Nicholas C. Smith

Specific lgG1 and lgG2 antibody responses of dogs to Leishmania infantum and other parasites

Sera from dogs naturally infected with Leishmania infantum were analysed for the IgG subclass specificity of their antibody response by ELISA. Dogs infected with L. infantum produced both IgGl and IgG2 antibodies with IgG2 being associated with asymptomatic infections and IgGl being associated with disease (symptomatic dogs, non‐ or low‐responsive to chemotherapy). The differential responses of IgG] and IgGl serum antibodies in asymptomatic and symptomatic dogs may indicate a dichotomous immune response to infection with L. infantum. To confirm this, on a broader scale, sera from dogs naturally exposed to an asymptomatic protozoan infection, Toxoplasma gondii, were also analysed as were sera from dogs exposed to the helminths, Dirofilaria immitis and Toxocara canis. Antibodies specific for T. gondii antigen detected in sera from 17 dogs were of the IgG2 subclass only. Both IgGl and IgG2 antibodies to D. immitis andl. canis were present in the sera of naturally infected dogs but IgGl appeared to be the predominant subclass. Furthermore, in dogs experimentally infected with T. canis, selective regulation ofIgG2 and IgGl responses was apparent since production of the two subclasses occurred at different times following infection, with IgGl levels declining as IgGl levels rose. Thus, the analysis of IgG subsets in parasitized dogs provides evidence of a dichotomous response to infection: IgGl is associated with asymptomatic protozoan infections and IgGl is associated with helminth infections and disease caused by protozoan infection.

The immunobiology of the innate response to Toxoplasma gondii

Toxoplasma gondii is a unique intracellular parasite. It can infect a variety of cells in virtually all warm-blooded animals. It has a worldwide distribution and, overall, around one-third of people are seropositive for the parasite, with essentially the entire human population being at risk of infection. For most people, T. gondii causes asymptomatic infection but the parasite can cause serious disease in the immunocompromised and, if contracted for the first time during pregnancy, can cause spontaneous abortion or congenital defects, which have a substantial emotional, social and economic impact. Toxoplasma gondii provokes one of the most potent innate, pro-inflammatory responses of all infectious disease agents. It is also a supreme manipulator of the immune response so that innate immunity to T. gondii is a delicate balance between the parasite and its host involving a coordinated series of cellular interactions involving enterocytes, neutrophils, dendritic cells, macrophages and natural killer cells. Underpinning these interactions is the regulation of complex molecular reactions involving Toll-like receptors, activation of signalling pathways, cytokine production and activation of anti-microbial effector mechanisms including generation of reactive nitrogen and oxygen intermediates.

Chasing the golden egg: vaccination against poultry coccidiosis

Eimeria species, of the Phylum Apicomplexa, cause the disease coccidiosis in poultry, resulting in severe economic losses every year. Transmission of the disease is via the faecal‐oral route, and is facilitated by intensive rearing conditions in the poultry industry. Additionally, Eimeria has developed drug resistance against most anticoccidials used today, which, along with the public demand for chemical free meat, has lead to the requirement for an effective vaccine strategy. This review focuses on the history and current status of anticoccidial vaccines, and our work in developing the transmission‐blocking vaccine, CoxAbic® (Netanya, Israel). The vaccine is composed of affinity‐purified antigens from the wall‐forming bodies of macrogametocytes of Eimeria maxima, which are proteolytically processed and cross‐linked via tyrosine residues to form the environmentally resistant oocyst wall. The vaccine is delivered via maternal immunization, where vaccination of laying hens leads to protection of broiler offspring. It has been extensively tested for efficacy and safety in field trials conducted in five countries and involving over 60 million offspring chickens from immunized hens and is currently the only subunit vaccine against any protozoan parasite to reach the marketplace.

