Superimposed visceral leishmanial infection aggravates response to Heligmosomoides polygyrus
Polyparasitism is the rule in all animal species, including humans, and has an important role in pathogenicity, diagnosis and control measures. Among them, co-infections by gastrointestinal helminths and protists are very prevalent under natural conditions but experimental infections are relatively scarce. Thus, despite the frequent association of visceral Leishmania infections and intestinal helminth parasitism the experimental co-infection has not been addressed. Heligmosomoides polygyrus, an intestinal nematode of mice, is related to other helminths causing important pathologies and is a model species for immunological studies. Mice are valuable experimental model for visceral leishmaniasis.
BALB/c mice infected with H. polygyrus (200 third-stage larvae, L3) were subsequently infected seven days later with Leishmania infantum (107 promastigotes) with the aim of determining the effect of the overinfection on the host response to the primary infection with the helminth.
Overinfection with the protist did not affect the establishment rate of the nematode but induced a higher fecal egg output. Helminth burdens in co-infected animals were significant at the end of the experiment. Early unspecific immune suppression induced by the nematode in mesenteric lymph nodes was not switched by L. infantum infection. Co-infection elicited a higher serum antibody (IgG1) response against the helminth.
Visceral leishmanial overinfection aggravated the early host response against primary infections with the intestinal helminth. This effect was evidenced by an increased longevity and higher production of non-protective antibodies.
KeywordsHeligmosomoides polygyrus Leishmania infantum Co-infections IgG1 IgG2a Lymphoproliferation ELISA BALB/c mice Helminth
enzyme-linked immunosorbent assay
eggs per gram
area under the curve
adult soluble extract
soluble leishmanial antigen
mesenteric lymph nodes
honestly significant difference
Gastrointestinal nematodiases are a major problem worldwide, both for human and animal health. Helminth infections are present in all species of wild and domestic animals, with different management practices and climates. Soil-transmitted helminthiases (STHs) are among the most common and persistent parasitic infections worldwide and it has been estimated that over three billion people are infected with one or more helminth species . In the veterinary arena these diseases have a strong impact on health status, productivity and therefore on the sustainability of animal production. Visceral leishmaniasis is the second most lethal parasitic disease for humans . Leishmania infantum zoonotic infection is found in the Mediterranean and Brazil where millions of dogs are naturally infected.
Multiparasite infections are the rule and the coexistence of more than one species in a host has significant effects on their pathogenicity, clinical course and design of control systems [3, 4]. Information is relatively scarce and only recently has a growing interest on the study of multiparasitism been observed [5, 6].
Heligmosomoides polygyrus (Nematoda: Strongylida), a natural parasite of mice intestines, is a species taxonomically related to those causing processes of relevance in humans (e.g. Necator, Ancylostoma) and in animal health and production (e.g. Ostertagia, Haemonchus). Primary exposure to H. polygyrus provokes a predominant Th2 response  accompanied by regulatory T cell (Treg) activation, thus failing to achieve an effective expulsion of the worms and causing chronic infections in most mouse strains [8, 9, 10]. The lack of protective response in primary infections is related to the immunosuppressive abilities of the nematode since an effective response appears when the primary infection is cleared (i.e. with drug treatment) . Immune interference between helminths and protozoans has been described and experimentally addressed in some combinations [12, 13, 14, 15, 16]. Cross-sectional studies have been carried out in human patients with cutaneous (caused by Leishmania braziliensis)  and visceral leishmaniasis  and helminth infections with conflicting results. However, the potential effect of a visceral Leishmania infection on previously infected hosts with intestinal helminths has apparently not been experimentally addressed. This lack of information is surprising, given the widespread distribution of helminths all over the world and the high prevalence of visceral leishmanial infections. Since H. polygyrus infections are a well established model of Th2 response in mice and L. infantum infection in mice elicits a mixed Th1/Th2 response [19, 20, 21], we administered an overinfection with L. infantum to BALB/c mice previously infected with H. polygyrus. Results obtained in this surrogate model showed that visceral leishmanial infection aggravated the primary response against the intestinal helminth.
