TECHNICAL FIELD THIS INVENTION relates to babesiosis. More particularly, this invention relates to the use of chemically-attenuated babesia parasites as a vaccine against babesia.

BACKGROUND

Babesiosis is a group of diseases and conditions of mammals caused by, or otherwise associated, protozoan protists of the genus Babesia. Babesiosis may occur in mammals such as cattle, buffalo, pigs, deer, dogs, sheep, donkeys, horses and humans, although without limitation thereto. Babesiosis is typically associated with severe haemolytic anaemia and a positive erythrocyte-in-saline-agglutination test indicating an immune-mediated component to haemolysis. Common sequelae include haemoglobinuria "red-water", disseminated intravascular coagulation and "cerebral babesiosis" caused by sludging of erythrocytes in cerebral capillaries. In some cases, babesiosis can prove fatal.

Human babesiosis is uncommon although some cases occur due to tick-borne Babesia divergens or Babesia microti, a parasite of small mammals. A few human cases have been fatal- Generally, in non-human mammals the main pathogeic species of Babesia include Babesia bovis, Babesia canis, Babesia bigemena, Babesia divergetis, Babesia ovis, Babesia av ta, Babesia occultans, Babesia eaballi and Babesia motasi.

The main parasites of bovine mammals are Babesia bovis, Babesia bigemena and Babesi divergent, which cause hemolytic anemia, such that an infected animal will also show pale to yellow mucous membranes due to the failure of the liver to metabolise excess bilirubin. In catde, babesiosis is also known as "tick fever" or "cattle fever" and is economically the most important arthropod-borne disease of cattle worldwide, with cattle populations in most areas of Australia, Africa, South and Central America and the United States at risk. Outbreaks of babesiosis can have considerable economic impact not only from mortality, but also calf abortions, loss of milk and/or meat production, the cost of control measures (such as acaricide treatments, vaccines and therapeutics) and negative impacts on cattle trade.

Babesia species are transmitted by ticks, which become infected when they ingest parasites in the blood of infected host mammals. The major vectors for B. bige ia are Rhipicephalus microplus and R. atm latus, while R. de ohratus, R. 2 geigyi, and R. everisi can also transmit this species. The major vectors for B. bovis are R. microplw and R. ammiaim, hut R. geigyi can also be a vector. B. divergent is transmitted mainly by Ixodes ricm ^' rn. B jakimovi may also be transmitted by an Ixodes species. Haemaphysalis punctata transmits B. major, Haemaphysalis longieornis transmits B. ovata, and Hya mma marginatum transmits B. o cultans.

The typical Babesia infection cycle begins when an infected tick sends sporozoites into a mammalian host while taking a blood meal. The sporozoites then invade red blood cells, where some transition from a trophozoite to a merozoite stage. Merozoites then asexually reproduce by binary fission, producing male and female gametes which may be ingested by a tick, where they undergo sporogony, producing sporozoites which are then transferred to a new mammalian host when the infected tick takes a blood meal. This repHcative blood-stage is approximately of about 10 hrs duration, compared to a much longer 24-48 hrs in other protozoan diseases such as malaria. It is also noted that there is no liver stage in babesiosis, unlike the case with malaria.

Babesia are maintained in cattle populations by asymptomatic carriers that have recovered from acute disease. B. bovis persists in cattle for years, and B bigemina survives for a few months. Recrudescence of parasitemia can occur at irregular intervals.

