In vivo comparison of susceptibility between Bos indicus and Bos taurus cattle types to Theileria parva infection

NDUNGU, S.G. BROWN, C.G.D. & DOLAN, T.T. 2005. In vivo comparison of susceptibility between Bos indicus and Bos taurus cattle types to Theileria parva infection. Onderstepoort Journal of Veterinary Research, 72:13–22 The objective of this study was to determine whether Bos taurus cattle differ form Bos indicus in their susceptibility to infection with the Muguga stabilate of Theileria parva and in their resistance to the resultant disease. Ten Friesians (B. taurus), ten improved Borans (B. indicus), ten unimproved Borans (B. indicus) and ten Zebus (B. indicus) born to dams from an East Coast fever (ECF) endemic area were inoculated with an infective dose50 dilution of T. parva Muguga stabilate 147. All the animals except one Friesian and one Zebu developed schizont parasitosis. All the improved Borans, nine of the Friesians, eight of the unimproved Borans and six of the Zebus developed a febrile response. Four of the improved Borans, four of the Friesians and three of the unimproved Borans died of theileriosis. No significant difference (P > 0.05) in the prepatent period occurred between the groups, but the Zebus had a significantly shorter duration of schizont parasitosis (P > 0.05) and took a significantly shorter time to recover (P > 0.05) than the other three groups. There was no significant difference in the two parameters between the other three groups. The study showed that three B. indicus breds and a B. taurus breed are equally susceptible to T. parva infection. However, Zebus born to dams from an ECF endemic area showed a better ability to control the course of disease than cattle from ECF free areas.


INTRODUCTION
Theileria parva is a protozoan parasite of the African buffalo (Syncerus caffer) which also infects cattle causing the disease East Coast fever (ECF) (Uilenberg 1976(Uilenberg , 1981)).It is transmitted transtadially by the three host tick Rhipicephalus appendiculatus.
The disease is prevalent in large areas of East and Central Africa where it causes major economic losses through morbidity and mortality.It is characterised by lymphocyte transformation into continuously dividing T. parva-infected lymphoblastoid cells (Hulliger, Wilde, Brown & Turner 1964) which infiltrate other lymphoid and non-lymphoid organs (Barnett 1960;Wilde 1967;De Martini & Moulton 1973).The non-lymphoid organs include the lungs whose infiltration is associated with severe pulmonary oedema (Irvin & Morrison 1987) resulting in asphyxiation and death of the affected animal.In susceptible animals morbidity and mortality may be in the region of 90 % (Brocklesby, Barnett & Scott 1961). 13 Onderstepoort Journal of Veterinary Research, 72:13-22 (2005) In  Veterinary Research, The objective of this study was to determine whether Bos taurus cattle differ form Bos indicus in their susceptibility to infection with the Muguga stabilate of Theileria parva and in their resistance to the resultant disease.Ten Friesians (B.taurus), ten improved Borans (B.indicus), ten unimproved Borans (B.indicus) and ten Zebus (B.indicus) born to dams from an East Coast fever (ECF) endemic area were inoculated with an infective dose 50 dilution of T. parva Muguga stabilate 147.All the animals except one Friesian and one Zebu developed schizont parasitosis.All the improved Borans, nine of the Friesians, eight of the unimproved Borans and six of the Zebus developed a febrile response.Four of the improved Borans, four of the Friesians and three of the unimproved Borans died of theileriosis.No significant difference (P > 0.05) in the prepatent period occurred between the groups, but the Zebus had a significantly shorter duration of schizont parasitosis (P > 0.05) and took a significantly shorter time to recover (P > 0.05) than the other three groups.There was no significant difference in the two parameters between the other three groups.The study showed that three B. indicus breds and a B. taurus breed are equally susceptible to T. parva infection.However, Zebus born to dams from an ECF endemic area showed a better ability to control the course of disease than cattle from ECF free areas.
The objective of this study was to compare the relative susceptibility and resistance to disease of cattle of the B. indicus and B. taurus breeds in order to determine whether the resistance is due to a lowered infectibility or a raised ability to recover from infection, and to relate these findings to the environmental background of the animals.To achieve this cattle of the European type of B. taurus and three different types or "breeds" of East African Zebu (B.indicus) cattle were infected with a standard dose of T. parva sporozoites.The Zebu cattle were (i) from dams which resided in an ECF endemic area; (ii) from an area known to be ECF free; and (iii) of improved Boran type, which had been maintained for generations free from ECF.

