Accepted Manuscript Title: Characterization of colostrum from dams of BLV endemic dairy herds Author: Ger´onimo Guti´errez Marina Lomonaco Irene Alvarez Fernando Fernandez Karina Trono PII: DOI: Reference:
S0378-1135(15)00090-5 http://dx.doi.org/doi:10.1016/j.vetmic.2015.03.001 VETMIC 6922
To appear in:
VETMIC
Received date: Revised date: Accepted date:
3-11-2014 27-2-2015 2-3-2015
Please cite this article as: Guti´errez, G., Lomonaco, M., Alvarez, I., Fernandez, F., Trono, K.,Characterization of colostrum from dams of BLV endemic dairy herds, Veterinary Microbiology (2015), http://dx.doi.org/10.1016/j.vetmic.2015.03.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Characterization of colostrum from dams of BLV endemic dairy herds
Gutiérrez, Gerónimo1*†, [email protected]
4
Lomonaco, Marina1†, [email protected]
5
Alvarez, Irene1,[email protected]
6
Fernandez, Fernando1, [email protected]
7
Trono, Karina1, [email protected]
8 †
These authors contributed equally to this work
an
9
us
cr
3
ip t
2
10 11
1
12
Ciencias Veterinarias y Agronómicas, INTA. Nicolás Repetto y De los Reseros s/n,
13
1686, Hurlingham, Buenos Aires, Argentina.
16 17 18 19 20 21
M
d te
15
Ac ce p
14
Laboratorio de Virus Adventicios, Instituto de Virología, Centro de Investigaciones en
22 23 24
*Corresponding author. Tel/Fax: +54 11 4621 9050
25
E-mail address: [email protected] (G. Gutiérrez)
26
1 Page 1 of 15
27
Abstract
28
Bovine Leukemia Virus (BLV) is endemic in Argentina, where the individual
30
prevalence is higher than 80% in dairy farms. The aim of this work was to find
31
preliminary evidence to know if the high level of infection of the dam would implicate a
32
higher challenge to her own offspring. We collected 65 sets of samples consisting of
33
dam’s blood and colostrum from two heavily infected dairy farms, and investigated the
34
correlation between the dam´s blood proviral load and the presence of provirus in
35
colostrum. We also described the dual antibody/provirus profile in the colostrum.
36
Provirus was detected in 69.23% of the colostrum samples, mostly from dams with a
37
high proviral load, 36/45 (80%). Colostrum proviral load was significantly higher in
38
dams with high blood proviral load (p < 0.0001). Provirus was detected in colostrum
39
samples all along the antibody distribution, even in those with a low amount of
40
antibodies. These results show that even when high blood proviral load dams offer
41
higher levels of infected cells to their offspring through colostrum they also offer higher
42
levels of protection of antibodies. On the contrary, low blood proviral load dams also
43
offer infected cells but a poor content of antibodies, suggesting that these animals could
44
play an important role in the epidemiological cycle of transmission.
46 47 48
cr
us
an
M
d
te
Ac ce p
45
ip t
29
Keywords: BLV; colostrum; proviral load; antibody
Introduction
49 50
Bovine Leukemia Virus (BLV) is endemic in Argentina, where the individual
51
prevalence is higher than 80% in dairy farms of the main productive areas of the
52
country (Gutiérrez et al., 2012). In the herds of these farms, about 10% of the calves are 2 Page 2 of 15
born infected, and blood proviral load reaches high levels (more than 1% of white blood
54
cells infected) in more than 40% of infected animals (Lomonaco et al., 2014). In this
55
work we investigated the correlation between the dam’s blood proviral load and the
56
presence of provirus in colostrum, in two heavily infected dairy farms. Concurrently, we
57
described the dual antibody/provirus profile in the colostrum of naturally infected dams.
58
Even when the effect of consuming colostrum from infected dams is still a matter of
59
controversy, the aim of this work was to find preliminary evidence to know if the high
60
level of infection of the dam would implicate a higher challenge to their own offspring.
us
cr
ip t
53
62
an
61
Materials and Methods
64
M
63
Farms and samples
d
65
The study was carried out using samples from 2 commercial dairy farms naturally
67
infected with BLV, coded as farm A and B (86.5% and 89.3% of individual prevalence,
68
respectively). Both farms had typical Holstein dairy herds, with about 350 milking cows
69
each. We collected 65 sets of samples (39 from Farm A and 26 from Farm B) including
70
whole blood and colostrum from dams at the peripartum period. Farm owners’ consent
71
was obtained before animal sampling. The procedures followed for extraction and
72
handling of samples were approved by the Institutional Committee for Care and Use of
73
Experimental Animals of the National Institute of Agricultural Technology (CICUAE-
74
INTA) under protocol number 35/2010, and followed the guidelines described in the
75
institutional Manual.
