1
Transmission of BLV between infected cows and their calves
during gestation periparturient period: a Pilotstudy in Chile,
Xth Region.
September 2008-March 2009, a research project as part of professional examination of Veterinary
Medicine, University of Utrecht.
Drs. J.J. van Arkel
Supervisors:
Dr. G.E. Monti DVM Msc (UACh)
Dr. M. Nielen (UU)
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
2
Index
Summary ................................................................................................................................................. 3
1. Introduction ......................................................................................................................................... 4
Virus ..................................................................................................................................................... 4
Transmission of the virus .................................................................................................................... 4
Detection of BLV .................................................................................................................................. 5
Antibody .............................................................................................................................................. 5
Antigen ................................................................................................................................................ 5
Genetic diversity.................................................................................................................................. 6
Virus Difficulties................................................................................................................................... 6
Objective of the study ......................................................................................................................... 6
2. Materials and Methods ....................................................................................................................... 8
Animals ................................................................................................................................................ 8
Sampling of blood................................................................................................................................ 8
ELISA .................................................................................................................................................... 8
PCR....................................................................................................................................................... 8
Interview.............................................................................................................................................. 9
Analyses and proceeding of the results .............................................................................................. 9
3. Results ............................................................................................................................................... 10
Frequencies of antibody detection ................................................................................................... 10
PCR..................................................................................................................................................... 11
Statistical analyses............................................................................................................................. 11
Interview............................................................................................................................................ 11
4. Discussion .......................................................................................................................................... 13
5. Conclusion ......................................................................................................................................... 15
Acknowledgements ............................................................................................................................... 15
Literature: .............................................................................................................................................. 16
Appendix 1 Test results ......................................................................................................................... 19
Appendix 2............................................................................................................................................. 23
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
3
Summary
Bovine Leukemia Virus (BLV) is a retrovirus that causes economic looses in dairy herds in Chile.
Infection is mostly due to blood related proceedings, where infected B-lymphocytes can transfer
form one cow to another. Less important, but also less understand is the possibility of a very early
infection during gestation and in the first days of life of a newborn calf. The objective of this study
was to estimate the infection rate of newborn calves in highly infected herds in the Xth region of
Chile. From 61 newborn calves and 55 of their dams; blood samples were collected and tested with a
commercial enzyme-linked-immunosorbent assay (ELISA). From this, 57,7 % positive calves and 45 %
of the dams tested positive. The correlation (Pearson-correlation) between them was .866 (sign.
<.001), including the observance of interfering maternal antibodies. To exclude the influence of
maternal antibodies 29 selected blood samples were retested by PCR. 15.4% (n=4) tested positive,
knowing that all their dams tested positive too. Due to less data and many interfering factors in
management and execution of this research it isn’t possible to deduce any reliable consequences for
the level of infection in newborn calves.
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
4
1. Introduction
Virus
Bovine Leukemia Virus (BLV) is a ubiquitous RNA retro-virus, genus Deltaretroviridae that infects the
B-lymphocytes of the bovine (Bos Taurus), and integrates at the DNA of B-lymphocytes (Kettman
1980). Infection could lead to a development of polyclonal expansion of B-lymphocytes known as
persistent lymphcytosis (PL). Just, a few animals will show various clinical signs of disease. Therefore
three different stages of expression of the infection exist; only infected animal or asymptomatic
course (≈65%), persistent lymphocytosis (≈ 29%) and lymphosarcoma (> 5%). (van der Maaten 1990).
PL is a subclinical stage that changes blood view, the lymphocyte count is significant higher compare
to negative animals. Lymphosarcoma is characterizing by various types of tumors in lymph nodes,
spleen and bone marrow. Tumors are found during clinical examination and at the slaughterhouse
during meat inspection.
