Immune responses to Neospora caninum and prospects for vaccination

Elisabeth A. Innes, Stephen E. Wright, Paul Bartley, Stephen Maley, David Buxton.
Moredun Research Institute, Pentlands Science Park, Edinburgh EH26 OPZ
E.mail: lee.innes@moredun.ac.uk

Reproductive failure in cattle is of major economic and welfare concern to producers worldwide. There are many different causes of reproductive loss, including infectious disease and an accurate diagnosis of the condition may be problematic. Neospora caninum is a recently recognised protozoan parasite that has been linked with causing bovine abortion in many countries worldwide (Dubey, 2003). The parasite is closely related to Toxoplasma gondii, an important zoonotic pathogen, also known to cause congenital disease (Buxton, 1990). In this paper we will discuss the disease in cattle with emphasis on understanding the host-parasite relationship leading to devising strategies to control bovine neosporosis.

The disease
Epidemiological studies in several countries have shown that cattle infected with Neospora caninum are three to seven times more likely to have an abortion compared with uninfected cattle, with the highest risk during a first pregnancy (Thurmond and Hietala 1997a; Moen et al, 1998; Wouda et al, 1998). Adult cattle rarely show clinical symptoms following infection and disease manifests in the placenta and developing foetus (Innes et al 2002; Buxton et al 2002). Clinical consequences of infection include abortion of the foetus, birth of a weak calf sometimes showing neurological symptoms or birth of a clinically normal but persistently infected calf (Dubey and Lindsay, 1996). The clinical outcome is likely to be related to the timing of infection during pregnancy (Innes et al, 2002). Economic losses associated with the disease include costs associated with loss of calf, fertility problems and increased calving interval, reduced milk production, reduced value of stock and increased likelihood of culling (Thurmond and Hietala, 1997b; Trees et al 1999; Dubey, 2003).

Transmission and life-cycle stages
Neospora caninum may be transmitted to cattle via consumption of feed or water contaminated with the oocyst stage of the parasite or by vertical transmission of the tachyzoite stage from dam to foetus during pregnancy (Dubey, 2003). Dogs have recently been identified as a definitive host of the parasite (McAllister et al 1998; Basso et al 2001). Oocysts may be shed in the faeces of acutely infected dogs that may acquire the infection through the consumption of infected bovine placentas (Dijkstra et al 2001). The oocyst stage of the parasite is thought to persist in the environment but currently little is known about the environmental conditions that may favour oocyst survival or the frequency of oocyst shedding by dogs (Dubey et al 2003). Following infection, the tachyzoite stage of the parasite actively invades host cells and multiplies by a process called endodyogeny resulting in many tachyzoites which burst from the cell ready to invade new cells and resume rapid multiplication (Dubey and Lindsay, 1996). Using this process the parasite can disseminate via the circulation throughout the host (Okeoma et al 2004). The parasite can only multiply within host cells and it is thought that under pressure from the immune response of the host, the parasite differentiates into the slower multiplying bradyzoite stage. Bradyzoites are usually observed within tissue cysts in neural tissues (brain and spinal cord) and this is thought to be how the parasite may cause persistent infection in cattle (Dubey and Lindsay, 1996). Vertical transmission from dam to foetus may occur following an exogenous challenge during pregnancy or may result following recrudescence of an existing persistent infection. A characteristic of bovine neosporosis is the high rate of vertical transmission estimated at between 78-95% (Pare et al 1996; Davison et al 1999). Interestingly, transplacental transmission can occur over consecutive pregnancies and congenitally infected heifers can transmit the parasite to their own offspring (Bjorkman et al 1996).

Do cattle develop natural immunity?
Cattle which have experienced an abortion due to neosporosis have a significantly decreased chance of having a repeat abortion due to the same infectious agent (Anderson et al 1995; Wouda et al 1998), implying that cattle can develop a degree of protective immunity against the parasite. Further evidence for this came from an investigation of a point source outbreak showing that those cattle which had evidence of prior exposure to N. caninum were less likely to abort compared with those undergoing a primary infection (McAllister et al 2000). While cattle may be able to develop some protective immunity to help prevent abortion, this immunity does not protect so well against vertical transmission as evidenced by the high rates of repeated vertical transmission seen in natural infection. Therefore a vaccination strategy to prevent/reduce abortion may a more feasible goal than to prevent vertical transmission.

In any host-parasite relationship a vast array of different immune responses are induced against the various life-cycle stages of the parasite. Some of these immune responses will be protective to the host, others protective to the parasite, some may cause pathology in the host and others may be largely irrelevant. In the following sections we will discuss the different roles of the host immune response and how this contributes to our understanding of the host-parasite relationship, disease pathogenesis and immunological strategies to control the disease.

