Research Article
Austin J Vaccines & Immunother. 2016; 3(1): 1009.
Immunization with Live Attenuated Schizonts against Tropical Theileriosis (Theileria Annulata Infection) Increases Antibody Titers in Chronically Infected Cattle in Sudan
Hussien MO1, Taha KM2, Enan KA3, Salih DA4 and El Hussein AM4*
1Department of Microbiology, Ministry of Higher Education and Scientific Research, Sudan
2Atbara Veterinary Research Laboratory, Animal Resources Research Corporation, Sudan
3Department of Virology, Central Laboratory, Ministry of Higher Education and Scientific Research, Sudan
4Central Veterinary Research Laboratory, Animal Resources Research Corporation, Sudan
*Corresponding author: El Hussein AM, Central Veterinary Research Laboratory, Animal Resources Research Corporation, El Amarat, Khartoum, Sudan
Received: February 15, 2016; Accepted: June 06, 2016; Published: June 09, 2016
Abstract
In Sudan, two isolates of T. annulata, namely Atbara and Hantoub were isolated and successfully attenuated in cell culture. An immunization trial to monitor the antibodies induced by attenuated T. annulata schizont candidate vaccine injection to crossbred cattle under field conditions was conducted in Atbara town, Northern Sudan during June and July, 2013. In this trial, 25 cattle of ages ranging from 1 month to 6 years were divided into five groups: one group as control and the other groups were inoculated with different doses of the candidate vaccine (0.05; 0.1; 0.5 and 1 million cells). Blood samples were collected before immunization and four weeks after. The antibody profile against T. annulata was assessed using ELISA and IFAT. All the animals were naturally seropositive using both techniques. Titration of antibodies using IFAT at four weeks after immunization showed significant increase in antibody titers against T. annulata. These results indicated that the candidate vaccine induced an increase of antibody titers in immunized animals even if they were seropositive because of natural infection.
Abbreviations
TBD: Tick-Borne Disease; T. annulata: Theileria annulata; GMEM: Glasgow Minimum Essential Medium; NaHCO3: Sodium Bicarbonate; TaSP: Theileria annulata Surface Protein; IFAT: Indirect Fluorescent Antibody Test
Introduction
Tropical theileriosis is a major Tick-Borne Disease (TBD), which affects cattle throughout a large geographical area in the world including North Africa, Southern Europe and a large part of Asia. Since several years, the total cattle population at risk was estimated to be 250 millions [1]. The disease is caused by an apicomplexan parasite: Theileria annulata, which is transmitted by several tick species belonging to the genus Hyalomma [2]. Tropical theileriosis is one of the most important TBDs of cattle in Northern Sudan; it represents a major constraint to the intensification of cattle farming [3]. Schizont attenuated vaccine is to date the most effective measure for controlling this disease [4-6].
Immunization using attenuated T. annulata schizont vaccines usually but not always result in a response that involves both cellular and humoral immune responses. Cellular immune response include both specific (MHC-1 restricted) and non specific (MHC-1 non restricted) T cell response [7,8]. Humoral (antibodies) response has also been shown to be protective against the sporozoites and merozoites stages of the parasite [9]. The presence of humoral immune response detected by either IFA or ELISA is taken as indirect evidence of stimulation of cellular immune response [10].
In the Sudan, two isolates of T. annulata, namely Atbara and Hantoub were isolated and successfully attenuated in cell culture [11,12], they were evaluated as vaccine candidates in a series of immunization and field challenge trials [13,14] (Sharieff et al. unpublished data).
Owing to the prevalence of tropical theileriosis in most of the Sudan, especially in exotic dairy cattle and their crosses with local type, the present trial was carried out to monitor the antibodies induced by attenuated T. annulata schizont candidate vaccine application to crossbred cattle under field conditions in Atbara town, a known endemic area in Northern Sudan during June and July, 2013.
Material and Methods
Cell culture vaccine
Atbara attenuated T. annulata schizont candidate vaccine was used at passage 98 of culture in Glasgow Minimum Essential Medium (GMEM) (Sigma, USA) supplemented with 1.875 g of Sodium Bicarbonate (NaHCO3), 12.5 ml of sterilized Hepes buffer, 20% foetal calf serum, 100 mg of Streptomycin and 100000 IU penicillin per liter.
Immunization of animals using different doses
Twenty five crossbred cattle aged between 1 month and 6 years were randomly divided into five groups of four animals (group 1 and 3), five animals (group 2 and 4) and seven animals (group 5). The experimental animals in groups 1, 2, 3 and 4 were inoculated subcutaneously with 0.05; 0.1; 0.5 and 1 million cells of Atbara vaccine candidate and group 5 was kept as unimmunized control group.
Blood sample collection
The day of immunization, all animals were examined for the presence of T. annulata IgG antibody using T. annulata Surface Protein (TaSP) ELISA. All samples with a percentage positivity values higher than 31.6 were considered positive [15]. On the other hand, titration of sera using Indirect Fluorescent Antibody Test (IFAT) was performed on the day of immunization and four weeks as described before [16].
Statistical analysis
All IFAT titers were log-transformed then comparisons between the titers on the day of immunization and four weeks after were performed by Tukey test using SPSS 20.0 for Windows Software package. A probability of 0.05 was used as cut-off value.
Results
On the day of immunization, all the experimental crossbred cattle were positive to T. annulata IgG antibodies using TaSP ELISA and IFAT. Titration of antibodies with IFAT after four weeks of immunization revealed significant increase in antibody titers (p<0.05) (Tables 1 & 2).
