Endemic stability

Endemic stability is defined as the state where the relationship between host, agent, vector and environment is such that clinical disease occurs rarely or not at all (Perry, 1996). Passively acquired resistance from colostrum lasts about 2 months but is followed by innate immunity (as defined under immunity) to babesiosis from 3 to 9 months of age (Mahoney & Ross, 1972; Mahoney, 1974). Therefore calves exposed to babesiosis during the first 6 to 9 months rarely show clinical symptoms and develop a solid long-lasting immunity (Dalgliesh, 1993). Under conditions of endemic instability, some animals will fail to become infected for a considerable period after birth and may therefore develop severe, life threatening disease if they are exposed later in life, depending on their breed (Callow, 1984).

Mahoney (1974) estimated that if at least 75% of calves were exposed to B. bovis infection by 6 to 9 months of age the disease incidence would be very low and a state of natural endemic stability would exist. One infected tick is sufficient to transmit B. bovis, but tick infection rates can be low and the rate of transmission to cattle is therefore usually slow. In an Australian field study, only 0·04% of larval ticks were infected with B. bovis if Bos taurus cattle were involved and even less in paddocks stocking Bos indicus cattle (Mahoney & Mirre, 1971; Mahoney et al. 1981). Tick infection rates with B. bigemina are usually higher (0·23% in the Mahoney & Mirre (1971) study). Therefore, transmission rates in this species are higher than B. bovis and endemic stability is more likely to develop to B. bigemina than to B. bovis in regions where both are present.

A simple mathematical model has been used to predict the level of stability in a herd (Mahoney & Ross, 1972; Mahoney, 1974). It utilises the rate at which infection occurs in calves (determined serologically at a specific age), thereby providing a means of estimating the status of B. bovis infections (Mahoney & Ross, 1972; de Vos & Potgieter, 1983). It has proved useful in predicting stability levels in European breeds but less so in other breeds. More recently, a disease prediction spreadsheet model was developed to more accurately calculate incidence risk from age-specific seroprevalence (Ramsay, 1997). The model predicts the proportion of animals in each age and sex class that would be affected by different severities of the disease, and the information is then converted into a herd model to estimate the number of animals in each severity class. This model has been used to predict the potential impact of Babesia spp. on some large properties in Australia and the cost/benefit of control measures (unpublished observations).

Breed resistance to babesiosis

Bos indicus breeds almost invariably experience milder clinical symptoms to primary B. bovis infections than Bos taurus breeds (Lohr, 1973; Callow, 1984; Bock et al. 1997a; Bock, Kingston & de Vos, 1999a). This phenomenon is thought to be a result of the evolutionary relationship between Bos indicus cattle, Boophilus spp. and Babesia (Dalgliesh, 1993).

Mahoney et al. (1981) compared transmission rates of B. bovis in Bos taurus and in three-eighths to half of Bos indicus crossbred cattle in Australia and concluded that, in an environment unfavourable for tick survival, stocking with Bos indicus or Bos indicus crossbred cattle will, over several seasons, almost lead to the disappearance of the ticks. However, Bock et al. (1999a) showed that if naïve Bos indicus cattle are moved to a paddock with a high resident Boophilus microplus infestation, Babesia transmission rates can initially be very high.

In Australia, B. bigemina is usually of lower pathogenicity than B. bovis (Callow, 1979) and there is some ambiguity in the literature concerning the relative susceptibility of Bos indicus and Bos taurus breeds to this parasite, but most studies conclude that Bos indicus are much more resistant (Lohr, 1973; Parker et al. 1985; Bock et al. 1997a, 1999b).

Innate immune mechanisms

Innate immunity is non-specific and includes factors such as host–parasite specificity, genetic factors, age of the host and the response of host cells (such as the mononuclear phagocyte system and polymorphonuclear leukocytes).

Most Babesia spp. are highly host specific and often splenectomy is needed to establish an infection in an unnatural host (Mahoney, 1972). Cattle with long standing infections of Babesia can relapse to the infections if splenectomised (Callow, 1977). Susceptible cattle that have been splenectomised develop much higher parasitaemias to primary Babesia infections than do intact cohorts. These observations imply that the spleen plays a major role in the immune response to Babesia spp. However the use of splenectomy to develop experimental laboratory models for infection has not always been successful (Frerichs, Johnson & Holbrook, 1969). Different breeds of cattle are known to have different susceptibilities to infection with B. bovis and B. bigemina (see Epidemiology – Breed Resistance). The genetically determined factors that might control the susceptibility of different cattle breeds have not been identified.

There is an age-related immunity to primary infection of cattle with B. bovis and B. bigemina (Riek, 1963). Young calves exhibit a strong innate immunity compared to adult cattle (Trueman & Blight, 1978; Goff et al. 2001). Initially this innate immunity was thought to be due to the passive transfer of protective antibodies in the colostrum of immune dams (Mahoney, 1967c). However, calves from non-immune mothers exhibit resistance to B. bovis and B. bigemina (Riek, 1963). Recently, it has been shown that the innate immune response of young calves to infection with B. bovis involves the early induction of interleukin (IL)-12 and interferon (IFN)-γ and the presence of inducible nitric oxide synthase (iNOS) mRNA expression in the spleen (Goff et al. 2001). In contrast, IL-12 and IFN-γ mRNA were induced later in the infection and iNOS was not induced in adult cattle.