Infection with BLV is suppose to a decrease of milk yield, condemned carcasses at the slaughterhouse
and an increased culling rate in the dairy farms. In the United States is BLV an important disease
which has an influence on the economic losses in the dairy sector. (Ott 2003) Dairy farming in Chile is
at this moment an important economic activity, especially in the southern regions (IXth and Xth),
where about 55% of all Chilean milk is produced (ODEPA 2008). BLV is in Chile common in dairy herds
of all sizes. In the nineties were different kinds of researches that recognize prevalence within herds
about 40 to 50%. It would be necessary to evaluate the most important epidemiological aspects that
might play a role in the transmission of BLV within herds. (Villouta 1990; Moraga 1998)
Transmission of the virus
The main transmission route of BLV is horizontal by infected blood lymphocytes. During bloodrelated management interventions like; injections, vaccinations and de-horning (DiGiacomo 1985;
Lassauzet 1990) are the risks of transmission high. Also blood transfer vectors (mosquitoes and flies)
(Webber 1988; Hasselschwert 1993), close contact with BLV-positive animals (Lassauzet 1991) and
iatrogenic ways are horizontal transmission routes. It’s necessary to keep in mind that just 0,1 µl of
blood with infected lymphocytes enough is to infect a negative animal. (Dimmock 1991)
Other transmission-routes that are proved under experimental are vertical transmission (dam to calf
during gestation) (Van der Maaten 1982; Thurmond 1983; Ohshima 1984; Meas 2001), milk and
colostrums feeding (Mattheus 1982; Straub 1976; Kenyon 1982; van der Maaten 1982) and
reproduction related interventions. Research with diagnostic methods based on the detection of
antibodies (ELISA and AGID) shows that this ways of infections are possible, but less important as the
blood related transmission. (Hopkins 1997) At this moment its unknown what the exact importance
is of the last mentioned transmission routes.
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
5
Detection of BLV
BLV can be detected in two different ways, direct (antigen) and indirect (antibody). Direct methods
are used to prove present of virus-integration at the DNA of the host cell, especially the pro-virus.
The indirect methods are bases on the principal of detecting antibodies. BLV infected animals could
be detected 14 to 57 days post-infection by detection methods based on indirect methods’, like
ELISA and AGID (Johnson 1987; Monti 2005).
Antibody
Agar gel immunodiffusion test (AGID) (Miller 1976) was one of the first serological tests for detection
of BLV infection. It detects antibodies against gp51 (surface glycoprotein) and p24 (virus core
protein), whereby the gp51 appears before than p24 and remains lifelong. The meantime for
obtaining a test result is between 48-72 h. AGID compared with other detection methods has a lower
sensitivity. AGID couldn’t used for milk samples to detect specific antibodies against BLV. Although
this disadvantages of AGID, it is still an important test for import/export control points in many
countries over the world.
ELISA (enzyme-linked immunosorbent assay) is a common serological test (Portelle 1983). The
difference with AGID is that ELISA is based on monoclonal antibody; therefore is it possible to detect
one of each specific proteins (p24 and gp51). Detection by gp51 is proved to be more sensitive than
by p24 virus core-protein (Klintevall 1994). A practical advantage is the process-time, which provides
a result in 2-3 hours. The improved sensitivity of ELISA made this test one of the most used test for
BLV. Specificity (SP) of 0,98 and sensitivity (SE) of 1,0 are declared for different kind of commercial
ELISA tests (Camargos 2007). Not only in blood are BLV-antibodies present, also in milk (Mammerickx
1985). There are nowadays commercial ELISA-kits available for detecting specific BLV-antibodies in
milk.
Antigen
PCR, and especially the modified nested PCR technique, are both for the detection of BLV relative
new. PCR and modified nested PCR technique recognize the BLVDNA that’s integrated at the DNA of
infected B-lymphocytes. (Klintevall 1991; Rola 2002) This characteristic part of the DNA makes the
test useful to detect animals which not develop an immune response yet (Nagy 2006). The level of
detection is declared to be lower as AGID and ELISA tests. At this moment there is not a definitive
sensitivity and specificity given of the PCR tests. The results are widespread (SE .63-.98) (SP .89-1.0)
(Nagy 2006, 2007). PCR tests are relative expensive and includes many laboratory acts. Therefore are
PCR and modified nested PCR technique not very useful at control points and in a national
eradication program, but useful for specific groups of cattle like periparturent cows with low levels of
antibodies, just infected cows and calves with interfering maternal antibodies. (Nagy 2006)
Other very sensitive tests used in the past are syncytium inhibition assay (SIA) and immune
peroxidase assay (IPAI), whereby IPAI is more sensitive than SIA (Jerabek 1979). Both tests are based
on virus neutralization. Problem is the long time of incubation and the costs for the specific cell-lines
that are used. This makes these tests less useful for a quick and commercial interesting detection of
BLV.
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
6
Genetic diversity
Beside transmission it is demonstrated that BLV-provirus has a genetic diversity (Sagata 1985). There
are nowadays 7 different strains already known. (Fechner 1997) The Belgium, Japanese and NorthAmerica strains are more or less comparable with each other, while the Australian sub-type shows
more differences. These four strains are the most common world-wide. There are also strains like
Argentine, Polish and Italian (Marsolais 1994; Bicka 2002). The difference is made by different pointmutations at the DNA of the pro-virus. The strains can be differentiated by using different restriction
strains during the with a Restricted Fragment Length Polymorphism (RFLP) process (Beier 2001), this
should give us a view in de herd-specific strains, family-specific strains and animal-specific strains. For
Chile, the Argentine strain important, possible in the context of international trade of livestock.
