Performance assessment of three tests applied in enzootic bovine

Romanian Biotechnological Letters
Copyright © 2014 University of Bucharest
Vol. 19, No. 5,2014
Printed in Romania. All rights reserved
ORIGINAL PAPER
Performance assessment of three tests applied in enzootic bovine leukosis
diagnosis
Received for publication, May 20, 2014
Accepted, September 21, 2014
EMIL TÎRZIU1, CICERONIS CUMPĂNĂȘOIU1, ILEANA NICHITA1,
GHEORGHE D. REMAN2, CRISTINEL SONEA2, MONICA ȘEREȘ1
1
”King Michael I of Romania” Banat’s University of Agricultural Sciences and
Veterinary Medicine Timișoara, Faculty of Veterinary Medicine, Immunology,
Immunopathology and Microbiology Department, Romania
2
Biotera University, Bucharest, Romania
Corresponding author: Prof. PhD Emil Tîrziu, tel. 0040256277113, mobile
0040728296801, fax +040256277118, E-mail: [email protected], Aradului Street
No. 119, 300645, Timișoara, Romania
Abstract
The aim of the present paper was the comparative assessment of two ELISA tests on serum
and milk samples and agar gel immunodiffusion test (AGID) regarding ability to detect enzootic
bovine leukosis (EBL) in Timis County, according to farm type, age, breed and stage of lactation,
and how these parameters affect the humoral immune response of positive cattle. Serum samples
were analyzed using blocking ELISA and AGID, and the milk samples by indirect ELISA,
calculating quality parameters of applied tests. The obtained results showed that AGID detected 43
positive cattle and the two ELISAs 51 positive animals. Performances of AGID were influenced
by farm type, age and serum antibody level. It was established that cattle aged 3-6 years were most
prone to develop EBL. The level of serum antibodies has been influenced by farm type, age and
lactation phase, and milk antibodies level strictly by the phase of lactation; the lowest values of
milk antibodies were registered between 2nd and 8th month of lactation. Consumption of
insufficiently heat-treated milk from BLV positive cattle in 2-8 months of lactation is associated
with increased risk of infection in humans.
Keywords: enzootic bovine leukosis, serum and milk ELISA, AGID, level of antibodies.
1. Introduction
Enzootic bovine leukosis (EBL) is a malignant neoplastic disease of the
reticuloendothelial system, specific for adult cattle, expressed by complex clinical signs
induced by various locations of neoplastic B cells aggregations (M. Spînu [1]). It is caused by
bovine leukemia virus (BLV), a retrovirus that belongs to the Deltavirus genus of the family
Retroviridae, related to human T-lymphotropic virus (HTLV) and simian T-lymphotropic
virus (F.A. Murphy et al. [2]). The genomes of BLV and HTLV I and II show 58, respectively
59% similarity regarding their nucleotide sequences (S. Dube et al. [3]).
Domestic cattle are natural hosts for BLV, and water buffaloes are considered wild
reservoir of the virus (N. Gillet et al. [4], Office International des Epizooties [5]). Natural
infection is usually asymptomatic (without clinical or hematological changes), approximately
one third of infected animals develop persistent lymphocytosis, and only 0.1-10% tumors
(Office International des Epizooties [5]). Even if it is a chronic disease associated with a
relatively low mortality, EBL causes significant economic losses due to morbidity and
increased susceptibility to other infections, decreased milk and meat production, costs of
prevention and control measures, including compensations, and restrictions on the sale and
export of dairy and meat products etc. (M. Spînu [1], L. Nuotio et al. [6], Z. Trainin & J.
Brenner [7]).
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Performance assessment of three tests applied in enzootic bovine leukosis diagnosis
BLV is horizontally transmitted, usually through the transfer of infected cells from blood
and milk (direct contact, iatrogenic transmission, transfer via hematophagous insects, and
milk consumption) (S.G. Hopkins & R.F. Digiacomo [8]). Transmission via colostrum is
considered negligible due to the protective role of maternal antibodies (S.M. Rodriguez et al.
