Trypanosoma cruzi: effects of anti-thymocyte serum in mice and neonatal thymectomy in rats

CF₁ mice were given eight injections of normal rabbit serum (NRS), Hanks' balanced salt solution (HBSS), or rabbit anti-mouse thymocyte serum (ATS) beginning 3 days prior to and at 3-day intervals subsequent to intraperitoneal (ip) inoculation with 5 × 10⁴ trypomastigotes of a Brazil strain of Trypanosoma cruzi. Markedly enhanced parasitemia, increased numbers of tissue stages (amastigotes), and higher mortality occurred in ATS-treated mice as compared to NRS- or HBSS-treated controls. Administration of three injections of ATS at 3-day intervals during the latter stages of acute Chagas' disease, i.e., when numbers of parasites were declining, resulted in a transitory relapse (increase in numbers) of blood and tissue parasites. No relapse occurred in mice when ATS was administered at 3-day intervals over a period of 15 days during the subacute stage of the disease, i.e., after parasites had disappeared from the blood.Parasitemia and mortality were enhanced in neonatally thymectomized rats when compared to that observed in sham-operated and unoperated control rats following ip injection of 2 × 10⁵ trypomastigotes of T. cruzi. Serum obtained from thymectomized and control rats 5 weeks after inoculation with T. cruzi at a time when the blood of all animals had become microscopically negative for parasites were equally protective in passive transfer experiments, while serum from uninfected controls gave no protection.Gamma globulin levels significantly increased in thymectomized as well as intact rats by the third to fourth week of infection with T. cruzi, reached maximum concentrations in 5–6 wk, and remained elevated significantly at the twelfth week post infection as compared with uninfected controls. No significant changes occurred in total serum proteins or α and β fractions of any group, infected or uninfected.Total circulating leukocytes, especially lymphocytes, were diminished in mice and rats subjected to treatment with ATS or neonatal thymectomy.These data clearly indicate that neonatal thymectomy of rats and ATS treatment of mice suppress the acquired immune response to T. cruzi. Further, passive transfer experiments in rats confirm the protective role of circulating antibody in acquired immunity to Chagas' disease.     

Vaccination with Plasmodium knowlesi AMA1 formulated in the novel adjuvant co-vaccine HT™ protects against blood-stage challenge in rhesus macaques

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading blood stage vaccine candidate. Plasmodium knowlesi AMA1 (PkAMA1) was produced and purified using similar methodology as for clinical grade PfAMA1 yielding a pure, conformational intact protein. Combined with the adjuvant CoVaccine HT™, PkAMA1 was found to be highly immunogenic in rabbits and the efficacy of the PkAMA1 was subsequently tested in a rhesus macaque blood-stage challenge model. Six rhesus monkeys were vaccinated with PkAMA1 and a control group of 6 were vaccinated with PfAMA1. A total of 50 µg AMA1 was administered intramuscularly three times at 4 week intervals. One of six rhesus monkeys vaccinated with PkAMA1 was able to control parasitaemia, upon blood stage challenge with P. knowlesi H-strain. Four out of the remaining five showed a delay in parasite onset that correlated with functional antibody titres. In the PfAMA1 vaccinated control group, five out of six animals had to be treated with antimalarials 8 days after challenge; one animal did not become patent during the challenge period. Following a rest period, animals were boosted and challenged again. Four of the six rhesus monkeys vaccinated with PkAMA1 were able to control the parasitaemia, one had a delayed onset of parasitaemia and one animal was not protected, while all control animals required treatment. To confirm that the control of parasitaemia was AMA1-related, animals were allowed to recover, boosted and re-challenged with P. knowlesi Nuri strain. All control animals had to be treated with antimalarials by day 8, while five out of six PkAMA1 vaccinated animals were able to control parasitaemia. This study shows that: i) Yeast-expressed PkAMA1 can protect against blood stage challenge; ii) Functional antibody levels as measured by GIA correlated inversely with the day of onset and iii) GIA IC₅₀ values correlated with estimated in vivo growth rates.     