Heligmosomoides polygyrus larvae were provided by M. Grueiro (Faculty of Pharmacy, UCM, Madrid, Spain) and the isolate was maintained in our laboratory by passage in susceptible mice every 6 months. The infective third-stage larvae (L3) were obtained by incubation of fecal material on filter paper disks placed on a Petri dish with distilled water at 22 °C for 7 days. The isolate of L. infantum (M/CAN/ES/97/10.445 zymodeme MON-1) was supplied by M. Domínguez (ISCIII, Madrid, Spain) and has been maintained as promastigotes by passage in RPMI 1640 medium (Lonza Group, Basel, Switzerland) at 26 °C supplemented with heat inactivated fetal bovine serum (30 min, 56 °C) (Sera Laboratories International, Horsted Keynes, UK) and 100 U/ml penicillin + 100 μg/ml streptomycin (BioWhittaker, Verviers, Belgium).
Mice, experimental design and follow-up
Female BALB/c mice (Harlan Laboratories Models SL, Barcelona, Spain) were housed in our facilities (No. ES280790000155) in polystyrene cages (4 animals per cage) at a controlled temperature of 22–25 °C with a 12 h light 12 h darkness cycle and received water and commercial rodent feed ad libitum. Mice were randomly allocated to four experimental groups of eight animals per group. G1 and G2 animals were infected with 200 L3 of H. polygyrus in 0.2 ml water, using a bucoesophagic catheter, on day 0 of the experiment. G1 and G3 animals were infected by intraperitoneal injection, on day 7 post-infection (pi), with 107 stationary promastigotes of L. infantum in 0.1 ml PBS. G4 was the uninfected control group.
Individual blood samples were obtained on day -1, 6, 14, 21, 28 and 35 pi with H. polygyrus by puncture of submandibular vein. The blood volume obtained was 50 μl/ mouse/sample day except for the days when animals were euthanatized (14 and 35 pi) when 150 μl were taken. Blood was allowed to clot and the sera preserved at -20 °C until used. Samples taken at the end point of the experiment were employed for ELISA determinations. Coproscopical analyses were performed every 3 days from the 9th day pi onwards. Mice were individually isolated for 30 min, their feces collected and egg counts performed by a modified flotation technique. The results were expressed as eggs per gram of feces (epg). Cumulative epg output was estimated using the trapezoidal method to determine areas under the curve (AUC) of the animals and groups. Mice were euthanatized (CO2 inhalation - isoflurane) on days 14 and 35 pi, 4 animals per group at each time point. Intestines, spleens and mesenteric lymph nodes were removed and employed for further determinations. Individual intestines were opened and placed in physiological saline solution, kept overnight at 37 °C and the worms were recovered and counted. Spleens were extracted under sterile conditions and weighed individually. To assess L. infantum infection, a spleen sample was employed to prepare smears, stained with May Grünwald Giemsa and microscopically examined.
Heligmosomoides polygyrus soluble extract (ASE) was obtained from adult helminths. Nematodes were cleaned with cold PBS containing protease inhibitors (Roche, Mannheim, Germany), subjected to 8 freeze-thaw cycles (-80 °C to room temperature), homogenized in a glass-in-glass homogenizer and centrifuged at 3000× g for 30 min at 4 °C. Soluble leishmanial antigen (SLA) was obtained by 10 freeze-thaw cycles of L. infantum promastigotes followed by centrifugation and recovery of the supernatant. The concentration of protein was measured  and the antigen extracts (SLA, ASE) stored at -80 °C until use.
