Developing vaccines to control protozoan parasites in ruminants: Dead or alive?

Veterinary Parasitology 180 (2011) 155–163

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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar

Developing vaccines to control protozoan parasites in ruminants: Dead or alive? Elisabeth A. Innes ∗ , Paul M. Bartley, Mara Rocchi, Julio Benavidas-Silvan, Alison Burrells, Emily Hotchkiss, Francesca Chianini, German Canton, Frank Katzer Moredun Research Institute, Pentlands Science Park, Edinburgh EH26 OPZ, United Kingdom

a r t i c l e Keywords: Protozoa Vaccines Livestock Live Killed

i n f o

a b s t r a c t Protozoan parasites are among some of the most successful organisms worldwide, being able to live and multiply within a very wide range of hosts. The diseases caused by these parasites cause significant production losses in the livestock sector involving reproductive failure, impaired weight gain, contaminated meat, reduced milk yields and in severe cases, loss of the animal. In addition, some protozoan parasites affecting livestock such as Toxoplasma gondii and Cryptosporidium parvum may also be transmitted to humans where they can cause serious disease. Data derived from experimental models of infection in ruminant species enables the study of the interactions between parasite and host. How the parasite initiates infection, becomes established and multiplies within the host and the critical pathways that may lead to a disease outcome are all important to enable the rational design of appropriate intervention strategies. Once the parasites invade the hosts they induce both innate and adaptive immune responses and the induction and function of these immune responses are critical in determining the outcome of the infection. Vaccines offer green solutions to control disease as they are sustainable, reducing reliance on pharmacological drugs and pesticides. The use of vaccines has multiple benefits such as improving animal health and welfare by controlling animal infections and infestations; improving public health by controlling zoonoses and food borne pathogens in animals; solving problems associated with resistance to acaricides, antibiotics and anthelmintics; keeping animals and the environment free of chemical residues and maintaining biodiversity. All of these attributes should lead to improved sustainability of animal production and economic benefit. Using different protozoan parasitic diseases as examples this paper will discuss various approaches used to develop vaccines to protect against disease in livestock and discuss the relative merits of using live versus killed vaccine preparations. A range of different vaccination targets and strategies will be discussed to help protect against: acute disease, congenital infection and abortion, persistence of zoonotic pathogens in tissues of food animals and passive transfer of immunity to neonates. © 2011 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author. E-mail address: [email protected] (E.A. Innes). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.05.036

The World Health Organisation and the Food and Agricultural Organisation estimate that a 50% increase in food production will be needed to feed an estimated world population of 9 billion people by 2050. There is therefore a

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real need to ensure efficient, sustainable and healthy food animal production to help meet this challenge. Livestock farming experiences considerable losses in production through infectious disease and developing preventative measures relating to disease control offers significant support for efficient and welfare friendly livestock production. Diseases of livestock caused by infection with protozoan parasites are the cause of significant production losses worldwide. Parasites such as Theileria annulata and Theileria parva cause acute often fatal disease in livestock in a large geographical area stretching from North Africa, Southern Europe and Asia and in sub Saharan Africa (Morrison and McKeever, 2006). Although indigenous breeds of cattle show some resistance to these pathogens, the susceptibility of imported high producing breeds of cattle are a major impediment to the development of the cattle industry and improved meat and milk production in these countries (Shkap et al., 2007; Dargouth, 2008). Neospora caninum is now recognised as a major cause of abortion in cattle worldwide resulting in significant economic losses to producers which include costs associated with abortion, premature culling and reduced milk yields (Dubey, 2003). While it is difficult to attribute precise economic losses associated with bovine neosporosis, estimates of annual losses to the dairy industries in Australia and New Zealand are in the region of $ 100 million annually (Reichel, 2000; Reichel and Ellis, 2006) and losses of Euro 9.7 million have been reported for dairy herds in Switzerland (Hasler et al., 2006). An economic modelling study of control options for neosporosis indicated that vaccination was likely to be the most cost-effective control strategy (Reichel and Ellis, 2009). Toxoplasma gondii is one of the most successful parasites worldwide capable of infected all warm blooded animals including humans. Infection with T. gondii is a major cause of reproductive failure in sheep and goats worldwide and can also cause serious disease in the developing foetus if pregnant women become infected for the first time during gestation (Innes and Vermeulen, 2006). Recent data from Europe applying new methodologies to calculate the impact of disease such as disability-adjusted life years (DALYs) placed T. gondii as one of the most significant causes of food-borne disease worldwide (Kortbeek et al., 2009). Cryptosporidium parasites cause acute gastrointestinal disease in neonatal livestock species worldwide resulting in significant economic and production losses (De Graaf et al., 1999a,b). These parasites may also cause serious disease in people, in particular those that are immunocompromised (Mead, 2010). Cryptosporidium oocysts may survive and persist in the environment for long periods and are resistant to normal water disinfection treatments making them a significant problem to control in drinking and recreational waters where their presence has been linked to major disease outbreaks in people (Mackenzie et al., 1994). Vaccines offer green solutions for diseases as they are sustainable, reducing reliance on pharmacological drugs and pesticides. The use of vaccines has multiple benefits

such as improving animal health and welfare by controlling animal infections and infestations; improving public health by controlling zoonoses and food borne pathogens in animals; solving problems associated with resistance to acaricides, antibiotics and anthelmintics; keeping animals and the environment free of chemical residues and maintaining biodiversity. All of these attributes should lead to improved sustainability of animal production and economic benefit. A vaccine may comprise living or dead pathogen material that is used to immunise a host to provoke the induction of immune responses that may help to protect the host when challenged with the live virulent organism. Vaccination works by mimicking a natural infection and thus stimulating protective immunity in the host animal. There are several types of vaccines: they can contain a weakened version of the original pathogen (live attenuated vaccines like the human oral polio or measles vaccine); a killed version of the original pathogen (killed vaccines like the human typhoid vaccine); or part of the pathogen (subunit vaccines like the human meningitis vaccine). More recently nucleic acid vaccines have been developed where the nucleic acid coding for an important pathogen gene is directly transcribed and translated by the host animal. A critical component in designing effective vaccines is to understand how the immune system is able to protect the host against a variety of different pathogens. The ability to both measure and induce immune responses in livestock using components of pathogens is an absolute requirement for rational vaccine design (Innes and Vermeulen, 2006; Innes, 2010). This paper will discuss approaches to control of some of the major protozoan pathogens of livestock through vaccination and how knowledge of the host-parasite interactions will help to inform selection of appropriate strategies and targets. 2. Live vaccine approaches Many protozoan parasites have complex life cycles within their host species and they have evolved different strategies to evade or work with the immune response of the host to enable them to survive, multiply and differentiate to new forms. Following initial contact and invasion by the pathogen the host’s defence mechanisms become activated and start to respond to the invading pathogen initially through the innate pathways which help direct the adaptive immune responses. The net outcome of the wide array of different immune responses may have different consequences for the host parasite relationship (Innes and Vermeulen, 2006). Some immune responses may help protect the host against the invading pathogen, other immune responses may cause disease through immunopathology, in particular if an inflammatory response occurs in a critical tissue such as the placenta or brain. Other immune responses may help the parasite become established in the host, e.g.: interferon gamma (IFN␥) and other proinflammatory cytokines are thought to be a trigger for T. gondii to differentiate into the bradyzoite stage which can persist within tissue cysts for the lifetime of the host (Frenkel, 2000).