Virus Difficulties
One aspect of BLV is important to keep in mind, the typical long period of latency of the virus,
between 1-8 years after infection. The period of latency is known as the period between infection
and infectioness of the virus. (Ketmann 1980) The virus gives no antibody response and will be in
such low concentrations that detection with a direct method (PCR) is possible outcome for early
detection. (Belak 1993, Nagy 2004) Because of the long period of latency, it’s hard to determine the
set point/age of infection. Nowadays seroconversion is a conformation that a cow is infected, but
there is no continuing relation between time of infection, latency and seroconversion.
Secondly is the possibility of leukocyte and antibody transmission by colostrum and milk, which
interfere with an early detection of BLV. (Kenyon 1982; Ferrer 1981) Colostrum contains antibodies
against BLV if the mother has or had an antibody response, due to an earlier infection. An infected
cow can even infected a calf with BLV infected lymphocytes by colostrum (Ferrer 1981). Other
literature suggests that colostrum BLV-antibodies could be preventive against an infection with BLV,
but it’s not proved in a field study (Van der Maaten 1981). Antibody transmission is interfering with
the used indirect detection methods until the decrease of maternal antibodies at the age of 2-6
months (van der Maaten 1981, Oshima 1984). So it will be not possible to detect young calves as
BLV-positive with the nowadays used techniques in different eradication programs. To make clear if
the calf is infected, vertical or horizontal with BLV, PCR could be the detection tool to answer this
question.
Objective of the study
The objective of this study is to estimate the incidence of vertical transmission in highly infected dairy
farms in Chile, X region (infection rate over 55%). A screening with ELISA and a conformation with
PCR, should give us a more realistic view of the percentage of vertical transmission instead of the use
of the commonly used serological methods as ELISA and AGID. Taking pre-colostral blood samples
should make it possible to discriminate between vertical and horizontal transmission. It should be
clear that vertical transmission only happened during gestation.
A second aim, uprising during this study is the infection rate of calves during their first days of life. It’s
known that calves could be infected, but it’s unknown when and what percentage is under field
conditions with different interfering factors. These interfering factors are in our opinion: separations
from the cow, feeding, housing and management interventions.
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
7
Hereby is the transmission through colostrum feeding an important route, it is known that
lymphocytes can transfer from mother to calf during the first 24 hours of life. The gut is not
completely closed at that moment; it will be possible that BLV-infected lymphocytes can `enter` the
blood stream of calves (Merck 2008).
Taking blood samples and screening with EILSA during the first 10 days of life shows the percentage
of likely infected calves. To test ELISA positive calves afterwards with PCR could show the real
percentages of infected calves at such a young age.
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
8
2. Materials and Methods
Animals
For this pilot-study it was used the voluntarily cooperation of 5 dairy farms in the Xth region of Chile,
with an average of 150 productive cows. The mean average of infection by antibody detection in
blood and milk was 55%, between the herds (Monti 2009 not published yet). Within the herds the
range varies between 30-85%. The animals were all mixed-breed dairy cows; there was no
discrimination on age or lactations. Selection of dams that could give birth during this period were
selected by expecting calving dates, based on the farmers own registration. During every visit of the
farm, monthly, were samples taken of calves at a maximum age of 10 days at the test date. Taking
pre-colostral was nearly impossible because of time-management and distance between university
and farm. Due to less data and practical reasons, like infrequent farm visiting, unknown birthdates
and running research of Monti et we also extracted 68 results of calves within an age of 10 days – 1
year. al. They are summarized in appendix…
Sampling of blood
The dams were twice screened, in the period September 2008-April 2009 as part of a running BLV
project of Monti et al. (Facultades Ciencias Veterinarias, Instituto Medicina Preventiva Veterinaria,
UACh, Valdivia, Chile). These results were used for the separation of the dams in two groups. Group 1
infected with BLV and Group 2 not infected with BLV.
