Seasonal effect on Anaplasma marginale infections of beef cattle in previously

flooding areas

Nantiya Saetiew1,2 ,Pacharathon Simking1,2, Sinsamuth Saengow3, Benjamas Kurajog3, Benjarat Yimming3,
Kepalee Saeng-chuto3, Wissanuwat Chimnoi3 , Chanya Kengradomkil3 , Sarawut Yangtara3, Sayamchai
Suksai3, Noppawa Thaprathom3, Churit Seneemanoma4, Kamonthip Penghirun4, Marc Desquesnes3,
Stephane Herder3, Serge Morand3 and Sathaporn Jittapalapong3

ABSTRACT
Anaplasmosis is an important tick-borne disease of cattle caused by the obligate
intraerythrocytic rickettsiae, Anaplasma marginale, distributed among livestock throughout Thailand.
In 2011, Thailand was suffered from the great flooding along Chao Phraya River areas. The objective
of this study was to investigate the seasonal effect on A. marginale infections of cattle in 3 previously
devastated flooding provinces including Nan, Nakhon Sawan, and Ayutthaya. A total of 569 blood
samples were randomly collected using the multistage cluster random sampling technique during dry
and rainy season; from November-December 2012 (dry season, n=241), and May-June 2013 (rainy
season, n=328). The molecular assay was employed by one set of primers for specific amplification,
and the sequence analysis of the major surface protein 4 (MSP4). The factors associated with
A. marginale infections were analyzed based on age, sex, provinces, and herd size. The overall
prevalence of A. marginale was found 14.9% (36/241) in dry season compared to 23.2% (76/328) in
rainy season. Nakhon Sawan had the higher prevalence at 33.3% (27/81) in dry period compared to
27.3% (30/110) in rainy period. For herd size factor, A. marginale infections were significantly found in
large herds (80.0%, 12/15) more than small herds (23.8%, 5/21) and A. marginale infections were
risen in the rainy season (87.5%, 7/8) compared to the infection in dry season (71.4%, 5/7). Animals
between 1-5 year of age had the highest infections (22.7%, 93/409) and an increasing prevalence
was also found in the rainy season (25.7%, 72/280).

Keywords: Anaplasma marginale, MSP4 Gene, Cattle, Flooding provinces, Seasonal effect, Thailand
e-mail address: fvetspj@ku.ac.th
1
Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140
2
Center of Excellence on Agricultural Biotechnology, Science and Technology Postgraduate Education and Research
Development Office, Commission on Higher Education, Ministry of Education. (AG-BIO/PERDO-CHE)
3
Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand
4
Ayutthaya Provincial Livestock Development Office
 INTRODUCTION
Anaplasmosis is an important hemoparasitic disease in livestock productions of the
developing countries. The disease is caused by the rickettsiae, Anaplasma marginale (order
Rickettsiales, family Anaplasmataceae), and characterized by severe anemia and jaundice without
hemoglobinemia and hemoglobinuria (Dumler et al., 2001). Other clinical signs are weight loss,
decreased milk production, abortions, and sudden death (Richey and Palmer, 1990). A. marginale
in cattle is transmitted by ticks, biting flies such as tabanus and stable flies, and blood contaminated
fomites (Kocan et al., 2003). A. marginale is a dot-like in host red blood cell so that it will multiply and
break cells causing anemia. Therefore, the conventional method for the diagnosis of Anaplasma
infection is the microscopic examination of stained blood smears. However, due to the low sensitivity,
this method is not recommended for the detection of subclinical animals or carriers with low
parasitaemia (Carelli et al., 2007). To increase in the high sensitivity and specificity of a test,
polymerase chain reaction (PCR) is one of the alternative diagnosis providing a powerful tool for
epidemiological investigations with the high accuracy of Anaplasma spp. infection (de la Fuente et
al., 2005a).
Bovine Anaplasmosis in Thailand has been reported for more than five decades
(Sithornnakoon et al., 1965; Teerapat, et al., 1965; Watanasin, et al., 1965 ; Arunyakanon et al., 1966;
Jittapalapong et al., 1988; Chethanond et al., 1995; Chaichanapunpol et al., 1996; Phrikanahok et
al., 2000; Fungfuang et al., 2006; Worasing, 2007; Sriwarothai et al., 2008; Yawongsa et al., 2010;
Saetiew et al., 2012), and their distributions have an impact on the economic losses of livestock
production. In 2011, Thailand suffered from the great flooding over 70% of the country particularly in
nearby Chao Phraya river areas including Nan, Nakhon Sawan and Ayutthaya. Flooding might
change the environment that affects vector ecology based on their hosts and habitats and this will
also affect some vector-borne pathogens such as Anaplasma spp. During rainy season, flies and
ticks are significantly increasing in number so that this will increase a possibility of A. marginale
transmission. The purpose of this study was to investigate the seasonal effect on A. marginale
infections of cattle in recently flooded provinces.