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Therefore with the wide range of potential outcomes of the host parasite interaction it is important to understand which immune responses are critical in host protection, i.e. those that will protect the host against the invading pathogen and those regulatory immune responses that protect against immune-mediated pathology. An effective vaccine will act to stimulate host protection without exacerbating immunopathology (Innes et al., 2005). In terms of induction of protective immunity against protozoan parasites, live vaccines have been highly successful. These vaccines work well for diseases such as Tropical Theileriosis, East Coast Fever and Toxoplasmosis where animals recovering from a primary infection with the pathogen develop good immunity to protect them against disease following a secondary challenge (Buxton and Innes, 1995; Morrison and McKeever, 2006). A live vaccination approach is also much more likely to induce T-cell mediated immune responses through correct intracellular processing and presentation of antigens in association with MHC class I and Class II antigens. T-cell responses are considered critical to protect against intracellular pathogens and live vaccination approaches more closely mimic the induction of both the innate and adaptive immune responses that would occur in natural infection and will therefore induce appropriate inflammatory and regulatory immune responses in the host animals. 2.1. Drug attenuated infection for T. parva Several strategies have been used to develop live vaccines against protozoal parasites. T. parva is transmitted by ticks and causes an acute often fatal lymphoproliferative disease in cattle (Irvin and Morrison, 1987). A live vaccination technique developed in the 1970s involved an infection and treatment strategy where cattle were inoculated with defined doses of cryopreserved sporozoites and a simultaneous administration of a long acting tetracycline (Radley et al., 1975). While treatment of animals with tetracyclines has little efficacy once clinical signs are apparent, research showed that administration of the drug at the time of infection would attenuate parasite development in the host allowing the immune system time to cope with the infection and the animals would be solidly immune to a homologous challenge (Brocklesby and Bailey, 1965; Morzaria et al., 1987). This system has been used for many years to successfully protect cattle in East Africa from the consequences of infection with T. parva. There are some practical constraints on widespread use of this vaccination strategy as there is a requirement to prepare batches of sprorozoites from infected tacks and to test their potency by titration in cattle. There is also a danger of contamination of the vaccine with other pathogens as it is prepared from material derived from cattle (Katzer et al., 2007). The vaccine requires a cold chain for distribution as the sprorozoites have to be administered rapidly after thawing from liquid nitrogen (Morrison and McKeever, 2006). 2.2. Theileria schizont infected cell line immunisation A different live immunisation strategy has been adopted to protect cattle against tropical theileriosis caused by

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infection with the parasite T. annulata. Following invasion of host leucocytes by Theileria sporozoites the parasites differentiate into the schizont stage and induce clonal proliferation of the infected leucocyte with synchronous multiplication of the parasite (Hulliger et al., 1964). This synchronous division of parasite and cell enables the parasite to proliferate and multiply while remaining in an intracellular location. The development of tissue culture methods where bovine leucocytes could be transformed in vitro using sporozoites and maintained by regular passage in the laboratory opened up the possibility of using Theileria transformed cell lines as vaccines (Tsur and Adler, 1962; Brown et al., 1973). Several different laboratories growing T. annulata transformed cell lines observed that long term culture in vitro resulted in attenuation of virulence and that immunisation of naïve cattle with such cell lines induced protective immunity to a virulent challenge (Pipano, 1977, 1981; Shkap et al., 2007; Dargouth, 2008). Attenuation is achieved when inoculated schizonts no longer produce clinical symptoms in cattle and no piroplasm stages of the parasite are detected (Shkap et al., 2007). Cell line vaccines have been used successfully in a number of countries, e.g.: Israel, Iran, Morocco, Tunisia, India, China and Uzbekistan to protect cattle against tropical theileriosis. The ability to cryopreserve Theileria infected cells allows the preparation of frozen master, working and production seed materials to prepare mass cultures for vaccination purposes. The production of cell lines vaccines to protect against T. annulata infection involves extensive culturing and animal testing to check for virulence and there is also the danger of indigenous ticks picking up the vaccine strains during feeding on vaccinated cattle. Some cloned T. annulata cell lines have been produced which have impaired ability to differentiate into merozoites and therefore may pose less of a risk for tick pick up in the field situation (Shiels, 1999). 2.3. MHC incompatibility between vaccine and host While cell line immunisation worked well for T. annulata it was not so successful with T. parva. A requisite of cell line immunisation is that the parasite must establish infection within the cells of the host in order to stimulate Tcell mediated protective immune responses. Therefore the intracellular schizont stage of the parasite from the immunising cell line must transfer and establish itself within the immunised animals own cells (Wilde et al., 1966; Emery et al., 1982). Following up these observations, studies were conducted to look at the effect of histocompatibility differences between cell line and recipient. It was observed that it was considerably easier to immunise cattle with autologous T. parva infected cell lines than allogeneic cell lines (Morrison et al., 1981). In experiments where the MHC class I antigens of the donor cell line were matched to the recipient animal successful induction of protective immunity occurred suggesting that there was a histocompatibility barrier to cell line immunisation with T. parva (Dolan et al., 1984). The MHC relationship between cell line and recipient was not found to be a barrier to immunisation with T. annulata (Innes et al., 1988).

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The answer to this difference may lie in the fact that T. parva parasites prefer to infect and multiply within Tcells, whereas T. annulata infects macrophages and B-cells (Spooner et al., 1989). Following inoculation of an allogeneic infected cell line, the animal makes a cellular graft rejection response directed against the MHC antigens of the donor cell line (Innes et al., 1989). Macrophages phagocytose whatever cell debris is left after the destruction of the foreign cells by the immune system. If this cell debris contains T. annulata macroschizonts then the transfer from inoculum to host is complete as T. annulata can survive and multiply within macrophages. However, in the same situation, T. parva macroschizonts are unable to survive within macrophages and parasite transfer to host cells would not take place and protective immunity would not be induced. Cell line vaccination has been used very successfully in many countries to protect against Tropical Theileriosis and although it requires a cold chain for its delivery and facilities to culture and maintain parasite stocks, the difficulties in superceding this vaccine approach with an alternative sub unit vaccine suggest that these live vaccines will continue to offer livestock producers a real practical solution to control this devastating disease (Dargouth, 2008). 2.4. Incomplete life cycle strains as vaccines for congenital toxoplasmosis Live attenuated strains of T. gondii have been used to immunise breeding ewes to help protect against congenital disease (Innes et al., 2009). T. gondii is recognised as a major cause of abortion in ewes in temperate regions of the world such as Europe, Scandinavia, New Zealand where climatic conditions favour survival of the infective oocyst (Buxton and Rodger, 2008). Cats are the definitive host of the parasite and shed millions of T. gondii oocysts in their faeces following a primary infection. Once shed the oocysts sporulate in the environment and are infectious to people or animals following accidental consumption. Disease may occur in the ewes if they become infected for the first time during pregnancy and the parasite may invade and establish within placental and foetal tissues (Buxton, 1990). Following infection with Toxoplasma, animals develop protective immunity against disease in a subsequent pregnancy therefore making vaccination a feasible option to control the disease (Innes and Vermeulen, 2006). Following ingestion of oocysts, T. gondii sporozoites excyst in the gut and invade and establish within the cells of the host. Within host cells the parasites differentiate into tachyzoites where they multiply by a process of endodyogeny finally resulting in rupture of the parasitized cell and release of the tachyzoites to invade and infect other cells (Ferguson, 2009). Following the initial invasion of the host cells by the parasite the innate and the adaptive immune responses are activated and the intracellular nature of the parasite means that cell mediated immune responses involving T-cells are important in protective immunity (Innes and Wastling, 1995). As a result of the activation of the host cell mediated immune response the parasite responds by differentiating into the slower replicating bradyzoite stage which persists within host tissues. Consumption of undercooked meat from T. gondii