Eimeria maxima gametocyte antigens: potential use in a subunit maternal vaccine against coccidiosis in chickens

Affinity-purified gametocyte antigens (APGA) from Eimeria maxima, emulsified in Freunds adjuvant, were injected intramuscularly into breeding hens on two or three occasions. As a result, progeny of the immunized hens were partially immune to infection with E. maxima, Eimeria tenella and Eimeria acervulina (with a reduction in total oocyst output of 45-63% as compared with progeny of untreated hens). Thus, APGA in Freunds adjuvant appears to have great potential as part of a maternally applied vaccine against coccidiosis. The ability of APGA to induce partial cross-species protection is most probably due to the existence of conserved epitopes in the different species as indicated by comparative Western blots of E. maxima and E. tenella. Surprisingly, Freunds adjuvant by itself also induced significant levels of maternal immunity to coccidiosis (with a 12-35% reduction in oocyst output in the progeny). In contrast to the purified antigens or Freunds alone, crude extracts from gametocytes as well as other developmental stages, induced little if any significant maternal immunity despite provoking the production of large amounts of parasite-specific IgG, including antibodies to APGA. This result indicates that a successful maternal vaccine against coccidiosis requires, in addition to good recognition of protective antigens, the exclusion of irrelevant antigens from the vaccine preparation.

Neospora caninum: a cause of immune- mediated failure of pregnancy?

Resistance to many intracellular protozoan parasites is dependent on T helper cell 1 cytokine responses. This has important repercussions for pregnant females because strong T helper cell 1 cytokine responses are incompatible with successful pregnancy. Thus, there are two possible consequences of infection with protozoans such as Leishmania major, Plasmodium falciparum and Toxoplasma gondii during pregnancy: (1) pregnancy is compromised; or (2) resistance to the parasite is compromised. The apicomplexan Neospora caninum is a parasite renowned for its association with abortion in cattle. Furthermore, a major route of transmission for this parasite is congenital. The evidence for the hypothesis that T helper cell 1 cytokines play a role in these events is reviewed here.

Heat Shock Protein 70 Is a Potential Virulence Factor in Murine Toxoplasma Infection Via Immunomodulation of Host NF-κB and Nitric Oxide

We propose that the 70-kDa heat shock protein (HSP70) protects virulent Toxoplasma gondii from the effects of the host by immunomodulation. This hypothesis was tested using quercetin and antisense oligonucleotides targeting the start codon of the virulent T. gondii HSP70 gene. Oligonucleotides were transiently transfected into two virulent (RH, ENT) and two avirulent (ME49, C) strains of T. gondii, significantly reducing HSP70 expression in treated parasites. Virulent parasites with reduced HSP70 expression displayed reduced proliferation in vivo, as measured by the number of tachyzoites present in spleens of infected mice. They also exhibited an enhanced rate of conversion from tachyzoites to bradyzoites in vitro. Our results implicate HSP70 as a means by which virulent strains of T. gondii evade host proinflammatory responses: when RAW 264.7 cells were exposed to parasites with reduced HSP70 expression, differential expression of inducible NO synthase (iNOS) and cell NO production were observed between infections with normal and HSP70-deficient T. gondii. iNOS message levels were significantly increased when host cells were infected with HSP70 reduced virulent tachyzoites and HSP70-related inhibition of iNOS transcription resulted in altered host NO production by virulent T. gondii infection. Virulent parasites expressing reduced levels of HSP70 initiated significantly more NF-κB activation in host splenocytes than infections with untreated parasites. Neither proliferative ability nor conversion from tachyzoites to bradyzoites was affected by lack of HSP70 in avirulent strains of T. gondii. Furthermore, avirulent T. gondii strains induced high levels of host iNOS expression and NO production, regardless of HSP70 expression in these parasites, and inhibition of HSP70 had no significant effects on translocation of NF-κB to the nucleus. Therefore, the 70-kDa parasite stress protein may be part of an important survival strategy by which virulent strains down-regulate host parasiticidal mechanisms.