Host protective immune responses
The tachyzoite stage of N. caninum actively invades and multiplies within various cells of the host (Hemphill, 1999). The intracellular location of the parasite suggests that cell-mediated immune responses are likely to play a significant role in protective immunity (Marks et al 1998). Interferon gamma (IFNg) and tumour necrosis factor alpha (TNFa) are known to significantly inhibit intracellular multiplication of N. caninum (Innes et al 1995; Yamane et al 2000). The cytokines IFNg and interleukin 12 (IL-12) were shown to be important components of protective immunity using mouse models of infection (Khan et al 1997; Bazler et al 1999) and IFNg knockout mice showed a significantly increased vulnerability to N. caninum infection (Dubey et al 1998). The importance of CD4+ T-cells in protective immunity was highlighted in a study where mice were treated in vivo with antibodies to deplete CD4+ or CD8+ T-cells prior to challenge with N. caninum (Tanaka et al 2000). In the group of mice where CD4+T-cells were depleted, all mice died within 30 days of the challenge, in contrast no mice died within this time period in the control group or the group where CD8+T-cells had been depleted (Tanaka et al 2000). In addition, N. caninum-specific CD4+ T-cells, from infected cattle, were able to directly lyse parasite infected autologous target cells in-vitro (Staska et al 2003).

While we still know comparatively little concerning induction, function and regulation of protective immune mechanisms against N. caninum parasites in cattle current data would support an important role for CD4+T-cells and pro-inflammatory cytokines such as IFNg.

Changes to the host-parasite relationship at different stages of pregnancy
Neosporosis is a disease that manifests during pregnancy where the developing foetus is particularly vulnerable. Various changes occur in the maternal immune response to enable the dam to support the pregnancy and prevent immunological rejection of the semi-allogeneic foetus (Raghupathy, 1997). These natural changes in the immune system may favour the parasite and help to explain disease pathogenesis in pregnancy. Relevant to our understanding of bovine neosporosis are studies examining cytokine regulation in pregnancy, in particular at the materno-foetal interface (Innes et al 2002). The pro-inflammatory cytokines such as IFNg and IL-12 are involved in the generation of Th1-type immune responses that may be damaging to the pregnancy (Tangri et al 1993; Wegman et al 1993; Entrican, 2002). The cytokine environment of the placenta favours more regulatory Th2-type cytokines such as IL-10, IL-4 and transforming growth factor beta (TGF-b) whose role is to counteract the inflammatory responses induced by the Th1-type cytokines (Entrican et al 2002).

Therefore the natural immuno-modulation occurring in the pregnant dam resulting in a bias towards Th2-type immune responses may limit her ability to control N. caninum multiplication and the Th1-type immune responses, known to protect against N. caninum may be detrimental to the pregnancy. A similar example of pregnancy related changes to the immune system affecting the host-parasite relationship is seen with Leishmania major infection in mice where the protective immune response is also associated with a Th1-type immune response. During pregnancy there was a reduction in the IFNg response and an increase in production of the more regulatory cytokines IL-4 and IL-10 that resulted in the pregnant mice being less able to control the infection compared to non-pregnant controls (Krishnan et al 1996).

A study examining cell-mediated immune responses in pregnant cattle infected with N. caninum noted that there was a significant reduction in the antigen-specific cell-proliferation and IFNg response around mid-gestation compared to pre-pregnancy or early gestation (Innes et al 2001). Levels of progesterone in pregnant cattle are also known to increase steadily from early to mid-gestation (Pope et al 1969) and progesterone in known to bias a T-cell response towards a Th2 phenotype (Kalinski et al 1997). These studies indicate the changing dynamics of the maternal immune response as gestation progresses that may influence the response of the parasite within the host. Epidemiological studies have suggested that most recorded cases of Neospora-associated abortion occur between 4-6 months of gestation (Anderson et al 1991; Thurmond and Hietala, 1997; Moen et al 1998; Gonzales et al 1999). The changes in the maternal immune response around this time may influence recrudescence of a persistent infection or the ability of the dam to control a new infection. Recrudescence of T. gondii infection is known to occur in HIV infected patients when the T-cell and IFNg response are diminished (Luft et al 1984).

Several studies using controlled experimental infections with N. caninum have shown that the timing of placental and foetal infection is important in determining the outcome, in general the earlier in gestation this occurs the more severe the consequences for the foetus (Barr et al, 1994; Buxton et al 1998, Williams et al 2000; Maley et al 2003; Macaldowie et al 2004). In the study outlined previously examining temporal changes to the maternal immune system during pregnancy (Innes et al 2001) the dams showed significantly higher antigen specific cell-proliferation and IFNg responses in early compared to mid-pregnancy. Therefore an infection occurring at this stage of pregnancy may invoke a strong Th1-type immune response that may in itself prove detrimental to the pregnancy. Recent data examining lesions in the placenta of cattle experimentally infected with N. caninum in early gestation has shown a strong maternal inflammatory response in those dams where foetal death had occurred (Macaldowie et al 2004). Further examination of the placental tissues has shown the presence of NK cells, CD4+, CD8+ and dg T-cells and IFNg associated with foetal death, as these responses were not seen in those infected cattle carrying live foetuses or in the uninfected control cattle (Maley et al, manuscript in preparation). It is known from other studies that direct administration of IFNg can induce spontaneous abortion in pregnant mice (Chaout et al 1990).