Animal group
Age group
(body weight in kg)
Dose
(.106 cells)
Pre-immunization
Post-immunization
Group 1
1 < < 3 months
(30)
0.05
0.000*
1A
80
640
1B
80
640
1C
160
640
1D
80
1280
Geometric mean
95
761
Group 2
≥ 4 months
(40)
0.1
0.000*
2A
80
640
2B
80
320
2C
80
320
2D
80
640
2E
80
640
Geometric mean
80
485
Group 3
2-5 years
(40<≤ 120)
0.5
0.001*
3A
80
640
3B
160
1280
3C
160
1280
3D
160
160
Geometric mean
134
640
Group 4
≥ 6 years
(≥ 200)
1
0.003*
4A
160
320
4B
320
640
4C
160
640
4D
80
640
4E
160
640
Geometric mean
160
557
Significant at p<0.05
Table 1: IFAT Ab-titers of crossbred cattle before and after immunization with different doses of attenuated Theileria annulata schizont candidate vaccine at passage 98 during June – July, 2013.
Animal group
Day 0
Day 28
P value
Group 5 (Control)
0.001*
5A
80
320
5B
80
320
5C
80
320
5D
160
320
5E
160
160
5F
80
640
5G
160
640
Geometric mean
108
353
Significant at p<0.05
Table 2: Antibodies titers in the control group determined at day 0 and day 28 of the experiment.
Pre-immunization mean antibody titers were significantly higher (p<0.05) in older animals (2 years and above) than in the youngest one (1 - 4 months) However, no significant difference (p>0.05) was noted between different age groups after immunization (Table 3).
Age group
Mean Ab titers before immunization ± SD
Mean Ab titers after immunization ± SD
1 < < 3 months
1.98 ± 0.2b
2.88 ± 0.2
≥ 4 months
1.90 ± 0.0b
2.69 ± 0.2
2-5 years
2.13 ± 0.2a
2.81 ± 0.4
≥ 6 years
2.20 ± 0.2a
2.75 ± 0.1
SD: Standard deviation a, b: letters in the same column followed by different superscripts are significantly different (p<0.05)
Table 3: IFAT log-titers from different age groups of crossbred cattle before and after immunization with attenuated Theileria annulata schizont candidate vaccine at passage 98.
Analysis of antibody titers of tested cattle before and after immunization showed that the youngest animals (1 - 4 months) had the highest antibody response (Table 4).
Group
Age
Dose
Proportional increase in titre
(Post-titre / pre-titre)
1
1 < < 3 months
0.5×105
8 (761/95)
2
≥4 months
1×105
6 (485/80)
3
2 – 5 years
5×105
4.8 (640/134)
4
≥ 6 years
10×105
3.5 (557/160)
5(control)
1month < < 6years
-
3.3 (353/108)
Table 4: Proportional increase in titre of crossbred cattle before and after immunization with different doses of attenuated T annulata schizont candidate vaccine at passage 98.
Discussion
A well-established technique to produce attenuated live vaccine against tropical theileriosis has been used in Iran [17], Turkey [18], Tunisia [19,20], Uzbekistan [21] and Sudan [7]. In the present study, pre-immunization low titers were observed mostly in the youngest cattle group (1 - 4 months). This may probably due to maternal antibodies and/or low natural challenge history. Interestingly, this age group showed the highest antibody response four weeks after immunization indicating that pre-existing antibodies may not interfere with vaccine intake. Whilst, the lowest antibody response was observed in old cattle (aged ≥ 6 years). This may also indicate that the 1 - 4 months old animals are the most suitable age group to target for vaccination.
Doses used in the current trial were based on prior experience in the field where sero-negative animals were used [9] (El Hussein et al., unpublished data). The present results showed that these doses could be injected safely to cattle in the field. Indeed, the antibody increase observed in the immunized animals, indicated that the use of the vaccine in seropositive cattle could afford protection in partially immune animals that may succumb to the disease because of the high field challenge in some areas of Sudan although no reports of vaccination trials in naturally infected animals with theileria or any other apicomplexan parasites are recorded however therapeutic immunization has been tried in cases of viruses e.g HIV, bacteria e.g Helicobacter pylori, H. mustelae and parasites e.g leishmania donovani. The vaccines used were well tolerated and immunogenic and in some instances such as H. mustelae infection the infecting bacteria was cleared [22-25].
Moreover, no untoward side effects were observed among vaccinated animals. El Haj, [9] reported also that this cell culture vaccine using comparable doses was able to protect crossbred animals against disease occurrence.
Conclusion
It could be concluded that the present live attenuated schizont T. annulata candidate vaccine can be used safely in apparently healthy animals without regard to their natural infection status. Moreover, this kind of vaccine is known to be safe and reversion to virulence has never been demonstrated and that natural repeated challenges in the field induce long-lasting immunity without need to revaccinate these animals [6,26]. It is highly recommended that mass production and distribution of the vaccine be encouraged from the available strains of vaccine seeds in the Sudan. This vaccine could be injected once to young calves regardless of their serological status. The presence of high infected tick population will then boost the immunity. Because of the economic importance of tropical theileriosis in Sudan it is reasonable that the concerned animal health authorities in the country should consider adoption such policy that would lower costs of disease control in addition to the environmental benefits. Finally and to the best of our knowledge this study represents the first report on the reaction of T. annulata seropositive animals to immunization with attenuated schizont vaccine. However, increased titer in non vaccinated animals can indicate low level tick transmission of the parasite during the trial period. This might have also induced a booster effect in the vaccinated animals.
Acknowledgement
This work was supported by the Deutsche Forschungsgemeinschaft German–African Cooperation Projects in Infectiology “Molecular epidemiology network for promotion and support of delivery of live vaccines against Theileria parva and Theileria annulata infection in Eastern and Northern Africa” (DFG SE862/2-1).
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