The first steps taken in the development of an effective vaccination strategy against bovine babesiosis followed observations that animals recovered from natural infection with Babesia were strongty protected against subsequent challenge. Further investigation indicated that the use of donor blood from recovered animals to infect recipient animals did not produce the severe form of the disease. Live vaccines based on attenuated B. bovis and B. bigemina strains are currently used to protect cattle against babesiosis. Nevertheless they have several drawbacks represented by the risk of virulence reversion, contamination with pathogens and the difficulties inherent in industrializing production. SUMMARY

Surprisingly, treatment of merozoites or red blood cells infected with Babesia merozoites, with the DNA-binding agent tafiiramyein chemically attenuates the merozoites sufficiently to enable administration of the merozoites or infected red blood cells for the purposes of immunization against babesiosis. Even more 3 surprisingly, this immunization is effective as a single dose in the absence of adjuvant. A further unexpected benefit is that the treated merozoites of a particular Babesia isolate, strain or species will immunize against infection by heterologous Babesia isolates, strains and species.

The invention is therefore broadly directed to an iraunogenic composition comprising chemically attenuated whole, blood-stage Babesia parasites, preferably in the absence of adjuvant.

A preferred approach includes the administration of attenuated blood-stage Babesia parasites, either as parasite-infected red blood ceils or as purified blood- stage parasites in the absence of adjuvant Even more preferably, a single dose of attenuated blood-stage Babesia parasites or infected red blood cells is sufficient to immunize against subsequent Babesia infection.

Preferably, the parasite-infected red blood cells are intact cells.

In one aspect, the invention provides a method of producing an immunogenic composition including the step of treating blood-stage Babesia parasites or red blood cells infected with said blood-stage Babesia parasites with tafuramycin A or an analog or derivative thereof to thereby produce said immunogenic composition.

Suitably, the method excludes the step of including an adjuvant.

In another aspect, the invention provides an immunogenic composition comprising blood-stage Babesia parasites or red blood cells infected with said Wood- stage Babesia parasites that have been treated with tafuramycin A or an analog or derivative thereof; and an immunologically acceptable carrier, diluent or excipient.

Suitably, the immunogenic composition does not include an adjuvant.

In yet another aspect, the invention provides an immunogenic composition for use in treating or preventing babesiosis, said immunogenic composition comprising blood-stage Babesia parasites or red blood cells infected with said blood- stage Babesia parasites that have been treated with tafuramycin A or an analog or derivative thereof; and an immunologically acceptable carrier, diluent or excipient.

Suitably, the immunogenic composition does not include an adjuvant.

In a further aspect, the invention provides a method of treating or preventing babesiosis, said method including the step of administering the immunogenic composition of the aforementioned aspect to an animal to thereby prevent or inhibit Babesia infection or treat an existing Babesia infection in said animal. 4

Preferably, the method includes administering a single dose of said immunogenic composition to thereby prevent or inhibit Babesia infection or treat an existing Babesia infection in said animal.

Suitably, the method does not include administration of an adjuvant.

Typically, according to the aforementioned aspects the blood-stage Babesia parasites include^ or are, merozoites, sc izonts, rings or trophozoites, although without limitation thereto.

The animal of the aforementioned aspects is preferably a mammal, inclusive of humans, bovines, deer, pigs, horses, sheep, goats, donkeys and dogs, although without limitation thereto.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises ^* ' and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Rectal temperatures post vaccination with 1 x 10 ⁹ iRBCs untreated (D1578); treated with 2 μΜ tafuramycin A (TfA; D1574); or 20 μΜ TfA (01577). Calf 1599 received 10* uninfected RBC

Figure 2: PC V post vaccination. The treatment of these calves was as described in Fig. 1.

Figure 3: Positive control parasitaemia percentage post vaccination.

Figure 4; Temperatures of animals vaccinated as in Fig 1 post challenge with

10 ⁷ iRBCs

Figure 5: Packed cell volume of animals as in Fig. I post challenge with 10 ⁷ iRBCs.

Figure d: Parasitaemia count (xlO* /mL) from jugular vein blood smear post challenge with lO ⁷ iRBCs . Animals are as described in Fig. 1.