Activated monocytes, macrophages and neutrophils provide the first line of defence during an infection with Babesia spp., employing antimicrobial agents, including reactive nitrogen intermediates (RNI), reactive oxygen intermediates (ROI) and phagocytosis. In addition, these cells secrete cytokines that regulate the inflammatory response. Macrophages stimulated by B. bovis produce IL-1β, IL-12, tumour necrosis factor alpha (TNF-α) and nitric oxide (NO) (Shoda et al. 2000).

Increased phagocytic activity of macrophages has been proposed as a mechanism for the elimination of B. bovis in cattle (Mahoney, 1972; Jacobson et al. 1993) with specific antibodies playing an important role as opsonins (Jacobson et al. 1993). During a primary B. bovis infection in cattle, peripheral blood monocytes display a suppressed phagocytic ability and neutrophils exhibit an increased phagocytic ability that coincides with peak parasitaemia (Court, Jackson & Lee, 2001).

Nitric oxide is an RNI produced by iNOS in macrophages, monocytes, neutrophils and endothelial cells during acute infections. In vitro experiments have suggested that NO can reduce the viability of B. bovis (Johnson et al. 1996) and that B. bovis merozoites (intact and fractionated) can induce the production of NO by monocytes/macrophages in the presence of IFN-γ and TNF-α (Stich et al. 1998; Goff et al. 2002). Indirect in vivo evidence suggests that NO may have a role in the pathology caused by B. bovis (Gale et al. 1998). Gale et al. (1998) administered aminoguanidine, an inhibitor of iNOS, to cattle during a virulent B. bovis infection and caused a reduction in parasitaemia and amelioration of anaemia and pyrexia.

Phagocytosis is closely linked with an oxidative burst and the release of ROI (superoxide anion, hydrogen peroxide and hydroxyl radicals) within the phagocyte (Auger & Ross, 1992). The oxidative burst reaction can be stimulated by cross-linking of receptors on the phagocyte surface, such as Fc receptors for IgG, and ROI are released within the phagosome and into the extracellular environment. In vitro experiments have shown that ROI (superoxide anion and hydroxyl radicals but not hydrogen peroxide) produced by activated macrophages are babesiacidal (Johnson et al. 1996). Furthermore, ex vivo-derived monocytes exhibit increased, and neutrophils display decreased, oxidative activity during a primary B. bovis infection (Court et al. 2001).

Acquired immune mechanisms

Hyperimmune serum (from cattle infected with B. bovis many times) or a mixture of IgG1 and IgG2 prepared from hyperimmune serum of cattle can be used to immunise naïve calves passively against B. bovis infection (Mahoney et al. 1979a). This protection is strain specific. Splenectomised calves given hyperimmune serum and challenged with B. bovis recovered as effectively as intact calves. Serum collected after a primary infection was less effective than hyperimmune serum for transferring immunity to naïve calves and it was thought that this could be related to the antibody isotype present in the serum. Following B. bovis infection, antibodies directed against protective and non-protective parasite antigens and host antigens are produced (Goodger, Wright & Waltisbuhl, 1985). Antibodies probably act as opsonins for increased phagocytosis rather than having a direct effect on the parasite's viability (Mahoney et al. 1979a) and antibodies are thought to be important effectors during a secondary infection (Mahoney, 1986). Immune complexes of babesial antigen, bovine immunoglobulin and C3 form following B. bovis infection (Goodger, Wright & Mahoney, 1981). The major immunoglobulin in the complexes was IgM but some IgG1 and IgG2 in lower concentrations was present. Both bovine IgG1 and IgG2 are capable of fixing complement, and bovine IgG2 is the superior opsonising antibody subclass (McGuire, Musoke & Kurtti, 1979). Following B. bovis infection, IgG1 and IgM, but not IgG2, complement-fixing antibodies have been detected (Goff et al. 1982). Antibody-dependent cell-mediated cytotoxicity may be involved in the resolution of B. bovis infection in cattle (Goff, Wagner & Craig, 1984).

Adoptive transfer studies have not yet been performed with cattle to investigate the definitive role of T cells in the immune response to B. bovis and B. bigemina. However, in vitro experiments performed with T cell lines and clones from immune cattle have demonstrated a role for T cells. Peripheral blood mononuclear leukocytes and helper T cell clones from immune cattle proliferate in response to B. bovis antigens (Brown et al. 1991; Brown & Logan, 1992). The cytokine response by these CD4⁺ helper T cell clones was either a Th1 (IL-2, IFN-γ and TNF-α) or Th0 (IL-2, IL-4, IFN-γ and TNF-α) response with no Th2 clones detected (Brown et al. 1993). Abundant helper T cell clones were isolated from cattle immunised with rhoptry-associated protein-1 (RAP-1) and in vitro these clones produced a Th1 cytokine profile with plentiful IFN-γ (Rodriguez et al. 1996). These Th1 clones promoted IgG2 production by autologous B cells in the presence of antigen. Supernatants from helper T cell lines contain IFN-γ and TNF-α that induce macrophages to produce NO (Stich et al. 1998).