The immunological response of CBA mice to Trypanosoma musculi: mechanisms of protective immunity

CBA mice which had recovered from infection with Trypanosoma musculi were immune to challenge with all strains of the homologous species that were tested but were still full susceptible to challenge with T. cruzi, T. brucei or T. evansi. A heavy challenge inoculum of T. musculi was cleared rapidly from the blood of mice which had recently recovered from infection but, in mice which had recovered 11 months earlier, the parasitaemia changed very little for 3–4 days but then fell abruptly within a few hours. Immunization with a parasite extract in multiple emulsion conferred a strong though not complete protection against homologous challenge.Serum from mice which had recovered from infection had a marked neutralizing effect in vitro on the infectivity of the homologous parasites although the numbers of live organisms were not reduced during the period of in vitro incubation. The test did not reveal antigenic differences among three isolates of the parasite.A summary is given of the sequence of events that is thought to make up the immune response of mice to T. musculi.     

In vitro studies of the rabbit immune system: III. T-cell requirement for initiation but not maintenance of anti-sheep erythrocyte plaque-forming cells

The in vitro anti-sheep erythrocyte plaque-forming-cell (anti-SRBC PFC) response of primed rabbit spleen cells can be abrogated by prior treatment with anti-thymocyte serum plus complement (ATS + C). The direct addition of ATS without C to stimulated cultures also reduces the response 60–90%, if the antiserum is added before the initiation of antigen-stimulated PFC production. However, the PFC response becomes refractory to ATS inhibition by 48 hr. This loss of ATS sensitivity is not caused by T cells themselves becoming refractory to ATS for the following reasons: (i) ATS + C treatment or ATS addition at 48 hr causes cytotoxic effects comparable to similar treatments at 0 hr; (ii) The responsiveness of cells incubated 48 hr before the addition of SRBC remains sensitive to ATS inhibition at that time. The loss of ATS sensitivity is also not the result of soluble T-cell factors which have accumulated in the medium, because the number of PFC in stimulated cultures continues to increase after treating with ATS + C and reculturing in fresh medium at 48 hr. We conclude that T cells are required to initiate the production of antibody by B cells but are not required to maintain the later proliferative events.     

The kinetics of local cytokine and galectin expression after challenge infection with the gastrointestinal nematode, Haemonchus contortus

Gastrointestinal nematode parasites undergo several developmental stages within their mammalian host, each presenting different antigenic challenges to the immune system. To examine the expression of different immune mediators over time, biopsy samples were collected from the cannulated abomasum (true stomach) of immune sheep at several times after a challenge infection with Haemonchus contortus L3s. IL-5 and IL-13 mRNA expression levels were significantly increased above saline-challenged control levels at 5 and 7 days post challenge, while IL-4 showed an earlier peak at day 2 post challenge. IL-5, IL-13 and IL-4, as well as IFN-γ mRNA levels, peaked at 7 days before decreasing to non-significant levels at 28 days post challenge. TNF-α followed a similar profile while there was a slight increase in TGF-β in both control and challenged sheep. There was a significant increase in galectin-14 mRNA in the L3 challenged compared with the saline challenged group at 7 days while both galectin-11 protein and mRNA levels increased significantly by day 3 post challenge, peaking at 5-7 days post challenge. Distinct correlations were observed between these immune parameters at different times after L3 challenge. The galectin-14 protein level at day 2 post challenge was the only measured mediator significantly negatively correlated with worm burden. These studies highlight the dynamic nature of the immune response during parasite infection and the need to consider the different life cycle stages involved.     

A new role for an old antimicrobial: lysozyme c-1 can function to protect malaria parasites in Anopheles mosquitoes

Background

Plasmodium requires an obligatory life stage in its mosquito host. The parasites encounter a number of insults while journeying through this host and have developed mechanisms to avoid host defenses. Lysozymes are a family of important antimicrobial immune effectors produced by mosquitoes in response to microbial challenge.