Ac ce p
te
66
76 77
Antibody testing 3 Page 3 of 15
78
Plasma antibodies against the whole BLV viral particle were detected by ELISA as
80
previously reported (Trono et al., 2001). The antibody titers were assayed by the end-
81
point dilution method using two-fold dilutions of sera. Titers were expressed as the
82
reciprocal of the dilution.
ip t
79
84
cr
83
BLV detection and proviral load quantification
us
85
Total DNA was extracted from whole blood using a DNA extraction kit (High Pure
87
PCR Template Preparation kit, Roche, Penzberg, Germany) according to the
88
manufacturer’s instructions. BLV proviral DNA was detected by nested PCR (Wu et al.,
89
2003), and the relative quantification of the proviral load (PVL) was assessed as
90
described by Lew et al., (2004) using the SYBR Green Detection technology. All
91
samples were tested in duplicate by using 50 ng of DNA as template. A fragment of the
92
BLV pol gene was amplified together with a fragment of the constitutive 18 S gene used
93
as reference. As calibrator for both reactions, we used 50 ng of DNA from fetal lamb
94
kidney (FLK) cells, containing four copies of BLV proviral DNA per cell (Van den
95
Broeke et al., 2001), in a final concentration of 1% in peripheral blood mononuclear
96
cells (PBMCs) purified from a non-infected cow, according to Hopkins and DiGiacomo
97
(1997) for aleukemic animals. The relative PVL was expressed as the ratio obtained by
98
the sample for the BLV gene in comparison to the 18 S reference gene, based on the
99
efficiency and the cycle threshold deviation from the internal control sample (Pfaffl,
100
2001). With this method, the relative PVL (ratio) of the calibrator sample was set to 1
101
and all samples were referred to it. Samples showing a ratio ≥ 1 were considered with
102
high PVL while samples showing a ratio < 1 were considered with low PVL according
Ac ce p
te
d
M
an
86
4 Page 4 of 15
103
to our own criteria. The reaction showed a limit of detection of 1 BLV-infected cell in
104
10,000 non-infected cells.
105
Results
ip t
106 107
We quantified BLV-specific antibodies and proviral load in 65 sets of samples, each set
109
of samples including blood and colostrum from BLV-infected dams. Provirus was
110
detected in 69.23% of the colostrum samples (Figure 1). Provirus-positive colostrum
111
samples were mostly from dams with a high blood proviral load, 36/45 (80%) (Figure
112
1). Of these cows, the vast majority 92.3% (36/39) presented provirus-positive
113
colostrum (Figure 1). In addition, colostrum proviral load was significantly higher in
114
dams with high blood proviral load (p < 0.0001). The amount of provirus in colostrum
115
was always below a ratio of 0.22 (Figure 2), more than 100-fold lower in mean than
116
proviral load in blood. Colostrum samples were categorized as Low (≤ 16), Medium (32
117
- 256) or High (≥ 512), depending on the level of antibodies; each category representing
118
21.54%, 61.54% and 16.92% of the samples, respectively (Figure 3). Provirus was
119
detected in colostrum samples all along the antibody distribution, even in those with a
120
low amount of antibodies. The prevalence of this detection was 21.43%, 77.5% and
121
100% for categories low, medium and high, respectively (Figure 3). Colostrum proviral
122
load was significantly lower in samples from the low category. Medium and high
123
antibody categories showed the lowest difference in proviral load (p = 0.048) (Figure 3).
Ac ce p
te
d
M
an
us
cr
108
124 125
Discussion
126
5 Page 5 of 15
The effect of consumption of colostrum within a BLV control strategy is still a matter of
128
discussion between farmers of dairy herds. During the 1980´s some works were
129
conducted that demonstrated the infective and also the protective potential of colostrum
130
from infected cows under experimental conditions (Ferrer and Piper, 1981; Van Der
131
Maaten et al., 1981; Lassauzet et al., 1989). Later, some groups published that the
132
consumption of colostrum was a risk factor for BLV within-herd transmission (Sprecher
133
et al., 1991; Meas et al., 2002). Nonetheless, other epidemiological studies have shown
134
the reduction of BLV incidence in concordance with the consumption of colostrum
135
from BLV positive dams, interpreting the feeding of colostrum to newborn calves as a
136
protective factor (Nagy et al., 2007; Kobayashi et al., 2010). Thus, the role of colostrum
137
on natural transmission is still unclear. In this work, we describe the dual profile of
138
antibodies and provirus in colostrum from naturally infected dams, and the relationship
139
of this profile with the level of infection in dams’ blood.