Cattle
Forty animals consisting of ten Friesians, ten improved Borans (herein referred to as Borans), ten unimproved Borans from an ECF-free area (herein referred to as non-endemic Zebus) and ten Zebus born to dams from an ECF-endemic area (herein referred to as endemic Zebus) were used.The animals were used when they were 1-1.5 years of age.
The Friesians were purchased from a farm in the Nakuru District of Kenya.The farm is located in an ECF-endemic area, but because of strict acaricidal control, no cases of ECF have been reported over many years.The Boran cattle originated from a farm in Laikipia district, also located in an ECFendemic area but where strict acaricidal control is practised.The unimproved Borans or endemic Zebus used in the experiment were purchased from cattle traders in Kitui district of Kenya.The animals had been brought south from Northern Kenya, where the vector tick for ECF, R. appendiculatus, and therefore the disease, does not occur (Walker 1974).
The endemic Zebus used in the experiment were Nandi Zebus (Mason & Maule 1960, Maule 1990).They were born in Muguga to dams purchased while pregnant from Sergoit Division of Uasin Gishu District where ECF is endemic.The animals were obtained from farms where no or minimal tick control is practised.
During the experiment the cattle were maintained at Muguga, which is an ECF-endemic area where strict tick control has to be practised.For this purpose the pour-on formulation flumethrin (Bayticol R pour-on, Bayer, Germany) (Stendel 1985) was applied along the backline of the animals from the occipitum to the base of the tail by means of a calibrated cylinder at the manufacturer's recommended dose of 1 mg/kg body mass every 10 days.
All animals were bled before purchase and the serum screened for T. parva schizont antibodies by the method described by Burridge & Kimber (1972).Only animals with antibody titres of less than 1:40 were selected for use in the experiment.
The animals were fed on hay and supplemented with ranch cubes (Unga Limited, Nairobi) and mineral licks (Macklic, Cooper Kenya Limited, Nairobi).They were dewormed once every month with levamisole hydrochloride (Nilzan, ICI, England).

Theileria parva parasite
A sporozoite stabilate of the Muguga stock of T. parva (Muguga) (Brocklesby et al. 1961) stabilate 147 was used to infect the cattle.This was stored in liquid nitrogen and had been prepared by Dolan, Young, Lossos, McMillan, Minder & Soulsby (1984) from infected R. appendiculatus ticks according to the method of Cunningham, Brown, Burridge & Purnell (1973) as modified by Radley (1978).It has been characterised by Dolan et al. 1984).
Before use, the vials containing the stabilate were removed from the liquid nitrogen and thawed rapid-ly by holding them in a waterbath at 37 °C.After thawing it was left to equilibrate at room temperature for 30 min.It was then diluted to 1:20 in Eagle's Minimum Essential Medium with Earle's Salts and Glutamine (Flow Laboratories, Irvin, Scotland) containing 3.5 % w/v bovine plasma albumin fraction v (Serve Feinbiochemica, GMBH Heindenbergh Co. Germany) and 7.5 % glycerol v/v to give an estimated 50 % lethal dose (LD50) of 1 ml for Boran cattle (Dolan et al. 1984).

Experimental design and parameters
The experiment was carried out in five replicate phases each consisting of eight animals, viz.two Friesians, two Borans, two non-endemic Zebus and two endemic Zebus.The animals were inoculated subcutaneously with 1 ml of the diluted stabilate immediately below and in front of the left ear.

Clinical monitoring (temperature response)
Each animal's rectal temperature was recorded every morning from the day of inoculation (day 0) until death supervened or day 28 for those animals that recovered.A temperature of 39.5 °C or above that was associated with schizont parasitosis was considered to be a significant clinical response.

Haematology
Blood was taken from the jugular vein of each animal into Vacutainer tubes containing the sodium salt of ethylenediamine tetra-acetic acid (EDTA) as anticoagulant.Total white blood cell (WBC) counts and red blood cell (RBC) counts were determined using an electronic particle counter (Coulter Electronics Inc., Florida, USA).Haemaglobin values were determined using a haemoglobinometer (Coulter Electronics Inc.) and the packed cell volume (PCV) was determined by the haematocrit method using a haematocrit centrifuge (MSE Scientific Instruments, Sussex, UK).Thin blood smears were employed to determine by light microscopy the WBC differential counts.These smears were fixed in methanol and stained in 10 % Giemsa stain (BDH Ltd, Essex, UK) in Giemsa buffer (pH 7.2) for 30 min.Two hundred cells were counted and the number of cells of different categories expressed as a percentage of the total white blood cells counted.

