First evidence of Anaplasma phagocytophilum in cattle parasitism in Benin: an emerging

pathogen transmitted by ticks

ADEHAN Safiou Bienvenu1,2, KOUNONZO Maurice Landry 2, BADAROU Kadidjatoulaï Opeyemi 1, DAH
NOUVLESSOUNONMèdéhouékon Marius Durand2, KANDE Souleymane 3, GBAGUIDI Armel Michel2, BOKO
K. Cyrille2, MADDER Maxime4,5, FAROUGOU Souaïbou 2

1
Institut National des Recherches Agricoles du Bénin, Laboratoire des Recherches Zootechnique Vétérinaire et Halieutique
(LRZVH) – Santé Animale et Ecotoxicologie,Abomey-Calavi, Bénin.
2
Université d’Abomey-Calavi, Ecole Polytechnique d’Abomey-Calavi (EPAC), Unité de Recherches sur les Maladies
Animales Transmissibles (URMAT), Abomey-Calavi, Bénin.
3
Centre International de Recherche-Developpement sur l'Elevage en zone Subhumide (CIRDES) Bobo-Dioulasso Burkina-
Faso.
4
Prince Leopold Institute of Tropical Medicine, Vector Biology Unit,Nationalestraat 155, 2000 Antwerp, Belgium.
5
University of Pretoria, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, Onderstepoort, 0002,
South Africa.

Corresponding author: ADEHAN Safiou Bienvenu, 03BP3058 Jéricho Cotonou Bénin, Tél : +229 61825585,
adehankarim@yahoo.fr

Abstract
Anaplasma phagocytophilum, a bacteria transmitted by ticks, is responsible for granulocytic
anaplasmosis; an emerging zoonosis that has never been reported nor identified previouslyin
Benin and in the West African sub-region. This study was designed to investigate the
prevalence of the disease and evaluated mortalities recorded at Kpinnou farm after importing
Girolando cattle from Brazil in 2014 but also to assess the prevalence of the emerging disease
in Benin in the year 2014 period. A total of 1427 ticks were collected including 5 ml of blood
from each of one hundred cattle. Microscopic diagnosis of the revealed the presence
ofRhipicephalus (Boophilus) spp., Rhipicephalus microplus, Amblyomma variegatum,
Rhipicephalusspp and Hyalomma spp. Blood analysis results revealed the occurrence of 55%
positive cases as for A. phagocytophilum, 66% for Theileria, 54% for Babesia and 64% for
Trypanosoma. Taking in to account efficiency of methods; for

100% of A. phagocytophilumreported as positive by microscopy appear to be similarly

positive with molecular analysis. Three (3) false negatives were detected for Trypanosomaand
four (4) for Theileria and Babesia. PCR showed greater sensitivity and specificity even with
microscopy showing appreciable specificity. This study concludes that Anaplasma
phagocytophilum as the primary suspect in the massive deaths observed in cattle in the study
area,

Keywords: Anaplasma phagocytophilum; ticks; zoonosis; cattle; Benin

1
Introduction

Ticks transmit various pathogens (protozoa, bacteria, and viruses), which cause a wide range
of animal and human diseases. Vertebrate animals play an essential role in the life cycle of
tick species, while humans are accessory hosts (Holly et al., 2016).

Bacteria of the Anaplasmataceae family are Gram-negative bacteria (Lewis et al., 2009;
Rejmanek et al., 2012; Medlock et al., 2013) and are distributed worldwide (Woldehiwet,
2010). As a result of increasing climatic, environmental changes including increase in world
trade, more and more vector-borne diseases tend to be listed as emerging multi-host diseases
(WHO, 2016). These changes have resulted in metamorphosis and altered distribution of
vector and host species (Holly et al., 2016).

Granulocytic anaplasmosis, caused by Anaplasma phagocytophilum is a vector-borne, non-

contagious bacterial infection that infects domestic and wild animals and primarily targets
neutrophil granulocytes (Jones et al., 2008; Blaňarová et al., 2014; Rejmanek et al., 2012).
The disease is transmitted by various vectors such as ticks (which are the main ones), biting
insects (Stomoxes and Tabanidae) but also biological transmission or even iatrogenic (needle,
blood transfusion, etc.), they then speak of mechanical transmission. (Chevalier, 2012).