140
The most interesting finding was that even when the dams with high blood proviral load
141
showed a higher prevalence of provirus-positive samples (Figure 1) and a higher
142
proviral load in colostrum, the provirus was detectable in all categories of colostrum
143
samples, even in those with a low amount of antibodies (Figure 3). If the colostrum
144
carries viable cells with provirus and shows poor antibody content, it could be a vehicle
145
for transmission, as with those shown in Figure 3 categorized as Low or Medium, that
146
count for more than 80% of the studied samples. Moreover, when bloody colostrum is
147
fed to newborn calves, as often occurs in the dairy system, the consequences could be
148
even worse, since the offer of infected blood cells would be even higher. In this context,
149
the susceptibility to infection could be modified not only by the presence, but also by
150
the amount of antibodies and provirus. The final result of the colostrum consumption is
151
unpredictable, and would depend on the dual antibody/provirus profile, together with
Ac ce p
te
d
M
an
us
cr
ip t
127
6 Page 6 of 15
the duration of antibodies in the calf, and also of the immediate future challenge posed
153
by the raw milk and the commingled infected newborn calves. A well-designed dose-
154
response trial could be modeled using these data as the base line to build experimental
155
groups that receive different antibody/provirus amounts in colostrum, and that should be
156
followed up to analyze the future incidence of infection. The positive correlation found
157
between the dams’ amount of antibodies in blood and colostrum (data not shown)
158
suggest that newborn calves could be supplemented with colostrum from a bank made
159
with secretions of highly infected dams to provoke a higher level of passive
160
immunology. The critical point should be the use of cell-deprived, frozen or heat
161
inactivated secretions to render them non-infectious as previously reported
162
(Baumgartener et al., 1976; Rubino and Donham, 1984; Kanno et al., 2014).
163
These results suggest that colostrum can constitute a vehicle for transmission, as
164
reported for HTLV, a similar complex Retrovirus that infects humans (Kinoshita et al.,
165
1984). The use of fresh raw colostrum from low proviral load dams is thus not as good
166
as we previously supposed, at least until the protective/infective dose of colostrum is
167
estimated. We can speculate that the consumption of colostrum with infected-cells and a
168
poor content of antibodies could play a critical role in BLV propagation during young
169
age. Van Der Maaten et al. (1981) showed that 106 mononuclear cells from an infected
170
cow can infect a susceptible newborn calf by the oral route, but cannot do so in the
171
presence of antibodies.
172
We consider that colostrum and milk could play an important role in the
173
epidemiological cycle of transmission and should not be unattended, especially during
174
the first two months of age when, after natural intake of colostrum from their dams, the
175
calves are fed with raw bulk tank milk from the dairy. The use of colostrum
176
supplementation from a pasteurized or frozen bank made with secretions from highly
Ac ce p
te
d
M
an
us
cr
ip t
152
7 Page 7 of 15
infected dams to finally provoke a high antibody titer in the calf could be a good
178
alternative to resist a future challenge. Finally, conversely to blood borne BLV
179
transmission, we can presume that dams with low proviral load could be those that pose
180
the highest risk of transmission by feeding colostrum or milk to their own offspring.
ip t
177
cr
181
Acknowledgements
183
This work was supported by Laboratorio de Virus Adventicios (INTA). All authors
184
were supported by INTA, G. Gutiérrez and I. Alvarez were also supported by Consejo
185
Nacional de Investigaciones Científicas y Técnicas (CONICET).
an
us
182
M
186
References
188
- Baumgartener L., Olson C., Onuma M., 1976. Effect of pasteurization and heat
189
treatment on bovine leukemia virus. J. Am. Vet. Med. Assoc. 169:1189-1191.
190
- Ferrer J.F., Piper C.E., 1981. Role of colostrum and milk in the natural transmission of
191
the bovine leukemia virus. Cancer Res. 41:4906-4909.
192
- Gutiérrez G., Carignano H., Alvarez I., Martínez C., Porta N., Politzki R., Gammella
193
M., Lomonaco M., Fondevila N., Poli M., Trono K., 2012. Bovine leukemia virus p24
194
antibodies reflect blood proviral load. BMC Vet. Res. 8:187.
195
- Hopkins S.G., DiGiacomo R.F., 1997. Natural transmission of bovine leukemia virus
196
in dairy and beef cattle. Vet. Clin. North Am. Food Anim. Pract. 13:107-128.
Ac ce p
te
d
187
8 Page 8 of 15
- Kanno T., Ishihara R., Hatama S., Oue Y., Edamatsu H., Konno Y., Tachibana S.,
198
Murakami K., 2014. Effect of freezing treatment on colostrum to prevent the
199
transmission of bovine leukemia virus. J. Vet. Med. Sci. 76:255-257.
200
- Kinoshita K., Hino S., Amagaski T., Ikeda S., Yamada Y., Suzuyama J., Momita S.,
201
Toriya K., Kamihira S., Ichimaru M., 1984. Demonstration of adult T-cell leukemia
202
virus antigen in milk from three sero-positive mothers. Gann. 75:103-105.
203
- Kobayashi S., Tsutsui T., Yamamoto T., Hayama Y., Kameyama K., Konishi M.,
204
Murakami K., 2010. Risk factors associated with within-herd transmission of bovine
205
leukemia virus on dairy farms in Japan. BMC Vet. Res. 6:1.