Methodology/Principal Findings

A mosquito lysozyme was identified as a protective agonist for Plasmodium. Immunohistochemical analyses demonstrated that Anopheles gambiae lysozyme c-1 binds to oocysts of Plasmodium berghei and Plasmodium falciparum at 2 and 5 days after infection. Similar results were observed with Anopheles stephensi and P. falciparum, suggesting wide occurrence of this phenomenon across parasite and vector species. Lysozyme c-1 did not bind to cultured ookinetes nor did recombinant lysozyme c-1 affect ookinete viability. dsRNA-mediated silencing of LYSC-1 in Anopheles gambiae significantly reduced the intensity and the prevalence of Plasmodium berghei infection. We conclude that this host antibacterial protein directly interacts with and facilitates development of Plasmodium oocysts within the mosquito.

Conclusions/Significance

This work identifies mosquito lysozyme c-1 as a positive mediator of Plasmodium development as its reduction reduces parasite load in the mosquito host. These findings improve our understanding of parasite development and provide a novel target to interrupt parasite transmission to human hosts.     

Participation of three lymphoid cell types in the in vitro activation of cell-mediated immunity to a syngeneic gross virus-induced lymphoma in rats.

W/Fu rats inoculated with the syngeneic Gross-virus induced lymphoma, (C58NT)D, had transient lymphocyte-mediated specific cytotoxicity against the tumor cells at 7–15 days after tumor injection. Spleen cells 40 days after immunization (spleen 40) were unreactive by a direct 4-hr ⁵¹Cr release assay, but activity appeared after in vitro culture of these spleen eclls by themselves for 18–24 hr. The nature of the cells involved in the activation of cytotoxicity and the characteristics of the effector cells themselves were studied. Significant differences were seen in the cell types involved in the activation phase and the effector phase. Activation appeared to require the cooperation of three cell types. Induction of activity was lost by treatment of cells with ATS plus complement, by passage over an EAC-column, or by treatment with carbonyl iron. Thus, T cells, CRL and macrophages were necessary for full activation of cytotoxicity in spleen 40. In contrast, after activation, only CRL seemed to be required for cytotoxicity, and treatment wih ATS or carbonyl iron had little effect. The effector cell detected after in vitro activation was quite distinct from that seen in the direct cytotoxicity assay with spleen cells at 10 days after tumor cell inoculation. The early, direct cytotoxic reactivity was dependent on T cells, being eliminated by treatment with ATS and complement but not by EAC columns or carbonyl iron. It appears therefore that the in vitro activation is a separate mechanism for cytotoxicity against tumor cells, rather than a simple recovery of T cells from in vivo inhibition.     

Control of IgE and IgG1 antibody production in mice.

The production of IgE and IgG1 was studied in untreated, thymectomized. splenectomized, anti-thymocyte serum-treated, or sublethally X-irradiated mice. Dinitrophenyl Ascaris and ovalbumin were used as antigens, and aluminum hydroxide was used as adjuvant. A suppression of IgE production was observed in adult thymectomized mice, although the kinetic pattern of the antibody response was the same as in control animals. IgG1 antibody production was not affected by thymectomy. Splenectomy did not change either IgE or IgG1 production. A single dose of rabbit anti-thymocyte serum (ATS) given 8 days after immunization inhibited IgE antibody production. The effect of ATS was dose dependent and also varied with the amount of antigen used, the immune response to high doses being more susceptible to the effect of ATS. No alteration in IgG1 production was caused by ATS even when IgE antibody formation was completely inhibited. When preceding immunization, sublethal irradiation enhanced IgE antibody formation and partially suppressed IgG1 production; applied after immunization, irradiation caused an enhancement of IgE production which was inversely proportional to the interval elapsed between the two procedures. On the other hand, the IgG1 antibody production was fairly resistant to the same treatment. The results suggest a clearcut separation between the mechanisms regulating IgE and IgG1 production in mice.     

