In Vitro Study on the Effect of Bee Venom on Some Cell ... · Five groups of 11-day-old embryonated chicken eggs were used to test the viability of LSDV. The inoculation was performed

Journal of Agricultural Science and Technology A 6 (2016) 124-135 doi: 10.17265/2161-6256/2016.02.006

In Vitro Study on the Effect of Bee Venom on Some Cell

Lines and Lumpy Skin Disease Virus

Samia Ahmed Kamal

Virology Department, Animal Health Research Institute (AHRI), Dokki, Giza 99999, Egypt

Abstract: Bee venom (BV) was used from long time ago in the medical field as treatment of chronic joint affections. In the recent decades, the screening process of new sources of antimicrobials discovers its high advantageous characteristics for combating various types of microbes, as well as trials to discover its anti-cancer medicinal fields. Lumpy skin disease virus (LSDV) causes disease in cattle of economic importance, and this work aimed to find treatment as well as alternative inactivant for LSDV. The use of bee venom as antiviral was experimented in this work and exhibited satisfied inhibitory effects on LSDV, meanwhile, the antigenic properties was still intact. The viability of virus was tested in tissue culture cells lines and in embryonated chicken eggs. According to doses and time of exposure, the cell lines of Hep-2 (human larynx carcinoma) and MCF7 (breast carcinoma cell line) were treated with different concentrations of BV and examined after 24 h post-inoculation. The Hep-2 and MCF7 cell lines were treated with various concentrations of BV in descending doses as follow: 25, 20, 15, 10, 5 and 0.5 ug/mL of BV. Then bee venom pathological effects on Hep-2 cells and MCF7 cells were observed, such as apoptosis, retarded growths and cytolysis. The results indicate the possibilities of using bee venom as anti-neoplastic and antiviral.

Key words: Bee venom, lumpy skin disease virus, anticancer, Hep-2 cells, MCF7, antiviral natural substances.

1. Introduction

Lumpy skin disease (LSD) acquired its name from

the skin lesions on the infected cattle. It is

characterized by formation of hard nodules on skin

and mucous membranes accompanied by fever,

lesions on the internal organs, general deterioration of

health with emaciation, enlarged lymph nodes, edema

of the skin, and sometimes death. LSD in cattle

usually was complicated by secondary infections

(bacteria, screw worm, other contaminant viruses

“orphan viruses”), which affected the productivity of

dairy farms and may cause deaths from complications

of secondary infections or from primary infections by

more pathogens endemic in the environment [1-4].

Lumpy skin disease virus (LSDV) (genus

Capripoxvirus, family Poxviridae) is stable, survives

at ambient temperature for long periods and is highly

resistant to inactivation, surviving in necrotic skin

nodules for up to 33 d or longer, in desiccated crusts

Corresponding author: Samia Ahmed Kamal, Ph.D.,

research fields: animal pathology and virology.

for up to 35 d and at least 18 d in air-dried hides. So, it

can remain viable for long periods in the environment

[5]. The virus is susceptible to sunlight and detergents

containing lipid solvents, but in dark environmental

conditions, such as contaminated animal sheds, it can

persist for many months. The virus is susceptible to

55 °C for 2 h and 65 °C for 30 min, but can be

recovered from skin nodules kept at -80 °C for 10

years and from infected tissue culture fluid stored at

4 °C for six months. It is affected by higher alkalinity

or acidity, and killed by ether (20%), chloroform,

formalin (1%) and some detergents, e.g., sodium

dodecyl sulphate [1, 6]. There is no specific antiviral

treatment available for LSDV-infected cattle. Two

vaccines, however, Neethling and Kenya sheep and

goat pox virus, have been used widely in Africa with

success [1]. Diagnosis of LSD usually depends on its

unique and characteristic clinical signs and its specific

macroscopic and microscopic histopathologic

appearance that never share resemblance with other

diseases. However, mild and subclinical forms require

D DAVID PUBLISHING

In Vitro Study on the Effect of Bee Venom on Some Cell Lines and Lumpy Skin Disease Virus