Blood samples of the dams and calves were taken by BD Vacutainer™ Precion Glide multi sample
system (BD Franklin Lakes, NJ USA). We used BD Vacutainer® Serum (BD Franklin Lakes, NJ USA) and
BD Vacutainer®, K2 EDTA 7,2 mg (BD Franklin Lakes, NJ USA) to collect two blood samples. Blood
samples of the calves were taken with 5 ml syringe (BD Franklin Lakes, NJ USA) and an 18G
Hypodermic needle (Nubeco Enterprises, Inc USA). We used Venoject® II Clot Act Z (Terumo Europe
NV Leuven, Belgium) and Venoject® II EDTA (K2) K2E (Terumo Europe NV Leuven, Belgium) to collect
the two blood samples.
Serum was kept in the fridge (4°C) until processing; whole blood was kept in the freeze (-20°C) until
processing.
ELISA
After manual separation of serum and clot was 1,0 ml of serum proceeded with the CHEKIT*
LEUCOSE SERUM (IDEXX Laboratories, USA) at the laboratory of the Universidad Austral de Chile,
Valdivia, Chile. Processes were followed by the IDEXX protocol.
PCR
One ml of whole blood was kept into the freeze until the PCR proceeding at the laboratory of the
Universidad Austral de Chile, Valdivia, Chile.
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
9
Oligonucleotide primers for PCR were designed according to sequence data published elsewhere
(Sagata et al., 1985). Primers corresponding to the env gene (Rice 1985) were selected, and env5032
5’ - TCT GTG CCA AGT CTC CCA GAT A - 3’, and env5099 5’ - CCC ACA AGG GCG GCG CCG GTT T - 3’
were used as forward primers. The reverse primers were env5521r 5’ - GCG AGG CCG GGT CCA GAG
CTG G - 3’, env5608r 5’ - AAC AAC AAC CTC TGG GAA GGG T - 3’. The sets env5099 and env5521 had
been established and described previously (Naif 1990; Naif 1992).
DNA was obtained from frozen blood collected with EDTA and was extracted using Jet QUICK kit
(Genmed, Germany). The first round of nested PCRs (Fechner 1996) was performed using env5032 /
env5608r as first primers; initial incubation of samples was at 72 ˚C for 2 minutes; denaturation took
place at 94 ˚C for 2 minutes followed by 50 amplification cycles consisting of denaturation at 95 ˚C,
30 seconds, primer annealing at 58 ˚C, 30 seconds and extension at 72 ˚C for 1 minute; final
extension took place at 72 ˚C for 4 minutes. The second round of amplifications was conducted using
the second pair of primers (env5099 / env5521r). The second round was the same as the first round
except that the primer annealing temperature was changed to 72 ˚C. A known positive and negative
control DNA sample was included in each test run, and samples showing a band migrating at 444
base pairs (bp) were considered as positive.
Interview
We used a questionnaire (appendix I) to learn more about the practical management of nursing
calves at the farms. The questions were based on high-risk management activities that contribute in
the transmission of BLV. Feeding, housing and interventions are the main activities that are related to
the transmission. (Lassauzet 1990) Appendix 1 concludes the interview and the results
Analyses and proceeding of the results
All test-results from this project and the project of Monti were saved in an MS-excel file. All unique
tubes match with an individual animal, including number of the animal (Identification) and date of
sampling. Based on the farmers databases were the expecting calving dates, birthdates, mother-calf
relations, ID and ages of the calves at moment of sampling documented. For the statistic -analyze it
was used: Win Episcope 2.0 and SPSS 16 for Windows.
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
10
3. Results
Frequencies of antibody detection
The results of all tested calves (n= 129) are proceeded in table 1. Results are known of 55 dams who
gave birth during this project.
# Calves
# Negative < 10 days
# Positive < 10
days
# Negative >
10 days
# Positive
> 10days
% Positive ELISAtest
A
25
9
15
1
0
60,00%
M
20
1
4
3
12
80,00%
S
78
10
7
42
19
33,33%
W
3
0
3
0
0
100,00%
T
3
2
1
0
0
33,33%
129
22
30
46
31
47,28 %
Farm
Total
Table 1 results of detection antibodies by an ELISA test at the individual farms, Xth region of Chile
Extracting results from the older calves, (n=77) between 10 days and 1 year old, proceed in
December and November 2008 shows an positive test result of 40,78 % with an average age of 90
(median 80) days. Negative calves had an average age of 156 (median 154) days.
Excluding calves older than 10 days (n=52) it was estimated that 57,69% tested positive by ELISA.
For the selection of BLV-positive and BLV – negative dams (n=55) where 2 groups formed: Group 1
contains 25 dams which are BLV-positive, based on at least 2 positive ELISA test-results and Group 2
contains 30 dams which are BLV-negative, based on at least 2 negative ELISA test-results.
