mann MA, Ubelaker JE, McCarthy JL. Wood B. A role for elevated plasma corticosterone in modulation of host response by Trichinella pseudospiralis. Parasite Immunol 1988;10:139-150. [14] Pozio E, Shaikenov B, La Rosa G, Obendorf DL. Allozymic and biological characters of Trichinella pseudospiralis isolates form free-ranging animals. J Parasitol 1992;78:1087-1090.
International Journal for Parasitology 28 (1998) 343-348
RESEARCH NOTE
Comparison of the inflammatory responses of mice infected with American and Australian Trichinda pseudospiralis or Trichinella
spiralis
K. Alford,” D.L. Obendorf,b T.M. Fredeking,’ G.L. Stewart”*
E. Haehling” and
“Center for Parasitology, Box 19498, The University of Texas at Arlington, Arlington, TX 76019, U.S.A. ‘Animal Health Laboratory, Mt Pleasant Laboratories, P.O. Box 46, Kings Meadows, Tasmania 7249, Australitr ‘Antibody Systems, Inc., 1901 Norwood Drive, Hurst. TX 76OJ4, U.S.A.
Received 16 June 1997; accepted 14 October 1997
Abstract
This study wasdesignedto determineif the Tasmaniandevil isolateof Trichineflu pseudospirulis suppresseci inflammation asdoesthe original isolate.White adult worm numberswere similar in al.1groups,lower enteritis occurredin devil isolate-infectedmicecomparedwith miceinfectedwith the original isolateof T. pseudospiru&QT with Tricknelfu spirnlis. Diaphragm muscleinflammationwasgreater in T. spiraks-infectedthan in mice infectedwith either isolate of T. pseudospiralis or concurrently infected with T. spiralis and either of the isolatesof T. pseudospiralis. Granukxna inflammationwaslower in miceinfectedwith either isolateof T. pseudospiralis comparedwith uninfectedor 7: spiralisinfected mice. The devil isolate down-regulatedinflammation more profoundly during the intestinal phasethan the musclephasecomparedto the original isolate, differenceswhich may be related to the biology of their natural hosts. ((2 1998Australian Society for Parasitology.Publishedby ElsevierScienceLtd. Key words:
Trichinella
spiralis;
Trichinella
pseudospiralis;
Myeloperoxidase: Enteritis; Myositis; String granuloma; Tasmanian devil:
Mouse
Endoparasites of vertebrates display an arsenal of immunoevasive strategies. Members of the genus Trichinella establish a long-term and intimate relationship with their vertebrate host [l] and the host responds to infection with a vigorous inflammatory reaction against all stages in the life-cycle of this parasite. While the encapsulating species of this genus (e.g., Trichinella spiralis) induce the host
*Corresponding author. Tel: (817) 272-2423;Fax:(817) 2722855; e-mail: [email protected]
to form a collagenous capsule around the muscIe larva to protect it from inflammation, the muscle larvae of Trichinella pseudospiralis, which are not enclosed within a protective capsule, down-regulate host inflammatory capacity during the muscle and enteric phases of infection [2]. This parasite was originally recovered from a raccoon (Pxoc):on iotor; [3]), and was occasionally isolated from sever4 different species of carnivorous wiid birds [4]. The recent discovery of a sylvatic cycle for T. pseudospiralis in Tasmanian dasyurids is the first observation of a consistent natural focus for this parasite
SOO20-7519198 $19.00+0.00 $2 1998 Australian Society for Parasitology. Published by ElsevimerScience Ltd. Printed in Great Britain PII: s002075199700184-7
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et al. / International
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and has demonstrated that it routinely infects an ancient group of marsupials [5], showing a high prevalence of infection among Tasmanian devils (Surcophifus havrisiz) and a lower rate of infection among other dasyurids. Here we have determined that this new isolate of unencapsulated Trichinelhz evades immunological destruction by host inflammatory response in a manner similar to that of the original isolate of T, pseudospiralis. Female S-lo-week-old ICR Swiss albino mice (Harlan SpragueDawley, Houston, TX, U.S.A.) were used in all experiments. Animals were maintained on a 12-h light-dark cycle and provided with food and water ad &turn. Trichinella spiralis has been maintained in ICR Swiss outbred mice since 1944 when it was isolated from a pig at Drake University in Iowa, U.S.A. Trichinella pseudospiralis (original raccoon isolate) was obtained in 1978 from Dr Dickson Despommier of Columbia University, New York, U.S.A. and has since been maintained in ICR Swiss outbred mice. The methods used for isolation of muscle larvae of all species of parasite and infection of hosts have been described previously [6]. The Tasmanian devil isolate of T. pseudospirulis was obtained from Tasmanian devil muscle tissue provided by one of the authors (Dr David Obendorf) and was recovered