125

rapid and reliable laboratory testing to confirm

diagnosis. Laboratory diagnosis of LSDV could be

achieved by different techniques, as electron

microscopy, egg inoculation, cell cultures, fluorescent

antibody test and serological tests. However,

polymerase chain reaction (PCR) was applied for

routine diagnosis, because it is more sensitive and

specific, and for rapid detection of LSDV in field

specimens [7, 8]. Hep-2 and MCF7 cell lines were

derived from cancer lesions and used in vitro in

research work. Bee venom (BV) from the sting of

honeybee (Apis mellifera L.) is traditionally used in

China, Korea and Japan for arthritis, tendinitis,

bursitis and other chronic conditions. It is reported to

have pro-inflammatory [9] and anti-inflammatory

effects [10-14]. Effective control of LSD requires

solutions focused on combating the LSDV by finding

remedy and safe vaccine.

Scientists usually screen the nature to find new

sources of antimicrobials, and cationic peptides are

major adventure in this field, while BV contains

cationic peptides beside many other valuable

substances that have synergistic effects. The aim of

this study was to study the pathologic effects of BV

on cell lines for safety concerns and the suitable doses,

in other hand, to study the effects of safe doses of BV

on LSDV as a step towards applications as antiviral.

2. Materials and Methods

2.1 Preparation of BV Stock Solution

The colonization is conducted at the apiaries of

Plants Protection Institute (PPI), Beekeeping

Department, Agriculture Research Center (ARC),

Egypt.

The electrical shock method has been used to

stimulate the bees to sting. The collector frame was

placed at the entrance of the hive and connected to a

device which supplies electrical impulses; when bees

receive a mild electric shock, they sting the surface of

the collector sheet, as they see this to be the source of

danger. The deposited venom between the glass and

the protective material was dried, then later scrapped

off by a razor blade and collected in a dark bottle in a

powder form. According to Mraz [15], BV powder

(crude) of dose 2 mg was dissolved in 2 mL tissue

culture media—minimum essential medium eagle

(MEME), and then filtered by 0.22 μm syringe filter.

That final concentration of the BV stock was 1,000

ug/mL (i.e., 1 uL = 1 ug) and kept at -20 °C.

Preparation of BV solution was performed according

to Kamal et al. [16].

2.2 LSDV and Hyperimmune Sera

The field strain of LSDV and hyperimmune serum

were kindly provided by Dr. Mohamed Hussein from

Animal Health Research Institute (AHRI). It was used

for cell culture adaptation and propagation [17].

Recent local isolate of LSDV from natural outbreak

in Damenhour city in 2014, was tested by AHRI and

confirmed by immunoflurescent test and RT-PCR.

2.3 Tissue Culture Cell Lines

Maiden-Darby bovine kidney cells (MDBK), Hep-2

and MCF7 were prepared and provided by Egyptian

Company for Production of Vaccines, Sera & Drugs

(VACSERA), Egypt. The MDBK cells were used for

adaptation of LSDV, virus titration as well as serum

neutralization test (SNT). Hep-2 and MCF7 cell lines

were used for studying histopathological effects of BV

on living cell. The cell lines of Hep-2 and MCF7 were

treated with different concentrations of BV and

examined after 24 h post-inoculation. The Hep-2 and

MCF7 cell lines were treated with various

concentrations of BV in descending doses as follow:

25, 20, 15, 10, 5 and 0.5 ug/mL of BV [16].

2.4 LSDV Titration and Determination of Infectious

Dose (ID50)

MDBK cell lines were used for determination of

virus’ infectivity titer. The 50% tissue culture

infectious dose of a virus (TCID50) was carried out by

traditional methods of virus quantification. Viral


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infectivity titer was evaluated according to the method

adopted by Reed and Muench [18].

2.5 Incubation of LSDV with BV

The stock mixture of inactivated LSDV was

prepared by adding BV to LSDV and left for 8 h at

room temperature (21 °C), then kept in refrigerator at

-20 °C [16].

2.6 LSDV Isolation from Natural Outbreak

Skin biopsies, comprising epidermis and dermis of

the nodular skin lesions, were collected from local

cattle in Al Behera Governorate during an LSDV

outbreak in 2014. Biopsy specimens were taken

aseptically and the incisions were sutured. Samples

were collected in 15 mL sterile tubes and stored at

-20 °C until use. The procedures were performed

according to Payment and Trudel [19].