[9]). In utero transmission was demonstrated in cattle with persistent lymphocytosis (S.G.
Hopkins & R.F. Digiacomo [8], M.L. Lassauzet et al. [10]).
Although experimental transmission has been demonstrated in a number of species,
including primates (M.J. Burridge [11]), only sheep, goats, rabbits and rats develop the
disease (leukemia) (N. Gillet et al. [4], A. Florins et al. [12], M. Mammerickx et al. [13], P.
Dimitrov et al. [14]).
Regarding risks to human health, a long time it has been considered that BLV is not
pathogenic to humans (Office International des Epizooties [5], T. Burmeister et al. [15], R.
Kettmann et al. [16]), but some new evidence contradicts this statement. Thus, anti-BLV
antibodies were detected in 12.5 to 74% healthy people evaluated (Gh. Nikbakht et al. [17],
G.C. Buehring et al. [18]), and 12.3% of tissue samples collected from seropositive people
contained BLV proviral DNA (Gh. Nikbakht et al.[17]). Also, proviral DNA was detected in
35.8% of breast tissue samples collected from women with breast cancer (M. Giovanna et al.
[19]). It was previously demonstrated that BLV can infect human myeloma cells (K.
Slavikova et al. [20]) and human cells of neural origin (C. Altaner et al. [21]), causing also
cytopathic effect in four cell lines of human origin (C.A. Diglio & J.F. Ferrer [22]). All this
suggests a possible implication of this virus in the development and evolution of various types
of cancer and neurological diseases in humans.
Due to the great economic and health importance, EBL is the subject of intensive
eradication programs, implemented nationally and worldwide, based on early detection of
positive cattle. Our country implements the measures specified in the Program of supervision,
prevention, control and eradication of animal diseases, of those transmissible from animals to
humans, animal welfare and environmental protection, identification and registration of
cattle, pigs, sheep and goats of National Sanitary Veterinary and Food Safety Authority
(NSVFSA [23]).
Early diagnosis is essential for EBL control, in this purpose being used enzyme linked
immunosorbent assays (ELISA on serum samples and milk) and agar gel immunodiffusion
test (AGID). These diagnosis methods led to the eradication of EBL in many European
countries, such as Spain (1994), Cyprus (1995), Ireland (1997), Luxembourg (1999), Austria
(2001), Switzerland (2005), United Kingdom (2006), Slovenia (2006), The Netherlands
(2007), Sweden (2007) and Slovakia (2008) (Office International des Epizooties [5]).
This study aims to evaluate two ELISAs, on serum and milk samples, and AGID in terms
of antibodies anti-BLV detection in cattle in Timis County, depending on farm type, age,
breed and lactation phase. Furthermore, the influence of the four mentioned parameters on
level of anti-BLV antibodies in serum and milk was assessed.
2. Material and methods
Cattle and biological specimens. We assessed 368 female clinically healthy cattle from
11 localities of the Timis County where, between 2008 and 2009 outbreaks of EBL were
declared (the epidemiological data were obtained through the courtesy of the Timis Sanitary
Veterinary and Food Safety Directorate). Cattle, selected based on a random cluster sampling
program, were:
- from households (n = 283) and private farms (n = 85);
Romanian Biotechnological Letters, Vol. 19, No. 5, 2014
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EMIL TÎRZIU, CICERONIS CUMPĂNĂȘOIU, ILEANA NICHITA, GHEORGHE D. REMAN,
CRISTINEL SONEA, MONICA ȘEREȘ
- aged between two and ten years, with the following categories: 2 years (n=51), 3 years
(n=67), 4 years (n=81), 5 years (n=67), 6 years (n=53) 7 years (n=31), 8 years (n=12) and 9
years old (n=6);
- Romanian Spotted Cattle - RSC (n=122), Romanian Black Spotted Cattle - RBSC
(n=96) and Holstein-RBSC crossbred (n=150);
- in different lactation phases: first month (n=54), 2-3 months (n=73), 4-6 months (n=55),
7-8 months (n=65), dry period (n=53) and 1-2 weeks before parturition (n=68).