DETAILED DESCRIPTION

The present invention is predicated at least partly on the discovery that a single dose of tafuramycin A-treated blood-stage Babesia parasite-infected red blood cells of cattle can effectively immunize against subsequent Babesia infection. 5

Tafuramycin A is a rationally designed, DNA binding and alkylating agent that binds A-T rich regions of DMA The efficacy of taftiramycm A in chemically attenuating Babesia parasites is unexpected, as compared to other protozoans such as Plasmodium, Babesia parasites typically have a relatively low proportion of A-T residues (about 58% compared to about 81% in Plasmadmm).

Accordingly, in one aspect, the invention provides a method of producing an immunogenic composition including the step of treating isolated or purified blood- stage Babesia parasites or red blood cells infected with said blood-stage Babesia parasites with tafuramycin A or an analog or derivative thereof, to thereby produce said immunogenic compositioa

In another preferred aspect, the invention provides an immunogenic composition comprising isolated or purified blood-stage Babesia parasites or red blood cells infected with said blood-stage Babesia parasites that have been treated with tafuramycin A or an analog or derivative thereof.

As used herein "Babesia" parasites are any pathogenic protists of the genus

"Babesia". The genus "Babesia" includes pathogenic species such as Babesia bovis, Babesia canis, Babesia bigemena, Babesia divergens and Babesia microti, although without limitation thereto.

Typically, the blood-stage Babesia parasites are, or include, any form of the parasite that exists within red blood cells, including but not limited to merozoites.

It will be appreciated from the foregoing that one embodiment of the invention relates to in vitro treatment of isolated or purified blood-stage Babesia parasites or red blood ceils infected with blood-stage parasites, with tafuramycin A or an analog or derivative of tafuramycin A. This treatment is effective to chemically attenuate the blood-stage Babesia parasites without killing the parasite, such as by inhibiting parasite replication. Typically the attenuated blood-stage Babesia parasites are not capable of proliferation, or are capable of only limited proliferation, following attenuation by treatment with tafuramycin A.

Tafuramycin A is a rationally designed, DNA binding and alkylating agent based on duocarmycins that comprises a stereocenter.

By "tafuramycin A analogs or derivatives" is meant any molecule structurally related to tafuramycin A which exhibits binding to AT-containing nucleotide sequences to thereby induce DNA damage. 6

Tafuramycin, tafuramycin A, analogs or derivafrves inclusive of non-chiral, cbiral and racemic analogs and derivatives of duocarmycin and CC-1065, non-chiral, chiral and racemic isomers, salts or solvates thereof are also described in International Publications WO2002/030894, WO2008/050140, WO2009/064908 and WO2012/162731, Chavda. et al„ 2010, Bioorganic ά Med, Chem. 18:5016-5024, Howard, et or/., 2002, Bioorganic & Med. Chem. 10:2941-2952, Pornell et at., 2006, Med Chem. 2:139, 2006 and United States Patent 6660742. Reference is particularly made to jreco-iso-cyclopropylfuranop^-eJindoline-T I (TH-IH- 49 or tafuramycin A) and ^co-cyclopropyItetrahydrofurano[2 _; 3-f]quinoline-TK'il (TH-IlI-151 or tafuramycin B) analogs of CC- 1065 and the duocarmycms as described in Howard et at, 2002, supra and Purnell et al, 2006, supra.

Achiral se O-hydroxy-aza-CBI-ΤΜί, a s^o-cyclopropy1pyrido[e]radolone (CPyl) compound, is an example of an analog of centanamcyin as described in Chavda et l _^ 2010, supra. Racemic and chiral 5-methylfuran analogs of tafuramycin A are described in Purnell et al., 2006, supra.