Serology
Blood for serology was collected from the jugular vein into serum Vacutainer tubes (Becton Dickinson, UK) 1 day before the parasite inoculation and once a week after inoculation until death supervened or day 35.Antibody titres were determined using the indirect fluorescent test (Burridge & Kimber 1972) using T. parva schizont-infected cells grown in cell culture as antigen.A titre of 1:160 or above was considered a positive response.

Parasitological observations and scoring
Needle biopsy smears were made daily from the left parotid lymph node, regional to the inoculation site, commencing from day 5 post-inoculation until day 28 from those animals that survivied this long.The smears were air-dried and fixed in methanol followed by staining in 10 % Giemsa's stain in Giemsa buffer (pH 7.2) for 30 min.The slides were differentiated in buffer and air dried before examination under a microscope (Leitz, Germany) using the x 50 and x 100 oil immersion objectives for the presence of schizonts.Once schizonts were detected, lymph node biopsy smears were also made daily from the contralateral lymph node, to determine the degree of parasite dissemination by calculating the proportion of infected lymphocytes per field.The degree of parasitosis was recorded on an arbitrary scale as described by Teale (1983):

Ma+
= schizonts detectable in one or only a few microscope fields: parasitosis < 1 % Ma++ = schizonts detectable in 50 % or more fields examined: parasitosis 1-5 % Ma+++ = schizonts observed in all or most of the fields examined: parasitosis > 5 % At the same time blood smears were made daily from blood obtained from the tip of the tail.These were fixed and stained as described for the lymph node biopsy smears.The blood smears were examined for the presence of intraerythrocytic piroplasms and the proportion of infected erythrocytes calculated.

Definition of disease reactions
Definition of disease reaction was, with some modifications, based on the recommendations of a workshop on ECF immunisation (Anon 1989).The day of recovery was considered to be the day after the last schizonts were detected and the febrile reaction had declined to < 39.5 °C.An "inapparent reaction" was where no parasites were seen and there were no apparent clinical signs.A "mild reaction" was considered to be one where low numbers of schizonts (Ma+) were detected with no or transient fever for less than 4 days.A "severe reaction" was one where schizonts were plentiful (Ma++ or Ma+++), a febrile response was observed for several days and the animal recovered.A "very severe reaction" was one where schizonts were plentiful (Ma++ or Ma+++), fever was persistent and the infection resulted in treatment of the animal or euthanasia in extremis.

Post mortem examination
Full necropsy examinations were carried out on all animals that died or were humanely destroyed during the experiment.Impression smears from the spleen, superficial lymph nodes, kidneys, lungs and adrenal glands were made, fixed in methanol and stained in 10 % Giemsa stain.They were examined by light microscopy for the presence of schizonts.
The presence of schizonts and oedema of the lungs led to the conclusion either that death was caused by theileriosis or that theileriosis necessitated the euthanasia.

Statistical analysis
Analysis of data was performed using the Kruskal-Wallis test (Fowler & Cohen 1990) unless otherwise stated.The Rankit test for normality (Fisher & Yates 1957;De Martin & Moulton 1973) was used to determine the normality of distribution of the data before statistical analysis.Comparison of parameters between any two groups was done using the Mann-Whitney U test (Fowler & Cohen 1990) while comparison of the number of animals that died or recovered was done using the Fisher Exact Test (Fisher 1958).