MATERIALS AND METHODS

1. Study areas
Nan, Nakhon Sawan, and Ayutthaya were assigned areas that have been flooded in 2011. Nan
was located along Nan river as one of the beginning of Chao Phraya river while Nakhon Sawan and
Ayutthaya were situated along Chao Phraya river. These 3 provinces were flooded at different
magnitude depending on their landscape (Figure 1).

Figure 1 Topographic map of Thailand demonstrating primarily the Chao Phraya River System.

2. Seasonal factors
Thailand has three official seasons including summer, rainy, and winter (dry) season. However,
because of global warming, seasonal period has been significant inconsistency. This will create
different adjustment of environment. Normally, the dry season in Thailand starts from October to
February and the rainy season begin in May and ending in September.
This study was designed to collect samples during rainy and dry season to compare seasonal
effects. Therefore, samples collected from Nov to Dec, 2012 were assigned as dry season while
samples collected from May to June, 2013 were represent the rainy season.

3. Blood samples
A total of 569 blood samples of beef cattle was collected between November 2012 and June
2013. Animals were bled from the jugular vein into sterile tubes containing EDTA as an anticoagulant
and citrate salt to preserve for PCR technique, kept on ice during transportation to the Department of
Parasitology, Faculty of Veterinary Medicine, Kasetsart University, and stored at -20ºC until analyzed.
All information of animals including age, sex, and herd size were recorded.
4. Molecular detection of A. marginale
4.1 DNA extraction
The DNA was extracted from blood sample following the protocol of Sambrook et al. (1989).
and stored at -20°C prior to use.
4.2 Amplification of MSP4 gene
Primers MSP43 (5’-CCGGATCCTTAGCTGAACAGGAATCTTGC-3’) and MSP45 (5’-
GGGAGCTCC TATGAATTACAGAGAATTGTTTAC-3’) were used to amplify an approximately 850 bp
fragment of the msp4 gene (de la Fuente et al., 2005a). Negative controls (distilled water) were used
every 10 sample to ensure the accuracy of the method. The first sample tested positive by PCR and
sequence analysis was subsequently used as the positive control. The MSP4 gene was amplified
from 2 μl DNA by PCR using 1 pmol of each primer. For reactions were performed in an automated
DNA thermal cycler for 40 cycles. After initial denature step of 2 min at 95ₒC, each cycle consisted of
a denaturing step of 30 sec at 95ₒC, an annealing step of 30 sec 60ₒC and extension step of 1 min at
72ₒC. PCR products were electrophoresed on 1% agarose gel. Positive PCR products were purified.
4.3 Sequencing analysis
Purified PCR products were sent to Bio Service Unit (NSTDA) for DNA sequencing and BLAST
analysis was used to identify genomic DNA.

5 Statistical analysis
The positive numbers of A. marginale infections were analyzed with factors including sex, age,
and herd. Chi-square and Number Cruncher Statistical System (NCSS) ver. 2000 (Kaysville, UT)
programs were used to assess differences in the prevalence and intensity of infection. Comparisons
were conducted
(i) Between sex; Male, Female
(ii) Among age groups ; < 1 year, 1-5 year, > 5 year.
(iii) Among provinces
(iv) Among herd size
Comparison between season results was assessed by calculating a statistic at 95%
confidence. Results were compared by use of a software program (Win Episcope, version 2.0)

RESULTS
Using MSP4 primer, A. marginale was detected and identified as the PCR product at 849 bp.
Among 3 provinces, the highest prevalence of A. marginale infections was significantly found in
Nakhon Sawan (33.3%, 27/81) in dry season (P<0.05). Seasonal effects on A. marginale infection
were found in Nan (OR=0.12 and 95%CI=0.03-0.51) and Ayutthaya (OR=0.29 and 95%CI=0.12-0.72).
Herd prevalence and sex factor were found not association with A. marginale infections. For herd
size, A. marginale infection were found in large herds (n≥20) more than of small herds
(n<20)(P<0.05) particularly in the rainy season. Animals between 1-5 year of age were found most
positives and affected by season (OR=0.56, 95%CI=0.33-0.96).

