Evaluation of filter papers as a novel method for transportation of

specimen for diagnosis of brucellosis in animals

A. 1
Prasad ,
V. S. 1
Bahekar ,
Heena 3
T, F. 1
Mukherjee , S. K. 1
Rana , K. S. N. L. 1
Surendra ,
2
G. K. Sharma , V. A. Srinivasan 2
1 National Dairy Development Board, R&D Laboratory, Gachibowli, Hyderabad 500032, Telangana, India
2 National Dairy Development Board, Anand 388001, Gujarat, India
3 Jawaharlal Nehru Technological University, Hyderabad 500085, Telangana, India

Introduction
 Bovine brucellosis, caused by Brucella
abortus, is a highly contagious zoonotic
disease affecting cattle and buffaloes
mainly resulting in infertility and abortion.
 The major impediment in the diagnosis and
surveillance of the disease, is the
requirement of cold chain for
transportation of the appropriate samples
from remote location to the laboratory.
 Cellulose based filter papers can be
considered as safe and convenient mode of
transhipment of clinical material.
Experimental Design
Filter papers were evaluated as the means for
transportation of serum and clinical
specimens without cold chain for diagnosis of
brucellosis through antibody detection by
ELISA and antigen detection by real-time PCR
B. Evaluation of Nobuto filter paper for
serological diagnosis of Brucella abortus by
I-ELISA
1. The protocol was optimised by using a
panel of reference serum samples. Direct
serum samples and filter elutes of the same
samples were tested simultaneously for
antibodies against Brucella abortus by I-
ELISA. The results obtained were
compared.
2. Briefly, 40µl of serum samples was spotted
onto Nobuto’s filter paper (Advantec, Japan
, Fig 2.) and eluted with 360µl of elution
buffer (1:10 dilution). Elutes of each
sample was evaluated for detection of B.
abortus antibody by indirect ELISA
(IDEXX, Germany).
3. A total of 300 serum samples from cattle
. elutes
and buffaloes of unknown infection status
and their respective filter elutes were tested
B. 1. Comparison of the ELISA data of
filter elutes with direct serum
samples revealed 5-14% coefficient of
variation.
2. A total of 144 samples were
turned positive by both test method
and 152 samples turned negative by
both methods. Four samples out 300
samples showed variability in the
test (Table 1 & Fig 4).
3.Comparative evaluation of spotted
filter papers with direct serum
sample from 300 animals revealed
97.27% and 100% sensitivity and
specificity respectively.
4. The strength of agreement between
the two methods was found to be very
good (Kappa, k= 0.934)
Table 1. Comparison of ELISA results
performed on direct serum samples and filter
Positive by Negative by
I-ELISA Results Filter paper Filter paper Total

respectively. by I-ELISA. Positive by Direct serum

144 4 148

Fig 2. Processing of Nobuto Filter paper for serum Negative by Direct serum
0 152 152

Materials and Methods samples Total 144 156 300

A. Evaluation of FTA® Elute card for Fig 4. Graphical representation of comparison

diagnosis of Brucella abortus by real-time between direct serum and filter paper

PCR
Percentage Positivity Value (S/P)

1. Initially for optimization of the test,

B. abortus 544 strain spiked in various
matrix viz. bacteriological culture media,
PBS, milk, nasal swabs (NS), lachrymal
swab (LS) and genital swabs (GS) of cattle
were spotted onto the FTA® elute card
(Fig. 1). DNA extracted from FTA® card
was tested by real-time PCR targeting
BCSP-31 gene of Brucella (Mukherjee
et.al.,2016).
2. A total of 182 samples (viz.,Milk, NS, GS,
LS and aborted samples) from 87
Brucella sero-positive cattle and buffaloes
were collected and spotted onto FTA®
cards (at farm/field), and subsequently
transported to the Laboratory for
screening. Elutes from FTA cards and
their respective direct clinical samples
were also processed simultaneously by
real-time PCR.
Fig. 1: Clinical sample (Blood) spotted on FTA®
Cards
Result
A. 1. The lower limit of detection (LOD) by
real-time PCR was found to be 10 bacteria
and 480pg DNA from FTA® card (Fig 3).
2. Screening of 182 FTA spotted clinical
samples tested by Real-time PCR, of
blood, aborted material, milk, NS, GS
from cattle and buffaloes with history of
reproductive disorder revealed 6.5%
positivity for Brucella.
3. The sensitivity of FTA® card for NS,
GS, LS and milk was 88.89%, 82.35%,
66.67% and 40% respectively in
comparison to detection from direct
samples .
4. The specificity of FTA card was found
to be 100% except for GS (98.57%).
5. The strength of agreement between
both the two methods was very good
(Kappa , k= 0.823)
Fig 3. Real-time PCR plot depicting the LOD
10 copies
Serum samples (Individual)
Conclusion
FTA® elute card and Nobuto’s filter paper
can be used for transportation of clinical
materials and serum samples in room
temperature for diagnosis of bovine
brucellosis by I-ELISA and molecular
technique respectively. These filter paper
offer safe and convenient alternative
approach for transport of Brucella
suspected clinical samples without
compromising in diagnostic specificity
and sensitivity.
References
1. Betsy B., Olsen S and Ewalt Darla. The use
of FTA cards in molecular diagnosis
techniques for Brucellosis. Agriculture
Research Service , 2016
2. Curry P.S., Elkin B.T., Campbell M.,
Nielsen K., Hutchins W., Ribble C., Kutz
S.J., (2011). Filter-paper blood samples for
ELISA detection of Brucella antibodies in
caribou. J Wild Dis 47:12–20.

A. Prasad, Scientist I, NDDB R&D Laboratory; E-Mail : aprasad@nddb.coop