Abstract: Hyperimmune serum against Lumpy Skin Disease Virus (LSDV) was prepared by inoculation of the virus in successive doses into susceptible two calves with complete and incomplete Freund’s adjuvant. Identity tests were applied by Agar Gel Precipitation Test (AGPT) and Indirect Fluorescent Antibody Technique (IFAT). Serum Neutralization Test (SNT) and ELISA tests were applied for titration and evaluation of the hyperimmune sera before conjugation with fluorescent. Total protein concentration of the prepared LSDV antisera was 0.8 g dL-1. Separation of anti-LSDV immunoglobulins IgG were done using ammonium sulphate followed by conjugation with fluorescein isothiocyanate at pH 9.6. The anti-LSDV IgG conjugated fluorescein sterile and was used to detect LSDV in the MDBK cells and gave good results to dilution 1/20 ,while the reference conjugate to 1/30.
INTRODUCTION
Lumpy skin disease is an infectious eruptive viral skin disease affecting cattle and caused by a Neethling strain of Capripox virus which is antigenically related to African sheep and goat pox viruses (Matthews, 1982; Abd EI-Razek, 2003). Fluorescent Antibody Test (FAT) was most widely employed in LSD and Sheep-Goat Pox (SGP) diagnosis (Davies and Otema, 1978; Office International Epizootic, 1989, 1992). The isolation of LSDV from cattle in Egypt was confirmed by FAT by using anti-Capripox virus hyperimmune sera conjugated with FITC (House et al., 1990), which mentioned that FAT was rapid, accurate and sensitive serological technique for the diagnosis of pox viral diseases by detection of the intracellular viral antigen, in addition, FAT was used for rapid detection of the viral antigen in infected tissue culture.
The aim of this study was to prepare local, specific and economic conjugated antisera with FITC against LSDV for rapid diagnosis of this virus to safe time and cost.
MATERIALS AND METHODS
Animals
Two calves of about 1 year old were used for the preparation of LSDV hyperimmune
serum. They were screened and proved that they are free from antibodies against
LSDV.
Five adult Albino Swiss mice were used for the preparation of liver powder according to Narin and Marrack (1964). The liver powder was used to remove the non-specific fluoresceince from the prepared conjugate.
Virus
Lumpy skin disease virus (Ismailia strain) screened MDBK, was kindly supplied
from FADDL through Dr. J. House, the virus isolated during outbreak of cattle
in 1989, at Ismailia governorate, Egypt.
It was propagated on lamb testicle cells, then propagated on MDBK cells for 60 passages (Aboul Saoud, 1996). The virus has a titre of 6 10 gl0 TCID50/mL.
Fluorescein Isothiocyanate
It was obtained from Sigma, USA.
Dialysis Bag
It was obtained from Sigma, USA.
Ammonium Sulphate
It was obtained from Sigma, USA.
Conjugates
Fluorescein-Labeled Affinity Purified Antibody to Bovine LgG (H+L)
It was kindly supplied from FADDL through Dr. J. House. It was used as a reference positive control conjugate to evaluate the locally prepared one.
Anti-Bovine IgG (Whole Molecule) Peroxidase Conjugate
It was obtained from ICN biomedicals, INC, California, USA and used in solid
phase ELISA.
Lumpy Skin Disease Virus Antigen and Antisera
Standard purified reference LSDV antigens and antisera were kindly supplied
from FADDL Plum Island, USA through Dr. J. House.
Cell Cultures (MDBK)
The cells were obtained from Ames, Iowa Laboratories, USA and were grown
and maintained as described by Manual of Methods for Virology.
Preparation of LSDV Hyperimmune Serum
LSDV hyperimmune sera were prepared according to the method of Puranchand
et al. (1985).
Evaluation of Prepared LSDV Hyperimmune Serum
Purity Test
In accordance with the United States Code of Federal Regulation (CFR). It was applied in Central Laboratory for Evaluation of Veterinary Biologics (CLEVB), Abbasia, Cairo.
Identity test
The LSDV antigen was identified by IFA using reference LSDV antisera as
described by Davies and Otema (1978).
Agar Gel Precipitation Test (AGPT)
It was applied according to the method described by Sharma
and Dhanda (1972) and Abd EI-Razek (1999).
Serum Neutralization Test (SNT)
This was carried on microtitre plate 96 well using cell culture method according
to House et al. (1990) to screen the test animals
before inoculation and to estimate the induced antibody titre in the prepared
hyperimmune serum.
Enzyme Linked Immunosorbent Assay (ELISA)
Solid phase ELISA (indirect method) was done according to the method described
by Williams (1987).