Jugular venous blood samples were taken using sterile tubes without anticoagulant and
serum separator. After clotting at 4°C for 5 hours, sera were obtained using standard
procedure (C. Jury et al. [24]): the tubes were centrifuged for 10 min. at 1200rpm, the
supernatant serum was transferred into another tube and centrifuged again, in the same
condition, and the obtained sera were transferred into labeled cryotubes. Cryotubes were kept
at -80oC until analysis.
Milk samples were collected using 10 ml sterile tubes, labelled in correspondence with
blood samples, and were defatted according to instructions manual provided by the
manufacturer of the milk ELISA kit: tubes were centrifuged for 15 min. at 2500g and skim
milk below the fat layer was transferred in labelled cryotubes. Cryotubes were kept at -80oC
until analysis.
AGID was conducted using the Kit for serological diagnosis of EBL (SN "Pasteur
Institute" SA, Bucharest, Romania). The kit contains the following reagents: lyophilized ELB
antigen, lyophilized ELB control (positive) serum and diluent. Agar gel and plate preparation,
distribution of reagents and sera, reading and interpretation of the results were performed
according to the standard procedure (Office International des Epizooties [5], J.M. Miller et al.
[25]). Briefly, gel diffusion plates (0.8% noble agar and 8.5% NaCl) with wells filled with
reagents and test sera were kept at room temperature in humid chamber for 72 h before the
final reading. Test sera were considered positive when the precipitation line with the antigen
was clear and equidistant from the wells and formed a line of identity with the control serum.
ELISA. Blocking ELISA for the detection of antibodies against BLV in cattle sera was
performed using ELISA Enzootic Bovine Leukosis Virus (BLV) Antibody Test (IDEXX
Laboratories, Inc., Netherlands). Instructions manual of the kit complies with the standard
procedure of Office International des Epizooties [5). Briefly, serum samples were thaw in
water bath at 37oC; buffer solution 2 was added in microtiter plate precoated with BLV gp51,
and after that test sera and positive and negative control were transferred; the plates were
incubated 60 min. at 37°C, washed, and conjugate diluted 1:100 in buffer solution 1 was
added in wells; before substrate addition, the plates were incubated 30 min. at 37°C and
washed; the reaction was stopped by adding stop solution; microtiter plates were read at 450
nm. For each sample, antibodies level (%E/P) was calculated: %E/P = (OD sample / OD of
positive control) × 100. Any sample whose % E/P was less than or equal to 100% was scored
as BLV negative.
Indirect ELISA for the detection of antibodies against BLV in cattle milk was performed
according to standard method of Office International des Epizooties [5), with some
modifications specified in the instructions manual provided by the manufacturer of DRG®
Bovine Leukemia Virus p24-gp51 Ab serum, milk kit (DRG International Inc., USA) used for
assessment. Briefly, the milk samples were thaw in water bath at 37oC and diluted 1:2 in
deionized water; diluted milk samples and negative and positive controls were added in
washed microtiter plates precoated with p24 and gp51 viral antigens and monoclonal
antibodies anti-BLV-p24 and anti-BLV-gp51; the plates were incubated for 60 min. at 37°C;
after washing, HRPO conjugate, diluted 1:100 in HRP conjugate buffer, was added; after
another incubation, in the same condition, and washing, substrate (equal parts of buffer A and
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Performance assessment of three tests applied in enzootic bovine leukosis diagnosis
buffer B) was added, and plates were incubated for 15 min. at room temperature; thereafter,
stop solution was added, and microtiter plates were read at 450 nm. A sample was scored
BLV negative if the OD value was below or equal to the average OD value of the negative
control plus 0.150 OD units (OD samples ≤ mean OD negative control + 0.150 OD units).