RESULTS
All the ten Borans, nine of the Friesians, eight of the non-endemic Zebus and six of the endemic Zebus developed a significant clinical febrile response.Four of the Borans, four of the Friesians and three In vivo comparison of susceptibility between Bos indicus and Bos taurus cattle to Theileria parva infection of the non-endemic Zebus developed very severe reactions and died of theileriosis.Four of the Friesians, six of the Borans, four of the non-endemic Zebus and three of the endemic Zebus developed severe reactions and survived.One of the Friesians, three of the non-endemic Zebus and six of the endemic Zebus had mild reactions while one of the Friesians and one of the endemic Zebus had inapparent reactions.Overall, the Borans manifested the most severe clinical disease followed by the Friesians, the non-endemic Zebus and the endemic Zebus (Table 1).
The febrile reactions followed a similar trend with the Borans having the highest mean maximum temperature (40.6 + 0.4 °C) followed by the Friesians (40.0 + 0.4 °C), the non-endemic Zebus (39.9 + 0.8 °C) and the endemic Zebus (39.9 + 0.6 °C).Comparatively, there was no significant difference (P > 0.05) in the time to onset of fever, duration of fever and development of peak fever between the groups.There was, however, a significant difference (P > 0.05) in the time it took to reach the peak fever reactions, with the endemic Zebus taking a significantly shorter time to reach these than the other three groups.There was no significant difference in the time it took to reach peak fever between the other three groups.In those that did recover the endemic Zebus also took significantly shorter time (P > 0.05) to recover (i.e.schizonts and fever disappeared) than the other three groups, and there was no significant difference between the other three groups in the time it took to recover.
The clinical manifestations of the disease in the Friesians, the Borans and the non-endemic Zebus were similar.The animals with severe or very severe reactions were listless and inappetent with laboured breathing, and lost condition very rapidly.They had petechial haemorrhages on the visible mucous membranes, were photophobic, and they developed a moist cough and corneal opacities of varying numbers and severity.Of the three Zebus that had severe reactions, only one was listless and inappetent and consequently lost condition.The other two were alert and retained their appetite and condition throughout the duration of the experiment, although they had a moist cough and petechial haemorrhages on the visible mucous membranes.The superficial lymph nodes became enlarged in all the animals that developed clinical disease.Using the Fisher Exact test there was a significant difference in the number of animals that died and conversely, the number of animals that recovered.Significantly more Friesians, Borans and non-endemic Zebus died of theileriosis than endemic Zebus P > 0.05).17   There was no significant difference in the number of animals that died between the other three groups.
All animals except one Friesian and one endemic Zebu became infected and developed schizont parasitosis.The Friesian, however, developed a piroplasm parasitaemia, but the endemic Zebu did not.Both animals also developed positive antibody titres indicating they had been infected.All the Friesians and Borans, nine of the non-endemic Zebus and eight of the endemic Zebus developed piroplasm parasitaemias.
There was no significant difference (P > 0.05) in the length of the prepatent period between the groups (Table 2).There was a significant difference (P > 0.05) in the duration of schizont parasitosis with schizonts being detectable in the endemic Zebus for a shorter time than in the other three groups.
There was no significant difference in the duration of parasitosis between the Friesians, the Borans and the non-endemic Zebus.There was no significant difference (P > 0.05) between the groups in the time to onset of piroplasm parasitaemia, and in the time to peak parasitaemia.
The haematological responses were similar in all the groups.There was a marked fall in the WBC counts in all the four groups.There was no significant difference (P > 0.05) in the pre-infection total WBC values between the groups (Table 3), but the Boran WBC values fell to significantly lower levels (P > 0.05) than those of the Friesians, the non-endemic Zebus or endemic Zebus.The fall in WBC counts was a panleukopenia involving lymphocytes, neutrophils, eosinophils and monocytes.In all cases there was a recovery of the WBC numbers from the third week of infection in the animals that recovered.For all the animals that died, the WBC counts fell and did not recover.The Friesians had significantly lower (P > 0.05) pre-infection PCV values than those of the endemic Zebu although all were within the normal range.The Friesians had significantly lower (P > 0.05) pre-infection haemoglobin concentrations than the other three groups.There was no significant difference in PCV values between the other three groups.The PCV values fell slightly during the course of disease, but remained within the normal bovine range in the groups.There was a slight fall in the RBC counts in all the animals groups, but no significant differences were observed in the pre-infection values and those that developed during the course of the disease.In individual animals, six of the Friesians, five of the non-endemic Zebus, two of the Borans and a single endemic Zebu showed variable degrees of anaemia with no signs of regeneration of the RBC.All these animals had severe reactions except the endemic Zebu which had prolonged parasitosis but no fever.
All surviving animals had seroconverted (i.e. had positive antibody titres to T. parva schizont antigen) by day 35.Only two of the animals which died, one Friesian and one non-endemic Zebu, had seroconverted.The animals that died showed similar lesions on necropsy.They had various degrees of pulmonary oedema, hydrothorax, hydroperitoneum and hydropericardium.The kidneys revealed the presence of "pseudoinfarcts" typical of ECF and the mucosa of the abomasum had erosions.Ecchymotic haemorrhages were evident on the epicardium and the serosal surface of the rumen.The lymph nodes were enlarged and haemorrhagic.The spleen was of normal size or slightly atrophic.Schizonts were observed in impression smears made from the spleen, lymph nodes, kidneys, adrenal glands and lungs.