Table 1 A comparison of prevalence and factors associated with A. marginale infection in cattle
between dry and rainy season.
Parameter Dry season Rainy season Total OR 95% CI
(Nov-Dec 2012) (May-June 2013)
Individual
Prevalence Province
Nan 2/70 (2.9%) 23/115 (20.0%) 25/185(13.5%) 0.12 0.03-0.52
Nakhon Sawan 27/81 (33.3%) 30/110 (27.3%) 57/191(29.8%) 1.33 0.71-2.49
Ayutthaya 7/90 (7.8%) 23/103 (22.3%) 30/193(15.5%) 0.29 0.12-0.72
Total 36/241 (14.9%) 76/328 (23.2%) 112/569(19.7%) 0.58 0.38-0.90
df=2, 2=33.24, df=2, 2=1.73, df=2, 2=19.01,
P=0.001 P=0.421 P=0.001
Herd
prevalence Province
Nan 2/8 (25.0%) 3/4 (75.0%) 5/12(41.7%) 0.11 0.01-1.78
Nakhon Sawan 3/4 (75.0%) 3/4 (75.0%) 6/8(75.0%) 1.00 0.04-24.55
Ayutthaya 2/8 (25.0%) 4/8 (50.0%) 6/16(37.5%) 0.33 0.04-2.77
Total 7/20 (35.0%) 10/16 (62.5%) 17/36(47.2%) 0.32 0.08-1.27
df=2, 2=3.52, df=2, 2=1.07, df=2, 2=3.23,
P=0.172 P=0.586 P=0.198
Herd size n<20 2/13 (15.4%) 3/8 (37.5%) 5/21(23.8%) 0.30 0.04-2.42
n≥20 5/7 (71.4%) 7/8 (87.5%) 12/15(80.0%) 0.36 0.03-5.11
Total 7/20 (35.0%) 10/16 (62.5%) 17/36(47.2%)
df=1, 2=6.28, df=1, 2=4.27, df=1, 2=11.08,
P=0.012 P=0.038 P=0.001
Age (year) <1 11/78 (14.1%) 1/27 (3.7%) 12/105(11.4%) 4.27 0.52-34.74
1-5 21/129 (16.3%) 72/280 (25.7%) 93/409(22.7%) 0.56 0.33-0.96
>5 4/34 (11.8%) 3/21 (14.3%) 7/45(15.6%) 0.80 0.16-3.99
Total 36/241 (14.9%) 76/328 (23.2%) 112/569(19.7%)
df=2, 2=0.49, df=2, 2=7.70, df=2, 2=2.32,
P=0.780 P=0.021 P=0.312
Sex Male 25/168 (14.9%) 59/232 (25.4%) 84/400(21.0%) 0.52 0.10-1.14
Female 11/73 (15.1%) 17/96 (17.7%) 28/169(16.6%) 0.46 0.22-1.14
Total 36/241 (14.9%) 76/328 (23.2%) 112/569(19.7%)
df=1, 2=0.001, df=1, 2=2.27, df=1, 2=1.42,
P=0.970 P=0.131 P=0.224
Rain (mm) Temp (oc), Relative Humidity (RH %)

Figure 2 Climatic data 2012 to 2013 for the study area.

