Trop Anim Health Prod (2014) 46:461–465

DOI 10.1007/s11250-013-0514-0

REGULAR ARTICLES

Risk factors for anaplasmosis in dairy

cows during the peripartum
Jenevaldo Barbosa da Silva &
Adivaldo Henrique da Fonseca

Accepted: 21 November 2013 / Published online: 4 December 2013

# Springer Science+Business Media Dordrecht 2013

Abstract Anaplasma marginale is endemic in tropical and Keywords Anaplasma marginale . Cattle . Diagnostics .
subtropical areas around the world. Some studies have sug- Peripartum . Serologic testing
gested that cows during peripartum may present a transient
immunosuppression state and development of clinical signs of
anaplasmosis. The aim of this study was to investigate the
relationship between some risk factors and the seroprevalence Introduction
of A. marginale in dairy cows during peripartum in Rio de
Janeiro, Brazil. The risk factors analyzed in association with Tick-borne diseases are important constraints in livestock
the prevalence of antibodies against A. marginale in dairy industry worldwide. Protozoa and rickettsia tick-borne dis-
cows were calving season, reproductive experience, breed eases, such as Babesia bovis, Babesia bigemina protozoa,
standard, tick infestations, stocking density, and milk yield. and Anaplasma marginale rickettsia parasites, are responsible
The antibodies against A. marginale were tested in indirect for great economic losses and both social and economical
enzyme-linked immunosorbent assays. A primary screening impacts in most regions, particularly in Africa, Australia,
using a 2 × k contingency table of the exposed variables with Asia, and South America.
the outcomes was performed. All variables for which p <0.20 A. marginale is the tick-borne pathogen most prevalent
were included in a fixed effects log regression. The risk factors in the world and is responsible for cattle morbidity and
investigated to anaplasmosis were calving (OR 2.61, IC 1.08– mortality in subtropical and tropical countries, including
7.63), breed standard (OR 3.83, IC 0.08–0.28), reproductive Brazil and other regions of Latin America (Vidotto et al.
experience (OR 33.7, IC 2.14–5.16), milk yield (OR 3.9, IC 1998). The obligate intracellular bacteria replicate inside a
2.24–7.03), Rhipicephalus microplus infestations (OR 10.3, parasitophorous vacuolar compartment bonded to erythro-
IC 0.05–0.17), and stocking density (OR 22.3, IC 0.05–0.17). cyte membrane. Clinical disease results from destruction of
Low titers of antibodies against A. marginale during peripar- host-infected erythrocytes followed by severe anemia.
tum had been characterized as a period previous to develop- Studies have suggested that animals older than 1 year
ment of clinical anaplasmosis. Thus, studies on anaplasmosis of age in advanced pregnancy or lactation are most affect-
should consider each farm as an epidemiological unit, where ed by clinical anaplasmosis (Jones et al. 1968) developing
environmental and immunological factors may influence the acute infection (Jones and Brock 1966). Such events may
endemic status of the pathogen. be related to immunosuppression associated with the peri-
partum period in cows (Kehrli et al. 1989). Peracute
anaplasmosis, characterized by a high mortality rate within
J. B. da Silva (*)
a few hours of clinical signs developing, is most frequent-
Faculdade de Ciências Agrárias e Veterinárias—UNESP, Rod. Carlos
Tonanni, km 05, 14870-000 Jaboticabal, São Paulo, Brazil ly encountered in purebred animals and high producing
e-mail: jenevaldo@hotmail.com dairy cows (Ristic 1968).
The aim of the study was to investigate the relation-
A. H. da Fonseca
ship between some risk factors and the seroprevalence
Universidade Federal Rural do Rio de Janeiro—UFRRJ, BR465, Km
07, 23890-000 Seropédica, Rio de Janeiro, Brazil of A. marginale in dairy cows during peripartum in Rio
e-mail: adivaldofonseca@yahoo.com de Janeiro, Brazil.
462 Trop Anim Health Prod (2014) 46:461–465