Structural analysis of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) intracellular domain reveals a conserved interaction epitope

Plasmodium falciparum-infected red blood cells adhere to endothelial cells, thereby obstructing the microvasculature. Erythrocyte adherence is directly associated with severe malaria and increased disease lethality, and it is mediated by the PfEMP1 family. PfEMP1 clustering in knob-like protrusions on the erythrocyte membrane is critical for cytoadherence, however the molecular mechanisms behind this system remain elusive. Here, we show that the intracellular domains of the PfEMP1 family (ATS) share a unique molecular architecture, which comprises a minimal folded core and extensive flexible elements. A conserved flexible segment at the ATS center is minimally restrained by the folded core. Yeast-two-hybrid data and a novel sequence analysis method suggest that this central segment contains a conserved protein interaction epitope. Interestingly, ATS in solution fails to bind the parasite knob-associated histidine-rich protein (KAHRP), an essential cytoadherence component. Instead, we demonstrate that ATS associates with PFI1780w, a member of the Plasmodium helical interspersed sub-telomeric (PHIST) family. PHIST domains are widespread in exported parasite proteins, however this is the first specific molecular function assigned to any variant of this family. We propose that PHIST domains facilitate protein interactions, and that the conserved ATS epitope may be targeted to disrupt the parasite cytoadherence system.     

S. mansoni Bolsters Anti-Viral Immunity in the Murine Respiratory Tract

The human intestinal parasite Schistosoma mansoni causes a chronic disease, schistosomiasis or bilharzia. According to the current literature, the parasite induces vigorous immune responses that are controlled by Th2 helper cells at the expense of Th1 helper cells. The latter cell type is, however, indispensable for anti-viral immune responses. Remarkably, there is no reliable literature among 230 million patients worldwide describing defective anti-viral immune responses in the upper respiratory tract, for instance against influenza A virus or against respiratory syncitial virus (RSV). We therefore re-examined the immune response to a human isolate of S. mansoni and challenged mice in the chronic phase of schistosomiasis with influenza A virus, or with pneumonia virus of mice (PVM), a mouse virus to model RSV infections. We found that mice with chronic schistosomiasis had significant, systemic immune responses induced by Th1, Th2, and Th17 helper cells. High serum levels of TNF-α, IFN-γ, IL-5, IL-13, IL-2, IL-17, and GM-CSF were found after mating and oviposition. The lungs of diseased mice showed low-grade inflammation, with goblet cell hyperplasia and excessive mucus secretion, which was alleviated by treatment with an anti-TNF-α agent (Etanercept). Mice with chronic schistosomiasis were to a relative, but significant extent protected from a secondary viral respiratory challenge. The protection correlated with the onset of oviposition and TNF-α-mediated goblet cell hyperplasia and mucus secretion, suggesting that these mechanisms are involved in enhanced immune protection to respiratory viruses during chronic murine schistosomiasis. Indeed, also in a model of allergic airway inflammation mice were protected from a viral respiratory challenge with PVM.     

Activation of the P2X7 receptor triggers the elimination of Toxoplasma gondii tachyzoites from infected macrophages

Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii, which is widespread throughout the world. After active penetration, the parasite is enclosed within a parasitophorous vacuole and survives in the host cell by avoiding, among other mechanisms, lysosomal degradation. A large number of studies have demonstrated the importance of ATP signalling via the P2X₇ receptor, as a component of the inflammatory response against intracellular pathogens. Here we evaluate the effects of extracellular ATP on T. gondii infection of macrophages. ATP treatment inhibits the parasite load and the appearance of large vacuoles in the cytoplasm of intracellular parasites. ROS and NO assays showed that only ROS production is involved with the ATP effects. Immunofluorescence showed colocalization of Lamp1 and SAG1 only after ATP treatment, suggesting the formation of phagolysosomes. The involvement of P2X₇ receptors in T. gondii clearance was confirmed by the use of P2X₇ agonists and antagonists, and by infecting macrophages from P2X₇ receptor-deficient mice. We conclude that parasite elimination might occur following P2X₇ signalling and that novel therapies against intracellular pathogens could take advantage of activation of purinergic signalling.     