Granulocytic anaplasmosis, also known as pasture fever, Anaplasma phagocytophilum

ehrlichiosis or large bluegrass disease was first described in ruminants in 1932 by MacLeod in
sheep. The latter demonstrated in Scottish sheep a feverish syndrome transmitted by ticks and
caused by bacteria. The latter was then named Rickettsia phagocytophila then Cytoecetes
phagocytophila and finally Ehrlichia phagocytophila until 2001 (Renard, 2018).

Ticks are blood-sucking arthropods that parasitize almost all vertebrates around the world and
can occasionally bite humans. They transmit many disease agents some of which are common
to humans and animals. Ticks and mosquitoes are the main vectors of diseases of humans and
animals (René, 2013). According to Madder et al., 2014, ticks have become a major concern
of medical and veterinary research for the past 150 years, and not only because of the direct
effects on their hosts, such as injury to their sites attachment, loss of blood by spoliation and
paralysis caused by toxins present in their saliva, but also because they are efficient vectors of
a wide variety of microorganisms (viruses, bacteria, protozoa, helminths).

Bovine anaplasmosis caused by Anaplasma phagocytophilum is largely unknown,

underestimated and never diagnosed in Benin. It is also an emerging zoonosis whose
ignorance in human medicine is comparable to that of the veterinary community.

The present study was carried out to study the prevalence of Anaplasma phagocytophilum in
cattle reared on different farms in different regions of Benin by trying to identify the main
possible risk factors or germs associated with the infection.

1- Material and methods

2
1.1- Study areas

This study was carried out from July to November 2014 over the entire national territory of
Benin in cattle farms to look for the presence of Anaplasma phagocytophilum and other
possible hemoparasites in blood samples but also for the presence of ticks on cattle body.
Ticks were therefore taken from one hundred cattle distributed randomly in twenty herds
located in five rural townships (Athiémé, Kétou, Tchaourou, Bassila and Gogounou). The
selected townships are included in three agro-ecological areas of Benin (Areas II, V and VIII)
giving a rate of three selected townships out of eight, they are recognized for holding large
cattle herds with heavy tick infestation in livestock (Adehan et al., 2016); Kpinnou livestock
state farm (FEK) and .Okpara livestock state farm (FEO), were then included in the sample
sites.

In each herd, five cattle aged four to five years visibly infested with ticks, including one male
and four females were selected. The sex, the presence of ticks and the breeding system
practiced were noted. The geographic coordinates of sites were also taken using a Garmin 3.6
brand GPS. Kpinnou

Benin is a country with three different climatic zones. The South is characterized by four
seasons: a mainrainy season (April to July), a maindry season (January to March), a
shorterdry season (November to December) and a shorterrainy season (August to October).
The Northern part has two seasons: a rainy season from June to October and a dry season
from November to May. It should be noted that during the dry seasons, a dry wind called
Harmattan blows from the interior of the continent towards the ocean. Concerning the
vegetation, there is a woody to grassy trend from south to north; corresponding to a variation
from anthropic dry forest relic to anthropic savannah. Widlife is diversified, taking in to
account small and big herbivorous and also carnivorous mammals; all include in the two
national parks. Due to transhumance, herds go through the country seasonally contributing to
microbial and parasitic spread.

1.2- Tick collection and blood sampling

On the body of each selected animal, all the visible ticks were collected and stored in 100 ml
tubes containing 70% alcohol. Blood was drawn from the jugular vein in Venoject tubes
containing Ethylene-Diamine-Tetra-Acetic (EDTA) and blood smears were taken (OIE,
2012). Next, identified wattman No. 3 filter papers were soaked with whole blood from each
animal and dried away from flies and other insects, then separated by No. 4 filter papers.
These filter paper samples were only taken from the four herds at Kpinnou's farm and were
then sent to the Prince Léopold Institute of Tropical Medicine (IMT) in Antwerp in Belgium
for molecular identification.

1.2.1- Tick identification

After collection, ticks were sent to the Transmissible Diseases Research Unit (URMAT) and
identified microscopically usingthe identification key from Walker et al., 2003. A first

3
stepenabled us to identify ticks down to genus level using a ×60 magnification stereoscope.
The second stepwas focused only on ticks of the genus Rhipicephalus (Boophilus) to
determine its different species under an electric microscope (Olympus) at ×100 magnification.