2.7 Embryonated Chicken Eggs Inoculation: Testing

BV Inactivation Ability of LSDV

Five groups of 11-day-old embryonated chicken

eggs were used to test the viability of LSDV. The

inoculation was performed via the chorio-allantoic

membrane (CAM) route. The dose of all samples was

0.1 mL/egg. The first group was infected with LSDV

from natural samples, the second embryonated

chicken eggs group infected with LSDV isolated in

tissue culture (Egyptian strain), the third group

(positive control) injected with BV stock solution and

the fourth group (test group) injected with LSDV

mixed with BV. The fifth group was kept as control

negative without injection. All groups were incubated

at 36 °C in CO2 incubators [19].

2.8 Virus Neutralization Test

Infective tissue culture material containing

disintegrated cells and maintenance medium was

centrifuged at 1,500 rpm for 10 min. Serial 10 fold

dilutions of the supernatant were made in Hanks

saline and mixed with equal volumes of undiluted

serum. Parallel dilutions were mixed with normal

serum. Contact was allowed to take place for 1 h at

37 °C, and after that, the mixture were inoculated onto

established monolayers in culture tubes, five tubes

being used per dilution. All tubes were examined daily

for cytopathologic effects (CPE) [19].

2.9 Agar Gel Precipitation Test (AGPT)

AGPT was performed for detecting the antigenic

properties of LSDV under experiment, the treated

virus with BV and the original viruses used in the

experiment [19].

3. Results and Discussion

3.1 Adaptation and Titration of LSDV in MDBK Cells

The titration results of LSDV in MDBK cells were

shown in Table 1. The highest level gives at the 10th

passages titre of log10(6.4)/1 mL and MDBK showed

satisfactory growth rate for LSDV. The highest

infectivity titer was used for the experiment.

3.2 Embryonated Chicken Eggs Inoculation

The results showed that both the control groups

(negative control and positive control) were normal

and still alive until the end of experiment. The test

group showed normal appearance without

pathological changes, which indicated the absence of

live viral particles. However, the other groups showed

pathologic changes in CAM (Fig. 1). This finding

indicates that LSDV is completely inactivated by

BV. The use of embryonated chicken eggs in testing the Table 1 Titration of different passages of LSDV on MDBK cells.

Virus passagesTime of CPE appearance (d)

Virus titre Log10(TCID50)/mL

3 3 5.2

6 2 5.4

8 3 6.0

10 3 6.4

CPE: cytopathic effect. TCID50: tissue culture infective dose 50 (this method evaluates an endpoint where 50% of the cel cultures are infected).


127

Fig. 1 The viral growths of LSDV in the form of thickening of CAM and pock lesions.

viability of LSDV is the most suitable method,

however, other methods were used as second choice.

The dose of 0.1 mL/egg was determined after testing

different sets of embryonated chicken eggs, and the

small dose was determined to avoid the direct toxicity

of BV on embryonated chicken eggs [17].

3.3 Morphological Examination of Hep-2 and MCF7

Cell Lines Treated with BV

3.3.1 Morphological Changes of Unstained Hep-2

Cell Lines

The unstained Hep-2 cells was treated with

different concentrations of BV while still attached to

the culture chamber and examined by the inverted

microscope. The morphological changes were shown

in Fig. 2. The apparent effects were graduated from

nearly destructed cells to lesser effects according to

concentration. In this experiment, the exposure time

was fixed (24 h). The cells were divided into eight

groups. In group 1 (control), non-treated cells showed

complete and intact sheet of cells (Fig. 2a). In group 2,

cells treated with 20 µg/mL BV showed incomplete

sheet with destructed cells (Fig. 2b). In group 3, cells

treated with 15 µg/mL showed destructed sheet (Fig.

2c). In group 4, cells treated with 10 µg/mL showed

incomplete sheet with some normal parts (Fig. 2d),

which indicated incomplete destruction of cells by BV.