Comparison between serum ELISA, milk ELISA and AGID. For each test, the
proportion of positive and negative results, related to the farm type, age, breed and lactation
phase of animals and relative to total samples assessed, were determined. The results of the
three tests, in terms of positive and negative animal number, were passed onto a 2 x 2
contingency table, which is used to calculate sensitivity, specificity, prevalence and predictive
values. Quality parameters were calculated using standard formulas, AGID being considered
reference test (K.Y. Choi et al. [26], E.T. González et al. [27]). Also, apparent prevalence (%
of positive samples only by serum and milk ELISA) and true prevalence (% of positive
samples by ELISA and AGID) and Youden's index (shows correlation between sensitivity and
specificity), and the correspondence between tests were determined (González et al. [27]).
Statistical analysis was performed using the Statistical Package for Social Sciences
(SPSS, version 21, Chicago, IL, USA). In order to establish the influence of farm type, age,
breed and lactation phase of assessed animals on tests performance and level of anti-BLV
antibodies in serum and milk, data were classified as nominal and quantitative. Chi-square
test was used to compare nominal variables. For quantitative data, mean, standard deviation,
minimum and maximum values were determined. Differences of the mean of various
categories were analyzed using one-way ANOVA test. Differences were considered
significant when P values were less than 0.05.
3. Results
The two ELISA tests on serum and milk samples were 100% concordant, detecting an
equal number of positive animals (n = 51) in total of 368 examined, with 8 more than the
AGID. The comparative results of the three tests, according to farm type, breed and age of
cattle are listed in Tables 1-3.
Table 1. Comparative results of AGID and serum and milk ELISAs according to farm type
No.
Farm type
1.
Households
Private
farms
Total
2.
No. of assessed
cattle
283
ELISA+
ELISA+
AGID+
AGIDNo.
%
No. %
34 12.01 7 2.47
85
368
9
43
10.58
11.68
1
8
1.17
2.17
ELISAAGID+
No.
%
0
0
0
0
0
0
ELISAAGIDNo.
%
242
85.51
75
317
88.23
86.14
Table 2. Comparative results of AGID and serum and milk ELISAs according to cattle age
No.
Age
No. of assessed
cattle
1.
2 years
51
ELISA+
AGID+
No.
%
3
5.88
ELISA+
AGIDNo.
%
1
1.96
2.
3 years
67
12
17.91
0
0
0
0
55
82.08
3.
4 years
81
21
25.92
0
0
0
0
60
74.07
4.
5 years
67
5
7.46
2
2.98
0
0
60
89.55
5.
6 years
53
2
3.77
1
1.88
0
0
50
94.33
6.
7 years
31
0
0
1
3.22
0
0
30
96.77
Romanian Biotechnological Letters, Vol. 19, No. 5, 2014
ELISAAGID+
No.
%
0
0
ELISAAGIDNo.
%
47
92.15
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EMIL TÎRZIU, CICERONIS CUMPĂNĂȘOIU, ILEANA NICHITA, GHEORGHE D. REMAN,
CRISTINEL SONEA, MONICA ȘEREȘ
7.
8 years
12
0
0
2
16.66
0
0
10
8.33
8.
9 years
6
0
0
1
16.66
0
0
5
8.33
TOTAL
368
43
11.68
8
2.17
0
0
317
86.14
Table 3. Comparative results of AGID and serum and milk ELISAs according to cattle breed
No.
Breed
No. of assessed
cattle
1.
2.
3.
RSC
RBSC
H- RBSC
TOTAL
122
96
150
368
ELISA+
AGID+
No.
%
15 12.29
14 14.58
14
9.33
43 11.68
ELISA+
AGIDNo. %
5
4.09
1
1.11
2
1.33
8
2.17
ELISAAGID+
No.
%
0
0
0
0
0
0
0
0
ELISAAGIDNo.