MAP Kinase Phosphatase-2 Plays a Key Role in the Control of Infection with Toxoplasma gondii by Modulating iNOS and Arginase-1 Activities in Mice

The dual specific phosphatase, MAP kinase phosphatase-2 (MKP-2) has recently been demonstrated to negatively regulate macrophage arginase-1 expression, while at the same time to positively regulate iNOS expression. Consequently, MKP-2 is likely to play a significant role in the host interplay with intracellular pathogens. Here we demonstrate that MKP-2^−/− mice on the C57BL/6 background have enhanced susceptibility compared with wild-type counterparts following infection with type-2 strains of Toxoplasma gondii as measured by increased parasite multiplication during acute infection, increased mortality from day 12 post-infection onwards and increased parasite burdens in the brain, day 30 post-infection. MKP-2^−/− mice did not, however, demonstrate defective type-1 responses compared with MKP-2^+/+ mice following infection although they did display significantly reduced serum nitrite levels and enhanced tissue arginase-1 expression. Early resistance to T. gondii in MKP-2^+/+, but not MKP-2^−/−, mice was nitric oxide (NO) dependent as infected MKP-2^+/+, but not MKP-2^−/− mice succumbed within 10 days post-infection with increased parasite burdens following treatment with the iNOS inhibitor L-NAME. Conversely, treatment of infected MKP-2^−/− but not MKP-2^+/+ mice with nor-NOHA increased parasite burdens indicating a protective role for arginase-1 in MKP-2^−/− mice. In vitro studies using tachyzoite-infected bone marrow derived macrophages and selective inhibition of arginase-1 and iNOS activities confirmed that both iNOS and arginase-1 contributed to inhibiting parasite replication. However, the effects of arginase-1 were transient and ultimately the role of iNOS was paramount in facilitating long-term inhibition of parasite multiplication within macrophages.     

Expression of genes in gastrointestinal and lymphatic tissues during parasite infection in sheep genetically resistant or susceptible to Trichostrongylus colubriformis and Haemonchus contortus

Resistance to an acute gastrointestinal nematode (GIN) infection is dependent on the ability of the host to recognise the parasite and mount a protective Th2 response. It is hypothesised that lambs which are genetically susceptible to GIN will differentially up-regulate Th1-type genes and therefore remain susceptible to chronic parasitism compared with genetically resistant lambs which will differentially up-regulate Th2-type genes and clear the parasite infection. Two selection flocks, in which lines of Merino sheep produced lambs genetically resistant or susceptible to GIN, were acutely challenged once or thrice with either Haemonchus contortus or Trichostrongylus colubriformis. Faecal-egg counts (FECs), and plasma and tissue anti-parasite (H. contortus or T. colubriformis) antibody isotype responses showed that resistant animals challenged three times with T. colubriformis established a protective Th2 response (negligible FEC, IgG1 and IgE) whereas susceptible animals required multiple challenges to establish a significant IgG1 response despite FECs remaining high. Trichostrongylus colubriformis elicited a more pronounced host response than H. contortus. RNA extracted from tissues at the site of each parasite infection and associated lymph nodes were interrogated by microarray and quantitative PCR analyses to correlate host gene expression to FECs and antibody responses. The IFN-γ inducible gene cxcl10 was up-regulated in the susceptible line of the Trichostrongylus selection flock sheep after a tertiary challenge with the parasites H. contortus and T. colubriformis. However, a uniform pattern of genes was not up-regulated in resistant animals from both selection flocks during both parasite infections, suggesting that the mode of host resistance to these parasites is different, although some similarities in host susceptibility were apparent.     