In group 5, cells treated with 5 µg/mL showed

moderate changes in the form of slowing cell growth,

thus it could be explained as BV acts at this

concentration biologically while cells tries to

overcome its toxic effects (Fig. 2e). However, with

lesser concentrations, the toxic effects disappeared

gradually and the biological effects were expressed as

changes in the growth rates. These biological changes

were observed clearly in the lower concentrations,

beginning from cells treated with 2.5 µg/mL, which

showed slight changes in the form of slowing in the

cell growth (Fig. 2f) (group 6). While cells treated

with 1 µg/mL BV showed complete sheet with slight

changes (Fig. 2g) (group 7). Consequently, the lesser

effects were observed in cells treated with 0.5 µg/mL

BV, which showed complete sheet with slight changes

(Fig. 2h) (group 8). These findings indicated that the

safest dose begins from concentration 0.5 µg/mL.

3.3.2 Morphological Changes of Unstained MCF7

Cell Lines

The inverted microscopy examination results of

unstained MCF7 cells treated with different

concentrations of BV were shown in Fig. 3. The time

of exposure was fixed (24 h) and it was divided into

six groups. In group 1, the control untreated cells

showed the normal sheet (Fig. 3a). In group 2, cells

treated with 20 µg/mL BV showed complete digestion

and cell remenants (Fig. 3b). In group 3, cells treated

with 15 µg/mL BV showed digestion (Fig. 3c). In

group 4, cells treated with 10 µg/mL BV showed

smaller and shrinkage cells (Fig. 3d). In group 5, cells

treated with 5 µg/mL BV showed some shrinkage (Fig.

3e). In group 6, cells treated with 0.5 ug/mL BV

showed normal picture (Fig. 3f).

3.4 Histopathological Results

3.4.1 Hep-2 Cells Stained by Hematoxylin and

Eosin (H&E) and Examined under Inverted

Microscope

The histopathological changes of stained Hep-2

cells treated with different concentrations of BV were

shown in Fig. 4. The time of exposure was fixed (24 h)


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(a) Untreated Hep-2 cell (b) Hep-2 cell treated by BV 20 µg/mL

(c) Hep-2 cell treated by BV 15 µg/mL (d) Hep-2 cell treated by BV 10 µg/mL

(e) Hep-2 cell treated by BV 5 µg/mL (f) Hep-2 cell treated by BV 2.5 µg/mL

(g) Hep-2 cell treated by BV 1 µg/mL (h) Hep-2 cell treated by BV 0.5 µg/mL

Fig. 2 Morphological changes of Hep-2 cell lines treated with different concentrations of BV (unstained ×20).


129

(a) Untreated MCF7 cell (b) MCF7 cell treated by BV 20 µg/mL

(c) MCF7 cell treated by BV 15 µg/mL (d) MCF7 cell treated by BV 10 µg/mL

(e) MCF7 cell treated by BV 5 µg/mL (f) MCF7 cell treated by BV 0.5 µg/mL

Fig. 3 Morphological changes of MCF7 cell lines treated with different concentrations of BV (unstained ×20).

and cells were divided into seven groups. In group 1,

non-treated cells showed normal pathway of mitotic

divisions in various stages; nucleoli were prominent

inside large vesicular nucleus and the cytoplasm was

abundant (Fig. 4a). In group 2, cells treated with 20

µg/mL BV showed cellular digestion (Fig. 4b). In


cellular digestion (Fig. 4c). In group 4, cells treated

with 10 µg/mL BV showed cellular digestion (Fig. 4d).

In group 5, cells treated with 5 µg/mL BV showed

cellular digestion (Fig. 4e). In group 6, cells treated

with 2.5 µg/mL BV showed cellular destruction (Fig.

4f). In group 7, cells treated with 1 µg/mL BV showed

apoptosis (Fig. 4g).