55 combinations of ELISA test-results of calves and their dams could show in table 2 the following
results.
Dam-Calf Combinations
Total births
55
Positive Dams (Group 1) (45%)
Dam and calf positive
Dam positive, calf negative
20
36,40%
5
9,00%
21
38,20%
9
16,40%
Negative dams (Group 2) (55%)
Dam and calf negative
Dam negative, calf positive
Table 2 Combinations of test-results dam -calf
Discrimination of sexes of the calves shows in table 4 no difference. Both groups’ male and female
have a positive combination, dam and calf percentage between 30% and 40%.
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
11
Male calves 40%
Total births
Total positive comb.
Total only calf
Total only mother
Total negative comb.
22
7
5
2
8
31,80%
22,70%
9%
36,40%
Female calves 60%
Total births
Total positive comb.
Total only calf
Total only mother
Total negative comb.
33
13
4
3
13
39,40%
12,10%
9,10%
39,40%
Table 3 Sex discrimination in prospect of dam -calf relation.
PCR
After testing with ELISA calves selected were for PCR-test, mostly based on positive outcome of
ELISA-test. It resulted in 29 results, which are represented in table 5. Four calves should be excluded
based on an age > 10 days.
PCR results
Calves
Average age
8,73
# Calves
29
Pos
8
Neg
21
% Pos
27,58%
Table 4 PCR Results
In combination with the results of the dams four calves tested positive (15,3 % resp.). All four had a
mother which tested positive for BLV-antibodies. One of the four calves tested negative for BLVantibodies with the ELISA test.
Statistical analyses
A Pearson-correlation (SPSS 16) test resulted only in a significant correlation between positive ELISA
test of the dam and calf at 0.886 (<0.01). There were no other correlations based on sex, age and
PCR-positive test results.
Beside the aim of the study the usage of an ELISA-test for the detection of BLV by calves was
evaluated. Preceeding the combinend ELISA test-results and PCR results (n=29) with Win Episcope 2.0
and presume that PCR-test is the golden-standard with a level of confidence of 99,5%. An sensitivity
of 87,50 (54,68;100) and specificity of 19,04 (0;43,10) was found. The positive predictive value is
29,17% (3,12;55,21), the negative predictive value is 80,00% (29,77;100)
Interview
All exact results (n=4 farms) are shown in Appendix 1. One farm was excluded, because we visited
this farm once. All high-risk management proceedings are summarized in table 5.
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
12
Subject
Score
Transmission route
Note:
(farms/total farms)
Feeding
Colostrum feeding by own
dam
(4/4)
Colostrum
Fresh milk
(0/4)
Milk
Discharged milk
(2/4)
Milk
Only for the male calves
Milkreplacer
(4/4)
Milk
All female calves were fed a milk
replacement for four weeks
Cleaning drinking facilities
(4/4)
Milk/colostrum rests
All farms clean their drinking
facilities 2 or more times a day. One
farm cleaned just once a day.
Seperation directly
(0/4)
Colostrum/milk
Seperation after a few hours
(1/4)
Colostrum/milk
Seperation after a few days
(3/4)
Milk or colostrum from
other cows
Individual housing
(1/4)
Prevent contact with BLVpositive animals
(Laussazet 1991)
Grouphousing
(3/4)
Contact with BLV-positive
animals
All calves were housed in groups > 5
animals.
Prevention for mosquito’s
(4/4)
Insect bites
There were still a lot of mosquito’s
during the summer.
Replace needles after a single
treatment
(4/4)
Blood on/in needle
What they answer, no proof of
practice.
Dehorning
(4/4)
Blood on hotspot
Ear tagging
(4/4)
Blood on eartag device
Remove of the
supermammair tits
(3/4)
Blood on scissors
Housing
Blood related interventions
Two farms used an device, which
make no contact with the calf.
Table 5 Summarized interview results
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
13
4. Discussion
Goal one was to obtain pre-colostral blood of newborn calves, to prove if there was an opportunity
for vertical transmission. Poor management and registration made it hard to obtain enough data to
prove infection between dam and calf. Calves were born in big pens with many other dams at the
end of gestation. There was no surveillance during birth and the following hours. Excluding two
samples we failed to collect blood samples before calves took colostrum of their dam. It’s even
doubtful if they suckle by their own dam because there were many more. Separated housing of the
calves was realized after 6 to 72 hours. Beside the possible infection route by colostrum it was not
even clear which blood related treatments all calves undergo, like vaccination, ear tagging, injections
and dehorning. Ear tagging, injections and vaccinations are treatments at risk for infection (Lassauzet
1990). In the summer period (December – February) where in some farms a lot of mosquito’s and
flies, which are known vectors for horizontal transmission of BLV (Dimmock 1991).
