Trichinella spiralis: Characterization and strain distribution of rapid expulsion in inbred mice

Appropriately immunized mice display a response that is biologically equivalent to rat rapid expulsion. Only two inbred strains ($\text{NFR}\text{N}\text{and}\text{NFS}\text{N}$ derived from NIH Swiss mice) have been shown to respond in this manner. Mice of the $\text{Balb}\text{c}$, CBA, $\text{A}\text{He}$, C₃H, SJL, or C₅₇Bl strains are “nonresponders” which require approximately twice as much intestinal exposure (in days) to Trichinella spiralis to elicit a response half as effective. Genetically, the responder is dominant, autosomal, and does not appear to be linked to the MHC. The characteristics of mouse and rat rapid expulsion of T. spiralis are not identical but share these features: initial rejection within 24 hr of challenge; a rejection efficiency >90%, from 1 to 5 weeks after the primary; induction of response does not require exposure to the complete infection; rapid expulsion is immunologically specific for preadults; adult worms are resistant. While a genetic basis for responsiveness exists in mice there is, as yet, no evidence for genetic control in rats. In both mice and rats, rapid expulsion is distinguished from the intestinal hyperreactivity associated with rejection of the primary infection by the kinetics and amplitude of the rejection of transplanted adult worms.     

Eimeria nieschulzi and Trichinella spiralis: Analysis of concurrent infection in the rat

The effects of concurrent primary infection of the rat with Eimeria nieschulzi and Trichinella spiralis on the number of oocysts of E. nieschulzi shed by the host and on the number, distribution, and fecundity of adult T. spiralis were analyzed. When rats were initially infected with E. nieschulzi followed 9 days later by infection with T. spiralis there occurred a significant decrease in the total numbers of adult worms in the small intestine, a significant shift in the position of these worms along the length of the small gut, a decrease in the fecundity of adult female worms, and a decrease in muscle parasitism when compared with rats infected with T. spiralis alone. When rats were initially infected with T. spiralis, followed 9 days later by infection with E. nieschulzi, there occurred a significant decrease in the numbers of oocysts shed over 24 hr on Days 7, 9, and 11 postinfection below that seen with rats infected only with Eimeria. These changes are discussed in terms of the enteropathophysiologic lesions and enteric inflammation known to occur during infections with these two parasites.     

Trichinella spiralis: expression of rapid expulsion in rats exposed to an abbreviated enteral infection.

Rats infected with Trichinella spiralis for the first week of the enteral infectious cycle displayed a strong rapid expulsion reaction during a challenge infection. The response was induced with equal facility in animals given low or high immunizing doses of infectious larvae (500 to 5000 larvae). Large challenge infections resulted in a 10–15% reduction in the efficiency of rejection as assessed 24 hr after challenge. Rats became primed to express rapid expulsion within the first week of primary infection whether the infection remained patent or not. However, maximum effectiveness was not realized until the second week after the initial infection. Once induced, the capacity to express rapid expulsion persisted for 6 weeks after the primary infection. Immunized hosts were capable of resisting two challenge infections spaced by periods of from 12 to 72 hr. This finding suggests that a mediator is not consumed by the initial response.     

Nippostrongylus brasiliensis: Effect of cyclophosphamide treatment on worm expulsion in rats

The precise immunological mechanisms associated with expulsion of the gastrointestinal nematode Nippostrongylus brasiliensis remain controversial. In order to investigate the effects of drug-induced immunosuppression on parasite burdens and expulsion, various regimens of cyclophosphamide were administered to parasitized Wistar rats. It was observed that both the number of worms established from an infective dose of 3000 larvae and the time of expulsion were markedly increased with higher doses of cyclophosphamide. Thus, at the highest sublethal level of treatment (100 mg/kg), 82% of the infective dose was recovered at Day 9 postinfection compared with 51% in nontreated controls. Furthermore, in such treated rats expulsion was delayed in 6 days beyond that of nontreated animals. As cyclophosphamide, at the levels used in the present study, is known to primarily effect B-cell function, the results support the view that antibody-mediated responses play an essential role in worm expulsion.     