Therefore while we know that Th1-type immune responses may be protective to the dam against N. caninum infection, this type of immune response induced in placental tissue may be highly detrimental to the foetus.These observations highlight how immune cytokines may have both a beneficial and detrimental effect on the host depending on their concentration and tissue location.

Development of foetal immunity
A further important influence determining the outcome of infection is the relative immunocompetence of the foetus at the time of challenge. The immune system of the foetus matures progressively throughout gestation (Osburn et al 1982). Studies examining foetal immune responses in cattle infected with N. caninum in early gestation have shown mitogenic responses in foetal spleen and thymus cells around day 100 of gestation but there was no evidence of antigen specific cellular or humoral immune responses at this stage (Innes et al, manuscript in preparation). Evidence of specific cell-mediated and humoral immune responses occurs around 4-7 months of gestation (Andrianarivo et al 2001; Almeria et al 2003; Bartley et al 2004). The increasing immunocompetence of the foetus as pregnancy progresses will enable the foetus to better control the parasite infection resulting in reduced disease severity.

Therefore the dynamics of the host-parasite relationship changes throughout pregnancy. Important factors influencing severity of disease in bovine neosporosis include the timing of the infection during pregnancy, the relative immunocompetence of the foetus and the various consequences of the maternal immune response being host protective, parasite protective and in causing immunopathology.

Control strategies
As dogs are known to play an important role in the transmission of the parasite and oocysts may persist for some time in the environment it is important to introduce farm management procedures to prevent or minimise faecal contamination of feedstuffs and water (Dubey, 2003). Efficient disposal of infected placentas, foetuses or still born calves will also help to minimise sources of contamination. Testing of animals prior to introducing them to the herd and culling of infected cattle may be an option depending on the level of infection within the herd. Various pharmaceutical agents have been tested in vitro and in-vivo and have shown some efficacy against the tachyzoite stage of the parasite (Lindsay et al 1994; Gottstein et al 2001). However, there are no drugs available that are effective in curing cattle of N. caninum infection and there may be problems arising from drug residues in milk from lactating cows (Dubey, 2003).

There is currently much interest in developing a control strategy against bovine neosporosis based on vaccination. The targets for such a control strategy would include prevention of Neospora-associated abortion and ideally prevention of vertical transmission of the parasite.

Induction of protective immunity
Encouraging studies in this area have shown that experimental infection of naïve animals prior to mating induced protective immunity against both abortion and vertical transmission of the parasite following challenge during pregnancy ( Innes et al 2001, Buxton et al 2001). In addition, persistently infected cattle were protected against a challenge that induced foetopathy in naïve control animals (Williams et al 2003).

A live vaccine preparation is likely to stimulate appropriate CMI responses against intracellular pathogens as it more closely mimics what is happening during natural infection and the parasite antigens are presented to the immune system in the correct context. There is interest in developing attenuated strains of the parasite that may be useful as vaccine preparations (Lindsay et al 1999). A highly successful commercially available vaccine to prevent toxoplasmosis in sheep utilises a live attenuated strain of T. gondii (Buxton and Innes, 1995). Drawbacks of live vaccines include a limited shelf-life and safety concerns therefore attention has also focussed on development of killed vaccines. The major challenges in designing an effective killed vaccine against an intracellular pathogen are to select relevant antigens and to deliver these antigens to the host to stimulate appropriate and long-lasting protective immune responses.

Selection of relevant antigens
Understanding protective host immune responses may be helpful in selection of relevant antigens. Antigens recognised by immune sera and also immune T-cells may prove to be useful vaccine candidates (Marks et al 1998; Hemphill, 1999; Staska et al 2005; Tuo et al 2005). In addition parasite antigens known to be involved in host cell invasion and survival are likely to be important (Hemphill 1999). Due to the complex interaction of the parasite and the bovine host involving different life-cycle stages a killed vaccine may have to comprise a cocktail of different antigens (Innes et al 2002).

Antigen delivery strategies
Live antigen delivery systems have been used to elicit immune responses against a wide range of pathogens. Recombinant virus vectors have been shown to stimulate specific CMI responses against other intracellular protozoan parasites (Honda et al 1998; Schneider et al 1998; Oliveira-Ferreira et al 2000).

