Acquired immunity in rats against Angiostrongylus cantonensis infection

Acquired immunity against Angiostrongylus cantonensis was induced by immunizing rats with somatic antigens from fifth-stage larvae and adult worms and live third-stage larvae. Rats immunized twice had significantly fewer worms than rats immunized three times. Fewer worms were recovered from rats immunized with 200 live third-stage larvae than from any other groups. Rats immunized with somatic antigens had higher enzyme-linked immunosorbent assay (ELISA) antibody levels than rats immunized with live larvae. Rats immunized with live third-stage larvae of Angiostrongylus cantonensis were more strongly protected against challenge infections (62-92%) than rats immunized with antigens extracted from fifth-stage larvae (0-30%) and adult worms (11-24%).     

Experimental infection routes of Angiostrongylus cantonensis in mice.

Stomach intubation is the most common method used in the experimental infection of animals with Angiostrongylus cantonensis. In order to compare the effectiveness of other possible transmission methods, groups of BALB/c mice were given infective third-stage larvae of A. cantonensis by different routes including intraperitoneal or subcutaneous injections, and penetration of anal mucosa, vaginal mucosa, conjunctival mucosa, lacerated skin, unabraded skin, foot pad and tail skin, while stomach intubation was used as control. Recovery of fifth-stage larvae was higher in mice inoculated with third-stage larvae subcutaneously. Successful infections were established through all experimental transmission routes except tail skin penetration. This study suggests that oral infection may not be the only route for the transmission of human angiostrongyliasis, and subcutaneous infection may be a better method for experimental infection.     

Effects of age and splenectomy on heavy infection of Angiostrongylus cantonensis in rats

This study was designed to determine the effects of age and the role of spleen in rats with heavy Angiostrongylus cantonensis infection. Young rats (8 weeks) infected with 100 larvae were found to have significantly higher worm recovery rate (75.0±6.6%) than the adult (6 months) (55.7±1.5%) and the aging ones (13 months) (57.6±4.0%). Moreover, the recovery rate in adult rats with 400 larvae (33.6±10.67%) was significantly lower than those with 100 larvae (55.7±1.53%) or 200 larvae (53.3±5.4%). The splenectomized young rats with 100 larvae had a significantly higher recovery rate (84.3±2.5%) than the intact (75.0±6.6%) or sham splenectomized ones (74.4±3.8%). Although titers of antibody against A. cantonensis increased with time, those against young adults were significantly higher before week 4 whereas those against adult worms become significantly higher since week 4. Titers in the splenectomized rats were also found to be significantly lower than those in the intact ones. These finding indicate that young rats are more susceptible to A. cantonensis. Crowding effect may occur in rats with heavy infections. The effects of splenectomy on the host are independent of the intensity of infection.     

Mechanisms of vaccine-induced protective immunity against Coxiella burnetii infection in BALB/c mice

To elucidate the mechanisms of vaccine-induced protective immunity against Coxiella burnetii infection, we compared the protective efficacy and immunogenicity between formalin-inactivated phase I vaccine (PI-V) and phase II vaccine (PII-V) in BALB/c mice. PI-V generated significant protection while PII-V did not confer measurable protection. Analysis of cytokine and subclass Ab responses indicated that both PI-V and PII-V were able to induce a Th1-dominant immune response but did not identify the component of host response that distinguished their ability to induce protective immunity. Interestingly, immunoblot analysis identified a difference between PI-V and PII-V vaccinates in antigenic recognition by specific Ab isotypes. The observation that PI-LPS elicited significant protection but PII-LPS did not confer measurable protection suggests PI-LPS may play a key role in PI-V-induced protection. Adoptive transfer of either immune sera or splenocytes mediated significant protection in naive BALB/c mice, supporting the notion that both humoral and cellular immunity are important for development of protective immunity. However, the evidence that immune sera and B cells were unable to control infection while T cells conferred significant protection in SCID mice supports the hypothesis that T cell-mediated immunity is critical for host defense against C. burnetii infection. This report presents novel evidence to highlight the importance of PI-LPS and Abs in protective immunity and has important implications for the design of new generation vaccines against Q fever.     