Trichinella spiralis: Immunization of rats with an antigen fraction enriched for allergenicity

Trichinella spiralis whole muscle larval extract was fractionated by gel filtration and anion-exchange chromatography, and the protein fractions were assayed for allergenicity by a footpad-swelling test in mice; IgE antibody levels in rats immunized with the fractions were determined by passive cutaneous anaphylaxis test in rats. By these methods, an allergenic fraction from T. spiralis was isolated. The fraction, F1-b, was shown to be monodisperse by analysis with SDS-PAGE, IEP, and isoelectric focusing, indicating that it is a single protein moiety with a molecular weight of approximately 45,000 and a pI of approximately 5.1. The Schiff-periodate test showed Fl-b to be a glycoprotein. Rats immunized with Fl-b had significantly fewer intestinal worms than did nonimmunized controls at 24 hr and 7 days after oral challenge with T. spiralis larvae.     

Trichinella spiralis: Comparison with an Arctic isolate

The muscle phase of Trichinella spiralis and of Trichinella sp. isolated in the Arctic was compared in experimental and wild animals. Reproductive capacity indices (RCI) of the Trichinella sp. isolate were significantly lower in laboratory rodents but were similar to T. spiralis in wild rodents. Sprague-Dawley rats were the most refractory to the Trichinella sp. isolate of all laboratory rodents. Outbred strains of mice were more susceptible to both T. spiralis and the Trichinella sp. isolate than inbred strains of mice. T. spiralis muscle larvae survived longer in mice and the survival of both T. spiralis and the Trichinella sp. isolate larvae was higher in female mice. While single pair interbreeding experiments showed reproductive isolation between T. spiralis and the Trichinella sp. isolate, multiple pair and transplant breeding experiments showed reproductive compatibility. Male and female infective larvae of T. spiralis and the Trichinella sp. isolate differed morphometrically, but a convergence in size of worms was observed after prolonged passages of the parasites in mice. Passaging history of the isolate and host species was found to have a significant effect on Trichinella morphology. It is proposed that the Trichinella sp. isolate is a physiological variant of T. spiralis and not a distinct species.     

Trichinella spiralis: Delayed rejection in mice concurrently infected with Nematospiroides dubius

The immune response of mice to the nematode Trichinella spiral's was markedly altered when the infection was superimposed upon an existing infection with Nematospiroides dubius. The expulsion of a primary infection of T. spiralis was delayed in such mice, and the worms persisted for at least 4 weeks longer than they did in control mice. The degree to which expulsion was suppressed was related to the number of N. dubius present. It would appear that both adult and larval stages of N. dubius can exert a suppressive effect, since the expulsion of T. spiralis was affected within days of a super-imposed (i.e., larval) N. dubius infection. When adult N. dubius were removed from mice 4 days before infection with T. spiralis, the mice expelled the latter parasite within the normal time, indicating that recovery from the suppressive effects of concurrent infection occurred rapidly. Concurrent infection with N. dubius appeared to affect both the afferent and efferent arms of the immune response to T. spiralis, since sensitization by, and memory of, prior infection were impaired and the expression of acquired immunity was inferior to that of controls.     

Trichinella spiralis: Rapid,immunologically influenced reduction of intestinal,sodium-coupled sugar transport in the rat