Frequency of BLV-positive calves within herds varies between 33,33% and 100%, based on a single
ELISA test. But as showed in static analyze obtained data from the ELISA-test has a low predicted
value. A positive predicted value low as 30% remarks that the opportunity to detect a real infected
calf is very low from this point of view. A positive ELISA result show more over success of transmission
of (maternal) antibodies during gestation and periparturient period.(Ferrer 1978, Johnson 1987).
Negative result is more reliable, because the negative predicted value is 80%. But al these data were
obtained from a small group (n=29).
If we took the PCR test as a golden-standard, suggested by Nagy (2003), for all sampled calves we can
doubt about perfection of the test. SE depends on design and execution of the primers (Marsolais
1994 and Amperse 2008 (not published)) especially for commercial sold PCR tests. Secondly less then
10% of the B-lymphocytes in adult cattle is infected with BLV. (Mirsky 1996), this influences the
chance of detection. Thirdly Kampen (2006) proves that during gestation B-lymphocytes will increase
from 1% - 2% until 10% to 20%. But the absolute amount of B-lymphocytes in newborn calves is
lower in the first week compare to older calves and adult cattle (Ayoub 1996), this will also influence
the chance of detection. At last the possibility of transmission of B-lymphocytes by colostrum and
their uptake in the first 24 hours of life is very less, because colostrum of cows contains less then 5%
B-lymphocytes (Reber 2006).
Our second aim, what is the infection rate is of calves in their first days of live become clear. As
showed in table one 56,69% of the calves are positive for detection of antibodies by ELISA. There’s no
comparable data on farm level previously publicized. But we couldn’t make a difference between
maternal antibodies and calves their own ones. It’s known that the detection level for maternal
antibodies of BLV is between two to six months (van der Maaten 1981, Oshima 1984). Preceding a
PCR-test, despite of previously mentioned criticism showed an infection rate of 15,6% in the first
days of live, whereby even one ELISA-negative calf was tested positive by PCR. Unfortunately there
was no possibility to obtain pre-colostral blood for PCR-testing. Infection rate is still more than
Thurmond (1983) suggested (6,4%) by vertical transmission (in utero) based on pre-colostral AGID
testing, but less then Piper (1979) under research facilities (10- 25%).
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
14
Sex-associated infection with BLV was not proven; there were no statistically differences in testing
with ELISA or PCR.
Testing calves between an age of 10 days and 1 year with ELISA marks that the average infected age
(100) is within the period of interfering maternal antibodies. ELISA isn’t able to detect that these
animals are infected, as previously mentioned lifetime of maternal antibodies is two to six months
(Oshima 1984). For definitive status of the animals it could be practical to retest these animals with
ELISA after six months of age or test all these animals with PCR.
The results of the interview based on four out of five farms, shows that during normal circumstances
and with knowledge of the transmission of BLV infection possible is directly after birth and the
following days. Beside this interview it became clear that group housing of pregnant dams in a big
pen at the end of gestation and during parturition is common. Colostrum feeding and blood related
interventions were possible the factors most at risk which the young calves were exposed to.
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
15
5. Conclusion
We proved that there are some infections with BLV either newborn or after parturition, but due to
less data and many interfering factors in management and execution of this research it isn’t possible
to deduce any reliable consequences for the level of infection in newborn calves.
But descriptive we can suggest that if more than 1 out of 7 calves (15%) are infected, based on the
PCR-test result, in their first days of live it’s recommended to do further research to the importance
of transmission in newborn calves, this could define the moment of infection in herds.
It’s necessary to develop protocols within herds for birth process, sampling and herd administration.
Also should specific risks in management, like colostrum-feeding, blood-related proceedings and
housing be clear before research start.
Acknowledgements
Thank you for help; G.E. Monti and family, O. Allocilla, B.Benavides, N.Nielen. Thanks for support;
Fundo Sichahue, Fundo Aromos - Soto, Fundo Muller, Fundo Winker, Fundo Twelle, Marcelo Gomez,
Ann Huenink and Ginger James
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
16
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18.