%
102
83.6
81
84.37
134
89.33
317
86.14
The eight cattle scored ELISA+AGID- had the following characteristics regarding:
- farm type: seven were from households and one from private farms;
- age: one was 2 years old and the remaining seven were aged 5 years and above;
- breed: five were RSC, one RBSC and two H-RBSC;
- lactation phase: five were in dry period and three in 7-8 months of lactation.
The majority of the positive detected cattle by both serum and milk ELISA were from
households (n = 41) RSC (n = 20) and aged between 3 and 5 years (n = 33).
Table 4 summarizes data used for the calculation of serum and milk ELISA quality
parameters. The columns refer to standard test (AGID) and the rows refer to the challenge
tests (serum and milk ELISA), that had the same results.
Table 4. 2 x 2 contingency table for quality parameters of serum and milk ELISAs calculation
ELISA
AGID
AGID +
AGID -
Total
ELISA +
a = 43
b=8
a+b = 51
ELISA -
c=0
d = 317
c+d = 317
Total
a+c = 43
b+d = 325
N=a+b+c+d = 368
After applying the standard formulas, the following values of the two ELISAs quality
parameters were obtained: sensibility = 100%, specificity = 97.53%, positive predictive value
= 84.31% and negative predictive value = 100%. The apparent prevalence was 13.85% and
true prevalence 11.68%. Youden’s index was 0.97 and correspondence between tests 84.31%
(Figure 1).
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Performance assessment of three tests applied in enzootic bovine leukosis diagnosis
Figure 1. Correspondence between serum and milk ELISAs and AGID tests
According to the farm type, there were significant higher OD and %E/P values (P =
0.003) of anti-BLV serum antibodies in cattle from private farms compared to positive cattle
from households (Table 5), but the level of milk antibodies was not significant affected by the
farm type (P=0.504).
As it can be seen from Table 6, age groups analyzed were characterized by varying levels
of serum anti-BLV antibodies, but the significant differences (P = 0.005) were recorded only
between 3 years old cattle on the one hand, and 4 years and 5 years old cattle, on the other
hand. The differences between these groups regarding milk antibodies were not significant (P
= 0.49 and 0.71). The other age groups were not statistically analyzed due to the low number
of positive cattle.
The differences in milk and serum antibodies level for the three local breeds were not
significant (P = 0.57) (Table 7). After adding a second discrimination element, respectively
farm type, we obtained significant differences (P 0.001) between H-RBSC from households
and farms in the serum antibody level (OD = 2.23±0.49 and %E/P = 125.24±28.16,
respectively OD = 3.10±0.22 and %E/P = 174.14±12.75). Significant differences (P 0.001
and P = 0.002) were also obtained between serum antibodies level of H-RBSC from farms on
the one hand, and RBSC (OD = 2.60±0.53 and %E/P = 145.15 ± 33.29) and RSC (OD =
2.60±0.53 and %E/P = 145.66±30.60) from households, on the other hand. Differences
between breeds considering milk antibodies level were insignificant, whether related or not to
the farm type.
Table 5. OD values and level of anti-BLV antibodies in serum and milk according to farm type
Farm type
Serum antibodies
No. of.