3.4.2 Stained MCF7 Cells Examined under Inverted

Microscopy

The histopathological changes of MCF7 cells

treated with different concentrations of BV were

shown in Fig. 5. The time of exposure was fixed (24 h)

and the cells were divided into four groups. In group 1,


130

(a) Untreated Hep-2 cell (b) Hep-2 cell treated by BV 20 µg/mL

(c) Hep-2 cell treated by BV 15 µg/mL (d) Hep-2 cell treated by BV 10 µg/mL

(e) Hep-2 cell treated by BV 5 µg/mL (f) Hep-2 cell treated by BV 2.5 µg/mL

(g) Hep-2 cell treated by BV 1 µg/mL

Fig. 4 Histopathological changes of stained Hep-2 cells treated with different concentrations of BV(H&E ×40).


131

(a) Untreated MCF7 cell (b) MCF7 cell treated by BV 20 µg/mL

(c) MCF7 cell treated by BV 15 µg/mL (d) MCF7 cell treated by BV 10 µg/mL

Fig. 5 Histopathological changes of stained Hep-2 cells treated with different concentrations of BV (H&E ×20).

non-treated cells showed normal cellular growth

(Fig. 5a). In group 2, cells treated with 20 µg/mL BV

showed digestion (Fig. 5b). In group 3, cells treated

with 15 µg/mL BV showed digestion (Fig. 5c). In


digestion (Fig. 5d).

3.5 Cytopathologic Effects of Safe Dose (0.5 ug/mL)

on Hep-2 Cell Lines and MCF7 Cell

Hep-2 Cells treated with dose 0.5 ug/mL showed

after 24 h vesicle inside the nucleus with irregular

edges (Fig. 6), and Hep-2 cells treated with dose 0.5

ug/mL after 48 h showed shrinkage of cells with dense

irregular chromatin and distorted nuclear contents (Fig.

7a). Some cells showed abnormal stain affinity of the

nucleus with reddish color, and the nucleus showed

destructed condensed chromatin inside intact nuclear

membrane. The cytoplasm was free from any vacuoles

with partially destructed cytoplasmic membrane (Fig.

7b). Some other cells showed dissolved chromatin

with intranuclear vesicles formations with completely

intact nuclear membrane and two identical

intranuclear dark stained inclusion bodies surrounded

by hallow zones. The other cell showed large

homogenous poorly stained nucleus with intact

nuclear membrane and cytoplasm pushed to one side

(Fig. 7c). Some cells showed completely dissolved

chromatin with completely intact nuclear membrane

with the disappearance of the cytoplasm (Fig. 7d).

Fig. 6 Hep-2 cells treated with 0.5 ug/mL after 24 h (H&E

×20).


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(a) (b)

(c) (d)

Fig. 7 Hep-2 cells treated with 0.5 ug/mL after 48 h (H&E ×20).

MCF7 cells treated with dose 0.5 ug/mL showed

after 24 h vesicular nucleus (Fig. 8), and MCF7 cell

lines treated with dose 0.5 ug/mL after 48 h showed

shrinkage of cells with dense irregular chromatin and

distorted nuclear contents (Fig. 9a). Some cells

showed fragmentation (Fig. 9b).

3.6 AGPT Result

AGPT was performed for detecting the antigenic

properties of LSDV under experiment, the treated

virus with BV and the original viruses used in the

experiment. This test showed that the antigenic

properties were still intact, which indicated by

attaching of BV inactivated LSDV to its specific

antibodies and forming well defined and strong line of

precipitate (Fig. 10). Other tested viable LSDV was

give similar results. However, if BV has destructive

effect on LSDV, this antigenic characteristic would

diminish, and also the use of safe doses was key to

maintain the antigenicity of inactivated LSDV by this

method [20].

BV was used from long time ago in the medical

field as treatment of chronic joint affections. In the

recent decades, the screening process of new sources

of antimicrobials discovers its high advantages

characteristics for combating various types of

microbes, as well as trials to discover its anti-cancer

Fig. 8 MCF7 cells treated with 0.5 ug/mL after 24 h (H&E

×20).


133

(a)

(b)

Fig. 9 MCF7 cells treated with 0.5 ug/mL after 48 h (H&E

×20).