1.2.2- Identification of hemoparasites

The smears produced were fixed with absolute methanol and then stained with a 10% May-
Grunwald Giemsa (MGG) solution. They were then observed under an Olympus optical
microscope at the objective 100 in order to identify the various hemoparasites present.

o Trypanosomes were identified according to morphological criteria which indicate the

size and position of the kinetoplast, the shape of the posterior end, the presence or not
of a free flagellum, the presence or not of an undulating membrane and the size (OIE,
2012).

o Babesia bigemina (Smith and Kilborne, 1892) is identified by its intraerythrocytic

forms which generally appear as pears measuring 4 - 5 x 2 µm. We sometimes observe round
shapes, 2 - 3 µm in diameter, and also irregular shapes. Pear shapes come in pairs (twin
shapes) forming an acute angle to each other.

o Babesia bovis (Starvovici, 1893) is small in size. Intra-erythrocytic forms are pear-
shaped, ring-shaped or irregularly shaped. The vacuolated annular forms (signet-ring) are
frequent. It measures 2.4 x 1.5 µm and has a central location. Twin forms form an obtuse
angle.

o Theileria mutans (Bettencourt, França and Borges, 1907) is widespread in Africa. It

causes benign theileriosis, "Turning sickness". The parasitological diagnosis is based on the
demonstration of schizonts ("Koch's blue bodies") in lymph node punctures and of piroplasms
in red blood cells. These piroplasms appear as multiform parasites represented by numerous
bacillary elements, comma, and a few rare oval shapes with masses of purplish red chromatin.
Their dimensions vary between 1.25 and 1.50 µm long axis on 0.5 and 0.75 µm minor axis.

o Anaplasma phagocytophillum (Theiler, 1910) is located inside leukocytes, mainly

neutrophils and possibly monocytes. In these white blood cells, the bacteria can either be
found as an elementary body isolated in the cytoplasm, or as a cytoplasmic cluster of
rickettsiae, also called a "morula". Because of the very low parasitaemia, it requires a long
and detailed analysis of the smear under the microscope.

1.3- Statistical analyses

The data were encoded in Excel software of WINDOW (version 2007). The statistical
processing was mainly carried out in R 3.3.1 software (R Core Team 2016, https://www.R-
project.org/). The analyses take into account, among other things, the achievement of:

o Simple correspondence factor analysis to describe associations between breeds or

genetic types and agro-ecological zones (or livestock systems) at the sample level;

4
 o Simple correspondence factor analysis to describe associations between different
parasites and agro-ecological zones (or farming systems) at the sample level;

o Calculus of prevalence rate of parasite using Bernouilli probapility estimation

approche

o A modeling aiming to assess the explanatory power of the prevalence rates of

Anaplasma phagocytophilum from the potential explanatory variables that are “agro-
ecological zones (or breeding systems)”, “Cattle breeds”, “Sex”, “Prevalence of Babesia ”, “
Prevalence of Theileria ”, and“ Prevalence of Trypanosoma ”.

o An analysis of proportion data according to generalized linear models was then

carried out using the GLM function (binomial family), starting from the saturated model. The
model was simplified using the step, update and anova (for deviance analysis) functions of R.
The model to be retained is the one for which the estimated parameters are significant and
which has the lowest values of the AIC.

2- RESULTS

2.1- Relative importance of the numbers of different sexes in the sample

The distribution of the sexes in the sample is directly linked tothe sampling method; the
sampling sex ratio being 1 male to 4 females. The analysis and interpretation of the influence
and the relative importance of the sex variable in the study will necessarily take into account
the link resulting from the sex ratio, in order to limit the biases likely to be generated by
making inferenceon the cattle populations concerned by the study in the five
townshipssampled (Figure 1).

2.2- The overall mapping of the breeds in the sample

Five breeds of cattle or genetic types were identified in the sample. These are the Borgou,
Girolando, Goudali, White Fulani breeds and the crossbreedgenetic type taking into account
subjects from the crosses Girolando x Azawack, Gir x Azawack, Girolando x Borgou and Gir
x Zebu peulh. The importance of each breed in the sample reveals a heterogeneity of their
distribution in the different sampled sites (Table I).