Larvicidal effect of flubendazole on Angiostrongylus cantonensis in mice with various worm burdens

Flubendazole given orally at 50 mg/kg to mice inoculated with 50, 250 or 500 larvae of Angiostrongylus cantonensis showed almost complete larvicidal effect, irrespective of the worm burden.     

Curcumin alleviates eosinophilic meningitis through reduction of eosinophil count following albendazole treatment against Angiostrongylus cantonensis in mice

Angiostrongylus cantonensis (A. cantonensis) is the most common cause of parasitic eosinophilic meningitis worldwide. By using an animal model of BALB/c mice infected with A. cantonensis, previous studies indicated that the anthelmintic drug, albendazole, could kill A. cantonensis larvae and prevent further infection. However, the dead larvae will induce severe immune responses targeting at brain tissues. To alleviate the detrimental effects caused by the dead larvae, we administered curcumin, a traditional anti-inflammatory agent, as a complementary treatment in addition to albendazole therapy, to determine whether curcumin could be beneficial for treatment. The results showed that although curcumin treatment alone did not reduce worm number, combined treatment by albendazole and curcumin helped to reduce eosinophil count in the cerebrospinal fluid, better than using albendazole alone. This alleviating effect did not affect albendazole treatment alone, since histological analysis showed similar worm eradication with or without addition of curcumin. Nevertheless, curcumin treatment alone and combined albendazole-curcumin treatment did not inhibit MMP-9 expression in the brain tissue. In conclusion, curcumin, when used as a complementary treatment to albendazole, could help to alleviate eosinophilic meningitis through suppression of eosinophil count in the cerebrospinal fluid.     

Endogenous IFN-gamma production is induced and required for protective immunity against pulmonary chlamydial infection in neonatal mice

Chlamydia trachomatis infection in neonates, not adults, has been associated with the development of chronic respiratory sequelae. Adult chlamydial infections induce Th1-type responses that subsequently clear the infection, whereas the neonatal immune milieu in general has been reported to be biased toward Th2-type responses. We examined the protective immune responses against intranasal Chlamydia muridarum challenge in 1-day-old C57BL/6 and BALB/c mice. Infected C57BL/6 pups displayed earlier chlamydial clearance (day 14) compared with BALB/c pups (day 21). However, challenged C57BL/6 pups exhibited prolonged deficits in body weight gain (days 12-30) compared with BALB/c pups (days 9-12), which correlated with continual pulmonary cellular infiltration. Both strains exhibited a robust Th1-type response, including elevated titers of serum antichlamydial IgG2a and IgG2b, not IgG1, and elevated levels of splenic C. muridarum-specific IFN-gamma, not IL-4, production. Additionally, elevated IFN-gamma, not IL-4 expression, was observed locally in the infected lungs of both mouse strains. The immune responses in C57BL/6 pups were significantly greater compared with BALB/c pups after chlamydial challenge. Importantly, infected mice deficient in IFN-gamma or IFN-gamma receptor demonstrated enhanced chlamydial dissemination, and 100% of animals died by 2 wk postchallenge. Collectively, these results indicate that neonatal pulmonary chlamydial infection induces a robust Th1-type response, with elevated pulmonary IFN-gamma production, and that endogenous IFN-gamma is important in protection against this infection. The enhanced IFN-gamma induction in the immature neonatal lung also may be relevant to the development of respiratory sequelae in adult life.     