The model of acquired immunity to B. bovis and B. bigemina focuses upon the pivotal role of CD4⁺ helper T cells in the immune response (Brown et al. 2001). These helper T cells produce cytokines, of which IFN-γ seems to be of major importance, which activate phagocytic cells and enhance antibody production by B cells.

B. bovis and B. bigemina cross protection

Protective cross species immunity against infection cannot be induced with B. bovis and B. bigemina (Smith et al. 1980). This is in agreement with Legg (1935) who found that B. bovis infection does not protect against B. bigemina challenge, but B. bigemina immunity does give some protection against B. bovis, the latter being further confirmed by Wright et al. (1987). The observation was exploited in Australia for protection against B. bovis for a short period in the 1930s, but was found to be unreliable (Callow, 1984).

Using a complement fixation test (CFT), (Mahoney, 1964) detected antibodies to B. bovis and B. bigemina for 7 and 4 months, respectively, following infection. Homologous antibodies were detected by indirect fluorescent antibody test (IFAT) for a much longer period of time following B. bovis infection than infection with B. bigemina (Smith et al. 1980). Smith et al. (1980) found that heterologous antibody cross-reactivity as measured by the IFAT following B. bovis or B. bigemina infection was restricted to the period during, and shortly after, recovery. Wright et al. (1987) also found that B. bovis antisera reacts with B. bovis and B. bigemina parasites in an IFAT but that B. bigemina antisera only reacts with B. bigemina parasites and not with B. bovis parasites. However, in the latter study it was unclear at what time after infection the sera were collected. In the IFAT, parasites and not the infected erythrocyte are stained (Wright et al. 1987).

In contrast to the IFAT, immunoblotting experiments demonstrated two-way cross-reactivity between B. bovis and B. bigemina (Wright et al. 1987). Antisera to B. bigemina and to B. bovis reacted strongly with homologous and heterologous antigens demonstrating that many antigens are common to both parasites (Wright et al. 1987). B. bigemina antisera recognised the same proteins in B. bigemina and B. bovis antigen extracts (Wright et al. 1987). However, B. bovis antisera recognised more proteins in the B. bovis than in the B. bigemina antigen extracts. Two way serological cross-reactivity between B. bovis and B. bigemina was demonstrated by (Mahoney, 1967a) using a CFT.

Duration of immunity

With Bos taurus cattle, Mahoney, Wright & Mirre (1973) found tick-transmitted B. bovis infection lasts for at least four years but with B. bigemina it is usually less than 6 months (Mahoney et al. 1973). Immunity to both parasites however, remained for at least four years. Tjornehoj et al. (1996) placed a great deal of importance on the correlation between the persistence of antibodies to B. bovis and the duration of immunity at the herd level. However, there is ample evidence in the literature suggesting the presence of antibodies is not necessarily an indication of immunity nor is the absence of detectable antibodies necessarily an indication of a lack of immunity. Mahoney et al. (1979a) clearly showed that while antibodies alone provide good homologous protection, the same did not apply in cross-protection between strains. They concluded that the protective mechanism of cross-immunity relied on priming of the host's immune system by the protective antigen(s) of the initial strain so that a secondary response against the heterologous strain occurred after challenge.

Johnston, Leatch & Jones (1978) showed that immunity of Bos indicus cross cattle to B. bovis lasted at least 3 years despite the fact that most of the trial cattle eliminated the infection during that time. Unfortunately, Johnston and co-workers did not monitor antibody levels in the cattle. In a different study Callow et al. (1974a,b) showed that indirect fluorescent antibody tests (IFAT) on sera from vaccinated cattle sterilised with Imidocarb were generally negative within six months of treatment, but this decrease in IFAT titre was not associated with a loss of immunity. In a long-term study of immunity in Bos taurus cattle to B. bovis, Mahoney, Wright & Goodger (1979b) found that 30% to 50% of the vaccinated or infected trial cattle became sero-negative in an indirect haemaglutination test during the trial period. Nevertheless, these cattle were still immune 4 years after initial vaccination or exposure. Therefore, it appears that a persistent, detectable antibody titre is not a prerequisite for immunity. However, it is a very effective indicator of recent infection either naturally or by vaccination.

Strain variation and persistence of infection

Isolates and selected strains of B. bovis and B. bigemina differ antigenically (Dalgliesh, 1993) and cross-immunity experiments have shown that recovered cattle are more resistant to challenge with the same (homologous) isolates than with different (heterologous) ones (de Vos, Dalgliesh & Callow, 1987; Dalgliesh, 1993; Shkap et al. 1994).

Antigenic variation is also known to occur during B. bovis and B. bigemina infections with recovered cattle retaining a latent infection varying from six months to several years with detectable recrudescence of parasitaemia occurring at irregular intervals during the latent phase of the infection (Mahoney & Goodger, 1969). Antigenic variation within the vertebrate host is thought to allow variant Babesia parasite populations to continue to adhere to endothelial cells thus avoiding splenic clearance and persist in the face of apparent immunity (Allred, 1995, 2001). Many species of Babesia establish infections of long duration in immune hosts. Allred (2003) suggested that antigenic variation, cytoadhesion/sequestration, host–protein binding, and induction of immunosuppression probably facilitate persistence in the individual immune host. He also suggested that monoallelic expression of different members of a multigene family might facilitate multiple infections of immune hosts, and population dispersal in endemic areas (Allred, 2003).