The Athiémé and Tchaourou sites have the particularity of each presenting an exclusive breed
(or genetic type), as they are State farms engaged in genetic improvement programs based on
specific breeds.

From the simple correspondence factor analysis of the sample data, it appears that the first,
the second and the third axes group together almost all of the information they contain. That is
41% of the information for the first axis, 41% also for the second axis and 17.81% for the
third axis. The projection of the variables in the factorial plane confirms a particular
association between the sampled sites and the cattle breeds (figure 2).

5
The Goudali and Borgou breeds are respectively associated with the communes of Bassila and
Gogounou while the Girolando race and the crossbreedsare respectively associated with the
communes of Athiémé and Tchaourou.

2.3- Prevalence rates of hemoparasites in Benin

2.3.1- Overall mapping of parasite prevalence in sampled cattle

All the sampled sites show cattle infected by the four different parasites (Table II). Simple
correspondence factor analysis yielded 80.29%, respectively; 16.29% and 3.42% of the
information for the first three factor axes. The projection of the variables in the factorial plane
highlights a specific distribution of the parasites according to the agro-ecological zones
(figure 3).

Athiémé district clearly appears to be a hotbed of Anaplasma while those of Gogounou and
Kétou are to some extent associated with Theileria and Trypanosoma and that of Tchaourou
should likewise be to some extent associated with Babesia. The agro-ecological sampled sites
are therefore differently infested by the four genera of parasites that are Anaplasma, Theileria,
Babesia and Trypanosoma.

2.3.2- Prevalence of Anaplasma phagocytophilum

The prevalence rate of Anaplasma phagocytophilum observed in the entire sample is 55%.
Nevertheless, variations in the prevalence rate seem to appear between the cattle sampled
according to the site (figure 4).

The prevalence rate of A. phagocytophilum in the sample from Kpinnou's farm is 100%; this
prevalence rate is significantly higher than that of all other samples. The sample from the
Okpara State Farm comes next with a prevalence rate of 65% followed at an intermediate
level by the sample of Bassila district (40%) and finally those from Gogounou and Kétou
district (with identical prevalence rates equal to 35%).

Deviance analysis leads to estimation of prevalence rates of Anaplasmosis at the levels of

each herd and agroecological area.

There is a link between the prevalence of Anaplasma phagocytophilum and the type of herd or
the agro-ecological area considered (p-value = 1,187.10-6). The herds at Kpinnou State Farm
are by far the most infested with Anaplasma phagocytophilum; they are followed by those
from Okpara State Farm; then by those of agro-ecological areas II and V including
respectively the districtof Gogounou on one hand and those of Bassila and Kétou on the other
hand. These towns have the lowest levels of Anaplasma phagocytophilum infestation.

In general, for all the concerned agro-ecological areas and substantially for the wholecountry,
the prevalence rates or the risks of an infestation by Anaplasma phagocytophilum are between
54,875% and 74,375% at an uncertainty rate of 0, 00125. The estimate of the prevalence rates
or risks of infestation by agro-ecological areas or by herd is given in Table III.

6
Furthermore, with regard to the analysis according to the variable "Sex", the prevalence rates
of Anaplasma phagocytophilum in the samples are 65% for male cattle and 52,5% for female
cattle. We can deduce that at the level of the herds of the State Farms of Kpinnou or agro-
ecological areas II and V including respectively the districtof Gogounou on one hand and
those of Bassila and Kétou on the other hand, the rate of Prevalence of Anaplasma
phagocytophilum does not depend on the sex of the bovine (X-squared = 0,56818, df = 1, p-
value = 0,451).

2.3.3- Prevalence of theileriosis in the different agro-ecological areas

The prevalence rate of Theileria mutans observed in the entire sample is 37%. But variations
in the prevalence rate seem to appear between the sampled cattle herds (figure 5).

Sampled Cattle in Tchaourou district have the highest prevalence rate for theileriosis (45%).
At an intermediate level of prevalence, come those of the of Athiémé, Gogounou and Kétou
(with respective prevalence rates of 40%, 40% and 35%), and finally at a lower level of
prevalence those of Bassila district (25%).

