Identification of In Vitro Culture Derived
Immunogenic Antigens from Local Isolates of Babesia bigemina
Asadullah Abid, Haroon Akbar, Sikandar Ali, Matiullah Khan, Adeel Mumtaz Abbasi, Zia ul Rehman and Imran Rashid*
Department of Parasitology, University of Veterinary and
Animal Sciences, Lahore 54000, Pakistan
*For correspondence: imran.rashid@uvas.edu.pk
Received 18 May 2021; Accepted
23 June 2021; Published 18 September 2021
Abstract
Bovine babesiosis is an economically significant
tick-borne disease in tropical and subtropical regions of the world.
Considering its importance, the current research was as an effort to identify
novel antigens of B. bigemina to control bovine babesiosis. The present
study evaluates the presence of immunogens (exoantigens) produced in vitro in
local isolates of B. bigemina. The blood from naturally infected cattle
calves, infested with ticks, showing signs of hemoglobinuria and elevated
temperature, were used in this study. It was examined under microscope for
intra-erythrocytic bodies, confirmed by PCR and used for in vitro
propagation of B. bigemina parasite using MASP culture technique. The
culture supernatants were harvested and pooled every 24 h for three days. These
supernatants were lyophilized, stored and rehydrated for SDS-PAGE and
immunoblotting. Two proteins of 65 kDa and >180 kDa were identified in
immunoblotting with the serum of a cow recovered from natural episode of
babesiosis caused by B. bigemina. The immunogens of Babesia can be
derived from in vitro cultivation. Furthermore, the diagnostic and
vaccine potential of the identified immunogens should be exploited for
developing rapid diagnostic kit and or a vaccine. © 2021 Friends Science
Publishers
Keywords: Babesia bigemina; Exoantigens; MASP;
SDS-PAGE; Western Blot
Introduction
Tick borne diseases (TBD) i.e., blood protozoan
diseases, pose a major threat to livestock sector. To meet the increasing
demand of meat, milk and other dairy products, majority of high-producing
cattle are being imported from other countries. It is, therefore, necessary to
check the current status of TBDs of cattle and buffalo in Pakistan to devise
their control (Atif et al. 2012; Jabbar et al. 2015). Babesiosis,
among TBDs, is responsible for considerable economic losses in crossbred cattle
in tropical and subtropical regions of the world (Passos et al. 1998).
The principal species causing bovine babesiosis are Babesia (B.)
bovis, B. bigemina and B. divergens as demonstrated by World
Organization for Animal Health (OIE). These parasites belong to phylum
Apicomplexa, order Piroplasmida and genus Babesia. It completes its life cycle
in vertebrate and an invertebrate host. Ixodidae tick, Rhipicephalus
microplus is a known-vector of B. bigemina, but the main source of
spread of disease is transportation of animals from infected areas. High level
of parasitemia in cattle is reported due to B. bigemina (Young and
Morzaria 1986). The most pronounced symptoms of the disease are elevated
temperature, anemia, dehydration, hemoglobinuria, and death. The recovered
animals remain carriers of the disease and become a source of spread when an
uninfected tick takes a blood meal (Alvarez et al. 2019). Diagnosis is
an important step to prevent the outbreak of babesiosis, but it is difficult to
detect by current conventional diagnostic methods due to less parasitemia
(Chaudhry et al. 2010).
Currently, the disease is being controlled by
chemotherapy, ticks control and vaccination. The
former two methods leave residues in meat and milk. Further, no chemoprophylaxis
schedule is made against the control of babesiosis due to less information on
prevalence of the parasites. The effective control for bovine
babesiosis is live attenuated vaccine but it has significant drawbacks
including danger of disease in immunostressed or immunocompromised animal,
maintenance of cold chain and a short storage life (Dominguez et
al. 2007). The animals may remain in
carrier state of disease and become a source of infection to healthy animals via tick vectors (Timms et al.
1984). Mishandling and lack of satisfactory vaccine are the major challenges
for the control of babesiosis (Montenegro-James et al. 1992). Nowadays,
vaccines are aimed to provide long lasting immunity without health risks, and
no withdrawal times.
During infection in vertebrate host, babesia parasite
sheds its merozoite surface coat that contains antigens. The parasite also
secretes proteins by their organelles, such as rhoptries and micronemes, to
invade host erythrocytes. Cattle can be immunized with these parasitic antigens
that are released during its proliferation (Wieser et al. 2019). With
the advent of Microaerophilous Stationary Phase (MASP) culture technique, the
presence of exoantigens is evident in in vitro culture supernatants. The
immunogenic exoantigens are a potential source of vaccine material (Patarroyo et
al. 1995). For the prophylactic control of babesiosis, one approach is the
use of immunogenic, culture-derived, organism-free exoantigens. The exoantigens
can potentially provide better protection against heterologous challenge and
apparently there is no risk of the animals moving to carrier state (Timms et
al. 1984). The parasitic antigens can also be helpful for diagnostic
purposes (Ristic and Arellano 1986). Keeping in view the importance of babesiosis,
the experiments in present study were designed with the aim to isolate and
identify exoantigen/s from parasitic culture media.