Babesia bovis infection

Cytokines and other pharmacologically active agents have an important function in the immune response to Babesia. The outcome is related to the timing and quantity produced, but their overproduction contributes to disease progress causing vasodilation, hypotension, increased capillary permeability, oedema, vascular collapse, coagulation disorders, endothelial damage and circulatory stasis (Wright et al. 1989; Ahmed, 2002). Although stasis is induced in the microcirculation by aggregation of infected erythrocytes in capillary beds, probably the most deleterious pathophysiological lesions occur in the brain and lung. This can result in cerebral babesiosis and a respiratory distress syndrome associated with infiltration of neutrophils, vascular permeability and oedema (Wright & Goodger, 1988; Brown & Palmer, 1999). Progressive haemolytic anaemia develops during the course of B. bovis infections. While this is not a major factor during the acute phase of the disease, it will contribute to the disease process in more protracted cases.

The acute disease generally runs a course of 3 to 7 days and fever (>40 °C) is usually present for several days before other signs become obvious. This is followed by inappetence, depression, increased respiratory rate, weakness and a reluctance to move. Haemoglobinuria is often present; hence, the disease is known as redwater in some countries. Anaemia and jaundice develop especially in more protracted cases. Muscle wasting, tremors and recumbency develop in advanced cases followed terminally by coma (de Vos & Potgieter, 1994). The fever during infections may cause pregnant cattle to abort (Callow, 1984) and bulls to show reduced fertility lasting six to eight weeks (Singleton, 1974). Cerebral babesiosis is manifested by a variety of signs of central nervous system involvement and the outcome is almost invariably fatal (de Vos & Potgieter, 1994).

Lesions include an enlarged soft and pulpy spleen, a swollen liver, a gall bladder distended with thick granular bile, congested dark-coloured kidneys and generalised anaemia and jaundice. Other organs may show congestion or petechial haemorrhages and occasionally there will be pulmonary oedema. The grey matter surface of the brain can appear pink. Acute cases will show haemoglobinuria, but this may be absent in subacute or chronic cases. Clinical pathology centres on a haemolytic anaemia, which is characteristically macrocytic and hypochromic. Haematological, biochemical and histopathological changes are described by deVos & Potgieter (1994).

Non-fatal cases may take several weeks to regain condition but recovery is usually complete. In subacute infections, clinical signs are less pronounced and sometimes difficult to detect. Calves that become infected before they attain nine months of age often develop subclinical infections only (Callow, 1984). Recovered cases remain symptomless carriers for a number of years with the duration of infection being breed dependent (Mahoney, 1969; Johnston et al. 1978).

Babesia bigemina infection

Pathogenesis is almost entirely related to rapid, sometimes massive, intravascular haemolysis (Callow, 1984). Coagulation disorders, cytoadherence and the hypotensive state seen in acute B. bovis infections are not features of B. bigemina infections (Wright & Goodger, 1988; Dalgliesh et al. 1995). With most strains of B. bigemina, the pathogenic effects relate more directly to erythrocyte destruction. Haemoglobinuria is present earlier and more consistently than in B. bovis infections and fever is less of a feature. Acutely affected cattle are usually not as severely affected as those with B. bovis infections. There is no cerebral involvement and recovery in non-fatal cases is usually rapid and complete. However, in some cases the disease can develop very rapidly with sudden and severe anaemia, jaundice and death, which may occur with little warning (Callow, Rogers & de Vos, 1993). Animals that recover from B. bigemina remain infective for ticks for 4 to 7 weeks and carriers for only a few months (Mahoney, 1969; Johnston et al. 1978).

Origin and purification of strains

Since 1990, three strains of B. bovis and one of B. bigemina (G strain) have been used to produce vaccines in Australia. Changes in the B. bovis vaccine strain were necessary due to periodic increases in the vaccine failure rate above an acceptable background level (Bock et al. 1992, 1995). Candidate low virulence B. bovis isolates were obtained from naturally infected, long-term carrier animals as described by (Callow, 1977). Contaminating haemoparasites such as Babesia, Anaplasma, Eperythrozoon and Theileria buffeli were eliminated using selective drug treatment combined with rapid passage in calves or culture (Dalgliesh & Stewart, 1983; Anonymous, 1984; Stewart et al. 1990; Jorgensen & Waldron, 1994).

Attenuation of parasites

Babesia bovis – The most reliable way of reducing the virulence of B. bovis involves rapid passage of the strain through susceptible splenectomised calves (Callow, Mellors & McGregor, 1979). The mechanism by which attenuation occurs is not fully understood, but may result from selective enrichment of less virulent parasite subpopulations or from down regulation of a virulence gene (Cowman, Timms & Kemp, 1984; Carson et al. 1990; Timms, Stewart & de Vos, 1990). Attenuation is seldom complete and reversion to virulence can occur following passage of parasites through ticks (Timms et al. 1990) or intact cattle (Callow et al. 1979). Attenuation is also not guaranteed, but usually follows after 8 to 20 calf passages (Callow, 1984).