Precipitation of the Immunoglobulin
It was carried out by using a saturated solution of amunonimn sulphate (75%)
according to Narin and Marrack (1964), the globulin concentration
was determined and adjusted to be 20 mg mL-1 in Phosphate Buffer
Solution (PBS).
Estimation of Total Protein
Measurement of the amount of total protein concentration of LSDV antiserum,
was done by calorimetric method using spectrum diagnostic total protein reagent
and read absorbance of serum at 540 nm, according to Cannon
et al. (1974) and Tietz (1994).
CALCULATION
Conjugation of the Prepared Immunoglobulins with FITC
The immunoglobulins were adjusted to be at least 20 mg mL-1 PBS,
then diluted in equal volume of chilled carbonate bicarbonate buffer pH 9.6.
One milli gram of FITC powder was dissolved in 1 mL cold carbonate-bicarbonate
buffer. Hundred microliter of diluted FITC were mixed with 100 mg immunoglobulins
in PBS and left for 24 h at 4°C for complete conjugation, then pH was adjusted
to 9 with sodium hydroxide solution. The non-specific FITC was removed by using
1% mice liver powder and dialyzed against several changes of PBS of pH 7.4 at
4°C.
Evaluation of the Prepared Immunoglobulins with FITC
Purity test
The prepared IgG with FITC was tested against bacterial, fungal, mycoplasma and extraneous virus contaminants.
Fluorescent Antibody Technique (FAT)
It was carried out according to Davies and Otema (1978),
preparation of infected and non-infected control cover slips cultured with MDBK
cells, different dilutions of conjugation were used up to 1/50.
RESULTS AND DISCUSSION
This study was designed to prepare hyperimmune sera against LSDV conjugated with fluorescein isothiocyanate to be used for virus diagnosis and titration in a trial to provide a specific local reagent to easily detection of LSDV to save time and costs.
The results of AGPT to prepared LSDV hyperimmune sera were found to contain specific LSDV precipitating antibodies (Table 1). These results were in agreement with these obtained by Sharma and Dhanda (1969, 1972) and Sharma et al. (1987).
The prepared LSDV hyperimmune sera was found to contain specific LSDV neutralizing
antibodies of a titre 32 and 400 for previous viruses respectively as detected
by SNT and ELISA. These results agreed with those obtained by Martin
et al. (1975), Sharma et al. (1987),
Office lnternational Epizootic (1989) and Agag et al.
(1992).
| Table 1: | Evaluation of prepared LSDV hyperimmune sera before conjugation |
| *1 Sterile: Free from contaminants (Bacterial, mycoplasma,
fungus and extraneous viruses), *2 +: Indirect presence of antibodies against LSDV, *3 : Titre of neutralizing antibody after successive inoculation, *4 S/P: ELISA titre of indirect method. Cut-off about 128, AGPT: Agar Gel Precipitation Test, IFAT: Indirect Fluorescent Antibody Technique, SNT: Senun Neutralization Test, NA: Neutralizing Antibody, ELISA: Enzyme Linked Immunosorbent Assay |
|
| Table 2: | Titration of prepared anti-LSDV-(IgG) fluorescein conjugate and reference anti-bovine IgG fluorescein conjugate |
| *1 Strong positive reaction, *2 Weak positive reaction, *3 Non-specific negative non-infected cells | |
| Fig. 1: | MDBK cells infected with LSDV and stained with anti- LSDV conjugated fluorescein, notice that apple green fluorescence |
The total protein in the prepared LSDV antisera was 0.8 g dL-1 and this amount of proteins was satisfactory to use in conjugation with fluorescein. These results were in agreement with Nowotony (1979). The total protein of the negative sera was less than 0.8 g dL-1. So, it was clear that the globulin as the immune protein forming the antibodies appeared to be higher than negative sera of the non-inoculated animals. These results are in agreement with those obtained by Kataria and Sharma (1993). The titre of conjugated LSDV. IgG antibodies reached 1:32 which of good titre to use for conjugation (Nowotony, 1979).
The conjugated anti-LSDV hyperimmune senun demonstrated typical fluorescence staining reaction (apple green fluorescein up to 1 :20) (Table 2).
Negative control cells showed dull green staining (negative reaction) when reacted with both conjugated at different dilution (Fig. 1). Moreover, detection of LSD viral antigen intracytoplasmic of infected cells were also recorded by Davies and Otema (1978), House et al. (1990) and Office Internatiol Epizootic (1992).
CONCLUSION
From this result, we can say that we could prepare anti-LSDV IgG conjugated with fluorescein of low price, rapid (within 1 h), good titre and good quality and quantity in pure form to be used for diagnosis of LSD infection and for identity evaluation of produced vaccine.