Identification of a Major Dimorphic Region in the Functionally Critical N-Terminal ID1 Domain of VAR2CSA

The VAR2CSA protein of Plasmodium falciparum is transported to and expressed on the infected erythrocyte surface where it plays a key role in placental malaria (PM). It is the current leading candidate for a vaccine to prevent PM. However, the antigenic polymorphism integral to VAR2CSA poses a challenge for vaccine development. Based on detailed analysis of polymorphisms in the sequence of its ligand-binding N-terminal region, currently the main focus for vaccine development, we assessed var2csa from parasite isolates infecting pregnant women. The results reveal for the first time the presence of a major dimorphic region in the functionally critical N-terminal ID1 domain. Parasite isolates expressing VAR2CSA with particular motifs present within this domain are associated with gravidity- and parasite density-related effects. These observations are of particular interest in guiding efforts with respect to optimization of the VAR2CSA-based vaccines currently under development.     

Leishmania amazonensis response to artemisinin and derivatives

The worldwide presence of Leishmania parasites increases in the poorest regions. Current leishmaniasis treatments are unsatisfactory due to resistance development, side effects and cost. Herein, we describe the in vitro activity of artemisinin (ART), artemether (ATM), artesunate (ATS) and dihydroartemisinin (DHA) against Leishmania amazonensis. Selected compounds were assayed in the animal model of cutaneous leishmaniasis in BALB/c mice. On intracellular amastigotes, similar activity (p > 0.05) was observed for ART, ATM and ATS (IC₅₀ = 15.0–19.2 μM), which were inferior (p < 0.05) respect to reference endoperoxide ascaridole (IC₅₀ = 11.5 ± 1.0 μM) and superior (p < 0.05) compared with reference drug Glucantime® (IC₅₀ = 30.1 ± 9.0 μM). In contrast, DHA (IC₅₀ = 38.5 ± 4.7 μM) showed higher IC₅₀ values (p < 0.05) than other artemisinins and ascaridole, but similar (p > 0.05) than Glucantime®; while deoxyartemisinin caused smaller inhibition (IC₅₀ = 88.9 ± 5.2 μM). Selectivity indexes of >13, 6, 11 and 1 were obtained for ART, ATM, ATS and DHA, respectively. In addition, the potential effect of ART and ATS was also demonstrated in the murine model, causing a significant reduction (p < 0.05) of the lesion size and parasite load regarding untreated animals and treated with vehicle. Effects of both artemisinins were comparable (p > 0.05) with Glucantime® and ascaridole-treated mice. In particular, artemisinin is recommended to further studies, which could be an advantage over the ascaridole endoperoxide and could be useful in endemic areas of parasite resistance to antimonials.     

Prevention of Clostridium difficile -induced ileocecitis with Bacteriophage

A bacteriophage specific for Clostridium difficile was examined for its ability to prevent ileocecitis in a hamster model. This species- and strain-specific bacteriophage was isolated from a lysogenic strain of C. difficile . Hamsters were maintained in sterile isolation cages to prevent the acquisition of C. difficile from the environment. Bicarbonate neutralization of gastric acidity was necessary for bacteriophage survival in the hamster's gastrointestinal tract. Bacteriophage recovery from the hamster cecum was 2×10⁴plaque forming units/mL of cecal contents 24 h after orogastric challenge with 10⁸plaque forming units/mL of bacteriophage. However, there was no bacteriophage recovery 48 h post challenge, indicating dissipation of bacteriophage from the hamster intestinal tract within this time frame. Twenty-four hours after being challenged with clindamycin, one group of hamsters was challenged with C. difficile followed by a single dose of bacteriophage (10⁸plaque forming units/mL). Two additional groups of hamsters received phage doses immediately after C. difficile challenge and subsequently thereafter every 8 h up to 48 and 72 h, respectively. The gastric acidity was neutralized with bicarbonate buffer preceding every bacteriophage treatment. Control animals that received only clindamycin and C. difficile died within 96 h after challenge while the majority of bacteriophage treated hamsters survived. Two weeks after stopping bacteriophage treatment, the surviving hamsters were re-challenged with clindamycin and C. difficile . All the hamsters died within 96 h indicating susceptibility of the surviving hamsters to C. difficile disease in the absence of bacteriophage treatment.     