Attenuation of Babesia spp. by irradiation has been attempted, but the results were variable (Purnell & Lewis, 1981; Wright et al. 1982). In vitro culture has also been used to attenuate B. bovis (Yunker, Kuttler & Johnson, 1987).

Babesia bigemina – Rapid passage in splenectomised calves is not a reliable means of attenuating B. bigemina (Anonymous, 1984) but the virulence of isolates decreases during prolonged residence in latently infected animals. This feature has been used to obtain avirulent strains by splenectomising latently infected calves and using the ensuing relapse parasites to repeat the procedure (Dalgliesh et al. 1981a). A single B. bigemina isolate (G strain) has been used in the Australian and South African vaccines since 1972 and the early 1980s, respectively.

Stabilates

The suitability of a strain for use in a vaccine can be determined by challenging vaccinated cattle and susceptible controls with a virulent, heterologous strain. Both safety and efficacy can be judged by monitoring fever, parasitaemia and depression of packed cell volume during the vaccine and challenge reactions (Timms et al. 1983a). Any candidate isolate must also be tested for freedom from potential contaminants.

After testing for virulence, immunogenicity and purity, suitable strains are preserved as master stabilates in liquid nitrogen. Polyvinyl pyrrolidone (MW 40000 Da) (Vega et al. 1985) is the preferred cryoprotectant as it is not toxic to the parasites at temperatures above 4 °C, allows intravenous inoculation and is safe to use (Standfast & Jorgensen, 1997). Dimethyl sulphoxide is a very effective cryoprotectant but its use was discontinued in Australia in 1991 because of the risk of toxicity to operators, recipient animals and parasites (Dalgliesh, Jorgensen & de Vos, 1990).

Propagation in splenectomised calves

Susceptible splenectomised calves receive inocula from stabilate banks and parasitised blood is collected for production of vaccine when the parasitaemias exceed preset limits. Passaging of B. bigemina in splenectomised calves is not recommended but B. bovis usually requires passaging to produce a sufficiently high parasitaemias for vaccine. B. bovis vaccine strains are not passaged more than 30 times (including the attenuation passages) to safeguard against diminished immunogenicity (Callow & Dalgliesh, 1980).

A sufficient volume of blood can be collected from a 4–6 month old calf to provide up to 25000 doses. To do this the calf is sedated, the jugular vein catheterised and blood collected into a closed system using a peristaltic pump. Heparin is a suitable anticoagulant. After collection of the blood, the calf is treated with a babesiacide and given supportive therapy. Depending on the volume of blood collected, the calf is also transfused using blood from a suitable donor. Due to the high cost and limited availability of suitable, health-tested donors, calves previously infected with B. bovis can be used to provide B. bigemina organisms. In Australia, quinuronium sulphate (de Vos & Potgieter, 1994) is used to treat the primary B. bovis infection because it has no residual effect and will not suppress the development of a subsequent B. bigemina infection. The risk of red cell agglutination can be prevented by washing the B. bigemina-infected red cells by centrifugation to remove agglutinating antibodies. This does not appreciably affect parasite viability (Standfast et al. 2003).

Vaccine specifications

Quality assurance

Control of outbreaks

Use of a vaccine in the face of an outbreak is common practice in Australia. Superimposing vaccination in this way on a natural infection will not exacerbate the condition, but will pre-empt the development of virulent infections in the proportion of the herd not yet exposed to field challenge. To prevent further exposure, the group should also be treated with an acaricide capable of preventing tick attachment from the time of diagnosis to 3 weeks after vaccination. Injectable or pour-on formulations of ivermectin and moxidectin (Waldron & Jorgensen, 1999) as well as fluazuron (Hosking et al. 2004) are highly effective acaricides but do not prevent transmission of Babesia.

Clinically affected cattle should be treated as soon as possible with a suitable babesiacide. In the case of a severe outbreak, it may be advisable to treat all the cattle with a prophylactic compound (e.g. imidocarb or diminazene) and to vaccinate them later when the drug residue will not affect vaccine parasite multiplication.

Cattle born in vector-infested regions

Any factor affecting the survival of the tick vectors will affect the risk of babesiosis occurring. Thus, increased tick numbers will increase the threat of disease until an endemically stable situation develops. Conversely, reduced tick numbers will increase the longer-term risk of babesiosis due to the reduced natural exposure of calves. For these reasons, cattle owners in endemic areas of Australia are advised to supplement natural exposure by vaccinating calves at weaning age. Vaccination is also recommended if cattle are being moved within the endemic area.

Susceptible cattle imported into vector-infested country or regions

Large numbers of cattle, predominantly of Bos taurus breeds are being imported into tropical, developing countries to upgrade local livestock industries. This practice has, in the past, led to significant losses due to tick-borne diseases, including babesiosis (Callow, 1977) if preventative measures were not taken. Most of the countries involved did not have access to an effective vaccine. Vaccination of naïve cattle moving from ‘tick-free’ to endemic areas within Australia is usually very effective. This practice has played a crucial role in making the livestock industries in these parts more sustainable and competitive in meeting market demand with regard to breed type.