by methods used for isolation of the original isolate of T. pseudospiralis [6]. The Tasmanian devil isolate was passaged through ICR Swiss outbred mice six times before being used in the experiments described here. The pepsin-HCl digestion method described by Stewart et al. [6] was used in recovery of muscle larvae on day 40 p.i. for determining muscle larva burdens in the diaphragm muscles of infected mice. In mixed infections, the numbers of encapsulated and unencapsulated muscle larvae were determined among 200 larvae examined on a compound microscope (40 x ) in each of three freshly isolated diaphragm muscles sandwiched between two glass microscope slides. The percentage of encapsulated and unencapsulated worms was used to determine the number of larvae isolated from the diaphragm muscle which were T. spiralis or T. pseudospiralis. In infection with a single species of parasite, mice were given 500 infective larvae of either isolate of T. pseudospiralis or 250 infective larvae of T. spiralis per OS. Concurrently infected
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animals received 250 infective T. spiralis larvae and 500 infective larvae of the devil isolate or original isolate of T. pseudospiralis. For determination of total number of adult worms, mice were given 250 infective larvae orally, and on days 6, 9, 11 and 14 p.i. the small bowel was removed from each of at least four mice, split lengthwise and incubated at 37°C in 25 ml of PBS for 1 h. Following incubation, adult worms were counted at 10 x on a stereoscopic microscope. Inflammation was measured in diaphragm muscle, small intestine and around cotton string implants by determining myeloperoxidase activity (a marker enzyme for granulocytes) following the recovery and homogenisation of diaphragm muscle [7] or mucosa from the entire small intestine [8], or following removal and homogenisation of the entire granuloma surrounding cotton string implants [9]. Myeloperoxidase activity was assayed by a modification [lo] of the method of Maehly and Chance [ 111. Protein determinations were by the spectrophotometric Coomassie blue binding technique of Sedmak and Grossberg [12]. Enteritis was measured on days 6, 9, 11 and 14 p.i. in the small bowel of each of four or five mice for each experimental group. Diaphragm inflammation was assessed in each of four mice from each group on day 25 p.i. The total number of larvae of each species was determined for diaphragm muscles from each of five or six mice for each species of parasite in both single and concurrent infections. Because the numbers of muscle larvae becoming established differed among each of the three species of parasite tested, total mean myeloperoxidase activity (mg protein)-‘min-’ (diaphragm inflammation) was divided by the mean number of larvae recovered from this muscle to calculate specific myeloperoxidase units per larva. To measure the systemic, non-specific suppression accompanying infection with each species of parasite under study, a l-cm section of sterile 5-ply cotton string was implanted subcutaneously (s.c.) as described by Stewart et al. [9] in each of five uninfected mice and in five mice from each of the three infection groups on day 10 p.i. All strings and associated granulation tissue were removed 5 days following implantation and inflammatory response was measured in terms of myeloperoxidase activity by the methods outlined above. Data were analysed by analysis of vari-
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Fig. 1. Enteritis ((pm01 H,Or decomposed) min’(mg protein))‘) in the mucosa of the small bowel recovered from mice infected for 9, 11 or 14 days with TrichineNu spiralis (Ts), the original isolate of Trichinellupseudospiralis (Tp) or the devil isolate of T. p.wudospirnlis (TTp). Standard errors are shown above each bar.
ante, followed by multiple range analysis. Differences between means were considered significant at P < 0.05. Inflammation in the small bowel of mice infected with either of the three parasites tested was undetectable on day 6 p.i. (data not shown). On days 9, 11 and 14 pi. inflammation in the small bowel of mice infected with the devil isolate of T. pseudospiralis was significantly reduced compared with that seen in mice infected with either the original isolate of T. pseudospiralis or T. spiralis (Fig. 1). Similar numbers of adult worms were present in the small intestines of mice in all three groups on days 6 and 9 pi. (Table 1). By day 11 pi. similar numbers Table 1 Mean number of adult worms recovered on days 6, 9, 11 and 14 pi. from the small intestines of mice infected with either Trichinella spiralis, the original isolate of T. pseudospiralis or the devil isolate of Trichinella pseudospiralis Day post-infection
Ts” (S.E.)