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18
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An epidemiological study of natural in utero infection with bovine leukemia virus (1983) 47-3 p. 316-319
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49. AMPERSE C. Comparison of the validity of different serological and molecular test for bovine leukemia virus in
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blood mononuclear cells at two subclinical stages of infection. Journal of Virology (1996) 70:4 p. 2178-2183
51. KAMPEN A.H., OLSEN I., TOLLERSRUD T. Lymfocyte subpopulations and neutrophil function in calves during the
first 6 months of life. Vet. Immunol Immunopath (2006) 113 p. 53-63
52. AYOUB I.A., YANG T.J. Age-dependent changes in peripheral blood lymphocyte subpopulations in cattle: A
longitudinal study. Development & comparative immunology (1996) 20-5 p. 353-363
53. REBER A.J., LOCKWOOD A., HIPPEN A.R., Colostrum induced phenotic and trafficking changes in maternal
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54. OSHIMA K., MORIMOTO N., KAGAWA Y., NUMAKUNAI S., HIRANO T., KAYANO H. A survey for maternal
antibodies to bovine leukemia virus (BLV) in calves born to cows infected with BLV Jpn. Journal Vet. Sci. (1984)
46-4 p. 583 – 586
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
19
Appendix 1 Test results
Until 10 days
Tubo ID
7449 903
7269 3584
7270 3585
7476 3596
7266
7267
7532
7277
Man 1 Zwart
Man 2 Wit
902
575
7455 3595
7397
7398
7450
7390
7391
7392
907
552
901
872
873
544
5861 3529
7482 3594
7396
7468
7387
7537
7388
7518
551
3593
905
920
870
2302
5870 3526
7389
7395
7485
7393
871
906
3592
549
5863 3524
7394
8563
7543
7386
550
3023
919
542
6359 866
7260
7481
7261
7265
7495
3582
3591
3583
3581
3590
Farm
age
t
a
a
a
m
m
t
s
a
s
s
t
s
s
s
a
a
s
a
s
m
s
w
a
s
s
a
s
a
s
w
m
s
s
a
a
a
a
a
0
1
1
1
2
2
2
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
5
5
5
6
6
6
6
7
7
7
7
8
8
8
8
8
Result ELISA Result PCR
1
1
1
1
1
1
0
1
0
0
0
0
0
1
1
0
0
0
1
1
1
1
1
1
0
0
0
1
1
0
1
0
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
1
0
0
1
0
0
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
20
6358 865
7262 8758
7263 8759
7517 2301
5876 1999
5878 2000
7264 8760
7276 916
7413 810
7474
7258
7480
7487
3588
8757
3587
3586
s
a
a
w
a
a
a
s
m
a
a
a
a
8
9
9
9
9
9
9
9
10
10
10
10
10
0
1
1
1
0
0
1
0
1
1
1
1
1
0
0
1
0
1
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
21
Age 10 days - 1 year
Tubo
ID
7413
7399
6359
6358
7259
7257
6368
7256
7255
7254
7251
7253
7252
6367
6363
6360
6362
6361
7414
6366
7401
6365
7406
7402
6364
7403
7404
6370
6369
6374
7415
7410
6371
6373
7412
7408
7405
6375
6378
Tubo
0810
5220
0866
0865
910
559
0864
558
557
556
908
555
554
0863
0862
0861
0860
0859
0808
0858
0806
0857
0807
0804
0856
0803
0802
0855
0854
0853
0899
0898
0852
0851
0896
0895
0894
0850
0849
ID
Farm
Age
Result ELISA
m
a
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
m
s
m
s
m
m
s
m
m
s
s
s
m
m
s
s
m
m
m
s
s
10
111
112
1
0
1
0
0
1
1
0
0
0
1
1
0
1
1
1
0
1
1
0
0
0
1
1
1
1
1
1
1
1
0
1
0
1
1
1
1
0
0
Age
Result
Farm
12
14
15
20
23
25
26
28
29
30
30
30
30
32
35
38
40
49
53
62
65
68
69
71
76
80
82
83
85
86
89
92
96
102
103
104
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
22
Elisa
7409
7407
6379
6377
7411
6372
6376
6501
6492
6491
6496
6494
6493
6511
6495
6500
6510
6499
6502
6497
6503
6508
6509
6460
6504
6506
6507
6498
6461
6505
6453
6490
6435
6441
6437
6419
6421
6446
0891
0892
0848
0847
0890
0846
0845
0844
0843
0842
0841
0839
0837
0838
0836
0835
0833
0832
0830
0831
0829
0826
0825
0821
0822
0820
0819
0818
0817
0814
0812
0811
0808
0807
0806
0804
0803
0801
m
m
s
s
m
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
114
114
115
119
120
121
128
136
139
141
143
153
155
155
163
176
200
211
214
214
224
228
229
243
243
244
247
251
253
258
264
270
285
296
297
302
306
312
1
0
1
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
1
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
23
Appendix 2
Results of the Interview of the farmers:
A: Alimento
A.1.:
De qué se alimentan los ternoros hasta el primer mes?