K strain B. bovis and G strain B. bigemina from Australia have been shown experimentally to be protective in South Africa (de Vos, Bessenger & Fourie, 1982; de Vos, Combrink & Bessenger, 1982); Sri Lanka (D. J. Weilgama, personal communication, 1986); Bolivia (Callow, Quiroga & McCosker, 1976) and Malawi (Lawrence et al. 1993). Vaccine containing these strains has also been used with beneficial results in countries in many parts of the world, including Zimbabwe and Swaziland in Africa, Venezuela and Ecuador in South America, Malaysia and the Philippines in Southeast Asia, and islands of the Caribbean. The feasibility of importing a vaccine strain that is known to be effective, of low virulence and free from contaminants to protect imported or local cattle should therefore be considered.

Hazards and precautions of live vaccine use

Potential spread of Babesia following vaccination

Concern is often expressed that natural spread of infection may occur from vaccinated to unvaccinated cattle. Australian evidence suggests that it is very unlikely that use of vaccine will introduce infection into a previously uninfected area. A 15 year study of the history of babesiosis outbreaks in Australia found no evidence suggesting that vaccination of cattle on a neighbouring property was the cause of an outbreak (Callow & Dalgliesh, 1980). On the other hand, inadequate tick control by a neighbour and spread of naturally-infected ticks was found to be a contributing cause. Similarly, there has been no evidence that use of a strain from one country resulted in the spread of infection in another. Tick numbers appear to be more relevant in transmission of Babesia than the presence of animal reservoirs of infection.

The current B. bovis strain (Dixie) used in Australia is known to be transmissible by Boophilus microplus under laboratory conditions and to increase in virulence following tick transmission (unpublished observations). Despite this, Bock et al. (1999a) offered circumstantial evidence that the presence of this strain in vaccinated cattle is unlikely to alter the dynamics of transmission of parasites under field conditions or constitute a significant risk to naïve cattle grazing with vaccinated cattle once vaccine induced parasitaemias have fallen to undetectable levels.

The G strain of B. bigemina used in Australia since 1972 (Dalgliesh et al. 1981a) is poorly, if at all, transmissible by Boophilus microplus (Dalgliesh, Stewart & Rodwell, 1981b; Mason, Potgieter & van Rensburg, 1986). Also, cattle infected with B. bigemina are reported to remain infective for ticks for only 4 to 7 weeks (Mahoney, 1969; Johnston et al. 1978) so if transmission to ticks occurs, it will be over a short period only.

Loss of viability

Stored in liquid nitrogen, a frozen vaccine will remain viable for many years but it loses viability very rapidly after thawing. If glycerol is used as cryoprotectant, a thawed vaccine can remain viable for only 8 hours at temperatures ranging from 4–30 °C. If dimethyl sulphoxide is used, a vaccine should preferably be used within 30 minutes although work in South Africa has indicated that thawed dimethyl sulphoxide-based vaccine remains infective for 8 hours if kept on melting ice (D. T. de Waal, personal communication). Chilled vaccines can remain viable for up to a week if stored at 4 °C. At higher temperatures, viability is lost rapidly.

Lack of protection

Since the introduction of a standardised method of production in Australia, live babesiosis vaccines have generally proved to be highly effective (Callow & Dalgliesh, 1980). In most cases, a single vaccination provided lasting, probably life-long immunity against field infections with antigenically different strains. However, troublesome failures of the Australian B. bovis vaccine occurred in 1966, 1976, and again in 1988–1990 (Bock et al. 1992), and were thought to be due to loss of immunogenicity brought about by frequent passaging of the vaccine strains in splenectomised calves (Callow & Dalgliesh, 1980). In each case, the problem was solved by replacing the vaccine strain. A similar loss of immunogenicity in a multi-passaged strain of B. bovis was reported in South Africa (de Vos, 1978). To prevent future recurrences of the problem, the number of passages of the vaccine strains of B. bovis is limited by frequently reverting to a master stabilate with a low passage number (Callow & Dalgliesh, 1980).

More recently (1992–1993), B. bovis vaccine failures were again reported in Australia despite restrictions on passage numbers and replacement of the vaccine strain (Bock et al. 1995). The occurrence of these failures did not correlate with time after vaccination. Bock et al. (1995) considered eight possible factors, and while the situation is complex and no simple cause is forthcoming, recent research emphasis has fallen on the immune responsiveness of the host and immunogenicity of vaccine parasite subpopulations (Dalrymple, 1993; Bock et al. 1995; Lew et al. 1997a,b). The B. bovis vaccine strain used in Australia since 1993 has been shown by PCR assay to contain two subpopulations. It was found to be more protective than higher passages of this strain that contained one subpopulation (Bock & de Vos, 2001).