Lymphocyte proliferation was determined using the tetrazolium salt (MTT) method  with some modifications. Mesenteric lymph nodes (MLN) and spleen were dissected individually and homogenized in medium RPMI 1640. The suspension was filtered (100 μm mesh), centrifuged at 300× g, 10 min, 4 °C and the pellet resuspended in medium with 10% fetal bovine serum, 100 U/ml of penicillin + 100 μg/ml streptomycin and 1% glutamine. Erythrocytes were lysed with red blood cells lysis buffer (0.15M NH4Cl + 10mM KHCO3 + 0.1M Na2EDTA, pH7.2). The viability was assessed with Trypan blue. Viable cells from MLN (2 × 106 cells/ml) and spleen (5 × 106 cells/ml) were seeded in 96-well flat bottomed plates (Corning, Corning, USA; 200 μl/well) and stimulated with Concanavalin A (GE Healthcare, Madrid, Spain; 5μg/ml), SLA and ASE (5 μg/ml for MLN; 10 μg/ml for spleen). The cells were incubated for 96 h at 37 °C in a humidified atmosphere of 5% CO2. Proliferation was determined by adding 50 μl MTT (5 mg/ml) (Sigma, St. Louis, USA) + 200 μl culture medium for 4 h at 37 °C. Absorbance was read at 570 nm. All determinations were performed in triplicate and negative controls were included. Results were expressed as stimulation index (SI): optical density (OD) of stimulated cell cultures/OD of unstimulated control wells. SI ≥ 1.5 were considered positive.
Enzyme linked immunosorbent assay (ELISA)
Serum specific antibody response (IgG1 and IgG2a) was determined by ELISA. Optimal assay conditions were determined in a checkerboard manner. Briefly, microtitre plates (Nunc, Roskilde, Denmark) were coated with 5 μg/ml ASE or SLA. Anti-mouse IgG1-HRP was from Nordic Immunology (Eindhoven, Netherlands) and HRP anti-IgG2a was from Southern Biotech (Birmingham, USA). Mice sera were diluted 1/800 and conjugate 1/2500 to determine IgG1 against ASE; 1/50 sera dilution and 1/1000 conjugate dilution to estimate anti-SLA response. IgG2a against ASE employed sera diluted 1/400 and 1/2000 dilution conjugate. Anti-Leishmania IgG2a was determined with 1/25 sera dilution and 1/2000 conjugate.
Intergroup differences were analyzed by t-test (adult helminthes, epg) or 2-way ANOVA (epg, spleen weight, lymphoproliferative response, ELISA results) followed by Tukey’s HSD test. Exact P-values, degrees of freedom (df) and analysis carried out are given using the APA format. The level of significance was set at P < 0.05. Figures were prepared with Graphpad Prysm 5.
Overinfection with L. infantum provokes higher fecal egg output of H. polygyrus-infected mice
Leishmania infantum overinfection does not affect establishment of H. polygyrus although it increases helminth survival
Spleen enlargement of co-infected mice relates to H. polygyrus infection
Early unspecific immune suppression elicited by H. polygyrus is not switched by L. infantum overinfection
Overinfection with L. infantum increased the antibody response to H. polygyrus
Under natural conditions, co-infections are the rule in all host species, including humans,  and they have a significant impact on disease course outcome, diagnosis and preventive measures . Helminth-protist interactions are very complex ranging from a lack of interference  to strong impairment of protective immunity . For the most part, some type of modulation/switching of immune response has been found in the co-infections so far explored. Direct comparison of the results is extremely difficult since the mechanisms involved and final outcome depend on the parasite species and also the schedule of infections (e.g. timing and doses administered). In our experiments, overinfection with L. infantum of previously infected mice with H. polygyrus strongly impaired the immune response against helminth infection. This interference was evidenced by a worsening of major parameters such as the apparent higher fertility of female worms in early patency and higher helminth burdens at the end of the experiment.