In a preferred embodiment, tafuramycin A or the analog or derivative is a compound of formula I:

wherein,

Ri is either MHR or OR, where R is selected from Ή, benzyl, benzyloxycarbonyl, 4-nitroberizyloxycarbonyl and T-n ethylpiperazmyl-N-carDonyl; 7

R ₂ is selected from H and Ci-e alkyl;

Ra is selected from H and alkyJ;

or ¾ and R3, form a fused ring selected from the group consisting of a benzene ring, a pyrrole ring, a pyridine ring, a furan ring and a 5-methylfuran ring, which fused ring may be optionally substituted with Cj^ alkyl, CF3 or Cw, alkyloxycarbcmyl;

Y is an alkylene radical selected from -€¾-, -C¾CH ₂ - or -CH ₂ C¾CH ₂ -; or Y may be -CH- to form a five-merabered ring with R4;

X is an electrophilic leaving group;

R4 is selected from H, Ci~$ alkyl, a -C¾- group bonded to Y to form a five- membered ring or a -CH ₂ - group bonded to the -CH ₂ - to which X is attached to form a six membered ring;

Rs is selected from the group consisting of:

(a)

where Yi and Y ₂ are independently selected from O and NH;

0»)

*

8

(h) 9

; and

(i) t-butoxy, benzyloxy and 9-fl«orenylmethyloxy.

In one particular embodiment of the compound of formula I;

Preferably, Ri is OH.

Preferably, R ₂ and R together form a furan ring.

Preferably, Y is -CH- and forms a five-membered ring with R .

Preferably, X is selected from chloro, bromo, iodo, mesylate, tosylate, thio, ammonium, Ci-¾ alkylsulfonyi and d-6 alkylsulfoxyl.

More preferably, X is selected from chloro, bromo, iodo, mesylate and tosylate.

Even more preferably, X is chloro.

Preferably, R4 is a -CH2- group bonded to Y to form a five membered ring structure.

Preferably, R ₅ is:

OMe M

A preferred form of this embodiment is a compound of Formula Π (mfuTamyci A):

In another embodiment of the compound of Formula 1:

Preferably, Ri is NH ₂

Preferably, R ₂ and Rj together form a benzene ring.

Preferably, Y is -CH-.

Preferably, X is selected from chloro, bromo, iodo, mesylate, tosylate, thio, ammonium,, alkylsulf nyl and alkylsulfoxyL

More preferably, X is selected from chloro, bromo, iodo, mesylate and tosylate.

Even more preferably, X is chloro.

Preferably, R* is H or C _1-6 alkyl.

Preferably, Rs is

A preferred form of this embodiment is a compound of Formula 111 (centanatrrycin):

(III)

En yet another embodiment of the compound of Formula I:

Preferably, Ri is OH.

Preferably, Rj and R3 together form a pyridine ring optionally substituted with Ci^ alkyl.

Preferably, Y is -CH-.

Preferably, X is selected from chloro, bromo, iodo, mesylate, tosylate, thio, ammonium,, alkylsulfonyl and Ci-β alkylsulfoxyl.

More preferably, X is selected from chloro, bromo, iodo, mesylate and tosylate.

Even more preferably, X is chloro.

Preferably, R is B or Ci^ alkyl.

Preferably, R* is

A preferred form of this embodiment is a compound of Formula IV (AS-VII Q4):

(IV)

Typical, although non-limiting concentrations of tafuramycin A or analogs or derivatives for treatment of blood-stage Babesia parasites, or red blood cells infected with Babesia parasites, are in the range of about 0.1 to 100 μΜ. Preferably, the concentration is in the range of about 0.5-50 μΜ, more preferably i tbe range about 1-40 μΜ or even more preferably i the range of about 2-20 μΜ. Particular concentrations include about 0.5 μΜ, i.O μΐνΐ, 1.5 μΜ, 2.0 μΜ, 3 μΜ, 4 μΜ, 5.0 μ , 6.0 μΜ, 7.0 μΜ, 8.0 μΜ, 9.0 μΜ, 10 μΜ, 1 J μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μ , 16 μΜ, 17 Μ, 18 μΜ, 19 μ , 20 μΜ, 25 μΜ and 30 μ , or in any range therebetween.

Treatment duration may be in the range 1 minute to 12 hours, preferably 10 minutes to 4 hours or more preferably about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5 or 2 hours, or any range therebetween.