Fig. 10 The positive result of AGPT. LSDV mixed with BV gave the strong line of precipitate.

medicinal fields. The viability of virus was tested in

tissue culture cells lines and in embryonated chicken

eggs. According to doses and time of exposure, BV

pathological effects on Hep-2 cells (human larynx

carcinoma) and MCF7 cells (breast carcinoma cell

line) were observed, such as apoptosis, retarded

growths and cytolysis. The results indicate the

possibilities of using BV in cancer treatment fields

and in vaccine preparations against LSDV, also in

therapeutic preparations to treat LSD illness.

The control of LSD without vaccination is

extremely difficult in endemic areas. In Egypt, cattle

vaccinated against the disease by using Kenyan sheep

pox culture vaccine could not stop the disease [21].

Accordingly, effective control of LSD requires not

only rapid and accurate laboratory diagnosis supported

by clinical findings [22], but also real solution focused

on combating the LSDV by effective vaccination and

treatment. In the present work, BV exhibited satisfied

inhibitory effects on LSDV, meanwhile, the antigenic

properties was still intact. Embryonated chicken eggs

inoculation is considered the best recommended

method required for LSDV isolation from the tissue

specimens, followed by several passages in the

specific tissue cultures [23]. BV contains various

peptides, including mellitin, apamin, adolapin and

enzymes as well as non-peptide components, such as

histamine, lipid and carbohydrates [24-26]. Also,

some components of BV can cause inflammation by

inducing interleukin (IL)-1β via p38 mitogen activated

protein kinase (MAPK), while others act as

anti-inflammatory by suppressing inducible nitric

oxide synthase (iNOS) and cyclooxigenase (COX)-2

via nuclear factor (NF)-kB. The naturally occurring

peptides of whole BV have various pharmacological

potencies, however, these components exhibit

anti-inflammatory activity in inflammatory cells [27,

28]. The major components of honey BV are mellitin,

phospholipase A2 (PLA2) and hyaluronidase [29].

The peptide mellitin comprises approximately 50% of

BV [30]. BV has antibacterial, anti-parasitic and

antiviral properties [31]. The PLA2 of honey bee and

snake venoms have potent anti-human

immunodeficiency virus (HIV) activities. These

PLA2s block HIV-1 entry into host cells through a

mechanism linked to PLA2 binding to cells [32]. The


134

inhibitory effects of BV on LSDV could be owned to

its enzymatic constituents, besides the cationic peptide

mellitin which comprises approximately 50% of BV

[30]. However, the PLA2s of honey bee have potent

anti-human immunodeficiency virus (HIV) activities.

These PLA2s block HIV-1 entry into host cells

through a mechanism linked to PLA2 binding to cells

[32]. Venoms induce a variety of immune response,

including both acute inflammatory responses, such as

mast cell degranulation, and adaptive immune

responses, such as T helper type 2 responses and IgE

production [33]. Venoms of honeybee (hymenoptera

species) including the Apis mellifera, cause

inflammation and allergy that succumbed to

individual variations. BV injected inside vertebrate

targets by stings leads to local inflammation in most

instance, however the severe allergic response takes

place in 3% of the population who are allergic to

hymenoptera venoms [34-36]. Therefore, one could

emphasize that the whole BV constituents play the

synergistic actions role on its therapeutic effects as

anti-neoplastic and antimicrobials.

4. Conclusions

From in vitro study and testing BV on cell lines, the

safest dose is 0.5 ug/mL. However, the time of

exposure is considered as the most important factor in

regarding toxic effects of BV on living cells.

Moreover, in vitro and in vivo conditions are different

in many aspects, because in vitro are restricted to the

given materials. While in vivo, the synergistic effects

along with many internal factors play major roles that

are included in the process of reactions to BV. The

biological effects inside the living organism may react

with higher doses than that determined in vitro, so

theoretically the safe doses in vitro are lower than that

could be used in vivo. The inhibitory effect of BV on

LSDV was achieved in this work at very lower

concentrations (0.5 ug/mL) and the antigenic

characteristics were still intact. It is possible to apply

BV for disinfecting health hazardous biological

laboratory wastes. The results emphasized that BV

may be good treatment for LSDV after determining

the suitable therapeutic doses.

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In Vitro Study on the Effect of Bee Venom on Some Cell ... · Five groups of 11-day-old embryonated chicken eggs were used to test the viability of LSDV. The inoculation was performed