DISCUSSION
In this study no differences were observed between B. indicus and B. taurus cattle or between any of the Zebu (B.indicus) groups in their infectibility with T. parva Muguga sporozoite stabilate.All the animals developed a patent schizont parasitosis except one of the Friesians and one of the endemic Zebus.Piroplasms, however, did develop in the blood of this Friesian indicating that the infection had established itself and both these animals seroconverted which confirmed this.The length of the prepatent period in the three B. indicus and the B. taurus cattle was the same, and as the prepatent period in T. parva infection is dose dependent (Jarret, Crighton & Pirie 1969;Cunningham et al. 1973;Radley, Brown, Burridge, Cunningham Pierce & Purnell 1974;Dolan et al. 1984;Buscher, Morrison & Nelson 1984;Mutugi et al. 1991) it is concluded that the animals used in this experiment must have received and effectively been infected by the same dose of the parasite.
The severity of disease is also dose dependent and in animals of equal susceptibility, it would be expected that their clinical reactions would be the same.This was not the case in this experiment.The Friesians and the Borans suffered from a markedly more severe disease than the endemic Zebus, while the non-endemic Zebus had a disease reaction mid-way between the Friesians and Borans and the endemic Zebus.Statistically, the Friesians and Borans were similar in their reaction to infection and the non-endemic Zebus and the endemic Zebus were similar to each other but different from the Friesian/Borans.(Irvin & Morrison 1987).These observations indicate that resistance to ECF in B. indicus cattle derived from endemic areas is not innate but acquired through selection of the more resistant members of the cattle population in ECF-endemic areas.This resistance is passed to the progeny since animals from endemic areas which have been kept for several generations under ECF-free conditions retain a high degree of resistance to ECF (Barnett & Bailey 1955b;Barnett 1957;Guilbride & Opwata 1963;Irvin & Cunningham 1981;Paling et al. 1991).In our study, the endemic Zebus showed high resistance to the disease despite of having had no prior contact with ECF throughout their lives.
The pathogenic effects of T. parva infection are due to its ability to induce uncontrolled proliferation of the infected lymphocytes (Hulliger et al. 1964;Malmquist, Nyindo & Brown 1970).The pattern of cellular changes during infection is similar in both B. indicus and B. taurus cattle (Morrison, Buscher, Murray, Emery, Masake, Cook & Wells 1981) and death of infected animals is brought about by this lymphoproliferation, membrane 'leakage' and infiltration of lymphoid and non-lymphoid organs by parasitized cells which interfere with the normal function of these organs.This is especially so in the lungs, where the infiltration is associated with severe pulmonary oedema which leads to asphyxiation (Irvin & Morrison 1987).The resistance of the endemic Zebus to ECF seems to be based on the greater efficiency of the immune mechanism to limit the proliferation of parasitized cells as exemplified by a shorter duration of parasitosis.How this is achieved and at what stage in primary infections, is not known.What seems certain is that the effect is not at the sporozoite infection stage since the prepatent period was the same for both the resistant and susceptible animals and, in effect, all cattle became infected.In field situations Barnett & Bailey (1955a), Young (1981) and Moll, Lodhing & Young (1984) observed that in ECF-endemic areas, calf mortality in indigenous animals due to ECF is very low in spite of morbidity approaching 100 %.Colostral antibodies are not protective (Mining 1992) and neither are serum antibodies (Cowan 1981).These observations lend support to our findings that Zebus from ECF-endemic areas are equally infectible as animals from ECF-free areas, but they have evolved immune mechanisms that efficiently control proliferation of parasitized cells.
In conclusion, no differences in infectibility or susceptibility to infection per se were observed between any of the cattle types used in this study.Moreover, B. taurus and B. indicus cattle from ECF-free areas suffered equally severe reactions when infection was induced with a 1:20 dilution of T. parva Muguga stabilate 147, a dose which was calculated to be an LD 50 in susceptible, improved Boran cattle.There was, however, a marked difference in resistance to ECF between animals (both B. indicus and B. taurus) originating from ECF-free areas and B. indicus originating from ECF-endemic areas, with the latter being markedly more resistant than the former.
Although the mechanism of this resistance is not known, it could be that these animals have greater populations of natural killer cells that inhibit the proliferation of infected cells.
vivo comparison of susceptibility between Bos indicus and Bos taurus cattle types to Theileria parva infection S.G.NDUNGU 1 , C.G.D. BROWN 2 and T.T. DOLAN 3 ABSTRACT NDUNGU, S.G.BROWN, C.G.D. & DOLAN, T.T. 2005.In vivo comparison of susceptibility between Bos indicus and Bos taurus cattle types to Theileria parva infection.Onderstepoort Journal of