positive
OD values
Antibodies level (%E/P)
cattle mean±SD Limits
mean±SD
Limits
Milk antibodies
OD values
mean±SD Limits
Households
41
2.54±0.56 1.79-3.44 142.42±31.52 100.2-191.9 0.73±0.31 0.30-1.45
Private
farms
10
3.10±0.22 2.68-3.45 174.14±12.75 150.6-194.0 0.66±0.24 0.30-1.04
Table 6. OD values and level of anti-BLV antibodies in serum and milk according to age of
positive cattle
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EMIL TÎRZIU, CICERONIS CUMPĂNĂȘOIU, ILEANA NICHITA, GHEORGHE D. REMAN,
CRISTINEL SONEA, MONICA ȘEREȘ
Age
category
No. of
positive
cattle
2 years
3 years
4 years
5 years
6 years
7 years
8 years
9 years
4
12
21
7
3
1
2
1
Serum antibodies
OD values
Antibodies level (%E/P)
mean±SD
Limits
mean±SD
Limits
1.92±0.21 1.79-2.17 107.90±12.09
100.67-121.86
2.29±0.60 1.79-3.17 128.39±33.87
100.2-177.3
2.89±0.57 1.79-3.45 162.24±25.74
100.5-194.05
2.93±0.42 2.16-3.44 163.92±24.11
120.97-191.91
2.54±0.22 2.39-2.81 142.75±12.85
134.02-157.51
2.78
155.99
2.71±0.71 2.20-3.21
151.9±39.95
180.15±123.65
1.8
100.57
-
Milk antibodies
OD values
mean±SD
Limits
0.98±0.16 0.75-1.22
0.73±0.32 0.41-1.34
0.81±0.32 0.39-1.45
0.67±0.17 0.48-0.93
0.44±0.11 0.41-0.57
0.3
0.73±0.32
0.5±0.96
0.72
-
Table 7. OD values and level of anti-BLV antibodies in serum and milk according to breed of
positive cattle
Serum antibodies
OD values
Antibodies level (%E/P)
mean±SD
Limits
mean±SD
Limits
Milk antibodies
OD values
mean±SD
Limits
20
2.59±0.59
1.79-3.42
145.15±33.29
100.67-191.70
0.783±0.33
0.41-1.45
RBSC
15
2.60±0.53
1.79-3.44
145.66±30.06
100.40-191.91
0.73±0.31
0.39-1.36
H-RBSC
16
2.77±0.54
1.79-3.45
155.80±30.98
100.20-194.05
0.63±0.22
0.30-1.04
Breed
No. of
positive
cattle
RSC
Anti-BLV serum antibodies reaches maximum values before parturition and during the
first month postpartum, are maintained at an average level between 2nd and 6th month of
lactation, and then declines in late lactation and during the dry period (Table 8, Figure 2).
This trend is not influenced by the farm type, age or breed, differences between groups being
insignificant. The difference between the mean values of serum antibodies in various phases
of lactation is significant (P 0.05), except for late lactation and dry period, when similar
values were obtained.
Table 8. OD values and level of anti-BLV antibodies in serum and milk according to lactation
phase of positive cattle
Serum antibodies
OD values
Antibodies level (%E/P)
mean±SD
Limits
mean±SD
Limits
Milk antibodies
OD values
mean±SD
Limits
6
2.98±0.33
2.61-3.44
166.77±18.84
146.50-191.91
0.98±0.16
0.75-1.22
13
2.80±0.46
1.79-3.45
157.28±26.25
100.51-194.05
0.56±0.06
0.43-0.64
10
2.58±0.35
2.203.147
145.05±20.03
123.65-176.60
0.46±0.11
0.30-0.65
5
2.24±0.58
1.79-3.07
125.98±29.75
100.40-172.60
0.46±0.07
0.39-0.55
12
2.29±0.67
1.79-3.07
128.74±38.08
100.20-183.33
0.81±0.12
0.643-1.04
5
3.25±0.12
3.12-3.45
148.46±31.40
175.22-194.05
1.37±0.06
1.27-1.45
Lactation
phase
No. of
positive
cattle
1th month
2nd-3rd
month
4th-6th
month
7th-8th
month
Dry
period
1-2 weeks
before
parturition
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Performance assessment of three tests applied in enzootic bovine leukosis diagnosis
Figure 2. Dynamics of milk and serum anti-BLV antibodies according to lactation phase (the
numeric values represents OD mean values)
Dynamics of anti-BLV milk antibodies is slightly different from that of serum antibodies:
high values are recorded before parturition and during the first month of lactation, the average
level in dry period and the minimum values during between 2nd and 8th month of lactation
(Table 8, Figure 2). These differences are statistically significant (P 0001).