Materials and Methods
The blood samples were collected from cattle calves
naturally infested with ticks and having signs of babesiosis. Thin blood smears
were prepared, fixed with absolute ethanol (2 min), stained with Giemsa (1:20
dilution for 30 min). The blood smears were observed under microscope (1000 ×
magnification) for the presence of intra-erythrocytic protozoans. The
parasitaemia was calculated by using following formula:
Polymerase
Chain Reaction (PCR)
PCR was performed using SimpliAmp Thermocycler and Taq
Polymerase (cat # TAQ005.500), specific primers were used to obtain product
size of 321 bp. The PCR mixture was prepared in a final volume of 20 µL.
Initial denaturation was given at 95ºC for 5 min, reaction was cycled for 35
times. Each cycle was started with denaturation at 95ºC for 30 sec, annealing
at 60ºC for 30 sec, extension step at 72ºC for 30 sec and a final elongation at
72ºC for 10 min was given. After that, these amplified DNA fragments were
analyzed on 1.5% agarose gel.
In vitro Culture
To obtain washed infected RBCs, the blood was
centrifuged at 1000 × g at 15ºC for 10 min. Supernatant, buffy coat and small
portion of upper red blood cells was discarded. The remaining sample was washed
with 1X PBS and used in culture of B. bigemina and as a source of
exoantigens. The parasites B. bigemina were cultivated by using MASP
culture technique (Levy and Ristic 1980). The atmospheric conditions were 5% CO2,
2% O2 and 93% Nitrogen.
Harvesting
Exoantigens
Soluble exoantigens were prepared according to method of
(Fish et al. 2008). The culture supernatant of B. bigemina was
harvested, according to method of (Nawaz 2018), every 24 h after culture was
placed. The supernatant was centrifuged at 6000 × g for 30 min then it was
filtered through 0.45 μm filter
to remove cell debris and extracellular parasites. This supernatant was
lyophilized and stored at 4ºC until use. The lyophilized supernatant was
rehydrated with distilled water prior to use in Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis (SDS-PAGE).
SDS-PAGE
Proteins were separated using methods of (Tsang et
al. 1983), all SDS-chemicals were obtained from BioRad Laboratories. After
casting the apparatus, two discontinuous gels were prepared at room temperature
having 5% stacking gel and 12% resolving gel. The gels, when solidified, were
placed in Tris-Glycine SDS buffer (Tank buffer). The samples were prepared
under reducing conditions using 2X Laemmli buffer (4% SDS, 20% glycerol, 0.004%
bromphenol blue, 0.125 M Tris-Cl, pH 6.8, 10% 2-mercaptoethanol) and an equal
volume of rehydrated culture supernatant. The samples were heated at boiling
temperature of water prior to loading in SDS-PAGE gel. Standard molecular
weight markers (Cat # 26616), ranges from 10 to 180 kDa, were used to check the
relative mobility of proteins. Initially 80 V, 200 mA for 30 min was applied to
linearize the samples on resolving gel, then 120 V, 400 mA for 150 min was
applied when samples were passing through resolving gel. The gel was stained
with Coomassie Brilliant Blue R-250, and de-stained with de-staining solution
(Glacial Acetic Acid, Methanol, and water) until the background was clear
(generally over 2 h) and observed for proteins.
Development
of Immunoblot
Proteins bands were transferred onto nitrocellulose
membrane (NCM) (Trans-Blot Turbo® Transfer SystemTM) for 7 min, 11
volts and 1.3 ampere. The gel and NCM was soaked in Tris-Buffered Saline Tween
(TBST). Proceeding transfer, the membrane was washed in TBST buffer. The NCM
was than blocked with 5% skimmed milk. The membrane was soaked in serum of
babesia recovered animal at 1:100 dilutions in blocking buffer. The membrane
was washed again in TBST buffer to ensure washing of spare antibodies. The
membrane was then treated with Rabbit anti-bovine IgG conjugated with Alkaline
Phosphatase (A0705 Sigma-Aldrich) at 1:3000 dilutions prepared in blocking
buffer. The membrane was washed again to ensure the removal of residual
secondary antibody. BCIP (5-bromo-4-chloro-3-indolyl phosphate; Ref no: 34042
Lot no: UH289227) was added over the membrane and placed in dark, frequently
checked until the visualization of bands over the membrane. The reaction was
stopped by diluting it with several volumes of distilled water. The membrane
was dried, photographed and stored for later use.
The schematic flow diagram for the identification of
exoantigen has been shown in Fig. 1.
Results
Microscopic Evaluation
The presence of intra erythrocytic bodies was seen and
3% parasitemia was observed in the blood of infected animals (Fig. 2).
PCR
The amplicon size was observed at 321 bp (Fig. 3) on
1.5% agarose gel, the control positive was developed in Molecular Laboratory,
Department of Parasitology, UVAS, Lahore, by (Umber et al. 2020).
MASP Culture
Increased parasitemia level leads to low oxygen
concentration that resulted in darkening of RBCs culture. The bright red color
indicates no or less proliferation of parasites in culture media, while a color
change indicates proliferation of babesia parasites in blood cells.