The tafuramycin A-treated blood-stage Babesia parasites may be used to prepare the immunogenic composition as an isolated, enriched, purified or partially purified parasite preparation, or the tafuramycin A-treated blood-stage Babesia parasites may be used to infect red blood cells, which are then used to prepare the immunogenic composition. Alternatively, red blood cells infected with blood-stage Babesia parasites may be treated with tafuramycin A and then used to prepare the immunogenic composition. In some embodiments, p BC may be administered as intact cells or as a lysate. Preferably, the pRBC are in intact form.

The pRBC may be obtained from blood of a parasite-infected animal prior to tafuramycin A treatment. Alternatively, to produce pRBC in vitro, non-infected red blood cells may be obtained from an animal and then infected in vitro with blood- stage Babesia parasites pre-treated with tafuramycin A, or with untreated blood-stage Babesia parasites so that the pRBC are thereafter treated with tafuramychi A.

Suitably, the dose of tafuramycin A-treated blood-stage Babesia parasites or red blood cells infected wi th said Babesi parasites (i,e pRBC) is capable of eliciting an immune response to subsequent infection by Babesia parasites. Preferably, the immune response is characterised by inducing a T cell response and, optionally, inducing B cells to produce detectable levels, or only low levels, of antibodies.

A typical dose of pRBC suitable for administration to cattle is no more than 10 ^u pRBC such as including 10 ^TO , W ⁹ , 10 ⁸ , 10 ⁷ , IQ ⁶ , lO ⁵ , 10 ⁴ , 10 ³ , 10* or lO RBC, or any range between any of these values.

In another embodiment, the dose may be in the form of purified blood-stage Babesia parasites. A typical dose suitable for adrninistrarion to cattle is no more than 10* ¹ pRBC such as including 10 ^1Q , 10 ⁹ , 10 ^s , 10 ⁷ , 10 ⁶ , 10 ^s , 10 ⁴ , 10 ³ , 10 ² or 10 pRBC, or any range between any of these values.

Accordingly, an aspect, the invention provides an immunogenic composition for preventing or treating babesiosis, said immunogenic composition comprising isolated or purified blood-stage Babesia parasites or red blood cells infected with said blood-stage Babesia parasites that have been treated with tafuramycin A or an analog or derivative thereof, said composition preferably excluding adjuvant.

In a further aspect, the invention provides a method of treating or preventing babesiosis, said method including the step of administering the immunogenic composition disclosed herein to an animal to thereby prevent or inhibit Babesia infectio or treat an existing Babesia infection in said animal, said method preferably excluding administration of adjuvant.

As used herein, "babesiosis" includes all forms of the disease caused by protozoan protists of the genus Babesia.

The genus "Babesia" includes pathogenic species such as include Babesia bovis, Babesia canis, Babesia bigemena, Babesia divergens, Babesia ovis, Babesia ovata, Babesia occultans, Babesia caballi and Babesia moiasi, although without limitation thereto.

In embodiments relevant to babesiosis in bovines, the causative Babesia species are typically esia bovis, Babesia bigemena and Babesia divergens. Suitably, for treatment of bovines, the immunogenic composition and method of prophylactic or therapeutic treatment of babesiosis comprises tafuramycin A-treated parasites of one or more strains or isolates of Babesia parasites such as Babesia bovis, Babesia bigemena and/or Babesia divergens, or pRBC infected with said Babesia parasites,

Babesiosis can include in non-specific flu-like symptoms, such as fever, chills, swears, headache, body aches, loss of appetite, nausea, or fatigue and other symptoms such as thrombocytopenia, low or unstable blood pressure and hemolytic anemia which can lead to jaundice and darkened urine, although without limitation tthereto.