infected food animals is a major route of transmission to people (Tenter et al., 2000) where the parasite may cause severe disease in the developing foetus in pregnant women, acute sometimes fatal infection in immunocompromised individuals and ocular disease and potentially behavioural disorders in immunocompetant individuals (McAllister, 2005). Therefore vaccines for T. gondii may have several different targets. These would include, development of vaccines to limit acute parasitaemia and thus protect against congenital toxoplasmosis; vaccines to limit the development of tissue cysts in food animals and therefore help protect against transmission to people and vaccines to help prevent oocyst shedding in cats to help limit environmental contamination and transmission of T. gondii to intermediate hosts (Innes et al., 2009). The S48 strain of T. gondii was originally isolated from an aborted ovine foetus in New Zealand and was passaged over 3000 times in laboratory mice initially to provide a source of antigen for diagnostic purposes. Over time the technicians in the lab noticed that the characteristics of the parasite had changed and this laboratory adapted strain had lost the ability to differentiate into bradyzoites and therefore would not persist in vivo (Buxton, 1993). These characteristics made the S48 strain an excellent candidate as a vaccine as it would undergo limited multiplication within the host animal thus stimulating appropriate cell mediated immune responses but it would not persist. The vaccine was found to protect ewes against Toxoplasma induced abortion in New Zealand (O’Connell et al., 1988; Wilkins et al., 1988). Further work looking in more detail at the immune responses induced following infection of sheep with the S48 strain of T. gondii found that the early immune responses involved interferon gamma (IFN␥) and CD4+ T-cells with a switch to CD8+ T-cells predominating during the recovery phase of an acute infection (Innes and Wastling, 1995). Studies on the duration of immunity following immunisation found that ewes were still solidly immune to challenge 18 months after inoculation of S48 T. gondii (Buxton et al., 1993). This live vaccine Toxovax® is currently the only commercial vaccine for toxoplasmosis worldwide. Similar to the cell line vaccines for T. annulata, this vaccine requires a cold storage for its delivery and has a short shelf life. In addition, as the mechanisms involved in causing the S48 strain to become an “incomplete” strain are not fully understood, there is always a chance that the strain could change its characteristics. 2.5. Live immunisation in bovine neosporosis While there are no live vaccines commercially available to help protect against bovine neosporosis the efficacy of such an approach has been tested experimentally. N. caninum is transmitted to cattle through ingestion of oocysts, shed by infected dogs, or by vertical transmission from dam to calf (Dubey, 2003). The disease manifests during pregnancy where Neospora tachyzoites in the circulation, invade and multiply within the placenta and then onto the foetus where the outcome can be foetal death or the birth of live congenitally infected animals (Buxton et al.,

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2002). A characteristic feature of N. caninum infection in cattle is the high rate of vertical transmission which often occurs over several generations and in successive pregnancies (Bjorkman et al., 1996; Davison et al., 1999). This high rate of vertical transmission may occur due a recrudescence of a persistent infection or be due to a new infection and it suggests that cattle do not develop very good immunity to protect against vertical transmission of the parasite (Innes et al., 2002). However, observations from field cases of bovine neosporosis suggest that cattle that have experienced abortion due to Neospora infection are less likely to abort again due to the same infectious agent (Wouda et al., 1998) and that cattle that had been previously exposed to Neospora were significantly less likely to suffer abortions during a point source exposure of the herd compared with naïve cattle (McAllister et al., 2000). Taken together, these observations suggest that cattle develop some immunity to the parasite following exposure and that vaccination may be a feasible option to control the disease. Infection of naïve cattle prior to mating with live N. caninum tachyzoites was found to protect against a challenge administered in mid-gestation that resulted in vertical transmission of the parasite in naive control cattle (Innes et al., 2001). A similar live infection prior to mating using a sheep model afforded protection against abortion following a challenge administered in mid gestation (Buxton et al., 2001). Administration of live tachyzoites prior to mating protected cattle against abortion following a challenge administered at day 70 of gestation (Williams et al., 2007). Interestingly in the same study another group of animals were immunised with a killed tachyzoite lysate material and this inoculum did not afford any protection against the challenge, emphasising the efficacy of the live vaccine approach in this context.

2.6. Attenuated strains of N. caninum There has also been interest in identifying strains of Neospora that are attenuated as regards virulence and pathogenicity in vivo as these could be useful vaccine strains. The NC-Nowra strain isolated from an infected calf in Australia was found to produce fewer clinical signs and lesions in the CNS of experimentally infected mice in comparison to a more virulent strain NC-Liverpool (Miller et al., 2002). Other Neospora isolates collected from cases in Spain have also been found to have attenuated virulence in vivo using mouse models (Regidor-Cerrillo et al., 2008). Repeated passage of Neospora tachyzoites in tissue culture resulted in attenuation of virulence in vivo (Bartley et al., 2006). These attenuated strains of Neospora may be potential candidates for live vaccination approaches to help protect cattle against Neospora associated abortion. A key requirement of these strains would be that they should not persist in the animals as there is such a high likelihood of vertical transmission with Neospora that there is a real danger that the “vaccine” strain may transmit to the foetus resulting in the birth of a congenitally infected animal.