Intermediate Hosts of Angiostrongylus cantonensis in Tenerife,Spain

The nematode Angiostrongylus cantonensis is the causative agent of human angiostrongyliasis, the main clinical manifestation of which is eosinophilic meningitis. Although this parasite has been found recently in its definitive rat host in Tenerife (Canary Islands, Spain), showing a widespread distribution over the north-east part of the island, there are no available data regarding which snail and/or slug species are acting as intermediate hosts on this island. Consequently, the objective of this work was to determine the possible role of three mollusc species, Plutonia lamarckii, Cornu aspersum and Theba pisana, as intermediate hosts of A. cantonensis in Tenerife. Between 2011 and 2014, 233 molluscs were collected from five biotopes where rats had been found previously to harbor either adult worms or antibodies against A. cantonensis, and the identification was carried out on the basis of morphological features and a LAMP technique. The prevalence of A. cantonensis larvae in the mollusc samples, based on morphological identification, was 19.3%, whereas 59 out of the 98 individuals (60.2%) analyzed by LAMP were positive. Positive results were obtained for the three mollusc species analyzed and two of the positive samples, both obtained from P. lamarckii, were confirmed as positive by 18S rRNA and ITS1 PCR. Sequence analysis of 18S rRNA PCR products showed 100% similarity with previously published A. cantonensis sequences. These results may be relevant from a public health point of view, since all the biotopes from which the samples were obtained were in inhabited areas or areas with human activity, but it is also important from the perspective of a possible transmission to other accidental hosts, such as dogs and horses, animals that are present in some of the areas analyzed.     

Novel recombinant parapoxvirus vectors induce protective humoral and cellular immunity against lethal herpesvirus challenge infection in mice

Orf virus (ORFV; Parapoxvirus ovis) was used to develop a novel vector system for the generation of effective and safe live vaccines. Based on the attenuated ORFV strain D1701-V, recombinants were produced that express the glycoproteins gC (D1701-VrVgC) or gD (D1701-VrVgD) of the alphaherpesvirus of swine, pseudorabies virus (PRV). Expression of gC and gD was also demonstrated on the surface of recombinant virus-infected murine cells that do not produce infectious ORFV. Single or combined immunization with the ORFV recombinants protected different mouse strains of a host species nonpermissive for ORFV against a fulminant, lethal PRV challenge infection equal to immunization with PRV live vaccine. Most notably, even a single immunization with D1701-VrVgC was protective, whereas two applications of D1701-VrVgD were required for immune protection. The higher protective capacity of D1701-VrVgC correlated with the induction of a strong specific humoral immune response. This suggestion was supported by transfer experiments using sera from recombinant-immunized mice, which resulted in partial gC but not gD antibody-mediated protection of the na?ve recipients. Remarkably, immunization of different immune-deficient mice demonstrated that the application of the PRV gC-expressing recombinant controlled the challenge infection in the absence of either CD4(+) or CD8(+) T cells, B cells, or an intact perforin pathway. In contrast, D1701-VrVgD-immunized mice lacking CD4(+) T cells exhibited reduced protection, whereas animals lacking CD8(+) T cells, B cells, or perforin resisted the challenge infection. The present study demonstrates the potential of these new vector vaccines to efficiently prime both protective humoral and cell-mediated immune mechanisms in a host species nonpermissive for the vector virus.     

Experimental double infection of Biomphalaria glabrata snails with Angiostrongylus cantonensis and Schistosoma mansoni

Two groups of Biomphalaria glabrata snails primarily infected with Angiostrongylus contonensis were secondarily exposed to infection with Schistosoma mansoni. To investigate any anatagonistic effect of the first infection on a superimposed one and to compare to singly and non-infected snails, a series of experiments was undertaken in which snails were individually exposed, variously, to 1,000 and 2,000 first-stage larvae of A. cantonensis and then to 5 and 10 miracidia of S. mansoni 1 day and 3 weeks later. Snails became infected with S. mansoni in both groups of snails with double infections and shed cercariae after the same incubation period as in the singly infected groups. The number of snails shedding cercariae simultaneously was similar in single and double infection groups during the first two weeks of shedding, after which this number decreased somewhat in doubly infected groups. Snails with double infection showed higher cumulative mortality rates than in snail groups with single infection with either A. cantonensis or S. mansoni. Therefore, initial infection of B. glabrata with A. cantonensis produced no inhibitory or retarding effect on subsequent infection of snails with S. mansoni.     