As used herein, "treating", "treat" or ''treatment" refers to a therapeutic intervention, course of action or protocol that at least ameliorates a symptom of the disease, disorder or condition after symptoms have at least started to develop. As used herein, ^preventing, "preve " or "prevention" refers to therapeutic intervention, course of action or protocol initiated prior to the onset of a symptom of the disease, disorder or condition so as to prevent, inhibit or delay or development or progression of the disease, disorder or condition or the symptom. Such preventative therapies may be referred to as "prophylaxis" or a "prophylactic" therapy. In a specific embodiment, immunization or vaccination is a form of preventative or prophylactic therapy.

One preferred, unexpected advantage of the present invention is that the tafuramycin A-treated blood-stage Babesia parasites of a particular isolate, strain or species may, upon administration to an animal, immunize or vaccinate against infectio by heterologous Babesia isolates, strains and/or species. By " etero gatis" pathogens means related pathogens that may be different strains or variants of a same or related species.

From the foregoing, it will also be appreciated that another preferred advantage of the present invention is that th immunogenic composition obviates the need for an adjuvant, whether as a component of the immunogenic composition or when administered together with the immunogenic composition. By "adjuvant is meant an agent which assists, augments or otherwise facilitates the elicitation of an immune response by an imraunogen. Non-limiting examples of excluded adjuvants include, Freund's adjuvant, aluminium hydroxide (ahim), aluminium phosphate, squalene, IL-12, CpG-oligonucleotide, Montanide ISA720, imiquimod, SBAS2, SBAS4, MF59, PL, Quil A, QS21 and iSCO s.

While it is preferred that adjuvant is absent from the immunogenic composition, it will be appreciated that other components such as immunologically acceptable carriers, diluents and/or excipients may be included. Typically, these include solid or liquid fillers, diluents or encapsulating substances that may be safety used in systemic administration. Depending upon the particular route of administration, carriers, diluents and/or excipients may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyois, alginic acid, isotonic saline, pyrogen- free water, wetting or emulsifying agents, bulking agents, coatings, binders, fillers, disintegrants, lubricants and pH buffering agents (e.g. phosphate buffers) although without limitation thereto. The immunogenic composition and method of prophylactic or therapeutic treatment of babesiosis may be administered to an animal in any one or more dosage forms that include tablets, dispersions, suspensions, injectable solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like.

Suitably, the immunogenic composition and/or method of prevention or treatment of babesiosis are effective against blood-stage Babesiosis. Blood-stage babesiosis is a stage where the Babesia parasite (e.g. merozoite) enters erythrocytes. In the blood stage, the parasite divides several times to produce new merozoites, which leave the red blood cells and travel within the bloodstream to invade new red blood cells. For the sufferer, blood-stage babesiosis is typically characterized by successive waves of fever arising from simultaneous waves of merozoites escaping and infecting red blood cells.

The immunogenic composition and method of prevention or treatment of babesiosis may elicit an immune response that is characterized as a CD4 ' T cell- mediated response (including solely CD4 ^r T celi-mediated responses and mixed CD4 ⁺ and CD8 ⁺ T cell-mediated responses), possibly with little or no antibody response. Preferably, the immunogenic composition and/or method immunize the animal to prevent, inhibit or otherwise protect the animal against subsequent Babesia infection, Preferably, a single dose of the immunogenic composition prevents, inhibits or otherwise protects the animal against subsequent Babesia infection.

As used herein "animar refers to any animal capable of infection by a Babesia parasite, particularly mammals inclusive of humans, bovines, pigs, deer, horses, donkeys, sheep and goats. As used herein, "havines" are members of the mammalian sub-family Bovinae and include cattle, buffalo, bison and yaks. Cattle include all breeds and sub-species of the genus Bos, including Bos indica and Bos taunts and hybrids thereof

So that preferred embodiments may be described in detail and put into practical effect, reference is made to the foDo ing non- limiting Examples.