Materials and methods mean number of larvae and nymphs); and high infestation
(above 31 gravid females and many larvae and nymphs). The
The study was conducted between the years 2008 and 2010 in stocking density was classified into low animal density (1 UA/
the dairy cow sector at the Seropédica Experimental Station, ha), medium density (2 AU/ha), and high density (5 AU/ha).
which belongs to the Agricultural and Livestock Research In order to detect IgG immunoglobulins of the humoral
Company of the State of Rio de Janeiro (Empresa de immune response from the animals naturally infected by A.
Pesquisa Agropecuaria do Estado do Rio de Janeiro; marginale, blood serum samples were subjected to ELISA
Pesagro-Rio) located in the Rio de Janeiro Metropolitan through the technique described by Machado et al. (1997) for
microregion (latitude 22°45′S, longitude 43º41′W, and alti- B. bovis adapted to A. marginale (Fig. 1). The absorbance was
tude 33 m). read at 405 nm in a micro-ELISA reader (Labsystems iEMS
Ninety cattle were selected by using a proportional strati- Reader MF).
fied sampling method, 46 of them had calved in the rainy The immunological response of each serum was estimated
season, and 44 the dry season. According to the reproductive in each dilution by determining the positive serum ratio (S/P),
experience of the animals studied, 20 were at their first lacta- considering positive and negative sera reference (Machado
tion, 22 at the second, and 24 at the third or subsequent et al. 1997). S/P values were grouped into ELISA levels
lactation. The breed distribution consisted of 20 pure (EL), which ranged from 0 (lowest level) to 9 (highest level).
Holstein crossbred, 30 crossbred blood (1/2 Holstein × 1/2 The maximum amplitude from EL was determined by the
Gir), 23 Girolando (5/8 Gir × 3/8 Holstein), and 17 pure Gir mean absorbance values of the negative group plus two stan-
by crossbreeding. dard deviations, as established by Machado et al. (1997). The
Blood sera from the cows were sampled during the last subsequent levels were determined by increment of 35 %.
5 weeks of pregnancy (450 samples), 1 week of calving (90 The risk factors analyzed in association with the prevalence
samples), and first 5 weeks of lactation (450 samples) To of antibodies against A. marginale in dairy cows were calving
better understand the associations between peripartum period season, reproductive experience, breed standard, tick infesta-
and the seroprevalence for A. marginale in dairy cows, we tions, stocking density, and milk yield. A primary screening
classified the peripartum period into three phases as follows: using a 2 × k contingency table of the variables was per-
weeks before calving (phase I), week of calving (phase II), and formed. All variables for p <0.20 were included in a fixed
weeks after calving (phase III). effects log regression. The operational procedures were per-
In order to evaluate the association of milk production with formed by the statistical software R Foundation for Statistical
A. marginale antibodies, the cows were grouped according to Computing, version 2.12.2 (2011).
their annual productivity. There were 27 cows producing less
than 1,500 kg of milk, 40 producing between 1,500 and
3,000 kg of milk and 23 producing more than 3,000 kg of Results
milk. To classify the Rhipicephalus microplus degree of in-
festations, the following criteria were considered: lack of adult Optical densities recorded to A. marginale in dairy cows as
ticks, nymphs, and larvae; mild infestation (between 1 and 10 measured by the ELISA test were 1.13±0.123 for the positive
gravid females and a few larvae and nymphs); moderate sera (n =15) and 0.138±0.013 for the negative sera (n =15).
infestation (between 11 and 30 gravid females ticks and a ELISA threshold was established based on the mean

Fig. 1 Serum blood collection

schedule in cows during the
peripartum
Trop Anim Health Prod (2014) 46:461–465 463