DISCUSSION
In Thailand, anaplasmosis is normally diagnosed by a conventional microscopic examination
(ME) of staining blood smear with some limitations (Liu et al., 2005). ME can only detect when
parasitaemia reach more than 106 infected erythrocytes per ml during acute anaplamosis but it was
limited to identify subclinical animal or carriers (Gale et al., 1996). For increasing the specificity and
sensitivity of PCR, the molecular technique has been developed to identify A. marginale DNA in the
infected animals (Carelli et al., 2007).
Anaplasma major surface proteins (MSPs) are involved in interactions between vertebrate
and invertebrate hosts (de la Fuente et al., 2005a). Therefore, MSP4 is likely to evolve more rapidly
than other genes because they are subjected to selective pressures exerted by host immune
systems. MSP4 is an immunodominant outer membrane protein with orthologs in all Anaplasma spp.
examined so far (de la Fuente et al., 2005b) and has been used as potential primers to detect
Anaplasma spp. infection (Hornok et al., 2012). MSP4 is also found in all stages of Anaplasma spp. in
both animals and vectors (Kocan et al., 2007).
The transmission of A. marginale is indirectly influenced by climatic conditions, soil, and
cattle biotypes, which all modulate the capacity of the land in order to support vector population
(Guglielmone, 1995). The hematophagous diptera was also considered as the potential mechanical
vectors for bovine Anaplasmosis (Yeruham and Braverman, 1981). During flooding in 2011, there
was a completely change of the vector population caused by less favorable climatic conditions
affected their biological cycle. Currently, the study areas were characterized by previously flooding
conditions that affect an ecology of potential vector such as ticks and blood sucking flies. These
current results demonstrated that A. marginale infection in dry and rainy season is independent of this
parameter (Table 1), and is consistent with previous studies (Atif et al., 2013).
Nan is a mountainous province consisted of Nan River that merge with Yom river to be the
Chao Phraya River. Therefore, Nan had only flash flooding that might have less effect on vector of
A. marginale. In Nan, the prevalence of A. marginale is found at 2.9%(2/70) and 20%(23/115) in the
dry and rainy seasons respectively. Comparatively, Teerapat and Dissamam (1965) reported the
prevalence of A. marginale of cattle in Nan at 40.0%(16/40). For seasonal effect, the increased
prevalence of A. marginale in the rainy season is associated with an increasing in vector population
(Mohanta, et al., 2011). The prevalence and population density of ticks were abundant in the rainy
season due to optimal the humidity. Consequentially, the rainy season favors ticks and blood sucking
flies building up to the high prevalence and density, while the dry season with low humidity and
temperature may have an adverse effect on tick and flies population (Islam et al., 2006).
Nakhon Sawan is located as the junction between the North and the Central region of
Thailand. Four major rivers of the North are merged at this area so that there are routinely flooded in
this area. Therefore, ecological habitats are modified with positively or negatively influenced vector
population. In Nakhon Sawan, the overall prevalence of A. marginale infection was 29.8% (57/191)
compared to 1.6% reported by Chaichanapunpol et al. (1996). This was indicated that flooding in this
area might enhance the survival of vectors via their ecology. However, no association between
seasonal effect and A. marginale prevalence were found. The high prevalence in Nakhon Sawan is
probably due to climatic condition and the management factors such as many vector habitats and
population (Figure 2). During the flooding, the farmers migrate their cattle into higher ground to keep
them safe and dry and some cattle might carry ticks and tick-borne disease. Ticks can multiply
resulting in an increased vector population leading to increase tick-borne disease. During flooding,
there was a limited area of pasture led to increase cattle density per area (Perez et al., 1994) and this
also was subsequently affected by tick population.
Ayutthaya is characterized by low land so that this area is normally flooded during the rainy
season. Therefore, all animals and vectors have been adjusted to the changed environment. The
prevalence of A. marginale infections in Ayutthaya is 15.5% compared to previous reports 48.4% by
Phrikanahok et al., (2000), 58.0% by Sriwarothai et al., (2008), 15.6% by Yawongsa et al., (2010) and
9.9% by Saetiew et al., (2012). The favor for infection was found in the rainy season (22.3%, 23/103)
higher than in the dry season (7.8%, 7/90). Flooding condition created the favor environment for ticks
and other insect vector. The migration of animals might bring ticks and their pathogens to
contaminate refuge or shelter areas.
After 2011 flooding, there was a trend of increasing prevalence of A. marginale in cattle.
The distribution of anaplasmosis was expected to change, as a result of environment modification
and animal migration. Climatic change may have an impact upon the epidemiology of anaplasmosis,
by affecting the distribution of ticks. Flooding may enhance the increased number of arthropods
involved in the biological or mechanical transmission of A. marginale and arthropod populations
usually rebound rapidly after extreme weather conditions. Populations of ticks are constantly
changed, and adjusted to inhabit new geographical areas which may contribute to the emergence
some tick-borne disease.

ACKNOWLEDMENTS
This research is supported by the Center of Excellence on Agricultural Biotechnology
(PERDO/2555-01), Science and Technology Postgraduate Education and Research Development
Office, Commission on Higher Education, Ministry of Education.

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