The number of helminths found at the beginning of the patency evidenced a high establishment rate (c.40%), in-line with previous observations in primary infections by H. polygyrus [24, 25]. It has been reported that a previous infection with Toxoplasma gondii led to a higher fecundity of H. polygyrus female worms 14 days pi . These results were comparable to those obtained by us with an unrelated protist species, L. infantum. Thus, a notable increase in fecal egg output was found in co-infected mice from day 15 to 21 pi without helminth number differences compared to the animals subjected to a single H. polygyrus infection (14 days pi). Moreover, we found at the end of the experiment (35 days pi) a significantly higher number of worms in co-infected mice. These findings are consistent with the partial abrogation of the Th2 response in H. polygyrus infections  and it has also been described in other co-infections with this helminth species [16, 26] irrespective of the experimental design. In H. polygyrus/Trypanosoma congolense co-infections, no differences were found in female helminth fertility [24, 27]. However, this experiment was not designed to determine the response in primary helminth infections but the acquired resistance after challenge. Our results suggest that the timing, order and protist species involved is apparently less relevant and in both cases immune response polarizes towards a Th1 type this resulting in both higher fecundity and longevity of helminths. The actual mechanistic basis of this interference is poorly known and deserves attention since it is much relevant within the framework of naturally infected hosts where the polyparasitism is the rule .
Single infection with L. infantum did not elicit significant changes in spleen weight in any of the experimental time points (1 week pi, 4 weeks pi). Intravenous infection of susceptible mice (e.g. BALB/c) with visceral Leishmania leads to self-limiting liver invasion and a progressive spleen parasitism. Thus, our results could be possibly related to the pi time elapsed . Mice infected with H. polygyrus alone or co-infected with L. infantum showed a notable increase in spleen weight, this being consistent with the immune response mounted against the nematode infection. It is noteworthy to indicate that co-infection with L. infantum actually caused an early reduction of spleen weights (14 days pi) compared to those seen in the mice with the helminth infection alone. This result supports the interference of the co-infection and the impairment of the characteristic Th2 protective response against the nematode [8, 29]. Poor anti-H. polygyrus specific lymphoproliferation in both lymph nodes and spleen was consistent with the unspecific immune suppression (ConA) elicited by nematode infection, particularly in the first parasitic stages (< 14 days pi). Conversely, the immune suppression in L. infantum-infected mice (G3) was seen in later stages (day 35 pi), perhaps related to the course of the infection . Visceral Leishmania infections cause a mixed Th1/Th2 response [19, 20, 21] and H. polygyrus is highly effective in blocking the protective Th2 immunity . In our experiment with primary helminth infections, high IgG1 anti-H. polygyrus levels were found after 35 days whereas no IgG2a was detected. This suggests a Th2 polarized response  and, in our experiment, L. infantum overinfection actually increased the antibody response against the helminth. A lack of correlation between IgG1 levels and protection (e.g. reduced parasite burden or fecal egg output) points towards the limited protective role of the antibodies detected by ELISA . Although not strictly comparable to our experimental approach, it has been observed that human patients co-infected with visceral Leishmania and helminths displayed no alteration of the course of leishmaniasis  whereas the clinical outcome of cutaneous infection by L. braziliensis was significantly influenced by helminth co-infection . It is possible that the initial clinical condition of patients, chemotherapeutic regimen, differential immune response against visceral and cutaneous Leishmania and the timing of infections (protist, helminth) could account for the variable response observed.
Considering all results together, our experiment confirmed the unspecific and specific immune suppression elicited by primary infection by H. polygyrus in mice. More importantly, overinfection with L. infantum of previously infected animals with the helminth aggravated the suppression, leading to higher parasite burdens, fecal egg output and longevity. Co-infection apparently polarized the immune response towards a non-protective Th1 type. However, no complete Th1/Th2 switch was found since co-infected mice developed a notable specific IgG1 response against Heligmosomoides. A lack of correlation between protection and IgG1 levels suggests the limited role played by antibodies in coping with helminth infection.
The authors thank the kind donation of H. polygyrus larvae by M. Grueiro (Faculty of Pharmacy, UCM) and L. infantum promastigotes culture by M. Domínguez (ISCIII, Madrid).
The authors thank the partial financial support by MINECO grant AGL2014-54049-R.
Availability of data and materials
All data corresponding to the results obtained in the experiments described are available from the authors.
MC and JMA conceived and designed the study. MC and MEGS performed the experiments. MC, MEGS and JMA discussed the results. JMA wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Experimental design and procedures were approved by the Ethical Committee (UCM) and Regional authorities (Comunidad de Madrid) (PROEX 169/15).
Consent for publication
The authors declare that they have no competing interests.
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