Polyclonal and specific antibodies mediate protective immunity against enteric helminth infection

Anti-helminth immunity involves CD4+ T cells, yet the precise effector mechanisms responsible for parasite killing or expulsion remain elusive. We now report an essential role for antibodies in mediating immunity against the enteric helminth Heligmosomoides polygyrus (Hp), a natural murine parasite that establishes chronic infection. Polyclonal IgG antibodies, present in naive mice and produced following Hp infection, functioned to limit egg production by adult parasites. Comparatively, affinity-matured parasite-specific IgG and IgA antibodies that developed only after multiple infections were required to prevent adult worm development. These data reveal complementary roles for polyclonal and affinity-matured parasite-specific antibodies in preventing enteric helminth infection by limiting parasite fecundity and providing immune protection against reinfection, respectively. We propose that parasite-induced polyclonal antibodies play a dual role, whereby the parasite is allowed to establish chronicity, while parasite load and spread are limited, likely reflecting the long coevolution of helminth parasites with their hosts.     

Immunoaffinity-isolated antigens induce protective immunity against larval Strongyloides stercoralis in mice

The objective of this study was to identify soluble protein antigens that would induce protective immunity against infective-stage larvae (L-3) of Strongyloides stercoralis in mice. Deoxycholate (DOC)-soluble proteins derived from L-3, adsorbed to aluminum hydroxide, induced protective immunity in BALB/c mice. The immunized mice generated parasite-specific IgG that could transfer passive immunity to na?ve animals. The protective antibodies bound to parasite antigens found in the muscles and nerve cords of the L-3. An IgG affinity chromatography column generated with IgG from the sera of DOC-immunized mice was used to purify specific larval antigens. Proteins were eluted from the affinity column with sizes of 80, 75, 61, 57, 43, and 32 kDa. This antigen pool stimulated both proliferation and IL-5 production by splenocytes recovered from mice immunized with live L-3. Vaccination of mice with the immunoaffinity-isolated antigens led to significant protective immunity, with 83% of challenge larvae killed. This study demonstrates that IgG-isolated proteins are candidate antigens for a vaccine against larval S. stercoralis.     

Angiostrongylus cantonensis: efficacy of albendazole-dexamethasone co-therapy against infection-induced plasminogen activators and eosinophilic meningitis

Angiostrongyliasis is one of the common causes leading to eosinophilic meningitis. Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) may play a role in the pathogenesis. Administration of steroid drugs has been reported to possibly relieve the symptoms of eosinophilic meningitis. This study evaluates the curative effects of albendazole-dexamethasone co-therapy on eosinophilic meningitis in BALB/c mice. Assay indicators for the therapeutic effect include worm recovery, histopathological score of meningitis, tPA, uPA, total protein, and leukocyte counts. The results show that the albendazole-dexamethasone co-therapy significantly decreased (P<0.05) these factors after treatment on day 5 post-inoculation (PI), in contrast to treatment on 15 PI. Thus, the timing of medication is important and is closely related to the anthelmintic efficacy of a drug. At the same dosage and days post-infection, the earlier administration shows better results. This study showed that albendazole-dexamethasone co-therapy is an effective approach for the treatment of parasitic meningitis.     

Angiostrongylus cantonensis: apoptosis of inflammatory cells induced by treatment with mebendazole or/and interleukin 12 in mice

Angiostrongylus cantonensis is the major cause of human eosinophilic meningoencephalitis. ICR mice were infected orally with 35 infective larvae and sacrificed at 4-14 days, 25 days or 32 days post infection (dpi) for pathological and immunocytochemical examinations. In the non-treated group, no apoptosis signal was found in the meninges or parenchyma of the brains (4-14 dpi). Only a few apoptotic cells were noticed at 25 dpi (3%) and 32 dpi (10%). In the groups, the animals were given a single dose of mebendazole (20 mg/kg, per os at various times) or injections of interleukin 12 (IL-12) (10 ng/daily, intraperitoneally), all the animals were sacrificed at 14 dpi; the number of apoptotic cells was increased (17-21%). In the group that received a single dose of mebendazole (4 dpi) in combination with IL-12 injections (4-13 dpi), mild meningitis was observed, and most of the infiltrated inflammatory cells were in the apoptotic program (55%). Taken together, apoptosis of the inflammatory cells (most were eosinophils) could be induced when the infected mice were treated with mebendazole or/and IL-12.     