SDS-PAGE Analysis
The SDS-PAGE analysis revealed many dense proteins in
all wells throughout the gel. Several proteins were seen overlapping each other
(Fig. 4). In sample 1, more prominent bands are present at 27, 32, 37, 45 and
60 kDa. In sample 2, these bands are present at 32, 36, 40, 68 and >180 kDa,
while in sample 3, the prominent protein bands can be seen at 40, 68 and
>180 kDa.
Development of Immunoblot
The blot was developed using serum of recovered animal
as a source of primary antibody, and a commercial secondary antibody conjugated
with Alkaline Phosphatase. BCIP, as a substrate show a colour change at 65 kDa
and > 180 kDa in supernatants.
Discussion
Fig. 1: A schematic flow diagram for identification of
Exoantigen/s
Fig. 2: a. Microscopic image of blood smear of a healthy animal while b. Microscopic image of blood smear of Babesia positive calf showing
intra-erythrocytic bodies
Fig. 3: PCR results on 1.5% agarose gel. WM indicates weight
marker, 1, 2 and 3 are PCR products. CP and CN are control positive and
negative, respectively
Being more economic, thin and thick Giemsa-stained
smears are prime choice for detection of blood protozoans, but these have many
drawbacks including false +ve/-ve results. Currently, highly sensitive and
specific molecular tools are being used for accurate diagnosis of Babesia at
species level (Alvarez et al. 2019), such as PCR based assays which are used for rapid
detection in live and dead animals (Singh et al. 2007). The molecular probes used in this
study were developed previously in our laboratory for the detection of B.
bigemina (Umber et al. 2020) and routinely used for the
evaluation of parasite in the blood of diseased animals. The molecular methods
are more analytical, sensitive and specific but these are not economic, require
higher expertise and technical skills.
Fig. 4: a. SDS-PAGE analysis of proteins in culture supernatant,
12% SDS-PAGE stained with Coomassie Brilliant blue R-250 b. Western Blot
analysis of supernatant proteins separated on SDS-PAGE, immunoblot was
developed using serum of B. bigemina recovered
cattle. M is weight marker, whereas 1, 2 and 3 are supernatant cultures
The
soluble antigens are efficient immunogens against bovine babesiosis for the
induction of protective immunity (James 1984). Exo-antigens are immunogens naturally
released from parasite into blood plasma or supernatant in case of in vitro cultures
(James 1989). The merozoite surface coat of Babesia has antigens, which are
responsible for production of antibodies that react with parasite and cause
lysis of merozoites. Exoantigens provide a potent, efficacious, and safe
control for immunoprophylaxis against bovine babesiosis (Montenegro-James
1989). The profile of exoantigen revealed two immunogenic
bands, at 65 kDa and greater than 180 kDa, identified by anti–B. bigemina
serum, collected from naturally infected animal with B. bigemina.
Further investigations are needed for identification of amino acid sequences,
protein classification and to understand the role of these immunogenic proteins
in parasitic life cycle. Our results indicate that these antigens can
potentially induce protective immunity as these were identified with anti–B.
bigemina serum from an animal recovered after a natural infection.
Antigens
produced in in vitro culture supernatants have been used in a commercial
vaccine against canine babesiosis (Pirodog®) (Moreau
et al. 1989). An improved vaccine (Nobivac® Piro) with
combination of different soluble antigens conferred greater protection against
heterologous infection (Schetters et al. 2007; Freyburger et al.
2011). The immunoprotective efficacy of in vitro produced B. bigemina
exoantigens have been reported (Beniwal et al. 1997). The present study
also corroborates the presence of novel exoantigens in in vitro culture
supernatants of B. bigemina. Since, the cultures require biological
components they have less hazard of contamination with extraneous agents than
live attenuated vaccines (Pipano 1997). Until optimal vaccines are developed,
the culture derived soluble Babesia immunogens may offer the best combination
of potency, efficacy and safety to fulfil the critical need for
immunoprophylactic control against bovine babesiosis.
Conclusion
The immunogens of Babesia can be derived from in
vitro cultivation. The diagnostic and vaccine potential of these identified
immunogens should be exploited for development of rapid diagnostic kit and/ or
a vaccine.
Acknowledgements
The research was conducted in Toxo and Cell Culture
Laboratories in Parasitology Department, UVAS, Lahore, Pakistan under
HEC-NRPU-7173 Funded Project.
Author Contributions
AA, HA and IR conceptualized, designed the experiment;
AA, MK, SA, AMA and ZR acquisition of Data, analysis, and interpretation,
drafting of manuscript; HA and IR supervised, proofread the experiment, and
give access to research components.
Conflict of Interest
The authors declare that they have no competing
interests.
Data Availability
Data presented in this study will be available on a fair
request to the corresponding author.
Ethics Approval
The study was approved from the Animal Welfare and Ethic
Society of University of Veterinary and Animal Sciences Lahore, Pakistan with
No. DR/ 496, Dated May 30, 2019.
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