Epithelium of isolated small intestinal segments were studied in Ussing-type chambers to detect physiological changes associated with rapid, immune rejection of Trichinella spiralis infective larvae. Electrophysiological parameters associated with Na⁺-coupled hexose transport were measured. Changes in transepithelial electrical potential difference (PD), resistance, and short circuit current (I_sc) due to the addition of actively absorbed β-methyl-d-glucoside (BMG) to the mucosal solution were determined. Measurements were made prior to and 30 min after primary and secondary infections. Animals were infected by intraduodenal inoculation. As the infective larval dose in primarily infected (nonimmunized) rats increased from 50 to 2000 larvae the magnitude of the rise in I_sc elicited by BMG decreased in a dose-dependent fashion, with 50 larvae per rat having no effect. In previously infected (immunized) rats challenged with a secondary inoculum, all doses, ranging from 50 to 2000 larvae per rat, decreased the BMG-stimulated change in I_sc by approximately 50%. The effect of 50 worms per rat in immunized hosts was equivalent to that produced by ~1600 worms in nonimmunized animals. Measurements of ¹⁴C-BMG mucosa-to-serosa flux confirmed that Na⁺-BMG cotransport was responsible for observed changes in I_sc. Results support the conclusion that changes in intestinal epithelial function are associated with larval challenge of immune rats.     

Trichinella spiralis: role of different life cycle phases in induction, maintenance, and expression of rapid expulsion in rats.

The capacity of different phases of the life cycle of Trichinella spiralis to induce rapid expulsion was examined. The phases examined included enteral preadults, enteral adults, and parenteral larvae. All had the ability to induce rapid expulsion although there were significant quantitative differences in their inductive capacity and in the kinetics of expression. Immunization with preadults required only a 48-hr enteral exposure to 2000 worms to induce strong rapid expulsion. In contrast rats required a 14-day exposure to adult worms to elicit a comparable response. After immunization with adults the reaction was demonstrable for only 2 weeks. Parenteral larvae produced only a weak rapid expulsion reaction by themselves and this response did not develop until some 8 weeks after challenge. When immunization with the enteral phases (preadult and adult) was combined with exposure to parenteral larvae a strong and enduring rapid expulsion reaction was observed. Phase specificity was also observed in the susceptibility of worms to the rapid expulsion response. The preadult phases, from infectious larvae to worms of up to 2 days of age were highly susceptible. Older worms, from 3 to 4 days old were not susceptible to rapid expulsion and could invade and establish themselves in the primed intestine for at least a 48-hr period without apparent adverse effects.     

Trichinella spiralis: Generation in the presence of rat serum of factors chemotactic for rat cells

Chemotaxis of rat peritoneal cells, of which the eosinophil was the predominant migratory cell type, toward incubates of Trichinella spiralis was studied using a modified Boyden chamber. Excysted muscle larvae, preadults, and adults were incubated in a buffered medium for 20 hr at 37 C. Worms were incubated alone or with serum or spleen cells, or both, from immune and nonimmune rats. Incubates of worm stages alone possessed no chemotactic activity as compared with incubation medium as a negative control and zymosan-activated serum as a positive control. Both normal and immune sera tested alone stimulated cell migration to the same degree. Incubates of spleen cells from either normal or immunized hosts did not show chemotactic activity. Chemotaxis caused by normal and immune sera were not altered by incubation with homologous spleen cells. Addition of larva, preadults, and adult worms to sera, however, enhanced chemotactic activity over sera alone. Chemotaxis caused by larvae plus immune sera was significantly greater than that stimulated by larvae plus normal sera. This difference decreased when preadults were substituted for larvae and was not observed when adult worms were used. Reversal of the chemical gradients showed that active cell migration caused by various incubates was due to Chemotaxis.     

Trypanosoma musculi and Trichinella spirails: Concomitant infections and selection for resistance genotypes in mice

Trypanosoma musculi infections were given to mice of different strains before, at the same time, and after an infection with 400 Trichinella spiralis. Examined parameters of the host response to T. spiralis were worm rejection, antifecundity responses, development of immunological memory, and muscle larvae burden. After dual infection, each mouse strain showed characteristic effects on resistance to T. spiralis. This was due to a dynamic interaction between the genes controlling rejection of T. spiralis and those influencing T. musculi growth. C3H mice develop high trypanosome parasitemias. This impairs worm expulsion and the development of memory to T. spiralis when Trypanosoma infections take place on the same day or 7 days before. The C57B1/6 mouse develops low parasitemias and T. musculi infections on the same day, or 7 days before T. spiralis, delaying worm rejection only slightly despite the overall weak capacity of B6 mice to expel worms. NFR-strain mice are strong responders to T. spiralis and also develop low parasitemias. Trypanosome infections on the same day, or after T. spiralis, produce a delay in worm rejection; the former is comparable to C3H mice. However, NFR mice alone showed enhanced rejection of worm when T. musculi infections preceded T. spiralis by 7 days. An unusual feature of C3H mice was that T. musculi infections 7 days before T. spiralis increased antifecundity responses at the same time that worm expulsion was inhibited. Trypanosome infections can therefore modulate distinct antihelminth immune responses in different directions simultaneously. The different outcomes of dual infections compared with single infections provides another selective mechanism by which genetic polymorphisms can be established and maintained in the vertebrate host.     