There is therefore no evidence of a dependence between the prevalence of theileriosis and the
different herds or agro-ecological zones (P-value = 0.7296). Thus, the prevalence rates of T.
mutans in the herds of the two state farms or in those of each of agro-ecological areas II and V
including respectively the commune of Gogounou on one hand and those of Bassila and
Kétou in the other hand cannot be considered as different (p-value = 0.7296). The prevalence
rate of T. mutans in the agro-ecological zones including each of the sampled sites, as well as
the average prevalence rate of theileriosis in Benin should therefore be estimated at 37%.
Therefore, the risks of infestation (or prevalence rates) for bovine theileriosis in Benin are
between 27.625% and 47.125% with an uncertainty level of 0.00125.

The analysis according to the variable "Sex" givesthe prevalence rates of Theileria mutans in
the samples as20% for male and 41.25% for female. We can deduce that at the level of the
herds of Kpinnou and Okpara State Farms or agro-ecological areas II and V including
respectively the townshipof Gogounou on one hand and those of Bassila and Kétou on the
other, the prevalence rate of Theileria mutans does not depend on the sex of the bovine (χ 2- =
2,2549, df = 1, p-value = 0.1332).

2.3.4- Prevalence of parasites of genus Babesia in the different agro-ecological areas

The prevalence rate of parasites of genus Babesia in all samples is 54%. Babesiosis
infestation levels in sampled cattle vary depending on the agro-ecological areas (figure 6).

Kpinnou farm has the highest prevalence rate of samples (75%) while the townships of
Bassila and Gogounou have the lowest prevalence rates (40%). Those of Tchaourou and
Kétou show intermediate prevalence rates of 65% and 50% respectively. The prevalence rates
of these parasites in the different herds or agro-ecological areas are almost equal (p-value =
0.09). At the traditional 5% threshold, there is no evidence of a statistical difference between
the prevalence rates in herds atstate farms level or between those in agro-ecological areas II
and V; but if we consider a 10%risk of error (1 in 10 chances of being wrong), the prevalence

7
rates in herds atstate farms level or in agro-ecological areas could be considered to be
different. We deduce that the average prevalence rate of babesiosis in cattle herds in Benin is
54% for a5% risk error.

Two species of Babesia were identified to beinfesting animals These are B. bigemina and B.
bovis, represented by respective prevalences of 21% and 33% at the whole sample level(all
agro-ecological areas combined). Thus, within infested animals, there is statistically no
evidence of a statistical difference between the prevalence rates of the two different Babesia
species (χ2- = 2,6667, df = 1, p-value = 0,1025) they are therefore fairly represented in the
population of infested cattle, there is just as much chance that an animal infested by a parasite
of the genus Babesia is infested with both B. bigemina and B. bovis.

On the whole, the average prevalence rate of babesiosis in agro-ecological areas including
each of the sampled sites in Benin is estimated at 54% with a confidence interval between
43,625% and 64,125% at 0.00125level of uncertainty. The risk of infection with babesiosis is
therefore relatively high in Benin.

Analysis on the basis of"Sex" gives prevalence rates of Babesia in the samples as65% for
male cattle and 51.25% for female cattle. We can deduce that at the level of Kpinnou and
Okpara State Farms or agro-ecological areas II and V including respectively Gogounou on
one hand and Bassila and Kétou on the other hand, the prevalence parasiterate of genus
Babesia in herds does not depend on the sex of bovine (X-squared = 0.72715, df = 1, p-value
= 0.3938).

2.3.5- Prevalence of parasites of the genus Trypanosoma in the different agro-ecological

areas

The prevalence rate of genus Trypanosoma in the entire sample is 64%. The levels of
infection in sampled cattle with trypanosomosis depends on the agro-ecological areas (figure
7).

The cattle sampled from Bassila and Gogounou townships are the least infested (60% and
55% respectively); those from agro-ecological areas including Kétou township and those from
Okpara State Farm have intermediate prevalence rates (65% for each of these sites). Kpinnou
State Farm has the highest prevalence rates of all the sites sampled (75%). However there isno
evidence of a dependence between the prevalence rates of trypanosomoses inthe different
herds and agro-ecological areas (p-value = 0.745). Thus, the prevalence of animal
trypanosomoses does not depend on the state farm or the agro-ecological area considered. We
deduce that the prevalence rate of animal trypanosomoses in Benin is 64%.