4. Discussion
We consider AGID as reference test because, according to the Program of supervision,
prevention, control and eradication of animal diseases of National Sanitary Veterinary and
Food Safety Authority (NSVFSA [23]), ELISA positive cattle are subject to confirmation by
the AGID test. If animals are ELISA positive and AGID negative, reconfirmation is done by
AGID, 30 days after the first blood sampling.
Comparative assessment of AGID and both serum and milk ELISAs, in terms of their
value in EBL diagnosis, revealed superiority of two ELISAs in relation to all monitored
parameters (farm type, age, breed and lactation phase). These findings are supported also by
numerous studies (E.T. González et al. [27], G. Florent et al. [28], G. Gutierrez et al. [29], P.
Have & R. Hoff-Jorgensen [30], K. Knapen et al. [31], M. Mammerickx et al. [32], K.
Murakami et al. [33], V.K. Nguyen & RF. Maes [34], C. Platzer et al. [35]).
As mentioned above, AGID detected eight less positive animals than ELISAs, seven from
households and one from private farms. This difference can only be explained by the low
level of serum anti-BLV antibodies in those samples (OD = 1.79±0.004 and %E/P =
100.59±0.25). These lower values also corresponded to the two lactation phases – 7-8 months
and dry period – in which AGID did not allow the detection of positive animals.
ELISAs superiority is reflected also in relation to age categories of tested cattle. Most
AGID false negative cattle belong to age groups over five years old. This confirms previous
findings, that the AGID detects a smaller number of BLV positive animals in the upper age
categories compared to ELISA (S.R. Pop [36]) and the maximum effectiveness of AGID
occurs in age ranges between 4 and 7 years (M. Mammerickx et al. [32], C. Platzer et al. [35],
S.R. Pop [36]).
The fact that most BLV positive cattle were aged between 3 and 6 years (40 out of 51)
suggests an age predisposition for the disease. Our findings are within the limits specified in
the literature, some authors considering that cattle aged 3-8 years are most prone to develop
EBL (M. Spînu [1], F.A. Murphy et al. [2]).
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CRISTINEL SONEA, MONICA ȘEREȘ
The absence of significant differences between the breeds regarding the number of
positive animals, did not allow us to establish, at least for cattle population studied, a breed
predisposition to EBL.
Sensitivity, specificity, positive predictive value and negative predictive value of the two
ELISA kits used are similar to those reported by Pop [36] and Sălceanu [37], for commercial
blocking ELISA kits compared to AGID in Bihor and Iași Counties.
The four quality parameters are prone to suffer variations depending on the type of ELISA
technique (indirect or blocking) used in the EBL diagnosis, as well as the target antibodies.
Diagnostic kits for simultaneous detection of two types of antibodies (anti-gp51 and p24)
have a low sensitivity and specificity; it also provides a large number of false positive and
false negative results which, in turn, affects positive and negative predictive values. This
phenomenon occurs because the tests allow, at least theoretically, an early diagnosis of the
disease, when the humoral immune response is dominated by anti-p24 antibodies. For
example, Leucokit kit-La Plata, LK-LP and Chekit Leucotest Bommeli AG have sensitivity of
about 97%, specificity of 76%, positive predictive value of 61% and negative predictive value
of 98% (E.T. González et al. [27]).
However, applying indirect ELISA on milk samples (using DRG® Bovine Leukemia Virus
p24-gp51 Ab serum, milk, DRG International Inc., USA), we obtained similar results to those
of blocking ELISA on serum (ELISA Enzootic Bovine Leukosis Virus (BLV) Antibody Test
(IDEXX Laboratories, Inc., Netherlands). The results accuracy can be explained by the fact
that the screening kit used allows the analysis of pools up to 15 milk samples compared to
other milk ELISA kits, which are able to detect anti-BLV antibodies in pools of 50-100
samples. Also, in our study, the milk samples were tested individually and diluted 1:2. The
majority of the indirect ELISA kits for detection of anti-p24 and gp51 antibodies contains
polyclonal antibodies, as opposed to DRG® Bovine Leukemia Virus p24-gp51 Ab serum, milk
used, in which microtiter plates are coated with monoclonal antibodies.