Strongyloides ratti: Dissociation of the rat's protective immunity into systemic and intestinal components

Analysis of the early stages of a challenge infection with Strongyloides ratti has shown that protection is expressed against the developing third-stage larval worms (L₃) and prevents the maturation to adulthood of most larvae. Challenge after an immunizing infection that was restricted to the parenteral L₃ migratory phase showed that some 10–40% of overall protection could be ascribed to systemic antilarval immunity. Some larvae were trapped in the skin at the site of injection whereas others failed to migrate to the head and lung of immune rats. Larvae arriving in the intestine at Days 3, 4, and 5 did not persist beyond Day 7 and 8. Studies using [⁷⁵Se]methionine-labeled L₃ showed a significant increase in fecal label in rats immunized by a complete infection. This loss did not occur to the same extent in rats immunized only with parenteral larvae. Significant rejection of worms transplanted to the intestine also indicated intestinal protection. The possible existence of large numbers of worms in a state of “arrested development” was excluded by their failure to appear after cortisone treatment and the absence of worm accumulation in radiolabeling studies. It is concluded that at least two responses operate against larval S. ratti, one is systemic and the other operates in the intestine against larvae in a manner that resembles the “rapid expulsion” rejection of Trichinella spiralis in immune rats.     

Nippostrongylus brasiliensis: Mast cell kinetics at small intestinal sites in infected rats

Mast cell kinetics during infection with the nematode Nippostrongylus brasiliensis were studied at various sites in the small bowel of rats and in heterotopically transplanted isografts of foetal small intestine placed under the kidney capsule. Infection produced an increase in the number of mast cells not only in the proximal jejunum, where most of the worms are located, but also in the distal ileum and in isografts of small intestine. However, globule leucocyte infiltration of the gut epithelium was confined to the proximal small intestine and did not occur in the distal ileum or isografts. These results show that the mast cell increase in the small bowel of N. brasiliensis-infected rats is a property of the whole organ, and is not restricted to sites of worm infection; but that in contrast, globule leucocyte infiltration of the epithelium is dependent upon the presence of worms within the bowel lumen.     

Trichinella spiralis: Alterations of blood chemistry in the mouse

Alterations in host blood chemistry during mouse trichinosis were studied. Mice infected with Trichinella spiralis showed depressions in serum glucose, serum pyruvate, serum total cholesterol, serum bilirubin, serum alkaline phosphatase, serum total proteins, serum total globulin, uric acid, albumin, and A/G ratio below levels found in uninfected animals. The concentrations of serum lactic dehydrogenase, serum glutamic-oxaloacetic transaminase, and serum creatine phosphokinase in infected mice were above that of uninfected mice. Hemoglobin, packed-cell volume (Hematocrit), creatinine, and calcium were at similar levels in infected and uninfected mice. These changes are discussed in terms of primary pathophysiologic lesions occurring in the gut and muscles of the trichinous host.     