206
- Lassauzet M.L., Johnson W.O., Thurmond M.C., Stevens F., 1989. Protection of
207
colostral antibodies against bovine leukemia virus infection in calves on a California
208
dairy.Can. J. Vet. Res. 53:424-430.
209
- Lew A.E., Bock R.E., Molloy J.B., Minchin C.M., Robinson S.J., Steer P., 2004.
210
Sensitive and specific detection of proviral bovine leukemia virus by 5' Taq nuclease
211
PCR using a 3' minor groove binder fluorogenic probe.J. Virol. Methods. 115:167-175.
212
- Lomonaco M., Alvarez I., Martínez C., Porta N., Merlini R., Carignano H., Gutiérrez
213
G., Trono K., 2014. Epidemiological features of BLV natural infection. Retrovirology
214
11(Suppl 1):P45.
215
- Meas S., Usui T., Ohashi K., Sugimoto C., Onuma M., 2002. Vertical transmission of
216
bovine leukemia virus and bovine immunodeficiency virus in dairy cattle herds.Vet.
217
Microbiol. 84:275-282.
218
- Nagy D.W., Tyler J.W., Kleiboeker S.B., 2007.Decreased periparturient transmission
219
of bovine leukosis virus in colostrum-fed calves. J. Vet.Intern. Med. 21:1104-1107.
Ac ce p
te
d
M
an
us
cr
ip t
197
9 Page 9 of 15
- Pfaffl M.W., 2001. A new mathematical model for relative quantification in real-time
221
RT-PCR. Nucleic Acids Res. 29:e45.
222
- Rubino M.J., Donham K.J., 1984. Inactivation of bovine leukemia virus-infected
223
lymphocytes in milk.Am. J. Vet. Res. 45:1553-1556.
224
- Sprecher D.J., Pelzer K.D., Lessard P., 1991. Possible effect of altered management
225
practices on seroprevalence of bovine leukemia virus in heifers of a dairy herd with
226
history of high prevalence of infection. J. Am. Vet. Med. Assoc. 199:584-588.
227
- Trono K.G., Pérez-Filgueira D.M., Duffy S., Borca M.V., Carrillo C., 2001.
228
Seroprevalence of bovine leukemia virus in dairy cattle in Argentina: comparison of
229
sensitivity and specificity of different detection methods. Vet. Microbiol. 83:235-248.
230
- Van den Broeke A., Cleuter Y., Beskorwayne T., Kerkhofs P., Szynal M., Bagnis C.,
231
Burny A., Griebel P., 2001. CD154 costimulated ovine primary B cells, a cell culture
232
system that supports productive infection by bovine leukemia virus. J. Virol. 75:1095-
233
1103.
234
- Van Der Maaten M.J., Miller J.M., Schmerr M.J., 1981. Effect of colostral antibody
235
on bovine leukemia virus infection of neonatal calves.Am. J. Vet. Res. 42:1498-1500.
236
- Wu D., Murakami K., Morooka A., Jin H., Inoshima Y., Sentsui H., 2003. In vivo
237
transcription of bovine leukemia virus and bovine immunodeficiency-like virus.Virus
238
Res. 97:81-87.
Ac ce p
te
d
M
an
us
cr
ip t
220
239
10 Page 10 of 15
Figure 1. Provirus-positive colostrum samples were mostly from dams with a high
240
blood proviral load. Dams´ blood proviral loads were plotted according to the detection
241
(or not) of provirus in their respective colostrum sample. Means were significantly
242
different (p ˂ 0.0001, Mann-Whitney test).
243
Figure 2. Blood proviral load was significantly higher than colostrum proviral load.
244
Proviral loads in dam´s blood and in their respective colostrum samples were plotted.
245
Means were significantly different (p ˂ 0.0001, Wilcoxon matched pairs test).
246
Figure 3. Provirus was detected in colostrum samples all along the antibody
247
distribution. Colostrum samples were categorized as Low (≤ 16), Medium (32 - 256) or
248
High (≥ 512), depending on the level of antibodies. Colostrum proviral loads were
249
plotted according to these antibody titer categories. Means were significantly different
250
(Mann-Whitney test).