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3. Killed vaccine approaches The success of the live vaccination approach against many intracellular protozoan parasites reflects the fact that protective immune responses involve the induction of CD4+ and CD8+ T-cell responses which require both endogenous antigen processing and presentation of antigens in association with MHC class I antigens as well as exogenous processing and presentation with MHC class II antigens. The challenge in developing effective killed vaccines is to select relevant parasite antigens and then deliver them to the immune system in such a form as to enable appropriate processing and presentation to the host immune system to induce effective innate and adaptive immune responses (Innes and Vermeulen, 2006). In this section we will review some of the approaches used to develop killed vaccines against protozoan parasitic diseases of livestock. 3.1. Theileria sporozoite antigens Immunodominant antigens on the surface of both T. parva and T. annulata sprorozoites have been identified using serum antibodies from immune cattle. Antibodies recognising these antigens were found to be capable of neutralising sporozoite infection of host cells (Morrison and McKeever, 2006). The T. parva antigen has a relative molecular mass of 67 kDa and is known as p67 (Nene et al., 1996). Recombinant forms of p67 have been tested experimentally in cattle and found to result in partial protection against acute challenge (Ballingall et al., 2004). Delivering the p67 antigen using live antigen delivery systems such as recombinant vaccinia (Honda et al., 1998) and Salmonella typhimurium (Gentschev et al., 1998) did not significantly improve efficacy of this vaccine approach. The immunodominant sporozoite gene identified for T. annulata, SPAG-1 is polymorphic between different parasite stocks (Katzer et al., 1994). Recombinant forms of the antigen have been tested in vivo and while they have been found to attenuate the effects of a virulent challenge, the vaccine preparations have not afforded good protection (Boulter et al., 1999). 3.2. Theileria schizont antigens Research into the critical protective immune responses in both T. parva and T. annulata infections of cattle have emphasised the importance of both CD4+ and CD8+ T-cells in immunity against the intracellular macroschizont stage of the parasites (Eugui and Emery, 1981; Morrison et al., 1987; Brown et al., 1989; Innes et al., 1989; McKeever et al., 1994). Induction of IFN␥ by immune CD8+ T-cells following antigen stimulation were employed as biological screening tools to identify T-cell immunodominant T. parva schizont antigens (Graham et al., 2006). The ability of these schizont antigens to induce protective immunity in cattle was tested using prime boost immunisation techniques with plasmid DNA or recombinant canary pox viruses followed by a boost with recombinant vaccinia viruses. While animals developed CD8+ T-cell IFN␥ responses, cytotoxic CD8+ T-cells responses were not induced as effectively. Immu-

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nisation using these protocols did modify the severity of disease following challenge which is likely to be due to effective induction of CD8+ T-cell responses (Graham et al., 2006). 3.3. Killed vaccines for T. gondii Killed vaccine approaches to help protect against T. gondii infection in farm animals have not proved as successful as the live vaccination approaches. Toxoplasma antigens incorporated into immunostimulating complexes (ISCOMS) did not induce protection against T. gondii induced abortion in sheep (Buxton et al., 1989). A crude fraction of T. gondii rhoptry proteins incorporated into an ISCOM adjuvant only afforded partial protection of pigs against development of T. gondii tissue cysts (Garcia et al., 2005). Protective immunity to T. gondii involves CD4+, CD8+ and IFN␥, therefore killed vaccine approaches would have to be able to induce these cell mediated immune responses in vivo to be effective. A recent study examined immune responses induced in pigs following intradermal inoculation with a T. gondii GRA-1-GRA-7 DNA cocktail (Jongert et al., 2008). The pigs developed strong specific humoral and Th1 type cell-mediated immune responses and IFN␥ which was encouraging for the development of novel killed vaccine approaches for T. gondii in food animals. The dense granule organelles of T. gondii are secretory vesicles that play a major role in the formation of the parasitophorous vacuole which enables T. gondii to parasitise and survive within host cells, therefore making these antigens attractive vaccine candidates (Cesbron-Delauw, 1994). 3.4. Killed vaccine for N. caninum A killed vaccine comprising Neospora tachyzoites formulated with an adjuvant (Havlogen), Bovilis® Neoguard is commercially available in USA, New Zealand and some other countries to help reduce Neospora associated abortion in cattle (Schetters et al., 2004). The vaccine is administered on 2 occasions prior to mating and during the first trimester of pregnancy, with recommended annual booster doses. Experimental studies using the vaccine preparation showed that the cattle did produce both a humoral and cell-mediated immune response following immunisation, however following challenge, all of the animals in the study whether vaccinated or controls had infected foetuses (Andrianarivo et al., 2000). This showed that under the challenge conditions used in this study the vaccine had not induced effective protective immunity. When the vaccine was tested using field challenge in Costa Rica it showed around 50% protection against Neospora associated abortions (Schetters et al., 2004). In New Zealand, again under field conditions an overall abortion rate of 4.3% was reported in vaccinated cattle compared to 5.7% in non-vaccinated cattle (Schetters et al., 2004). Killed lysate antigens from Nc-Nowra strain of Neospora were used to immunise cattle, but did not protect against abortion following challenge (Williams et al., 2007).

3.5. Antigen identification There has been considerable research interest in identifying relevant Neospora antigens that may be used as killed vaccine candidates. Parasite antigens that may be involved in invasion and survival in the host such as those secreted from micronemes, rhopteries or dense granules or those antigens that are abundant on the surface of tachyzoites have been highlighted (Hemphill et al., 1999; Jenkins, 2001; Ellis et al., 2003; Mercier et al., 2005). Understanding critical protective immune responses may also be important in selecting relevant antigens. Several studies have shown that antibodies specific for certain Neospora antigens can inhibit cell invasion, at least in vitro (Nishikawa et al., 2000; Haldorson et al., 2006). As T-cells are known to be important in protective immunity to N. caninum several research groups have also used bovine T-cells as bio-indicators to select relevant Neospora antigens (Marks et al., 1998; Staska et al., 2005; Tuo et al., 2005). As N. caninum, like T. gondii, is an obligate intracellular pathogen and protective immunity involves induction of cell mediated immune responses a major challenge in developing a killed vaccine will be to deliver the selected antigens in such a way as to stimulate appropriate immune responses. Some promising studies have been reported using live vector systems such as recombinant vaccinia virus (Nishikawa et al., 2001) and Brucella abortus expressing some microneme and dense granule antigens of Neospora (Ramamoorthy et al., 2007) using mouse models of N. caninum infection. 3.6. Passive transfer of immunity to cryptosporidiosis Cryptosporidiosis is an important infectious disease of young farm livestock and humans worldwide where infection with Cryptosporidium parasites can cause a severe, sometimes fatal diarrhoeal illness (Casemore et al., 1997). There are few if any effective therapies or treatments for Cryptosporidiosis on the farm although Halocur® is now licenced in the UK for the prevention of diarrhoea caused by Cryptosporidium parvum in newborn calves. However it has to be administered by a veterinary surgeon as it is contraindicated in calves that already have diarrhoea due to its toxicity in dehydrated animals. Cryptosporidium infection can occur in young neonates 2–10 days after birth and the main clinical sign, i.e.: profuse watery diarrhoea is accompanied by shedding of large numbers of sporulated oocysts which can be infective for other animals (De Graaf et al., 1999a). As animals may be exposed to oocysts immediately they are born an active vaccination approach to prevent disease is unlikely to be successful. In addition as young ruminants have a syndesmochorial placenta which does not allow the transplacental transfer of maternal immunoglobulin, neonates are born without antibodies. The young ruminants obtain immunoglobulin from the mother via colostrum as they can absorb the antibodies across the intestine for the first 48 h after birth (Watson et al., 1994). Therefore as the young ruminants may have circulating maternal antibodies against Cryptosporidium it may be difficult to induce specific immune responses through vaccination.