Angiostrongylus cantonensis: Biogenic amines in the lungs of infected rats

Levels of histamine, serotonin, norepinephrine, and dopamine were estimated sequentially in rats parasitized by the lungworm, Angiostrongylus cantonensis, between 30 and 75 days postinfection. The highest level of histamine in the infected lungs was 52.19 μg/g wet wt tissue, 13 times higher than the level found in control rats. The level of serotonin rose from the normal level of 6.41 to 10.27 μg/g wet wt tissue after the worms had lodged in the pulmonary artery for 15 days. There were no changes in norephinephrine or dopamine. Studies of host cell response to infection revealed that the increased histamine and serotonin levels corresponded to a rise in the lung population of mast cells, suggesting that these cells produced the amines.     

Trichinella spiralis: genetic basis for differential expression of phase-specific intestinal immunity in inbred mice

Responsiveness of mouse strains after phase-specific immunization with Trichinella spiralis is compared. Two strains ($\text{NFR}\text{N, NFS/N}$) showed strong overall responsiveness. The response type could be characterized in phase-specific terms as: strongly anti-adult, weakly to moderately anti-preadult, and strongly antifecundity. By comparison, congenic mice of the C₅₇B1 $\text{10}\text{Sn}$ background (B10·A, B10·D₂, B10·S, B10·Q) displayed poor total responses that could be characterized as: weakly anti-adult, very weakly anti-preadult, weakly anti-fecundity after preadult immunization, and mixed (weak and strong) after adult immunization. The $\text{C}{}_3\text{H}\text{HeJ}$ mouse appeared to be intermediate between the B10·BR and the $\text{NFR}\text{N}$ strains in overall responsiveness. Genetic determinants of anti-preadult or anti-adult responses of $\text{NFR}\text{N}$ strain mice were dominant over their B10 congenic counterparts as shown in F₁, crosses of $\text{NFR}\text{N}$ × B1O·BR mice. Since the $\text{NFR}\text{N}$ (predominantly H-2^q) and the $\text{NFS}\text{N}$ (H-2^S) are both strong responders, while the B10·Q(H-2^q) and B10·S (H-2^S) are weak, it is suggested that the major genes controlling anti-preadult and anti-adult responses are not linked to the major histocompatibility complex. However, variations in anti-adult immunity and anti-fecundity in the B10 congenic mice (B10·Q and B10·S are the strongest responders) suggest that minor genes linked to the MHC exert some control over these responses. Some evidence was obtained for gene complementation as the F₁ cross of $\text{NFR}\text{N}$ and $\text{NFS}\text{N}$ mice responded more vigorously than the parental lines. We conclude that multiple genes determine anti-T. spiralis intestinal responses in mice. The major genes are unlinked to the major histocompatibility complex whereas several minor genes are linked.     

Trichinella spiralis: immunity and inflammation in the expulsion of transplanted adult worms from mice.

The technique of implanting adult Trichinella spiralis into the intestines of mice has been used to assess the contributions of direct, anti-worm immunity and of intestinal inflammation to worm expulsion. The survival after transfer of worms exposed to an effective adoptive immunity in donors was no different from that of worms taken from control donors. Worms taken from donors 8 days after infection, i.e., shortly before the onset of expulsion, showed no increased susceptibility to an immunity adoptively transferred to the recipient mice. When worms were implanted into mice responding to a prior, oral infection they were expelled rapidly. This expulsion was independent of the age of the worms transferred and took place at the same time as the expulsion of the existing infection.     

Trichinella spiralis: Proteins and antigens isolated from a large-particle fraction derived from the muscle larva