Recombinant vaccinia viruses constructed to express the antigens Nc-SRS2 or NcSAG1 were able to induce protective immunity against acute N. caninum infection in non-pregnant mice (Nishikawa et al 2001a) and were also able to induce protection against abortion in a pregnant mouse model (Nishikawa et al 2001b). In both cases the best protection was achieved using the recombinant vaccinia virus expressing the NcSRS2 antigen.

Crude lysate antigen prepared from N. caninum tachyzoites has been tested using different adjuvant preparations in attempts to induce protective immunity in mice. The use of non-ionic surfactant vesicles as an adjuvant exacerbated encephalitis and clinical neurological disease in immunised mice (Bazler et al 2000) and administration of antigen with Quil A or ISCOMs resulted in enhanced protection (Lunden et al 2002). Administration of a crude tachyzoite lysate with ImmuMAXSRä adjuvant protected against vertical transmission of N. caninum in a pregnant mouse model (Liddell et al 1999). Protective immunity was also induced in mice using specific recombinant antigens, NcSRS2 incorporated into ISCOMs (Pinitkiatisakul et al 2005) and NcMIC3 antigen with the Ribi adjuvant system (Cannas et al 2003a).

DNA vaccination
With DNA vaccines the host is injected with DNA incorporated into a plasmid containing sequences encoding the antigens of interest. An advantage of DNA vaccination is the way that the plasmid is taken up and processed by antigen presenting cells resulting in the induction of both cell-mediated and humoral immune responses (Reyes-Sandoval and Ertl, 2001). This is of particular importance when trying to design vaccines against intracellular pathogens. Cytokines and immunostimulatory DNA sequences can be co-expressed to help modulate the type of immune response required (Sakai et al 2003).

Mice vaccinated intramuscularly (im) with a eukaryotic expression plasmid containing NcSRS2 or NcSAG1 cDNA inserts and then boosted using the recombinant antigens were better protected against N. caninum challenge than those mice receiving only recombinant antigen (Cannas et al 2003b). A further study showed direct immunisation of Balb/c mice with plasmid DNA encoding NcGRA7 or NcsHSP33 protected against congenital infection with N. caninum (Liddell et al 2003).

CpGs (oligodinucleotides) are known to activate Th1 type immune responses and pro-inflammatory cytokines and are thought to be useful adjuvants to enhance the immune response to vaccines against intracellular infections (Klinman, 2003, Mutwiri et al 2003). Addition of the CpG adjuvant to the vaccination of mice with plasmid DNA expressing NcGRA7 significantly improved protection (Jenkins et al 2004).

Killed vaccine trials in cattle
A killed N. caninum preparation combined with a POLYGEN™ adjuvant was used to vaccinate heifers at 35 and 63 days of gestation (Andrianarivo et al 2000). The cattle were challenged with a combined i.v/i.m inoculation of live N. caninum tachyzoites four weeks after the second inoculation. Following vaccination, the cattle developed specific humoral and cell-mediated immune responses and after challenge there was a boost to the antibody response but not to the cell-mediated immune response. All of the challenged heifers, either vaccinates or controls had infected foetuses indicating that under the challenge conditions used in this study the vaccine preparation had not successfully protected the cattle (Andrianarivo et al 2000).

A commercial vaccine, Bovilis ® Neoguard, Intervet comprising a killed Neospora tachyzoite preparation formulated with an adjuvant, SPUR® is currently commercially available in certain countries. The vaccine is administered sc on two occasions, 3-4 weeks apart in the first trimester of pregnancy. Data on the efficacy of the vaccine under field trial conditions showed that the vaccine had some protective effect against abortions occurring at 5-6 months of gestation in cattle in Costa Rica whereas, a similar study in dairy cattle from New Zealand resulted in no definite conclusions on the ability of the vaccine to protect cattle (Schetters et al 2004).

Concluding remarks
Recent data from controlled experimental infections of pregnant cattle is helping us to understand the complex dynamics of the host-parasite relationship in bovine neosporosis and to determine why some cattle abort their foetuses while others produce clinically healthy, albeit congenitally infected calves. Additional studies looking at induction of protective immune responses has given encouragement to the possibility of controlling the disease by vaccination. However there are still several challenges to overcome. It is important that the vaccine is designed in such way as to induce protective immune responses without exacerbating pathology. In addition, further work needs to be done to determine the immunological implications of cattle becoming infected with the parasite in-utero when their immune systems are still developing and being born persistently infected with the parasite. Does this somehow compromise their ability to develop effective immunity against N. caninum later in life and does this in part explain the high rates of repeated vertical transmission observed in natural infection? This would have important implications in devising a vaccination strategy as it may prove to be more efficacious to target the vaccine to naïve cattle and cull out those that are congenitally infected.

Acknowledgements
The authors would like to acknowledge the support of the Scottish Executive Environmental and Rural Affairs Department.

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