Vector control

Vector control was first used successfully to control and eventually eradicate Babesia from the USA (Pegram, Wilson & Hansen, 2000). Because, in Africa, babesiosis forms only part of very important complexes of ticks and tick-borne diseases, intensive, usually government-regulated tick control programmes have been used for many years. The situation in other continents is much less complex than in Africa but where babesiosis is endemic, disease control rather than eradication is generally the only realistic option. Eradication of the tick vectors (the so-called minimum disease situation) is a permanent solution to the problem but is rarely considered practical, environmentally sustainable or economically justifiable on either a national or a local basis.

Natural endemic stability

Natural endemic stability can seldom be relied on as a disease control strategy. Firstly, in endemic areas, climatic effects, genetic make-up of hosts and management strategies, inevitably have a major effect on the rate of transmission and ultimately on the likelihood of endemic stability developing. Secondly, endemic stability is an economic concept that incorporates risk management and loss thresholds. The climatic, animal and management parameters that allow endemic stability can change on a seasonal, let alone on an annual, basis. For example, a dry season can drastically affect tick numbers and parasite transmission rates to produce a generation of susceptible cattle. Thirdly, the model for endemic stability was developed in Australia and the Americas where the disease/vector interactions are relatively simple. The African situation is more complex and less predictable with four main diseases, several vectors, presence of game reservoirs and a larger range of susceptibility of bovine breeds.

In a study of dairy farms in Boophilus microplus-endemic areas of Australia, Sserugga et al. (2003) found 74% of herds of farmers who allowed a ‘few’ ticks to persist, assuming their cattle would be protected from tick-borne disease, had insufficient exposure to confer herd immunity, and a high risk of tick fever outbreaks. Leaving a ‘few’ ticks, although it is likely to have some protective effect, therefore cannot be considered a satisfactory approach to controlling the disease. Presumably, because farmers underestimated the numbers of ticks needed or would not allow sufficient ticks on their cattle to achieve endemic stability.

A serological survey in 1996 of 7067 unvaccinated weaner cattle 6 to 12 months of age on 115 properties in officially Boophilus microplus-infested areas of Northern Australia indicated that the average percentage of animals seropositive for B. bovis and B. bigemina per herd was only 4% and 11%, respectively (Bock et al. 1997b). Given the generally high Bos indicus content and extensive management of these herds, the risk that this represents is reduced, but difficult to ascertain. However, in some areas, the risk appeared to have been very high and significant losses would be expected especially where the Bos taurus content of the herd was increased.

Animal genetics

Certain breeds of cattle, notably Bos taurus breeds, are known to be more susceptible to primary B. bovis infection (Bock et al. 1997a). Bock et al. (1999a) showed pure-bred Bos indicus cattle had a high degree of resistance to babesiosis, but crossbred cattle were sufficiently susceptible to warrant the use of preventive measures such as vaccination. Similarly, genotype also plays a role in the development of protective immunity with Bos taurus breeds more likely to show a deficient immunity. An investigation of 62 reports of B. bovis vaccine failures in Australia showed that all were in cattle with no greater than 3/8 Bos indicus infusion and 85% were in pure Bos taurus herds (Bock et al. 1995). As discussed under breed susceptibility (see above), the use of cattle with more than 50% Bos indicus content will greatly reduce the impact of babesiosis.

Integrated control

Livestock industries need to take a pragmatic approach to management of the ticks and diseases associated with them. In the long term, an effective outcome can be achieved by integrating the strategic use of acaricides, exploitation of endemic stability, the application of vaccines in endemically unstable conditions and the use of tick-resistant breeds of cattle (Norval, Perry & Hargreaves, 1992; Perry et al. 1998).

Recombinant/subunit vaccines

Rapid advances in our understanding of mechanisms of immunity to many protozoa and the development of molecular tools for generating recombinant vaccines suggest that this is the future direction for protozoal vaccine development (see the chapter by Bishop et al. in this Supplement). However our lack of understanding of immune mechanisms to primary and secondary infection and the reality that many protozoa have developed elaborate mechanisms (e.g. antigen variation) for evading host immunity remain obstacles to developing effective vaccines using this technology (Jenkins, 2001).

Several attempts have been made to develop recombinant or subunit Babesia vaccines (Reduker et al. 1989; Wright et al. 1992; Dalgliesh, 1993; Brown & Palmer, 1999), to overcome the inherent deficiencies of vaccines based on blood or blood extracts. Wright et al. (1992) systematically tested biochemically fractionated merozoite antigens in immunisation and challenge trials using Quil A as an adjuvant and found 3 partially protective antigens (11C5, 12D3 and T21B4/RAP-1). These antigens were secreted proteins, not abundant and were not serologically immunodominant (Wright et al. 1992).