cr
us
an
M
d te Ac ce p
251
ip t
239
11 Page 11 of 15
Highlights
te
d
M
an
us
cr
ip t
Provirus was detected in 69.23% of colostra, even in those with low antibody titers 80% of provirus-positive colostra were from dams with high blood proviral load Colostrum proviral load was more than 100-fold lower than blood proviral load Consuming colostrum with infected-cells and a poor content of antibodies may be a risk
Ac ce p
251 252 253 254 255 256 257
12 Page 12 of 15
Ac
ce
pt
ed
M
an
us
cr
i
Figure 1
Page 13 of 15
Ac
ce
pt
ed
M
an
us
cr
i
Figure 2
Page 14 of 15
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 15 of 15
S0378-1135(15)00090-5 http://dx.doi.org/doi:10.1016/j.vetmic.2015.03.001 VETMIC 6922
To appear in:
VETMIC
Received date: Revised date: Accepted date:
3-11-2014 27-2-2015 2-3-2015
Please cite this article as: Guti´errez, G., Lomonaco, M., Alvarez, I., Fernandez, F., Trono, K.,Characterization of colostrum from dams of BLV endemic dairy herds, Veterinary Microbiology (2015), http://dx.doi.org/10.1016/j.vetmic.2015.03.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Characterization of colostrum from dams of BLV endemic dairy herds
Gutiérrez, Gerónimo1*†, [email protected]
4
Lomonaco, Marina1†, [email protected]
5
Alvarez, Irene1,[email protected]
6
Fernandez, Fernando1, [email protected]
7
Trono, Karina1, [email protected]
8 †
These authors contributed equally to this work
an
9
us
cr
3
ip t
2
10 11
1
12
Ciencias Veterinarias y Agronómicas, INTA. Nicolás Repetto y De los Reseros s/n,
13
1686, Hurlingham, Buenos Aires, Argentina.
16 17 18 19 20 21
M
d te
15
Ac ce p
14
Laboratorio de Virus Adventicios, Instituto de Virología, Centro de Investigaciones en
22 23 24
*Corresponding author. Tel/Fax: +54 11 4621 9050
25
E-mail address: [email protected] (G. Gutiérrez)
26
1 Page 1 of 15
27
Abstract
28
Bovine Leukemia Virus (BLV) is endemic in Argentina, where the individual
30
prevalence is higher than 80% in dairy farms. The aim of this work was to find
31
preliminary evidence to know if the high level of infection of the dam would implicate a
32
higher challenge to her own offspring. We collected 65 sets of samples consisting of
33
dam’s blood and colostrum from two heavily infected dairy farms, and investigated the
34
correlation between the dam´s blood proviral load and the presence of provirus in
35
colostrum. We also described the dual antibody/provirus profile in the colostrum.
36
Provirus was detected in 69.23% of the colostrum samples, mostly from dams with a
37
high proviral load, 36/45 (80%). Colostrum proviral load was significantly higher in
38
dams with high blood proviral load (p < 0.0001). Provirus was detected in colostrum
39
samples all along the antibody distribution, even in those with a low amount of
40
antibodies. These results show that even when high blood proviral load dams offer
41
higher levels of infected cells to their offspring through colostrum they also offer higher
42
levels of protection of antibodies. On the contrary, low blood proviral load dams also
43
offer infected cells but a poor content of antibodies, suggesting that these animals could
44
play an important role in the epidemiological cycle of transmission.
46 47 48
cr
us
an
M
d
te
Ac ce p
45
ip t
29
Keywords: BLV; colostrum; proviral load; antibody
Introduction
49 50
Bovine Leukemia Virus (BLV) is endemic in Argentina, where the individual
51
prevalence is higher than 80% in dairy farms of the main productive areas of the
52
country (Gutiérrez et al., 2012). In the herds of these farms, about 10% of the calves are 2 Page 2 of 15
born infected, and blood proviral load reaches high levels (more than 1% of white blood
54
cells infected) in more than 40% of infected animals (Lomonaco et al., 2014). In this
55
work we investigated the correlation between the dam’s blood proviral load and the
56
presence of provirus in colostrum, in two heavily infected dairy farms. Concurrently, we
57
described the dual antibody/provirus profile in the colostrum of naturally infected dams.
58
Even when the effect of consuming colostrum from infected dams is still a matter of
59
controversy, the aim of this work was to find preliminary evidence to know if the high
60
level of infection of the dam would implicate a higher challenge to their own offspring.
us
cr
ip t
53
62
an
61
Materials and Methods
64
M
63
Farms and samples
d
65
The study was carried out using samples from 2 commercial dairy farms naturally
67
infected with BLV, coded as farm A and B (86.5% and 89.3% of individual prevalence,
68
respectively). Both farms had typical Holstein dairy herds, with about 350 milking cows
69
each. We collected 65 sets of samples (39 from Farm A and 26 from Farm B) including
70
whole blood and colostrum from dams at the peripartum period. Farm owners’ consent
71
was obtained before animal sampling. The procedures followed for extraction and
72
handling of samples were approved by the Institutional Committee for Care and Use of
73
Experimental Animals of the National Institute of Agricultural Technology (CICUAE-
74
INTA) under protocol number 35/2010, and followed the guidelines described in the
75
institutional Manual.