With regard to the risk levels specific to each species, T. congolensehas a prevalence rate of
21%, while T. vivax and T. theileri have respective prevalence rates of 27% and 16%. Based
on infection in affected animals, there is no statistical difference between the prevalence rates
of the three different Trypanosoma species (χ2- = 2.8438, df = 2, p-value = 0.2413). There is
just as much chance that an animal infected with a parasite of genus Trypanosoma will be also
be infected with T. vivax, T. congolense or T. theileri as well.

8
On the whole, the average prevalence rate of trypanosomosis in agro-ecological areas
including each of the sampled sites or substantially in Benin is estimated at 64% with a
confidence interval between 53.875% and 73..375% at 0,00125 of uncertainty. The risks of
trypanosome infestation are therefore relatively high in Benin.

The analysis on the basis of variable "Sex"gives the prevalence rates of Trypanosoma in the
samples as 60% for male cattle and 65% for female cattle. We can deduce that at the herdlevel
, Kpinnou and Okpara State Farms or agro-ecological areas II and V including respectively
the township of Gogounou on one hand and those of Bassila and Kétou on the other, the
prevalence rate of genus Trypanosoma does not depend on the sex of the bovine (X-squared =
0.024414, df = 1, p-value = 0.8758).

2.4- Factors Influencing the prevalence rate of Anaplasma phagocytophilum

The saturated model taking in to account agro-ecological areas, breeds, sex of the animals as
well as the presence or absence of the parasites of genusTheileria, Babesia and Trypanosoma
does not make it possible to identify factors likely to explain the prevalence of Anaplasma
within the cattle throughout the study area. It is therefore not statistically possible to link the
prevalence of Anaplasma to agro-ecological factors (or the production system), nor to race or
sex. However, the model explaining the prevalence of Anaplasma bythe presence or absence
of parasites fromgenusTheileria, Babesia and Trypanosoma is significant (Table IV).

It therefore appears that the prevalence of Anaplasma is as high as the bovine is infested with
trypanosomes on one hand and when on the other hand this prevalence is as high as the
bovine is infested of Theileria but not infested of Babesia. Taken together, these results reveal
that Anaplasma parasitism is strongly supported by a community of associatedparasites; it
underlines the danger of the situation since trypanosomiasis is endemic in most agro-
ecological areas in Benin.

2.5- Agreement and inconsistency of results according to the diagnostic method

The proportion of discordant and concordant results for the diagnosis of parasites by
microscopy and by PCR as well as the estimates of the minimum chances of accurately
identifying the species of parasite by microscopy in the event of a positive test are
summarized in the tables below :

Table : Number of coherant versus opposite microscopy and PCR test’s results (Number of
cases out of 20)

Anaplasma Theileria Babesia Trypanosome

Positive 20 8 15 15
concordant
results
Negative 0 12 5 5
concordant
results

9
3- DISCUSSION

Various bacterial diseases are caused by bacteria and transmitted by vectors. The latter are
hematophagous arthropods which ensure active, and therefore biological, transmission of an
infectious agent from one vertebrate to another, which in our study are mainly ticks. These
diseases can exclusively affect humans or animals, but it is not uncommon for them to be
zoonoses or anthropozoonoses.

In many countries, including Benin, the livestock sector faces a major constraint related to
animal health. Bacteria of the genus Anaplasma are among the pathogensthat are of great
public health interest. Their phylogenic affiliations have evolved a lot and are still evolving.
They parasitize the blood cells of many animal species including humans. Their ability to
cross species barrier complicate their study and often delay their identification. Anaplasma
phagocytophilum (Ehrlichia phagocytophila) is one of the family members with mant
unidentified members. The disease cycle is trixene, made up of its definitive host, reservoir
and vector, all of which can be the source of the infecting parasite (Chevalier, 2002). In
addition, the epidemiology of vector-borne diseases depends on: reservoirs, susceptible
populations, vectors, infectious agents, but also on human activities, the environment and
climatic conditions. Because this bacterium is transmitted by ticks, it is all in one biotope
disease and seasonal.