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Calves experimentally immunised with killed C. parvum oocysts showed reduced levels of diarrhoea and oocyst shedding compared with control calves (Harp and Goff, 1995). However, the vaccine did not prove to be efficacious when tested under field conditions (Harp and Goff, 1998). Another more promising strategy is to immunise dams a few weeks prior to parturition to generate hyperimmune colostrum containing high titres of specific antibodies. A group of Holstein cows were immunised three times in late gestation with recombinant C. parvum, C7 protein containing the 101C terminal of the P23 antigen. Colostrum was collected from these “immune” cows along with control cows and the different colostrums were fed to groups of calves. The calves were then challenged orally with C. parvum oocysts and the calves receiving the immune colostrum were protected against diarrhoea and showed a significant reduction in oocyst shedding compared to control animals (Perryman et al., 1999). This approach as well as being of benefit to control disease in livestock will also benefit public health through reducing environmental contamination with Cryptosporidium oocysts (Jenkins, 2004). A recent study looked at the antibody responses in calves fed colostrum from dams vaccinated with a recombinant C. parvum oocyst surface protein rCP15/60 (Burton et al., 2011). This study showed that the calves had measurable quantities of the specific antibody in their serum indicating that the passive immunity approach to vaccination may be suitable to help prevent cryptosporidioses in livestock (Burton et al., 2011). The genome of C. parvum has recently been published along with analysis of the expressed protein repertoire of the excysted oocyst/sporozoite antigenic material (Sanderson et al., 2008). This may help to identify new vaccine targets that may be directed against important parasite antigens that are involved in host invasion and survival. However the most feasible approach to vaccinate young neonatal farm livestock against cryptosporidiosis is likely to involve the passive immunisation approach (De Graaf et al., 1999b). 4. Concluding remarks Protozoan parasitic diseases of livestock cause significant economic losses to producers worldwide and in the case of zoonotic pathogens, also pose a considerable public health risk. Their intracellular habitat and complex life cycles within the livestock host species presents a challenge for developing immunological control strategies. As discussed above considerable efforts have gone into understanding the host protective immune responses and discovering the precise parasite antigens that induce these protective responses. However, it has proved difficult to deliver these protective antigens within an adjuvant or other delivery vector to induce appropriate priming of the immune response that will protect livestock against field challenge with the wild type pathogens. On the other hand, considerable success in controlling protozoal livestock diseases has been achieved using a live vaccine approach. The beauty of using a live vaccine is that is does all the hard work for you as it induces the appropriate innate and adaptive immune responses required to induce long term protective immunity. Critics of live vac-

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cines cite safety risks, short shelf life and the need for a cold chain to deliver the vaccines in the field. However, these are all problems that are surmountable especially as live vaccines have a distinct advantage over killed counterparts, they actually work! Effective vaccines are desperately needed to help protect livestock, it is estimated that one animal dies every 30 s from East Coast fever. This need, combined with solutions being available in the form of live vaccination approaches has caused many researchers and animal health companies to re-evaluate the use and delivery of live vaccines to combat protozoal diseases of livestock. A recent example is the work of a private company VetAgro based in Tanzania that is working together with the Maasai cattle herders and have successfully vaccinated over half a million cattle against East Coast fever reducing mortality in their herds by 95% (Di Giulio et al., 2009). The live vaccine, based on the infection and treatment method discovered in the 1970s, has recently been registered in Africa by GALVmed, the global alliance for livestock veterinary medicines, funded by the Bill and Melinda Gates Foundation. Therefore if we are serious about delivering solutions to control these important intracellular protozoal diseases of livestock we should perhaps turn our attention back to live vaccines and consider how we may improve sustainable production of quality antigenic material, safety and delivery in the field. Conflict of interest statement None of the authors involved in this manuscript have any financial or personal relationships with other people or organisations that could inappropriately influence their work. Acknowledgement The authors would like to acknowledge the support of the Scottish Government, the Moredun Foundation Innovation Fund and Creative Science Company. References Andrianarivo, A.G., Rowe, J.D., Barr, B.C., Anderson, M.L., Packham, A.E., Sverlow, K.W., Choromanski, L., Loui, C., Grace, A., Conrad, P.A., 2000. A POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation failed to prevent foetal infection in pregnant cattle following i.v/i.m. experimental tachyzoite challenge. Int. J. Parasitol. 30, 985–990. Ballingall, K.T., Lutai, A., Rowlands, G.J., Sales, J., Musoke, A.J., Morzaria, S.P., McKeever, D.J., 2004. Bovine leucocyte antigen major histocompatibility complex class II DRB3*2703 and DRB3*1501 allelles are associated with variation in levels of protection against Theileria parva challenge following immunisation with the sporozoite p67 antigen. Infect. Immun. 72, 2738–2741. Bartley, P.M., Wright, S., Sales, J., Chianini, F., Buxton, D., Innes, E.A., 2006. Long term passage of tachyzoites in tissue culture can attenuate virulence of Neospora caninum in vivo. Parasitology 133, 421–432. Bjorkman, C., Johansson, O., Stenlund, S., Holmdahl, J., Uggla, A., 1996. Neospora species infection in a herd of dairy cattle. J. Am. Vet. Med. Assoc. 208, 1441–1444. Boulter, N., Brown, D., Wilkie, G., Williamson, S., Kirvar, E., Knight, P., Glass, E., Campbell, J., Morzaria, S., Nene, V., Musoke, A., d’Oliveira, C., Gubbels, M.J., Jongejan, F., Hall, R., 1999. Evaluation of recombinant sporozoite antigen SPAG-1 as a vaccine candidate against Theileria annulata by the use of different delivery systems. Trop. Med. Int. Health 4, 71–77.

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E.A. Innes et al. / Veterinary Parasitology 180 (2011) 155–163