Tp” (SE.)
TTp” (SE.)
Day 6 Day 9 Day 1I Day 14
105 (23)b 59 (3l)b 49 (13)d 10 (8)d
132 (35)b 98 (25)b 15 (6)“* 4 (6)’
141 (22) 65 (28) 38 (6)* 1 (I.2)d
“Ts = T. spiralis; Tp = T. pseudospiralis; of T. pseudospiralis. bn=5;
TTp = the devil isolate
“n= 10; *n=4.
*Differed significantly from the other two groups (P
of adult worms were recovered from mice infected with the devil isolate or with T. spiraiis, while the number of adult worms present in the bowel of mice infected with the original isolate of T. pseudospiraiis was significantly less than in the former two groups, By day 14 p.i., le,ss than 10 worms were found in the intestines of mice from any of the three infection groups. Inflammation in the diaphragm muscles of mice expressed as specific myeloperoxidase units per larva (Fig. 2) was dramatically reduced in muscle infected with the original isolate compared to that seen in muscle infected with the devil isolate. Compared to mice infected with T. spiralis alone (Fig. 2). diaphragm inflammation in mice infected concurrently with the devil isolate of T. pseudospiralis and T. spiraiis or the original isolate of T. pseudospiralisand T. spiralis was similar in the former group and reduced by 83% in the latter group. Mean total, muscle larvae of both species in concurrent infection, and mean total muscle larvae of T. pseudospirah and T. spiralis recovered from diaphragms of mice concurrently infected with 2: spiralis and the original isolate of T. pseudsopiralis were significantly greater than those recovered from mice infected with the devil isolate of 7’. pseuduspiralis and T. spiralis (Table 2). Inflammation measured in the granuloma forming around se. implanted cotton string was reduced in mice infected with the devil isolate of T. pseudospiralis by 51% and that of mice infected with the original
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800
700
1 T
EXPEWMENTK INFECTION
GAOUP
Fig. 2. Myositis presented as myeloperoxidase activity per larva ((nmol HZO, decomposed)(mg protein))‘min-‘larva-‘) in homogenates of diaphragm muscle recovered from mice infected with Trichinella spiralis (Ts), original isolate of Trichinella pseudospiralis (Tp), the devil isolate of T. pseudospiralis (TTp), or infected concurrently with T. spiralis and the original isolate of T. pseudospiralis (Tsf Tp), or with T. spiralis and the devil isolate of T. pseudospiralis (Ts+TTp). Standard errors are shown above each bar (n = 4).
Table 2 Mean total number of larvae per diaphragm muscle from mice concurrently infected with Trichinella spiralis and the original isolate of T. pseudospiralis or T. spiralis and the devil isolate of T. pseudospiralis
Ts+Tp Mean total muscle larvae of both species recovered Mean total muscle larvae of T. spiralis recovered Mean total muscle larvae of T. pseudospiralis
(S.E.pb Ts+TTp
1742 (307)
623 (143)*
1036 (182)
346 (98)*
706 (125)
277 (52)*
(S.E.)+
recovered
“Ts + Tp = T. spiralis and the original isolate of T. pseudospiralis; Ts +TTp = T. spiralis and the devil isolate of T. pseudospiralis. %=5.
*Differed significantly from Ts + Tp (P < 0.05).
isolate by 43% of that seen in mice infected with T. spiralis (Fig. 3). Based on differences between uninfected mice and infected mice in total myeloperoxidase activity around implanted string and the total number of muscle larvae recovered from
GROilP
Fig. 3. Granuloma inflammation, shown as myeloperoxidase activity ((pm01 H,Oz decomposed)min-‘(mg protein)-‘), around cotton string implanted S.C.in uninfected mice (UI), mice infected with Trichinellu spiralis (Ts), with the original isolate of Trichinella pseudospiralis (Tp), or with the devil isolate of T. pseudospiralis (TTp). Standard errors are shown above each bar (n=5).