The Role of the P2X7 Receptor in Infectious Diseases

ATP is an extracellular signal for the immune system, particularly during an inflammatory response. It is sensed by the P2X7 receptor, the expression of which is upregulated by pro-inflammatory cytokines. Activation of the P2X7 receptor opens a cation-specific channel that alters the ionic environment of the cell, activating several pathways, including (i) the inflammasome, leading to production of IL-1β and IL-18; (ii) the stress-activated protein kinase pathway, resulting in apoptosis; (iii) the mitogen-activated protein kinase pathway, leading to generation of reactive oxygen and nitrogen intermediates; and (iv) phospholipase D, stimulating phagosome-lysosome fusion. The P2X7 receptor can initiate host mechanisms to remove pathogens, most particularly those that parasitise macrophages. At the same time, the P2X7 receptor may be subverted by pathogens to modulate host responses. Moreover, recent genetic studies have demonstrated significant associations between susceptibility or resistance to parasites and bacteria, and loss-of-function or gain-of-function polymorphisms in the P2X7 receptor, underscoring its importance in infectious disease.

P2X7 Receptor-Mediated Killing of an Intracellular Parasite, Toxoplasma gondii, by Human and Murine Macrophages

The P2X7R is highly expressed on the macrophage cell surface, and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this study, we show that macrophages from people with the 1513C (rs3751143, NM_002562.4:c.1487A>C) loss-of-function P2X7R single nucleotide polymorphism are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knockout mice (P2X7R−/−) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production.

A Selective Review of Advances in Coccidiosis Research

Coccidiosis is a widespread and economically significant disease of livestock caused by protozoan parasites of the genus Eimeria. This disease is worldwide in occurrence and costs the animal agricultural industry many millions of dollars to control. In recent years, the modern tools of molecular biology, biochemistry, cell biology and immunology have been used to expand greatly our knowledge of these parasites and the disease they cause. Such studies are essential if we are to develop new means for the control of coccidiosis. In this chapter, selective aspects of the biology of these organisms, with emphasis on recent research in poultry, are reviewed. Topics considered include taxonomy, systematics, genetics, genomics, transcriptomics, proteomics, transfection, oocyst biogenesis, host cell invasion, immunobiology, diagnostics and control.

Maternal transfer of antibodies induced by infection with Eimeria maxima partially protects chickens against challenge with Eimeria tenella

Infection of breeding hens with Eimeria maxima induces production of Eimeria-specific IgG antibodies which are transferred to hatchlings via the egg yolk and confer a high degree of maternal immunity against homologous challenge and partial immunity to infection with another important species, Eimeria tenella. As an example, in an experiment using hatchlings from eggs collected between days 28 and 39 after infection of the hens with 20,000 sporulated E. maxima oocysts, control chicks (challenged with 100 sporulated oocysts) excreted 6.8 +/- 1.2 million (mean +/- S.E., n = 10) or 5.8 +/- 1.2 million (n = 8) oocysts of E. maxima or E. tenella, respectively, compared to 0.9 +/- 0.4 million (n = 5) E. maxima oocysts or 2.2 +/- 0.4 million (n = 9) E. tenella oocysts excreted by hatchlings of infected hens. This represents an 87% reduction in oocyst excretion with regard to E. maxima and a 62% reduction in oocyst excretion with regard to E. tenella in the progeny of the infected hens. In another experiment, eggs were collected from days 28 to 37 and again from days 114 to 123 after infection of the hens with E. maxima and hatchling oocyst excretion rates were 82% and 62%, respectively, reduced for E. maxima and 43% and 41%, respectively, reduced for E. tenella in the progeny of hens infected with E. maxima compared to the progeny of uninfected hens. ELISA and Western blot analyses of maternally-derived IgG revealed a high degree of cross-reactivity to antigens of E. maxima and E. tenella.(ABSTRACT TRUNCATED AT 250 WORDS)