Ac ce p
te
66
76 77
Antibody testing 3 Page 3 of 15
78
Plasma antibodies against the whole BLV viral particle were detected by ELISA as
80
previously reported (Trono et al., 2001). The antibody titers were assayed by the end-
81
point dilution method using two-fold dilutions of sera. Titers were expressed as the
82
reciprocal of the dilution.
ip t
79
84
cr
83
BLV detection and proviral load quantification
us
85
Total DNA was extracted from whole blood using a DNA extraction kit (High Pure
87
PCR Template Preparation kit, Roche, Penzberg, Germany) according to the
88
manufacturer’s instructions. BLV proviral DNA was detected by nested PCR (Wu et al.,
89
2003), and the relative quantification of the proviral load (PVL) was assessed as
90
described by Lew et al., (2004) using the SYBR Green Detection technology. All
91
samples were tested in duplicate by using 50 ng of DNA as template. A fragment of the
92
BLV pol gene was amplified together with a fragment of the constitutive 18 S gene used
93
as reference. As calibrator for both reactions, we used 50 ng of DNA from fetal lamb
94
kidney (FLK) cells, containing four copies of BLV proviral DNA per cell (Van den
95
Broeke et al., 2001), in a final concentration of 1% in peripheral blood mononuclear
96
cells (PBMCs) purified from a non-infected cow, according to Hopkins and DiGiacomo
97
(1997) for aleukemic animals. The relative PVL was expressed as the ratio obtained by
98
the sample for the BLV gene in comparison to the 18 S reference gene, based on the
99
efficiency and the cycle threshold deviation from the internal control sample (Pfaffl,
100
2001). With this method, the relative PVL (ratio) of the calibrator sample was set to 1
101
and all samples were referred to it. Samples showing a ratio ≥ 1 were considered with
102
high PVL while samples showing a ratio < 1 were considered with low PVL according
Ac ce p
te
d
M
an
86
4 Page 4 of 15
103
to our own criteria. The reaction showed a limit of detection of 1 BLV-infected cell in
104
10,000 non-infected cells.
105
Results
ip t
106 107
We quantified BLV-specific antibodies and proviral load in 65 sets of samples, each set
109
of samples including blood and colostrum from BLV-infected dams. Provirus was
110
detected in 69.23% of the colostrum samples (Figure 1). Provirus-positive colostrum
111
samples were mostly from dams with a high blood proviral load, 36/45 (80%) (Figure
112
1). Of these cows, the vast majority 92.3% (36/39) presented provirus-positive
113
colostrum (Figure 1). In addition, colostrum proviral load was significantly higher in
114
dams with high blood proviral load (p < 0.0001). The amount of provirus in colostrum
115
was always below a ratio of 0.22 (Figure 2), more than 100-fold lower in mean than
116
proviral load in blood. Colostrum samples were categorized as Low (≤ 16), Medium (32
117
- 256) or High (≥ 512), depending on the level of antibodies; each category representing
118
21.54%, 61.54% and 16.92% of the samples, respectively (Figure 3). Provirus was
119
detected in colostrum samples all along the antibody distribution, even in those with a
120
low amount of antibodies. The prevalence of this detection was 21.43%, 77.5% and
121
100% for categories low, medium and high, respectively (Figure 3). Colostrum proviral
122
load was significantly lower in samples from the low category. Medium and high
123
antibody categories showed the lowest difference in proviral load (p = 0.048) (Figure 3).
Ac ce p
te
d
M
an
us
cr
108
124 125
Discussion
126
5 Page 5 of 15
The effect of consumption of colostrum within a BLV control strategy is still a matter of
128
discussion between farmers of dairy herds. During the 1980´s some works were
129
conducted that demonstrated the infective and also the protective potential of colostrum
130
from infected cows under experimental conditions (Ferrer and Piper, 1981; Van Der
131
Maaten et al., 1981; Lassauzet et al., 1989). Later, some groups published that the
132
consumption of colostrum was a risk factor for BLV within-herd transmission (Sprecher
133
et al., 1991; Meas et al., 2002). Nonetheless, other epidemiological studies have shown
134
the reduction of BLV incidence in concordance with the consumption of colostrum
135
from BLV positive dams, interpreting the feeding of colostrum to newborn calves as a
136
protective factor (Nagy et al., 2007; Kobayashi et al., 2010). Thus, the role of colostrum
137
on natural transmission is still unclear. In this work, we describe the dual profile of
138
antibodies and provirus in colostrum from naturally infected dams, and the relationship
139
of this profile with the level of infection in dams’ blood.