EXAMPLES

Tafuramyein A B. bevit attenuation pilot trial

The aim of this trial was to assess infectivity, virulence and immunity induced by Dixie strain B. hovis treated with Tafuramycin A (TFA) compared to the current live attenuated vaccine Dixie strain B. b&w to gauge the plausibility of conducting a larger scale research trial involving susceptible intact yearling or adult cattle.

Methods

Attenuation method

Blood was taken from B.hovis infected calves into EDTA collection tubes. Thin blood smears were made to determine parasrtaemia. The blood was centrifuged at 1800 rpm for 8 minutes. Supematants were removed.

Tafuramycin A (TFA98) was prepared fresh from 2 mM stock solution.

Dilution of the stock solution in medium was necessary as the PET solution caused lyses of red blood cells.

I) TFA98 200μΜ solution: 1/10 dilution of stock solution in RPMI-

1640.

2) TFA9 20μΜ solution: 1/100 dilution of stock solution in RPMI- TFA98 (20 icromol r) parasite treatment #1

(3x) 300μ1 of parasitized red blood cells were diluted in 9 mL of serum free RPMI-1640. 1 mL of TFA98 200μΜ solution was added. This gave a final concentration of TFA98 of 20 μΜ.

TFA98 (2 micromolar) parasite treatment #2

(3x) 300 μϊ, of parasitized red blood cells were diluted in 9ml of serum free RPMI-1640. Iml of TFA98 20 μΜ solution was added. This gave a final concentration of TFA98 of 2 μΜ.

Untreated parasitised red h od cells

(3x) 300 μL· of parasitized red blood cells were diluted in 9 mL of serum free RPMI-1640. This is a wild-type parasite control without attenuating agent.

Untreated normal red blood cells

(3x) 300 μL of normal red blood cells were diluted in 9mL of serum free RPMI-1640. This is a negative control without attenuating agent.

Treated parasitized red blood cells (pRBC), untreated parasitized red blood cells (RBC) and normal red blood cells (nRBC) were incubated for 40 mmutes at 37°C in a CO; incubator in 25cm ² vented tissue culture flasks. Flasks were rotated every 10 mmutes to ensure even distribution. Following incubation, the parasitized red blood ceils were transferred into separate 50mL centrifuge tubes and the tubes filled up to 50mL widi RPMI-1640. They were centrifuged at 1500 rpm for 5 minutes. Supernatants were removed and the tubes filled up to 50mL with RPMI- 1640. Tubes were incubated at 37°C in a C0 ₂ incubator for 20 minutes. At the completion of this incubation, tubes were centrifuged at 1500 rpm for 5 minutes. The supernatants were removed and the tubes filled up to 50 mL with PBS. Following a final centrifugation at 1500 rpm for 5 minutes, supernatants were removed and pRBC pellet was resuspended in original volume. After an accurate cell count m a hemacytometer the required amount of pRBC for the appropriate immunizing dose was removed and diluted m 10 mL PBS. Tubes were centrifuged at ISOOrpra for 5 minutes before dilution to the appropriate immunizing dose of I x 10 ⁹ pRBC in 0.9% sodium chloride (6 mL). Trial method

Four Charolais cross Shorthorn splenectoraised calves (aged 4 to 6 months), derived from Tick Fever Centre's (TFC) breeder herd, were included. Refer to Table 1 for inoculation protocols. The two animals inoculated with TFA attenuated Dixie strain B. bavis were selected based on exemption from slaughter due to previous T ei!eria spp. sterilisation with unregistered products (primaquine and buparvaquone).

On 18 Mar 2013 calves were given the above inoculations intravenously.

However due to inaccurate parasttaemia counts (2 x 10 ⁷ iRBCs) inoculations were repeated on 20 March 2013 with the correct dose.

Eight weeks later, each calf was challenged with homologous Dixie strain B, tovfr (l l 0 ⁷ ) intravenously on 13 May 2013 (see Table 2).