Reduced fecundity of Hymenolepis nana due to thymus-dependent immunological responses in mice

Reduced fecundity of Hymenolepis nana due to thymus-dependent immunological responses in mice. International Journal for Parasitology16: 81–85. The fecundity of the dwarf tapeworm, Hymenolepis nana, was enhanced in congenitally athymic nude CD-1 (ICR) mice but depressed to the same degree as in phenotypically normal littermates when they were reconstituted with thymocytes before infection. The reduction in fecundity of this parasite was clear only when H. nana were recovered from those strains of mice which demonstrate a “late response” against luminal cysticercoid challenge before the established worms become fully mature (within 15 days of initial immunizing egg inoculation). The fecundity of H. nana in dd mice, which acquire the late response within 30–40 days, was greatly advanced than that in BALB/c or CD-1 (ICR) mice and somewhat better than even that in the nude CD-1 (ICR) mice and appeared to be little, or not at all, depressed. The fecundity and longevity of this parasite, highly variable among mouse strains, is discussed in terms of variations in the rapidity of expression of protective immunity against the lumen phase.     

The response of lambs to vaccination and challenge with Trichostrongylus colubriformis: Effect of plane of nutrition on,and the inter-relationship between,immunological responsiveness and resistance

Wagland B. M., Steel J. W., Windon R. G. and Dineen J. K. 1984. The response of lambs to vaccination and challenge with Trichostrongylus colubriformis: effect of plane of nutrition on, and the inter-relationship between, immunological responsiveness and resistance. International Journal for Parasitology14: 39–44. Merino lambs weaned at 8 weeks of age were fed either ground and pelleted (high plane, HP) or chopped (low plane, LP) lucerne hay ad libitum to achieve an approximate 2-fold difference in liveweight gain. When aged 17 and 21 weeks, 15 of the 20 lambs in each diet group were vaccinated with 80,000 irradiated T. colubriformis larvae. At 25 weeks, vaccinated and unvaccinated lambs were treated with anthelmintic and one week later challenged with 30,000 normal larvae. Four weeks after challenge the animals were killed for worm counts. After vaccination HP lambs had higher titres of antibodies to the parasite and after challenge had lower worm egg outputs, and lower worm burdens than LP lambs. Immunological responsiveness (serum titre of complement-fixing antibodies against worm antigen) and manifestations of resistance (eggs produced per female worm per day and percent protection calculated from worm counts) were significantly correlated within dietary groups. Percent protection and egg production per female worm were highly correlated (r = ?0.81) in individual animals pooled over dietary groups, suggesting that both manifestations of resistance respond to essentially the same immunological mechanism. Failure to obtain significant correlation between weight gain pre-vaccination and immunological and resistance parameters indicated that animal production and resistance to infection are not genetically linked. Negative correlation of weight gain during the vaccination period with serum antibody titre at challenge suggests that the developing immune response competes with weight gain for limited physiological resources of the animal.     

In vitro and in vivo effects of the phytohormone inhibitor fluridone against Neospora caninum infection

Neospora caninum causes abortion and stillbirth in cattle. Identification of effective drugs against this parasite remains a challenge. Previous studies have suggested that disruption of abscisic acid (ABA)-mediated signaling in apicomplexan parasites such as Toxoplasma gondii offers a new drug target. In this study, the ABA inhibitor, fluridone (FLU), was evaluated for its action against N. caninum. Production of endogenous ABA within N. caninum was confirmed by ultra-performance liquid chromatography–tandem quadruple mass spectrometry. Subsequently, FLU treatment efficacy was assessed using in vitro. Results revealed that FLU inhibited the growth of N. caninum and T. gondii in vitro (IC₅₀ 143.1 ± 43.96 μM and 330.6 ± 52.38 μM, respectively). However, FLU did not affect parasite replication at 24 h post-infection, but inhibited egress of N. caninum thereafter. To evaluate the effect of FLU in vivo, N. caninum-infected mice were treated with FLU for 15 days. FLU treatment appeared to ameliorate acute neosporosis induced by lethal parasite challenge. Together, our data shows that ABA might control egress in N. caninum. Therefore, FLU has potential as a candidate drug for the treatment of acute neosporosis.