Today we see an expansion of vector-borne diseases resulting from the intensification and
globalization of trade, climate change, and changes in interactions between humans and their
environment. These diseases are of real importance, both through the zoonotic role of some,
and through their economic impact on animal health (Zehnter, 2014). Ticks are considered to
transmit the greatest number of pathogens compared to other arthropod vectors (Baneth, 2014;
Beugnet and Marié, 2009; Heyman et al., 2010). Several of these pathogens are of both
veterinary and medical significance causing various diseases, including anaplasmosis,
babesiosis, Lyme borreliosis, ehrlichiosis and rickettsiosis (Baneth, 2014; Heyman et al.,
2010). Animal movements due to international trade associated with climate change and many
other factors are causing changes in the distribution of tick species and the disease agents they
transmit (Aydin and Bakirci, 2007). Regular studyof tick species and associated infectious
agents is therefore essential for epidemiological control. From numerous studies already
carried out, A. phagocytophilum is mainly transmitted by ticks of genus Ixodes(not identified
among those found during our study). Howeverother generaof Anaplasma vector ticks have
also been described. These are Dermacentor, Rhipicephalus and Amblyomma (Rymaszewska
and Grenda, 2008). Thus, I. ricinus, I. persulcatus, I. scapularis, Rh. Sanguineus, Rh. purse,
Rh. turanicus, A. americanum, D. variabilis, D. anderson, D. auratus, D. silvanum, Ha.
lagrangei, Ha. leporispalustris, Ha. longicomis, Ha. concinna, Ha. punctata, Ha.
megaspinosa, Hy. marginatum, Hy. asiaticum and Hy. detritum have been confirmed as
vectors for Anaplasma in various studies (Parola and Raoult, 2001; Goethert and Telford,
2003; Kim et al., 2003; Parola et al., 2003; de la Fuente et al., 2004; Stafford III, 2004;
Kawahara et al., 2006; Kim et al., 2006; Shpynov et al., 2006; Rymaszewska and Grenda,
2008; Yoshimoto et al., 2010; Jiang et al., 2011; Meng et al., 2012; Bonnet et al., 2013;
Palomar et al., 2015).

10
Examination of ticks collected in the present study allowed us to identify tick species such as
Amblyomma variegatum, Rhipicephalus microplus, Rhipicephalus spp., Hyalomma spp., R.
decoloratus and R. annulatus. Ixodes ricinus, the vector most implicated in Europe and
America in the transmission of A. phagocytophilum (Satta et al., 2011), was not identified in
any of the five sites of interest in this study.

Woldehiwet and Ristic (1993) have shown that the presence of granulocytic anaplasmosis
caused by A. phagocytophilum in areas where the I. ricinus tick has not been found.This
suggests the existence of other arthropod vectors, a thesis which corroborates our results. In
addition, other biting arthropods such as stomoxes and tabanids have been encountered.

3.1- Prevalence of different hemoparasites

Of the 100 blood samples analyzed, 55 tested positive for Anaplasma phagocytophilum; ie a
prevalence rate of 55%. Other parasites such as Theileria mutans, Babesia bigemina, Babesia
bovis, Trypanosoma vivax, Trypanosoma congolense and Trypanosoma theileri were also
encountered during analysis of blood samples with rates respectively 39%, 22% and 33%. ,
27%, 21% and 16%. A large proportion of animals collected during the study showgood
health trend, but some of them had arthritis and edema ofthe limbs. These findings
corroborate those of Ben Said andal. (2014); Guyot andal., (2011) and Stuen andal., (2013)
who reported that clinical signs associated with tick-borne fever and pasture fever included a
sudden onset of high fever, anorexia, dullness, arthritis, edema of the limbs and weight loss.

Alsoaccording to Ben Said et al. (2014) and Guyot et al. (2011), infection with A.
phagocytophilum can also induce an immune deficiency in ruminants leading to secondary
opportunistic infections such as those with Staphylococcus aureus, Pasteurella spp. and
Listeria monocytogenes (Carrade et al., 2009). This would explain the cases of co-infections
observed in Kpinnou State Farm animals.

The farms of Kpinnou and Okpara have the particularity of each sheltering only one breed (or
genetic type) of cattle. This is as a result of the farms being involved in genetics
selectionprograms based on specific breeds.