Following vaccination, the calves were monitored daily for 14 days by measuring rectal temperatures, microscopic examinatio of Giemsa-stained tail tip smears for parasitaemia and packed cell volume measurements from EDTA blood samples collected from the jugular vein. The EDTA samples were also stored frozen for later B> bovis PC - this continued twice weekly from Day 15 to 52. Serum samples were collected on day 0 and day 28 to determine seroconversion by B. hovis ELISA. A blood sample collected into lithium heparin was also collected on day 0 and leucocytes extracted and stored for later cell mediated immunity (CM I) evaluation in conjunction with post-challenge samples.

Following the challenge inoculation, the calves were again monitored daily for 23 days (then every second day until Day 29) by rectal temperature and microscopic examination of tail-tip (and jugular as appropriate) smears for parasttaemia. Packed cell volume (PCV) measurements were taken from blood collected from jugular vein EDTA samples until Day 14; then directly from the coccygeal artery/vein from Da 15 onwards until there was evidence of PCV recovery. EDTA blood samples were collected daily until Day 14 and stored frozen for later PCR analysis. Lithium heparin samples were also collected at day 0, 3, 7 and 14 post challenge inoculation for later CM1 evaluation.

Results Vaccination

Only the positive control (D1 78) showed microscopic evidence of B. hovis or any associated clinical effect. This calf developed maximum parasitaemia (97 x lQ ⁶ /raL (or 1.26% PPE)) and corresponding maximum temperature of 40.8°C (normal range 38.0-39.5°C) on Day 8 (Figures 1 and 3). Further, minimum PCV of 24%, representing a 24% reduction from pre-inoculation values, occurred on Day 10 (Figure 2). The positive control was treated with 5% quinuronrum sulphate (1 mg/kg subcutaneously) on Day 8, prior to onset of any marked clinical distress.

The two calves inoculated with TFA washed i BCs and the negative control showed no evidence of Dixie strain B. bovis on microscopic examination; and temperatures and PCVs remained relatively stable and within normal limits.

Challenge

The negative control (D1599) developed the greatest maximum PCV depression (35%) and maximum parashaemia count (43.0 xl 0 ⁶ /mL) compared to the other calves post challenge inoculation (Table 3; Figures 5 and 6). These findings correlated with pyrexia at 40.3°C on Day 8 post challenge (Figure 4). This calf spontaneously recovered without treatment within four days from Day 10 when a rapid decline in parasitaemia occurred (Figure 6). The PCV subsequently increased and plateaued from Day 15 at the lower end of the normal range, still 1 % below the pre-inocnlation value. The rectal temperature also returned to within normal limits from day 12, with occasional fluctuation.

The positive control remained negative tor B. bovis on microscopic examination, and maintained the most stability in temperature and PCV readings (Table 3; Figures 4 and 5).

The TFA 20 μΐ οϊβ _^ calf ( 1577) also experienced pyrexia at 40.6°C on Day 12 post challenge. Instead of a sudden decline in PCV (as in the negative control calf)' * gradual reduction in PCV was seen in this calf to a minimum of 24% on Day 20-21 , representing a PCV depression of 25%. This was associated with a persistent low level parasitaemia until Day 21 (with microscopic evidence of B. bovte on thick tail-ti smear only). The PCV remained stable between 24-25% from Day 21 onwards and began to slowly rise on Day 29.

The TFA 2 pmol/L calf (D1574) developed a greater and more persistent parasitaemia than the TFA 20 μιηοΙ/L calf, in which B. bevts iRBCs were evident in the jugular vein blood smear once only (on Day 7) (Figure 6). Moreover, both were sustantially leas than the negative control (Table 3). In addition, the TFA 2 μπτοΙ/L calf s PCV and temperature parameters remained within normal limits throughout the monitoring period (Figures 4 and 5), although PCV did reduce by 18% compared with pre-inoculatiort values. Interestingly, both TFA calves showed a similar slow decline in PCV from Day 9-25 which is most likely associated with the persistent parasitaemias.

Throughout the specification, the a m has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated without departing from the present invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.