Table 1 Positive serum for A. marginale than during the peripartum, respectively. In
ratio (S/P) values EL S/P Sample number
the rainy season, the cows had 2.3 times more chance to be
grouped into ELISA
levels (0–9) 0 0.000–0.150 293 seropositive. Among the breed standard studied, B. indicus
1 0.151–0.203 163 animals showed a risk of 6.52 times more to be seropositive
2 0.204–0.275 223 than Bos taurus.
3 0.276–0.372 138 In the milk yield group, animals with low production had
4 0.373–0.503 38 3.9 times more chance to be seropositive than animals with
5 0.504–0.680 22 high milk production. Cows at their first lactation showed a
6 0.681–0.919 8 risk of 33.7 times more to be seropositive than animals at their
7 0.920–1.241 1 second or more lactation.
8 1.242–1.676 293 The stocking density demonstrated that animals kept in a
EL Elisa levels, S/P 9 >1.676 163 high-density system had 22.3 times more chance to be sero-
positive serum ratio positive than animals kept in a low-density system. Cows with
low tick infestations showed 10.3 times more chance to be
absorbance of the negative sera plus 35 % over this value as seropositive than animals with high tick infestations.
described by Wilson et al. (1984) (Table 1). The results from multivariate analysis are shown in Table 3.
All variables studied were significant (p <05) in univariate According to this model, only the variables breed, milk yield,
analysis (Table 2). In pregnancy and lactation, the animals stocking density, and R. microplus infestations were signifi-
showed a risk of 2.61 and 4.33 times more to be seropositive cant. Bos indicus animals had 2.00 times more probability for

Table 2 Risk factors to anti-A.

marginale antibody IgG concen- Risk factors N P (%) χ2 OR p value CI 95 %
trations in dairy cows during
peripartum, Pesagro-Rio, Brazil, Peripartum
2008–2010 (1) Pre-calvingb 400 17 – – – –
(2) Calving 80 8 5.0 2.61 0.025 [1.08–7.63]
(3) Post-calving 400 26 8.5 0.60 0.003 [0.42–0.86]
Calving season
(1) Dryb 440 27 – – – –
(2) Rain 440 14 25.1 2.3 0.000 [0.29–0.60]
Breed standard
(1) B. indicus b 165 41 – – – –
(2) F1 165 10 43.2 2.49 0.000 [0.08–0.28]
(3) Girolando 165 29 19.8 1.71 0.000 [0.13–0.50]
(4) B. taurus 385 12 0.69 6.52 0.405 [0.40–1.44]
Reproductive experience
(1) 1ª lactationb 220 35 – – – –
(2) 2ª lactation 220 18 16.9 2.50 0.000 [1.57–4.00]
(3) 3ª lactation 330 14 33.7 3.31 0.000 [2.14–5.16]
(4) >4ª lactation 110 16 12.4 2.75 0.000 [1.50–5.20]
Milk yield
(1) <1,500b 220 36 – – – –
(2) >1,500 <3,000 495 16 36.7 3 0.000 [2.05–4.40]
(3) >3,000 165 12 27.2 3.90 0.000 [2.24–7.03]
N number of animals evaluated,
P(%) prevalence of seropositivity Tick infestations
for A. marginale, χ 2 chi-square, (1) Lowb 65 54 – – – –
OR odd ratio, CI confidence (2) Medium 312 30 52.6 2.70 0.000 [0.17–0.38]
interval
(1) High 503 10 85.5 10.3 0.000 [0.05–0.17]
1ª First Lactation
Stocking density
2ª Second Lactation
(1) Lowb 200 74 – – – –
3ª Third Lactation
(2) Medium 560 54 149.6 2.61 0.000 [0.08–0.17]
>4ª Fourth Lactation
b
(1) High 320 12 212.4 22.3 0.000 [0.03–0.07]
Reference value
464 Trop Anim Health Prod (2014) 46:461–465