Proteins and antigens derived from a large-particle fraction of muscle larvae of Trichinella spiralis (i.e., the S₃ fraction) were characterized in terms of their molecular weights, isoelectric points, carbohydrate contents, electrophoretic mobilities, antigenicity, and their ability to induce protection in mice. Gel filtration on Sephacryl S-200 yielded 5 major peaks of material while electrophoresis in polyacrylamide gel with sodium dodecyl sulfate revealed a minimum of 28 proteins ranging in MW from 11,000 to 200,000. Analytical isoelectric focusing on acrylamide gel yielded 37 bands of protein, while the periodic acid-Schiff reaction performed on a similar gel revealed 22 glycoproteins. Most proteins were within a pI range of 4.0–7.0, while all of the glycoproteins had pI ranging from 4.0 to 6.5. Immunoelectrophoresis of the S₃ fraction using hyperimmune rabbit serum demonstrated a minimum of 19 precipitin arcs, while crossed immunoelectrophoresis yielded 16 peaks. These determinations were made on several batches of material isolated in the same fashion and gave the same results. Preparative isoelectric focusing yielded 30 fractions. These fractions were assayed for the presence of antigens, then pooled and tested for their ability to induce protection in mice against an oral challenge infection. Fused rocket immunoelectrophoresis of all 30 fractions revealed the presence of a minimum of 18 antigens with pI ranging from 4.0 to 9.0. The pooled fractions (i.e., 1–9; 10–20; 21–30) all protected mice against oral challenge infection, while fraction 5 (pI = 4.3) protected best.     

Nippostrongylus brasiliensis: Peripheral leukocyte responses and correlation of basophils with blood histamine concentration during infection in rats

Rats infected on Day 0 with 3000 infective L₃ larvae of Nippostrongylus brasiliensis, and uninfected controls, were monitored daily through Day 23 postinfection for changes in peripheral leukocytes and blood histamine concentrations. A generalized leukocytosis was observed between Days 7 and 18, the period leading up to and immediately following the time of expulsion of adult worms from the small intestine. The total number of lymphocytes was elevated between Days 11 and 17 post-infection; however, there was no change in the percentage of lymphocytes relative to other white blood cell types. The total number and percentage of monocytes were no different from controls, with the exception of Day 5 postinfection. On that day, there was a significant elevation in the number (614/mm³ blood in infected rats, as compared to 160/mm³ blood in controls) and relative proportion (2.7% of total leukocytes in infected animals, compared to 0.8% in controls) of monocytes, coinciding with the termination of the pulmonary migration of larvae. A period of moderate neutrophilia occurred between Days 7 and 12, but this was not accompanied by any changes in the proportion of neutrophils. A biphasic eosinophil response was observed. An early elevation of eosinophils occurred between Days 3 and 5, corresponding to the period of larval migration through the lungs. A second period of eosinophilia began on Day 11, when worm expulsion was beginning, and continued through Day 19, i.e., beyond the period of worm expulsion. Basophilia was observed as early as Day 6 after infection, rising to a peak on Day 13 (6.8% of total leukocytes in the infected animals, as compared to 0.5% in controls), and declining thereafter, but remaining above control levels until termination of the experiment on Day 23. The histamine content of blood samples, as determined by an enzymic-isotopic assay, closely paralleled the development and decline of basophilia; histamine levels also peaked on Day 13 postinfection (422.5 pg histamine/mm³ blood in infected rats, compared to 66.0 pg histamine/mm³ blood in controls). As basophilia progressed during the course of infection, there was a decline in the amount of histamine per basophil. In uninfected rats and during the first week after infection, basophils contained about 1.5–2.0 pg histamine per cell. In the third week of infection, there was about 0.6 pg histamine per basophil. The time course of the basophilia suggests that these cells may be involved in the expression of immunity to N. brasiliensis.     

Dirofilaria immitis: Parasite-specific humoral and cellular immune responses in experimentally infected dogs

Humoral and cellular immune responses to adult antigens of Dirofilaria immitis were evaluated in experimentally infected dogs during the chronic phase of infection. All infected dogs had significantly elevated IgG (enzyme-linked immunosorbent assay) and IgE (passive cutaneous anaphylaxis) titers against D. immitis adult antigens. However, there was little difference between infected dogs and uninfected controls in cellular-immune responses to D. immitis adult antigen or phytohemagglutinin as assessed by the lymphocyte transformation assay. Although neither cellular nor humoral responses correlated with worm burdens, cellular responses among infected dogs correlated inversely with IgG titers to D. immitis adult antigen. These results are consistent with observations in other nematode and trematode systems which suggest that in chronic tissue helminth infections there is suppression of cellular immune responses to parasite antigens while humoral responses to the same antigens remain relatively preserved.