mice (mean total larvae per mouse: 13 410 + 1926, IZ = 5 for devil isolate; 20 275 ) 2248, n = 5 for original isolate) it was possible to assessthe relationship between reduction in granuloma myeloperoxidase activity seen in infected mice and the number of muscle larvae present in the host. Accordingly, in mice infected with the original isolate of T. pseudospiralis, string granuloma inflammation was reduced below that seen in uninfected mice by 938 f 102 myeloperoxidase units for every 1000 larvae present in the host. A similar reduction was seen in mice infected with the devil isolate of T. pseudospirah (1006 ) 95 myeloperoxidase units per 1000 larvae). Suppression of host inflammatory response during the intestinal and muscle phases of infection with the original isolate of T. pseudospirulis is essential for the survival of adult worms and the unencapsulated muscle larvae, respectively [ 131. Suppression of enteritis in mice infected with the devil isolate of T. pseudospiralis was much greater than that seen in infection with the original isolate of the parasite. Identification of the molecule(s) and mechanism(s) by which the devil isolate suppresses inflammation in the enteric compartment would be of considerable value and interest in characterising the arsenal of immunoevasive strategies employed
K. Alford
et al. / International
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by this parasite and in evaluating this substance for application in the treatment of enteric autoinflammatory disorders. Although inflammation per larva in diaphragm muscles of mice infected with the devil isolate was less than half that seen in mice infected with T. spiralis, it was four times greater than inflammation per larva in muscles from mice infected with the original isolate of T. pseudospiralis. Significantly fewer muscle larvae in hosts infected with the devil isolate could reflect a lack of acclimation in this parasite to the murine host. On the other hand, this effect may be a function of the inverse relationship between levels of muscle inflammation and establishment of muscle larvae [7. 131. High levels of host plasma corticosterone are seen during infection with the original isolate of T. pseudospiralis and are the basis for reduced myositis seen in animals infected with this parasite [9, 131. If a similar mechanism is employed by the devil isolate to suppress myositis, it may be applied with greater caution since in Tasmania this parasite infects predominantly animals that feed on carrion, exposing them to a wide variety of pathogens that could easily kill an immunocompromised host. Compared with the original isolate, the devil isolate was less capable of down-regulating inflammatory reaction in host muscle. This is clearly seen in mice infected concurrently with the devil isolate of T. pseudospiralis and T. spiralis, where it was apparent that the devil isolate was unable to reduce the development of myositis in response to T. spiralis. Infection with T. spiralis has never been reported for wild or domestic animals in Australia [Sj. Therefore, it is unlikely that the devil isolate of T. pseudospiralis has had an opportunity to adapt to sharing a host with T. spiralis. Interspecific competition in the muscles or gut of the host between the devil isolate and T. spiralis or the reduced ability of the devil isolate to suppress myositis may be the basis for reductions in muscle larvae of both species of parasite in concurrently infected mice [7, 131. The devil isolate failed to reduce T. spiralis-induced infected animals. myositis in concurrently However, inflammation around cotton string implants in mice infected with the devil isolate was reduced to a level similar to that seen in mice infected with the original isolate of T. pseudospiraiis. Myositis in diaphragm muscles from mice
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infected with the devil isolate is four times greater than that seen in mice infected with the original isolate of T. pseudospiralis and, as in infections with T. spiralis [7], might recruit inflammatory cetls into infected muscle and away from cotton string implants, accounting for a portion of the reduction seen in inflammation to implants in mice infected with the devil isolate of T. pseudospiralis. The original isolate of T. pseudospiralis occupies a wide geographic range [14] in which it has most commonly been recovered from carnivorous birds [4]. The migratory habits of this group of hosts and their ability to cross great distances unimpeded by natural barriers that would block quadrupeds is probably the basis for the wide geographic distribution of the original isolate of T. pseudospiralis. Since the tolerances for flight are narrower than those for mobility on land, it would be in the best interests of a parasite that primarily infects birds to avoid induction of high levels of myositis [7’j. This may be the basis for the more potent systemic anti-inflammatory effects seen during infection with the original isolate of this parasite. Acknowledgements--The authors acknowledge financial support of this project provided by Antibody Systems Inc., Bedford, TX. U.S.A.
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