The search for vaccine candidate antigens has focused mainly on merozoite surface antigens that are functionally relevant and immunodominant in naturally immune cattle, as well as conserved among strains. Candidate antigens identified include B. bovis merozoite surface antigen 1 (MSA-1) (Hines et al. 1995), B. bovis merozoite surface antigen 2c (msa-2c) (Wilkowsky et al. 2003), and B. bovis and B. bigemina RAP-1 (Wright et al. 1992; Norimine et al. 2002). Immunization of cattle with recombinant MSA-1 induced antibodies that were capable of neutralizing merozoite invasion of erythrocytes, however the immunized cattle were not protected against virulent challenge (Hines et al. 1995). MSA-2c has been identified as highly conserved among B. bovis strains and bovine antibodies to recombinant MSA-2c were able to neutralize the invasion of erythrocytes by merozoites indicating a functional role for this antigen (Wilkowsky et al. 2003). However, this antigen has not yet been tested in cattle to determine its protective efficacy. RAP-1 is a 60 kDa antigen of Babesia that is recognised by antibodies and T cells from naturally immune cattle (Rodriguez et al. 1996; Norimine et al. 2002). Native and recombinant B. bovis RAP-1 confers partial protection against homologous challenge (Wright et al. 1992). Native B. bigemina RAP-1 also induces partial protection against challenge infection (McElwain et al. 1991).

Results to date suggest that vaccines based on single antigens or even several antigens in combination do not confer the level or duration of cross-protection provided by living vaccines. Most work has centred on merozoite antigens but sporozoite antigens need also to be evaluated (Brown & Palmer, 1999). Unfortunately, the majority of experimental immunization trials use homologous challenge infections that do not reflect the situation in the field where heterologous virulent challenge would occur. Even a multicomponent recombinant vaccine may not provide long-term protection against field strains of Babesia spp. which have been shown to be capable of considerable genetic variation (Dalrymple, 1993; Lew et al. 1997a). The difficulty is to identify antigens that are targeted by the host's protective immune response across all strains, induce an appropriate long-term memory response and deliver them to the animal in a way that is cost effective. A subunit vaccine that protects against clinical signs, but allows for limited parasite replication may be an ideal strategy for protecting susceptible individuals (Jenkins, 2001). Yet, no recombinant vaccine for bovine babesiosis has been registered for use in any country and it is unlikely that one will be available in the near future.

Another avenue of research for the development of an alternative to the use of a live vaccine is DNA vaccine adjuvants. Recognition of foreign DNA (in particular unmethylated CpG motifs in DNA) by the innate immune system is a relatively recent discovery. CpG motifs in DNA derived from B. bovis-stimulated B cells to proliferate and produce IgG (Brown et al. 1998) and activated macrophages to secrete IL-12, TNF-α and NO and may provide an important innate defence mechanism to control parasite replication and promote persistent infection (Shoda et al. 2001).

In vitro culture-derived vaccines

In vitro culture methods reviewed by Pudney (1992) are also used to provide B. bigemina and B. bovis parasites for vaccine (Jorgensen et al. 1992; Mangold et al. 1996). These techniques are not widely used for production of vaccines, but have proven to be valuable research tools. In countries where it is difficult to obtain sufficient numbers of disease-free, susceptible donor calves, and materials and facilities for tissue culture are available, in vitro production of vaccines may be a viable option. At the National Institute of Agricultural Technology in Argentina, over 50000 doses of B. bovis and B. bigemina vaccine are produced using in vitro culture and sold to cattle producers each year (A. A. Guglielmone personal communication). Some studies found neither virulence or immunogenicity of Babesia vaccine strains were appreciably modified by short-term (3 months) maintenance in culture (Jorgensen, de Vos & Dalgliesh, 1989a; Timms & Stewart, 1989). However, more recent observations using PCR of polymorphic genetic markers have shown that proportions of B. bovis sub-populations changed with long-term continuous cultivation (Lew et al. 1997b). These drifts may not be significant but indications are that protection provided by culture-modified B. bovis strains may be inferior to that of parasites not exposed to a culture environment (Bock et al. 1995). Until more information is available, use of long-term cultures in the production of vaccines is not recommended unless facilities are available to monitor changes in parasite populations and to test for immunogenicity.

Non-living vaccines

Non-living vaccines would overcome many of the inherent difficulties in production, transport and use of live vaccines. One of the earliest attempts to induce protective immunity in cattle against B. bovis infection using a non-living vaccine was by Mahoney (1967b). The inoculation of cattle with killed parasites mixed with Freund's complete adjuvant partially protected against homologous challenge. Cell culture-derived exoantigens of B. bovis and B. bigemina have been extensively studied and proposed for use as vaccine in developing countries with reported promising results (Montenegro-James, 1989; Montenegro-James et al. 1992; Patarroyo et al. 1995). Other studies have shown the level and duration of protection conferred by these antigens against heterologous challenge was considerably less than those of live vaccines (Timms, Stewart & Dalgliesh, 1983b). Jorgensen et al. (1993) evaluated the use of pre-vaccination with homologous exoantigens as a method of reducing the risk of reactions following vaccination with live Babesia vaccine. This technique reduced the parasitaemia and development of anaemia but not the fever which sometimes follows the use of this vaccine.

Other approaches to the development of a non-living vaccine include fractionating merozoite antigens either by an empirical approach (Wright et al. 1992) or by continuous flow electrophoresis (Stich et al. 1999; Brown et al. 2001) the identification of antigens involved in the invasion process (Palmer & McElwain, 1995) and the identification of T cell and B cell epitopes of antigens that stimulate T helper cell clones (Wilkowsky et al. 2003).

A killed B. divergens vaccine has been prepared in Austria from the blood of infected calves (Edelhofer et al. 1998), but little information is available on the level and duration of the conferred immunity.