Correspondence between AGID and ELISAs was 84.31%, comparable to values reported by
other authors (E.T. González et al. [27], V.K. Nguyen & RF. Maes [34], H.M. Naif et al. [38]).
Youden’s index (probability of correct classification independent of prevalence) was 0.97,
close to the optimal upper limit (Y = 1), which demonstrates a high diagnosis value of ELISA
Enzootic Bovine Leukosis Virus (BLV) Antibody Test (IDEXX Laboratories, Inc.,
Netherlands) and DRG® Bovine Leukemia Virus p24-gp51 Ab serum, milk (DRG International
Inc., USA).
The more intense humoral immune response in BLV positive cattle from private farms
can be attributed to fodder supplements (minerals, vitamins, probiotics, prebiotics, etc.),
whose general stimulating effects of the immune system are widely recognized (C.M. Cope et
al. [39], J.W. Spears [40]). This intense immune response should be regarded with some
caution, because a high level of anti-BLV antibodies not necessarily means an optimal
humoral immune response to the virus (Z. Trainin & J. Brenner [41], H. Ungar-Waron et al.
[42]). The phenomenon is explained by some "defects" in the structure of immunoglobulins
M and G synthesized in BLV presence, which prevents them from reacting effectively with
viral structures (Z. Trainin et al. [43]).
The age influence on serum antibodies level should be also regarded with caution, because
the majority of the positive cattle from private farms belong to age categories with elevated
levels of anti-BLV antibodies. This phenomenon was also found in case of local breeds, the
differences between their antibodies level being statistically significant only if farm type is
associated in the analysis.
One aspect that deserves a special attention is the dynamics of anti-BLV antibodies
presence in milk. Their level drops significantly from the second month of lactation and
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Performance assessment of three tests applied in enzootic bovine leukosis diagnosis
reached minimum values in the peak of lactation. As mentioned above, milk from BLV
infected cattle, particularly of those with persistent lymphocytosis, contains viable virus (C.J.
Kuckleburg et al. [44], G. Gutierrez et al. [45]), so the transmission via milk consumption is
possible (S.G. Hopkins & R.F. Digiacomo [8]). It was also demonstrated that BLV present in
food products of bovine origin can not be completely inactivated by pasteurization or cooking
(G.C. Buehring et al. [18]). Thus, low levels of neutralizing antibodies in milk and, possibly,
structural defects of immunoglobulins (Z. Trainin et al. [43]), promotes human infection,
phenomenon with potential risks for developing cancer and neurological disorders (M.
Giovanna et al. [19], C. Altaner et al. [21]).
5. Conclusions
AGID does not allow detection of BLV seropositivity in cattle with low titers of anti-gp51
antibodies regardless of age, breed or lactation phase. ELISAS allowed detection of a higher
number of animals with EBL compared to AGID test, taken as reference. Cattle aged 3 to 6
years old are more prone to develop EBL. The humoral immune response in BLV positive
cattle from private farms is more intense, at least quantitatively, than in positive cattle from
households. The level of milk anti-BLV antibodies is conditioned by the lactation phase.
Consumption of insufficiently heat-treated milk from BLV positive cattle in 2-8 months of
lactation is associated with increased risk of infection in humans.
Abbreviations
BLV – bovine leukemia virus
EBL – enzootic bovine leukosis
ELISA – enzyme linked immunosorbent assay
AGID – agar gel immunodiffusion test
HTLV – human T-lymphotropic virus
H-RBSC – Holstein-Romanian Black Spotted Cattle crossbred
OD – optical density (absorbance)
RBSC – Romanian Black Spotted Cattle
RSC – Romanian Spotted Cattle
%E/P – level of anti-BLV serum antibodies
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