 Leche de vaca (no tratadas o de descarte)
 Sustituto lácteo
 Leche de descarte
All the farmers use a milkreplacer during the first month for their female calves. The male-calves will
also be fed with discharged milk.
A.2.a Por cuantos o cuantas tomos luego de nacer los terneros reciben calostro?
Mean for 2 days (2/4), but it depends on the possibility to split calf and cow. Some farms will split
after 6 hours max (1/4) other after 3 days, or more (1/4). It depends also on the fitness of the calves
b. Recibe calostro de:
 Fresco de su madre
 Fresco de cualquier madre
 Congelado de un banco de calostro
All farmers use fresh colostrum from the mother.
A.3. Como alimenta a los terneros?
 Tetillas/mamaderas
 Baldes
It depends on the management. Most of the farms use replacement nipples (3/4). Some use only
drinking buckets for the male calves.
A.4.Con qué freceuencia limpian el sistema de alimentación?
Most of the farmers clean after ever y time they use the bucket or nipples. (3/4) One farm cleans just
1 time a day.
A.5 Como lavan el sistema de alimentación? (Procedimiento)
Hot water and desinfection (chloride)
B. Instalaciones
B.1. Cuando se sepera el tenero de la madre?
12 hrs (1/4)
2 days (2/4)
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
24
3 days (1/4)
B.2 Como se alojan los teneros?
 Individual
 Individual por 5-10 dias, luego en grupos
 Grupos
Individual por 5-10 dias (1/4)
Groups (3/4)
B.3 Cuantos animals por grupo?
 1-3
 4-5
 >5
> 5 (3/4)
4 (1/4)
B.4 Qué criteros se utilize para ele agrupamiento? (edad, peso etc.)
Sex of calves (4/4) and fitness (1/4)
B.5. Con que frecuencia cambian la cama y limpian el establo de los teneros?
 Luego de cada tenero/grupo
 Una vez al semana
 Una vez al mes
 Cuando está muy sucio
 Cuando tengo tiempo
2 farms put in all lodges everyday new straw or other bedding.
1 farm cleans every 2 days
1 farm once a week
B.6 Controlan contra mosquitos y moscas?
 Si, pour-on
 Si, repelent por fumigación
 Otro:______
 Nada
2 farms use pour-on and 2 farms use smoke or repellent
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
25
C Intervenciones
C.1. tabla
Intervenciones
Fundo
1
2
3
4
Areteo
Si
Dia 3
Si
Si
Inyecciones *
Si
si
Si
Si
Descorne
Si
8 semanas
Si
No machos,
si teneras
Cirugías (castración,
tetillas, etc.)
No
Tetillas, si
Si
Si, tetillas
* = Vacunación, medicación y vitaminas con agujas
For dehorning, all the farmers use an hotspot
C.2 Limpian o desinfectan los instrumentos para aretear?
 Si, entre cada ternero
 Si al final del trabajo
 No
Yes after every calf (2/4)
No, but we use an ear tagger that make no contact with the calf. (2/4)
C. 3 Cambia las agujas?
 Si, entre cada de tenero
 Si, luego de cada de tipo de medicación
 Luego de cada ronda de inyecciones
 Si, pero al final del día
 Cuando la aguja está sucia o defectuosa
All the farmers changes needles after they use them for one calf!
C.4 Qué sistema de decorne utilizan?
Hotspot (heater) (4/4)
C.5 Hacen alguna maniobra quinrúgica masiva? (castración, recorte tetillas, etc.)?
Yes (3/4)
Remove of the supermammair tits. (3/4)
Castration of the males. (1/4)
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.
26
The truth, during our farm visits showed sometimes other things then the answers of the interviews.
We saw a lot of used needles and changing was not always done. A lot of dirty stables, with flies, high
contact level between the animals and not cleaned ear taggers were specific attention points.
Otherwise to prevent horizontal transmission of all kind of microbiological risk were the calves
housed apart from the heifers and dairy cows.
Transmission of BLV between infected cows and their calves during gestation periparturient period: a Pilotstudy in Chile, Xth Region. Van
Arkel, J.J., Monti, G.E., 2009.