140
The most interesting finding was that even when the dams with high blood proviral load
141
showed a higher prevalence of provirus-positive samples (Figure 1) and a higher
142
proviral load in colostrum, the provirus was detectable in all categories of colostrum
143
samples, even in those with a low amount of antibodies (Figure 3). If the colostrum
144
carries viable cells with provirus and shows poor antibody content, it could be a vehicle
145
for transmission, as with those shown in Figure 3 categorized as Low or Medium, that
146
count for more than 80% of the studied samples. Moreover, when bloody colostrum is
147
fed to newborn calves, as often occurs in the dairy system, the consequences could be
148
even worse, since the offer of infected blood cells would be even higher. In this context,
149
the susceptibility to infection could be modified not only by the presence, but also by
150
the amount of antibodies and provirus. The final result of the colostrum consumption is
151
unpredictable, and would depend on the dual antibody/provirus profile, together with
Ac ce p
te
d
M
an
us
cr
ip t
127
6 Page 6 of 15
the duration of antibodies in the calf, and also of the immediate future challenge posed
153
by the raw milk and the commingled infected newborn calves. A well-designed dose-
154
response trial could be modeled using these data as the base line to build experimental
155
groups that receive different antibody/provirus amounts in colostrum, and that should be
156
followed up to analyze the future incidence of infection. The positive correlation found
157
between the dams’ amount of antibodies in blood and colostrum (data not shown)
158
suggest that newborn calves could be supplemented with colostrum from a bank made
159
with secretions of highly infected dams to provoke a higher level of passive
160
immunology. The critical point should be the use of cell-deprived, frozen or heat
161
inactivated secretions to render them non-infectious as previously reported
162
(Baumgartener et al., 1976; Rubino and Donham, 1984; Kanno et al., 2014).
163
These results suggest that colostrum can constitute a vehicle for transmission, as
164
reported for HTLV, a similar complex Retrovirus that infects humans (Kinoshita et al.,
165
1984). The use of fresh raw colostrum from low proviral load dams is thus not as good
166
as we previously supposed, at least until the protective/infective dose of colostrum is
167
estimated. We can speculate that the consumption of colostrum with infected-cells and a
168
poor content of antibodies could play a critical role in BLV propagation during young
169
age. Van Der Maaten et al. (1981) showed that 106 mononuclear cells from an infected
170
cow can infect a susceptible newborn calf by the oral route, but cannot do so in the
171
presence of antibodies.
172
We consider that colostrum and milk could play an important role in the
173
epidemiological cycle of transmission and should not be unattended, especially during
174
the first two months of age when, after natural intake of colostrum from their dams, the
175
calves are fed with raw bulk tank milk from the dairy. The use of colostrum
176
supplementation from a pasteurized or frozen bank made with secretions from highly
Ac ce p
te
d
M
an
us
cr
ip t
152
7 Page 7 of 15
infected dams to finally provoke a high antibody titer in the calf could be a good
178
alternative to resist a future challenge. Finally, conversely to blood borne BLV
179
transmission, we can presume that dams with low proviral load could be those that pose
180
the highest risk of transmission by feeding colostrum or milk to their own offspring.
ip t
177
cr
181
Acknowledgements
183
This work was supported by Laboratorio de Virus Adventicios (INTA). All authors
184
were supported by INTA, G. Gutiérrez and I. Alvarez were also supported by Consejo
185
Nacional de Investigaciones Científicas y Técnicas (CONICET).
an
us
182
M
186
References
188
- Baumgartener L., Olson C., Onuma M., 1976. Effect of pasteurization and heat
189
treatment on bovine leukemia virus. J. Am. Vet. Med. Assoc. 169:1189-1191.
190
- Ferrer J.F., Piper C.E., 1981. Role of colostrum and milk in the natural transmission of
191
the bovine leukemia virus. Cancer Res. 41:4906-4909.
192
- Gutiérrez G., Carignano H., Alvarez I., Martínez C., Porta N., Politzki R., Gammella
193
M., Lomonaco M., Fondevila N., Poli M., Trono K., 2012. Bovine leukemia virus p24
194
antibodies reflect blood proviral load. BMC Vet. Res. 8:187.
195
- Hopkins S.G., DiGiacomo R.F., 1997. Natural transmission of bovine leukemia virus
196
in dairy and beef cattle. Vet. Clin. North Am. Food Anim. Pract. 13:107-128.
Ac ce p
te
d
187
8 Page 8 of 15
- Kanno T., Ishihara R., Hatama S., Oue Y., Edamatsu H., Konno Y., Tachibana S.,
198
Murakami K., 2014. Effect of freezing treatment on colostrum to prevent the
199
transmission of bovine leukemia virus. J. Vet. Med. Sci. 76:255-257.
200
- Kinoshita K., Hino S., Amagaski T., Ikeda S., Yamada Y., Suzuyama J., Momita S.,
201
Toriya K., Kamihira S., Ichimaru M., 1984. Demonstration of adult T-cell leukemia
202
virus antigen in milk from three sero-positive mothers. Gann. 75:103-105.
203
- Kobayashi S., Tsutsui T., Yamamoto T., Hayama Y., Kameyama K., Konishi M.,
204
Murakami K., 2010. Risk factors associated with within-herd transmission of bovine
205
leukemia virus on dairy farms in Japan. BMC Vet. Res. 6:1.