Table 3 Final logistic regression model applied to anti-A. marginale The lowest concentration of IgG at calving is probably due
antibody IgG concentrations in dairy cows during peripartum, Pesagro-
to the passive transfer of IgG1 to colostrum. According to
Rio, Brazil, 2008–2010
Sasaki et al. (1976) and Tizard (2004), a decrease in IgG
Risk factors Class p value OR CI 95 % immunoglobulin concentration in bovine blood during peri-
partum period can be associated by the massive transfer of
Peripartum Pre-calvingb – – –
these to the colostrum in the week before calving. However,
Calving 0.839 1.07 [0.552–2.074]
according to Jonsson (2006), these results suggest that lacta-
Post-calving 0.284 0.80 [0.535–1.200]
tion has some impact on the animals' resistance. Moreover,
Seasonal Dryb – – –
periparturient hormonal changes also contribute towards im-
Rain 0.067 0.72 [0.513–1.023]
paired immune function. Among other hormones, the serum
Breed B. taurus b – – –
levels of progesterone, estrogen, and cortisol change dramat-
F1 0.929 1.02 [0.582–1.807] ically at calving (Wettemann 1980; Detilleux et al. 1995;
Girolando 0.080 1.68 [0.939–3.014] VanKampen and Mallard 1997).
B. indicus 0.012 2.41 [1.209–4.816] A transient immunosuppression state in cows during
No. lactation 1ª lactationb – – – peripartum might contribute to the maintenance of the
2ª lactation 0.390 1.22 [0.776–1.911] rickettsia A. marginale infection in the herd through
3ª lactation 0.477 1.18 [0.740–1.897] transplacental transmission. Studies by Potgieter and
4ª lactation 0.577 0.87 [0.548–1.397] Van Rensburg (1987) and Ribeiro et al. (1995) suggest
Milk prod <1,500b – – – that transplacental transmission may occur in chronically
>1,500<3,000 0.900 0.98 [0.621–1.572] infected cows. Thus, we believe that a decrease of the
>3,000 0.000 0.219 [0.107–0.447] circulating antibodies levels during calving might lead
R. microplus Lowa – – – the occurrence of acute anaplasmosis and creates favor-
Medium 0.019 1.66 [1.085–2.556] able conditions for transplacental transmission.
High 0.020 2.11 [1.122–3.951] In the rainy season, Pérez et al. (1994) observed a higher
incidence of clinical anaplasmosis. The prevalence of antibod-
OR odd ratio, CI confidence interval
ies against A. marginale during the rainy season might be
1ª First Lactation
associated with a rise in vector population. However, in con-
2ª Second Lactation
trast to Pérez et al. (1994), we found no significant change (p
3ª Third Lactation
>0.05) of tick populations throughout the year, probably due
4ª Fourth Lactation
b
to steady climatic conditions during the dry and rainy seasons
Reference value within a range suitable for the R. microplus survival.
According to other reports, breed was an important risk
antibody response to A. marginale than B. taurus. Dairy cows variable associated to anaplasmosis. Bock et al. (1997) studies
with higher milk production showed 0.78 times more chance in calves infected with virulent A. marginale indicated that all
to be seropositive than animals with lower milk production, genotypes of calves were susceptible to infection. Among
and animals with R. microplus infestations were 2.11 times animals, five out of ten animals 100 % B. indicus, seven out
more probability for antibody response to A. marginale than of ten animals 50 % B. indicus, eight out of ten animals 25 %
uninfected animals. In addition to the associations already B. indicus , and ten out of ten animals 100 % B. taurus
mentioned, seroprevalence was greater at stock density. The required treatment against A. marginale. The authors conclud-
dairy cows kept in a high-density system showed 3.28 times ed that all breeds exposed to A. marginale are at risk for
more chance to be seropositive than animals kept in a low- disease development. Nevertheless, in B. taurus animals, this
density system. risk is more frequent than in B. indicus.
Generally, primiparous cows tend to produce colostrum
with reduced antibody levels (Elfstrand et al. 2002), and the
Discussion amount of antibodies increases after first lactation. Less
amount of colostral immunoglobulins result in a large amount
In terms of symptoms, clinical anaplasmosis is more com- of the circulating immunoglobulins.
monly encountered in cattle older than 1 year of age (Jones There was a clear positive association between milk yield
et al. 1968). Jones and Brock (1966) suggested that carrier and seroprevalence for A. marginale in herds. This result is in
cows in advanced pregnancy and or lactation may relapse and accordance with Ristic (1968) who observed that peracute
develop signs of acute infection. According to Kehrli et al. infection by A. marginale, characterized by a high mortality
(1989), these events may be involved in the mechanisms of rate within a few hours, is more frequent in high producing
immunosuppression associated with the peripartum period. dairy cows.
Trop Anim Health Prod (2014) 46:461–465 465

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