Brocklesby, D.W., Bailey, K.P., 1965. The immunization of cattle against East Coast Fever (Theileria parva infection) using tetracyclines: a review of the literature and a reappraisal of the method. Bull. Epiz. Dis. Afr. 13, 161–168. Brown, C.G.D., Stagg, D.A., Purnell, R.E., Kanhai, G.K., Payne, R.C., 1973. Infection and transformation of bovine lymphoid cells in vitro by infective particles of Theileria parva. Nature 245, 101–103. Brown, W.C., Sugimoto, C., Grab, D.J., 1989. Theileria parva: bovine helper T cell clones specific for both infected lymphocytes and schizont membrane antigens. Exp. Parasitol. 69, 234–248. Burton, A.J., Nydam, D.V., Jones, G., Zambriski, J.A., Linden, T.C., Cox, G., Davis, R., Brown, A., Bowman, D.D., 2011. Antibody responses following administration of a Cryptosporidium parvum rCP15/60 vaccine to pregnant cattle. Vet. Parasitol. 175, 178–181. Buxton, D., 1990. Ovine toxoplasmosis: a review. J. R. Soc. Med. 83, 509–511. Buxton, D., 1993. Toxoplasmosis: the first commercial vaccine. Parasitol. Today 9, 335–337. Buxton, D., Innes, E.A., 1995. A commercial vaccine for ovine toxoplasmosis. Parasitology 110, 11–16. Buxton, D., McAllister, M.M., Dubey, J.P., 2002. The comparative pathogenesis of neosporosis. Trends Parasitol. 18, 546–552. Buxton, D., Rodger, S.M., 2008. Toxoplasmosis and neosporosis. In: Aitken, I.D. (Ed.), Diseases of Sheep. , 4th ed. Wiley-Blackwell, Hoboken, pp. 112–118. Buxton, D., Thomson, K.M., Maley, S., Wright, S., Bos, H.J., 1993. Experimental challenge of sheep 18 months after vaccination with a live (S48) Toxoplasma gondii vaccine. Vet. Rec. 133, 310–312. Buxton, D., Uggla, A., Lovgren, K., Thomson, K., Lunden, A., Morein, B., Blewett, D.A., 1989. Trial of a novel experimental Toxoplasma iscom vaccine in pregnant sheep. Br. Vet. J. 145, 451–457. Buxton, D., Wright, S., Maley, S.W., Rae, A.G., Lunden, A., Innes, E.A., 2001. Immunity to experimental neosporosis in pregnant sheep. Parasit. Immunol. 23, 85–91. Casemore, D.P., Wright, S.E., Coop, R.L., 1997. Cryptosporidiosis: human and animal epidemiology. In: Fayer, R. (Ed.), Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Racon, pp. 65–92. Cesbron-Delauw, M.F., 1994. Dense granule organelles of Toxoplasma gondii: their role in the host parasite relationship. Parasitol. Today 10, 293–296. Dargouth, M.A., 2008. Review on the experience with live attenuated vaccines against tropical theileriosis in Tunisia: considerations for the present and implications for the future. Vaccine, G4–G10. Davison, H.C., Otter, A., Trees, A.J., 1999. Estimation of vertical and horizontal transmission parameters of Neospora caninum infections in dairy cattle. Int. J. Parasitol. 29, 1683–1689. De Graaf, D.C., Vanopdenbosch, E., Ortega-Mora, L.M., Abbassi, H., Peeters, J.E., 1999a. A review of the importance of cryptosporidiosis in farm animals. Int. J. Parasitol. 29, 1269–1287. De Graaf, D.C., Spano, F., Petry, F., Sagodira, S., Bonnin, A., 1999b. Speculation on whether a vaccine against cryptosporidiosis is a reality or fantasy. Int. J. Parasitol. 29, 1289–1306. Di Giulio, G., Lynen, G., Morzaria, S.P., Oura, C., Bishop, R., 2009. Live immunisation against East Coast fever—current status. Trends Parasitol. 25, 85–92. Dolan, T.T., Teale, A.J., Stagg, D.A., Kemp, S.J., Cowan, K.M., Young, A.S., Groocock, C.M., Leitch, B.L., Spooner, R.L., Brown, C.G.D., 1984. A histocompatibility barrier to immunization against East Coast Fever using Theileria parva infected lymphoblastiod cell lines. Parasit. Immunol. 6, 243–250. Dubey, J.P., 2003. Neosporosis in cattle. J. Parasitol. 89, 42–56. Ellis, J.T., Morrison, D.A., Reichel, M.P., 2003. Genomics and its impact on parasitology and the potential for development of new parasite control methods. DNA Cell. Biol. 22, 395–403. Emery, D.L., Morrison, W.I., Buscher, G., Nelson, R.T., 1982. Generation of cell-mediated cytotoxicity to Theileria parva (East Coast Fever) after inoculation of cattle with parasitized lymphoblasts. J. Immunol. 128, 195–200. Eugui, E.M., Emery, D.L., 1981. Genetically restricted cell-mediated cytotoxicity in cattle immune to Theileria parva. Nature 290, 251–254. Ferguson, D.J., 2009. Toxoplasma gondii: 1908–2008, homage to Nicolle, Manceaux and Splendore. Mem. Inst. Oswaldo Cruz. 104, 133–148. Frenkel, J.K., 2000. Biology of Toxoplasma gondii. In: Ambroise-Thomas, P., Peterson, E. (Eds.), Congenital Toxoplasmosis. Springer-Verlag, France, pp. 9–25. Garcia, J.L., Gennari, S.M., Navarro, I.T., Machado, R.Z., Sinhorini, I.L., Freie, R.L., Marana, E.R., Tsutsui, V., Contente, A.P., Begale, L.P., 2005. Partial protection against tissue cyst formation in pigs vaccinated with