206
- Lassauzet M.L., Johnson W.O., Thurmond M.C., Stevens F., 1989. Protection of
207
colostral antibodies against bovine leukemia virus infection in calves on a California
208
dairy.Can. J. Vet. Res. 53:424-430.
209
- Lew A.E., Bock R.E., Molloy J.B., Minchin C.M., Robinson S.J., Steer P., 2004.
210
Sensitive and specific detection of proviral bovine leukemia virus by 5' Taq nuclease
211
PCR using a 3' minor groove binder fluorogenic probe.J. Virol. Methods. 115:167-175.
212
- Lomonaco M., Alvarez I., Martínez C., Porta N., Merlini R., Carignano H., Gutiérrez
213
G., Trono K., 2014. Epidemiological features of BLV natural infection. Retrovirology
214
11(Suppl 1):P45.
215
- Meas S., Usui T., Ohashi K., Sugimoto C., Onuma M., 2002. Vertical transmission of
216
bovine leukemia virus and bovine immunodeficiency virus in dairy cattle herds.Vet.
217
Microbiol. 84:275-282.
218
- Nagy D.W., Tyler J.W., Kleiboeker S.B., 2007.Decreased periparturient transmission
219
of bovine leukosis virus in colostrum-fed calves. J. Vet.Intern. Med. 21:1104-1107.
Ac ce p
te
d
M
an
us
cr
ip t
197
9 Page 9 of 15
- Pfaffl M.W., 2001. A new mathematical model for relative quantification in real-time
221
RT-PCR. Nucleic Acids Res. 29:e45.
222
- Rubino M.J., Donham K.J., 1984. Inactivation of bovine leukemia virus-infected
223
lymphocytes in milk.Am. J. Vet. Res. 45:1553-1556.
224
- Sprecher D.J., Pelzer K.D., Lessard P., 1991. Possible effect of altered management
225
practices on seroprevalence of bovine leukemia virus in heifers of a dairy herd with
226
history of high prevalence of infection. J. Am. Vet. Med. Assoc. 199:584-588.
227
- Trono K.G., Pérez-Filgueira D.M., Duffy S., Borca M.V., Carrillo C., 2001.
228
Seroprevalence of bovine leukemia virus in dairy cattle in Argentina: comparison of
229
sensitivity and specificity of different detection methods. Vet. Microbiol. 83:235-248.
230
- Van den Broeke A., Cleuter Y., Beskorwayne T., Kerkhofs P., Szynal M., Bagnis C.,
231
Burny A., Griebel P., 2001. CD154 costimulated ovine primary B cells, a cell culture
232
system that supports productive infection by bovine leukemia virus. J. Virol. 75:1095-
233
1103.
234
- Van Der Maaten M.J., Miller J.M., Schmerr M.J., 1981. Effect of colostral antibody
235
on bovine leukemia virus infection of neonatal calves.Am. J. Vet. Res. 42:1498-1500.
236
- Wu D., Murakami K., Morooka A., Jin H., Inoshima Y., Sentsui H., 2003. In vivo
237
transcription of bovine leukemia virus and bovine immunodeficiency-like virus.Virus
238
Res. 97:81-87.
Ac ce p
te
d
M
an
us
cr
ip t
220
239
10 Page 10 of 15
Figure 1. Provirus-positive colostrum samples were mostly from dams with a high
240
blood proviral load. Dams´ blood proviral loads were plotted according to the detection
241
(or not) of provirus in their respective colostrum sample. Means were significantly
242
different (p ˂ 0.0001, Mann-Whitney test).
243
Figure 2. Blood proviral load was significantly higher than colostrum proviral load.
244
Proviral loads in dam´s blood and in their respective colostrum samples were plotted.
245
Means were significantly different (p ˂ 0.0001, Wilcoxon matched pairs test).
246
Figure 3. Provirus was detected in colostrum samples all along the antibody
247
distribution. Colostrum samples were categorized as Low (≤ 16), Medium (32 - 256) or
248
High (≥ 512), depending on the level of antibodies. Colostrum proviral loads were
249
plotted according to these antibody titer categories. Means were significantly different
250
(Mann-Whitney test).
cr
us
an
M
d te Ac ce p
251
ip t
239
11 Page 11 of 15
Highlights
te
d
M
an
us
cr
ip t
Provirus was detected in 69.23% of colostra, even in those with low antibody titers 80% of provirus-positive colostra were from dams with high blood proviral load Colostrum proviral load was more than 100-fold lower than blood proviral load Consuming colostrum with infected-cells and a poor content of antibodies may be a risk
Ac ce p
251 252 253 254 255 256 257
12 Page 12 of 15
Ac
ce
pt
ed
M
an
us
cr
i
Figure 1
Page 13 of 15
Ac
ce
pt
ed
M
an
us
cr
i
Figure 2
Page 14 of 15
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 15 of 15