Effects of spleen cells and serum on transfer of immunity to Strongyloides venezuelensis infection in hypothymic (nude) mice

The course of Strongyloides venezuelensis infection in congenitally hypothymic (nu/nu) mice and their heterozygous thymus-bearing littermates (nu/+) was followed. Unlike the infected nu/+ mice, the nu/nu mice were unable to expel the worms until the end of the observation period (98 days post-infection). In addition, about three times as many eggs were counted at the peak level of infection in faeces of the infected nu/nu mice in comparison with the nu/+ mice. No acquired resistance to rechallenge was observed among the nu/nu mice. Auto-reinfection within the infected nu/nu mice could not be supposed in the present study. The worm expulsion mechanism was generated by nu/nu mice which had been given syngeneic spleen cells from intact +/+ mice. The expulsion of adult worms, as well as the protection against migrating larvae, occurred anamnestically when spleen cells from immune +/+ mice were transferred. The serum transfer, however, only caused a retardation of larval migration. The results support the hypothesis that direct worm immunity and worm expulsion are a T cell-dependent phenomenon.     

Gamma delta T cells facilitate adaptive immunity against West Nile virus infection in mice

West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, and gammadelta T cells are involved in the protective immune response against viral challenge. We have now examined whether gammadelta T cells contribute to the development of adaptive immune responses that help control WN virus infection. Approximately 15% of TCRdelta(-/-) mice survived primary infection with WN virus compared with 80-85% of the wild-type mice. These mice were more susceptible to secondary challenge with WN virus than the wild-type mice that survived primary challenge with the virus. Depletion of gammadelta T cells in wild-type mice that survived the primary infection, however, does not affect host susceptibility during secondary challenge with WN virus. Furthermore, gammadelta T cells do not influence the development of Ab responses during primary and at the early stages of secondary infection with WN virus. Adoptive transfer of CD8(+) T cells from wild-type mice that survived primary infection with WN virus to naive mice afforded partial protection from lethal infection. In contrast, transfer of CD8(+) T cells from TCRdelta(-/-) mice that survived primary challenge with WN virus failed to alter infection in naive mice. This difference in survival correlated with the numeric and functional reduction of CD8 memory T cells in these mice. These data demonstrate that gammadelta T cells directly link innate and adaptive immunity during WN virus infection.     

Cellular and humoral immunity against vaccinia virus infection of mice

Despite the widespread use of vaccinia virus (VV) as a vector for other Ags and as the smallpox vaccine, there is little information available about the protective components of the immune response following VV infection. In this study, protection against wild-type VV was evaluated in mice with respect to the relative contributions of CD8(+) T cells vs that of CD4(+) T cells and Ab. C57BL/6 mice primed with the Western Reserve strain of VV mount significant IgM and IgG Ab responses, specific cytotoxic T cell responses, IFN-gamma responses in CD4(+) and CD8(+) T cells, and effectively clear the virus. This protection was abrogated by in vivo depletion of CD4(+) T cells or B cells in IgH(-/-) mice, but was not sensitive to CD8(+) T cell depletion alone. However, a role for CD8(+) T cells in primary protection was demonstrated in MHC class II(-/-) mice, where depleting CD8(+) T cells lead to increase severity of disease. Unlike control MHC class II(-/-) mice, the group depleted of CD8(+) T cells developed skin lesions on the tail and feet and had adrenal necrosis. Adoptive transfer experiments also show CD8(+) T cells can mediate protective memory. These results collectively show that both CD4(+) and CD8(+) T cell-mediated immunity can contribute to protection against VV infection. However, CD4(+) T cell-dependent anti-virus Ab production plays a more important role in clearing virus following acute infection, while in the absence of Ab, CD8(+) T cells can contribute to protection against disease.