crude rhoptery proteins of Toxoplasma gondii. Vet. Parasitol. 129, 209–217. Gentschev, I., Glaser, I., Goebel, W.I., McKeever, D.J., Musoke, A.J., Heussler, V.T., 1998. Delivery of the p67 sporozoite antigen of Theileria parva using recombinant Salmonella: secretion of the antigen enhances specific antibody responses in cattle. Infect. Immun. 66, 2660–2664. Graham, S.P., Pelle, R., Honda, Y., Mwangi, D.M., Tonukari, N.J., Yamage, M., Glew, E.J., de Villiers, E.P., Shah, T., Bishop, R., Abuya, E., Awino, E., Gachanja, J., Luyai, A.E., Mbwika, F., Muthiani, A.M., Ndegwa, D.M., Njahira, M., Nyanui, J.K., Onono, F.O., Osaso, J., Saya, R., Wildmann, M., Fraser, C., Maudlin, C.M., Gardner, I., Morzaria, M.J., Loosmore, S.P., Gilbert, S., Audonnet, S.C., van der, J.C., Nene, B.P., Taracha, V.E., 2006. Theileria parva candidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 103, 3286–3291. Haldorson, G.J., Stanton, J.B., Mathison, B.A., Suarez, C.E., Baszler, T.V., 2006. Neospora caninum: antibodies directed against tachyzoite surface protein NcSRS2 inhibit parasite attachment and invasion of placental trophoblasts in vitro. Exp. Parasitol. 112, 172–178. Harp, J.A., Goff, J.P., 1995. Protection of calves with a vaccine against Cryptosporidium parvum. J. Parasitol. 81, 54–57. Harp, J.A., Goff, J.P., 1998. Strategies for the control of Cryptosporidium parvum infection in calves. J. Dairy Sci. 81, 289–294. Hasler, B., Stark, K.D., Sager, H., Gottstein, B., Reist, M., 2006. Simulating the impact of four control strategies for the control of Neospora caninum in Swiss dairy cattle. Prev. Vet. Med. 77, 254–283. Hemphill, A., Fuchs, N., Sonda, S., Hehl, A., 1999. The antigenic composition of Neospora caninum. Int. J. Parasitol. 29, 1175–1188. Honda, Y., Waithaka, M., Taracha, E.L.N., Duchateau, L., Musoke, A.J., McKeever, D.J., 1998. Delivery of Theileria parva p67 antigen to cattle using recombinant vaccinia virus: IL2 enhances protection. Vaccine 16, 1276–1282. Hulliger, L., Wilde, J.K.H., Brown, C.G.D., Turner, L., 1964. Mode of multiplication of Theileria in cultures of bovine lymphocytic cells. Nature 203, 728–730. Innes, E.A., 2010. Vaccination against Toxoplasma gondii: an increasing priority for collaborative research? Expert Rev. Vaccines 9, 1117–1119. Innes, E.A., Andrianarivo, A.G., Bjorkman, C., Williams, D.J.L., Conrad, P.A., 2002. Immune responses to Neospora caninum and prospects for vaccination. Trends Parasitol. 18, 497–504. Innes, E.A., Bartley, P.M., Maley, S., Katzer, F., Buxton, D., 2009. Veterinary vaccines against Toxoplasma gondii. Mem. Inst. Oswaldo Cruz. 104, 246–251. Innes, E.A., Millar, P., Brown, C.G.D., Spooner, R.L., 1989. The development and specificity of cytotoxic cells in cattle immunised with autologous or allogeneic Theileria annulata infected lymphoblastoid cell lines. Parasit. Immunol. 11, 57–68. Innes, E.A., Ouhelli, H., Oliver, R.A., Simpson, S.P., Brown, C.G.D., Spooner, R.L., 1988. The effect of MHC compatibility between parasite infected cell line and recipient in immunisation against tropical theileriosis. Parasit. Immunol. 10, 47–56. Innes, E.A., Vermeulen, A.N., 2006. Vaccination as a control strategy against the coccidial parasites Eimeria, Toxoplasma and Neospora. Parasitology 133, 145–168. Innes, E.A., Wastling, J.M., 1995. Analysis of in vivo immune responses during Toxoplasma gondii infection using the technique of lymphatic cannulation. Parasitol. Today 11, 268–271. Innes, E.A., Wright, S.E., Bartley, P.M., Maley, S., MacAldowie, C., EstebanRedondo, I., Buxton, D., 2005. The host–parasite relationship in bovine neosporosis. Vet. Immunol. Immunopathol. 108, 29–36. Innes, E.A., Wright, S.E., Maley, S., Rae, A., Schock, A., Kirvar, E., Bartley, P.M., Hamilton, C., Carey, I.M., Buxton, D., 2001. Protection against vertical transmission in bovine neosporosis. Int. J. Parasitol. 31, 1523–1534. Irvin, A.D., Morrison, W.I., 1987. Immunopathology immunology and immunoprophylaxis of Theileria infections. In: Soulsby, E.J.L. (Ed.), Immune Responses in Parasitic Infections: Immunology, Immunopathology and Immunoprophylaxis. CRC Press, Boca Raton, FL, pp. 223–274. Jenkins, M.C., 2001. Advances and prospects for subunit vaccines against protozoa of veterinary importance. Vet. Parasitol. 101, 291–310. Jenkins, M.C., 2004. Present and future control of Cryptosporidiosis in humans and animals. Expert Rev. Vaccines 3, 669–671. Jongert, E., Melkebeek, V., De Craeye, S., Dewit, J., Verhelst, D., Cox, E., 2008. An enhanced GRA1-GRA7 cocktail DNA vaccine primes anti-Toxoplasma immune responses in pigs. Vaccine 26, 1025–1031. Katzer, F., Carrington, M., Knight, P., Williamson, S., Tait, A., Morrison, W.I., Hall, R., 1994. Polymorphism of SPAG-1, a candidate antigen for inclu-

E.A. Innes et al. / Veterinary Parasitology 180 (2011) 155–163 sion in a subunit vaccine against Theileria annulata. Mol. Biochem. Parasitol. 67, 1–10. Katzer, F., Ngugi, D., Schnier, C., McKeever, D.J., 2007. Influence of host immunity on parasite diversity in Theileria parva. Infect. Immun. 75, 4909–4916. Kortbeek, L.M., Hofhuis, A., Nijhuis, C.D., Havelaar, A.H., 2009. Congenital toxoplasmosis and DALYs in the Netherlands. Mem. Inst. Oswaldo Cruz 104, 370–373. Mackenzie, W.R., Hoxie, N.J., Proctor, M.E., Gradus, M.S., Blair, K.A., Peterson, D.E., Kazmierczak, J.J., Addis, D.G., Fox, K.R., Rose, J.B., Davis, J.P., 1994. A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N. Engl. J. Med. 331, 161–167. Marks, J., Lunden, A., Harkins, D., Innes, E.A., 1998. Identification of Neospora antigens recognised by CD4+ T-cells and immune sera from experimentally infected cattle. Parasit. Immunol. 20, 303–309. McAllister, M.M., 2005. A decade of discoveries in veterinary protozoology changes our concept of “subclinical” toxoplasmosis. Vet. Parasitol. 132, 241–247. McAllister, M.M., Bjorkman, C., Anderson-Sprecher, R., Rodgers, D.G., 2000. Evidence of a point source exposure to Neospora caninum and protective immunity in a herd of beef cows. J. Am. Vet. Med. Assoc. 217, 881–887. McKeever, D.J., Taracha, E.L.N., Innes, E.A., MacHugh, N.D., Awino, E., Goddeeris, B.M., Morrison, W.I., 1994. Adoptive transfer of immunity to Theileria parva in the CD8+ fraction of responding efferent lymph. Proc. Natl. Acad. Sci. U.S.A. 91, 1959–1963. Mead, J.R., 2010. Challenges and prospects for a Cryptosporidium vaccine. Future Microbiol. 5, 335–337. Mercier, C., Adjogble, K.D., Daubener, W., Cesbron-Delauw, M.F., 2005. Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexan parasites? Int. J. Parasitol. 35, 829–849. Miller, C.M., Quinn, H., Windsor, P.A., Ellis, J.T., 2002. Characterisation of the first Australian isolate of Neospora caninum from cattle. Aust. Vet. J. 80, 620–625. Morrison, W.I., Buscher, G., Murray, M., Emery, D.L., Masake, R.A., Cook, R.H., Wells, P.W., 1981. Theileria parva: kinetics of infection in the lymphoid system of cattle. Exp. Parasitol. 52, 248–260. Morrison, W.I., Goddeeris, B.M., Teale, A.J., Groocock, C.M., Kemp, S.J., Stagg, D.A., 1987. Cytotoxic T-cells elicited in cattle challenged with Theileria parva (Muguga): evidence for restriction by class I MHC determinants and parasite strain specificity. Parasit. Immunol. 9, 563–578. Morrison, W.I., McKeever, D.J., 2006. Current status of vaccine development against Theileria parasites. Parasitology 133, 169–187. Morzaria, S.P., Irvin, A.D., Taracha, E., Spooner, P.R., Voigt, W.P., Fuginaga, T., Katende, J., 1987. Immunisation against East Coast fever: the use of selected stocks of Theileria parva for immunization of cattle exposed to field challenge. Vet. Parasitol. 23, 23–41. Nene, V., Musoke, A., Gobright, E., Morzaria, S.P., 1996. Conservation of the sporozoite p67 vaccine antigen in cattle-derived Theileria parva stocks with different cross-immunity profiles. Infect. Immun. 64, 2056–2061. Nishikawa, Y., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2000. Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells. Int. J. Parasitol. 30, 51–58. Nishikawa, Y., Inoue, N., Xuan, X., Nagasawa, H., Igarashi, I., Fugisaki, K., Otsuka, H., Mikami, T., 2001. Protective efficacy of vaccination by recombinant vaccinia virus against Neospora caninum infection. Vaccine 19, 1381–1390. O’Connell, E., Wilkins, M.F., Te Punga, W.A., 1988. Toxoplasmosis in sheep. II. The ability of a live vaccine to prevent lamb losses after an intravenous challenge with Toxoplasma gondii. N. Z. Vet. J. 36, 1–4. Perryman, L.E., Kapil, S.J., Jones, M.L., Hunt, E.L., 1999. Protection of calves against cryptosporidiosis with immune bovine colostrum induced by a Cryptosporidium parvum recombinant protein. Vaccine 17, 2142–2149. Pipano, E., 1977. Basic principles of Theileria annulata control. In: Henson, J.B., Campbell, M. (Eds.), Theileriosis. Report of a Workshop Held in Nairobi, Kenya. IDRC, Ottawa, pp. 55–65.

163

Pipano, E., 1981. Schizonts and tick stages in immunization against Theileria annulata infection. In: Irvin, A.D., Cunningham, M.P., Young, A.S. (Eds.), Advances in the Control of Theileriosis. Martinus Nijhoff, The Hague, pp. 242–252. Radley, D.E., Young, A.S., Brown, C.G.D., Burridge, M.J., Cunningham, M.P., Musisi, F.L., Purnell, R.E., 1975. East Coast fever: 2. Cross-immunity trails with a Kenyan strain of Theileria lawrencei. Vet. Parasitol. 1, 43–50. Ramamoorthy, S., Sanakkayala, N., Vemulapalli, R., Jain, N., Lindsay, D.S., Schurig, G.S., Boyle, S.M., Sriranganathan, N., 2007. Prevention of vertical transmission of Neospora caninum in C57BL/6 mice vaccinated with Brucella abortus strain RB51 expressing N. caninum protective antigens. Int. J. Parasitol. 37, 1531–1538. Reichel, M.P., 2000. Neospora caninum infections in Australia and New Zealand. Aust. Vet. J. 78, 258–261. Reichel, M.P., Ellis, J.T., 2006. If control of Neospora caninum infection is technically feasible does it make economic sense? Vet. Parasitol. 142, 23–34. Reichel, M.P., Ellis, J.T., 2009. Neospora caninum—how close are we to development of an efficacious vaccine that prevents abortion in cattle? Int. J. Parasitol. 39, 1173–1187. Regidor-Cerrillo, J., Gomez-Bautista, M., Pereira-Bueno, J., Aduriz, G., Navarro-Lozano, V., Risco-Castillo, V., Fernandez-Garcia, A., Pedraza-Diaz, S., Ortega-Mora, L.M., 2008. Isolation and genetic characterization of Neospora caninum from asymptomatic calves in Spain. Parasitology 135, 1651–1659. Sanderson, S.J., Xia, D., Prieto, H., Yates, J., Hoises, M., Kissinger, J.C., Bromley, E., Lal, K., Sinden, R.E., Tomley, F., Wastling, J.M., 2008. Determining the protein repertoire of Cryptosporidium parvum sporozoites. Proteomics 8, 1398–1414. Schetters, T., Dubey, J.P., Adrianarivo, A., Frankena, K., Romero, J.J., Perez, E., Heuer, C., Nicholson, C., Russell, D., Weston, J., 2004. Intervet symposium: bovine neosporosis. Vet. Parasitol. 125, 137–146. Shiels, B.R., 1999. Should I stay or should I go now? A stochastic model of stage differentiation in Theileria annulata. Parasitol. Today 15, 241–245. Shkap, V., de Vos, A.J., Zweygarth, E., Jongejan, F., 2007. Attenuated vaccines for tropical theileriosis, babesiosis and heartwater: the continuing necessity. Trends Parasitol. 23, 420–426. Spooner, R.L., Innes, E.A., Glass, E.J., Brown, C.G.D., 1989. T. annulata and T. parva infect and transform different bovine mononuclear cells. Immunology 66, 284–288. Staska, L.M., Davies, C.J., Brown, W.C., McGuire, T.C., Suarez, C.E., Park, J.Y., Mathison, B.A., Abbott, J.R., Baszler, T.V., 2005. Identification of vaccine candidate peptides in the NcSRS2 surface protein of Neospora caninum by using CD4+ cytotoxic T lymphocytes and gamma interferonsecreting T lymphocytes of infected Holstein cattle. Infect. Immun. 73, 1321–1329. Tenter, A.M., Heckeroth, A.R., Weiss, L.M., 2000. Toxoplasma gondii: from animals to humans. Int. J. Parasitol. 30, 1217–1258. Tsur, I., Adler, S., 1962. Cultivation of Theileria annulata schizonts in monolayer tissue cultures. Ref. Vet. 19, 224–225. Tuo, W., Fetterer, R.H., Davis, W.C., Jenkins, M.C., Dubey, J.P., 2005. Neospora caninum antigens defined by antigen-dependant bovine CD4+ T-cells. J. Parasitol. 91, 564–568. Watson, D.L., Colditz, I.G., Gill, H.S., 1994. Immunological effector mechanisms in ruminants. In: Wood, P.R., Willadsen, P., Vercoe, J.E., Hoskinson, R.M., Demeyer, D. (Eds.), Vaccines in Agriculture: Immunological Applications to Animal Health and Production. CSIRO, Australia, pp. 21–36. Wilde, J.K.H., Hulliger, L., Brown, C.G.D., 1966. Some recent East Coast fever research. Bull. Epiz. Dis. Afr. 14, 29–35. Wilkins, M.F., O’Connell, E., Te Punga, W.A., 1988. Toxoplasmosis in sheep: III. Further evaluation of the ability of a live Toxoplasma gondii vaccine to prevent lamb losses and reduce congenital infection following experimental oral challenge. N. Z. Vet. J. 36, 86–89. Williams, D.J., Guy, C.S., Smith, R.F., Ellis, J., Bjorkman, C., Reichel, M.P., Trees, A.J., 2007. Immunisation of cattle with live tachyzoites of Neospora caninum confers protection against fetal death. Infect. Immun. 75, 1343–1348. Wouda, W., Moen, A.R., Schukken, Y.H., 1998. Abortion risk in progeny of cows after a Neospora caninum epidemic. Theriogen 49, 1311–1316.