The prevalence rate due to Anaplasma phagocytophilum in the FEK sample is 100%
and is significantly higher than that seen in all other samples. It is first followed by that of the
Okpara Breeding Farm (FEO) which is 65%, then that of the Bassila sample which is 40%
and finally those of the Gogounou samples and of Ketou which are identical and equivalent to
35%. These results testify to the susceptibility of this animal species to infection by A.
phagocytophilum, although no particular clinical sign has been demonstrated. The lower
prevalence observed in both Gogounou and Kétou could be due to several factors such as
bioclimatic conditions, low intensity of tick infestation, infesting tick species and tick control
practices. Moreover, since these areas are cotton production areas, the use of large quantities
of insecticides by cotton growers could lead to reducetick populations.

Ben Said et al. (2014) carried out a first serological study on Anaplasma
phagocytophilumprevalence in dromedaries (Camelus dromedarius) in Tunisia. The results

11
obtained showed prevalence rates of 29,2% in 226 dromedaries tested; or 66 positive subjects.
This is close to the rates of 35% obtained with the samples from Gogounou and Kétou cattle
herds.

Prevalence of infection with A. phagocytophilum is 36.7% in traditional farms versus a

minimum of 82.5% in productive farms with high health monitoring and control system. This
difference in prevalence between the two types of farming shows that the type of activity
seems not to influence the rate of infection and that the health control of productive farms
remains insufficient and needs to be strengthened. These results corroborate those of Leblond
et al., (2005) as well as Hansen et al., (2010) concerning equine granulocytic anaplasmosis,
which showed that infection by A. phagocytophilumwas not correlated with type of breeding.

All parasite borne infections associated with Anaplasmosis are of high importance even if the
prevalence rates of each show no particular differences according to agro-ecological areas.In
the study area, the two species of Babesia seen show the same risk of infection just as the
three species of Trypanosomes. In the particular case of trypanosomosis, it has been reported
that T. vivax is transmitted by all species of tsetse flies and also by mechanical vectors such as
Stomoxysand Tabanids (Magalie, 2006).

The average prevalence rate of T. mutans at the level of agro-ecological areas including each
of the sampled sites, as well as the average prevalence rate of theileriosis in Benin calculated
from the data is 37%. Therefore, the infection risks (or average prevalence rates) of bovine
theileriosis in Benin are calculated from the data as includes between 27.625% and
47.25%with an uncertainty levelof 0.00125.

The average prevalence rates for babesiosis in the different herds or in the different agro-
ecological areas are almost equal (p-value = 0.09). At the traditional 5% threshold, there is no
evidence of a statistical difference between the prevalence rates in the herds of state farms or
in those of agro-ecological areas II and V, but if one fixes a risk of error of 10% (1 chance in
10 of being wrong), the prevalence rates in the herds of state farms or agro-ecological areas
could be considered as different (quasi dependence between the prevalence and the herd or
the agro-ecological area). We deduce that the average prevalence rate of babesiosis in cattle
herds in Benin is 54% on average.

The studies carried out have made it possible to identify two species of Babesia in the
infected animals; These are B. bigemina and B. bovis, represented by respective prevalences
of 21% and 33% at the level of the entire sample (all agro-ecological areas combined). Thus,
within infected animals, there is statistically no evidence of a statistical difference between
the prevalence rates of the two different species (X - squared = 2.6667, df = 1, p-value =
0.1025); they are therefore fairly represented in the population of infected cattle, there is just
as much chance that an animal infected by a parasite of the genus Babesia is infected with
both B. bigemina and B. bovis

In total, the average prevalence rate of babesiosis in agro-ecological zones including each of
the sampled sites or substantially in Benin is calculated as 54% with a confidence interval

12
between 43.625% and 64.125% at the level of uncertainty of 0.00125. The risks of infestation
with babesiosis are therefore relatively high in Benin.

This study revealed the presence of three species of trypanosomes namely T. congolense, T.
vivax and T. theileri in the study area with the predominance of T. vivax followed by T.
congolense and finally T. theileri. This predominance can be justified by the fact that T. vivax
is transmitted by all species of tsetse flies and also by mechanical vectors such as stomoxys
and tabanids (Magalie, 2006).

Regarding the risk levels specific to each species, T. congolenseintervenes prevalence rate is
calculated as 21%, while T. vivax and T. theileri have respective prevalence rates of 27% and
16%. Therefore, within infested animals, there is no evidence of a statistical difference
between the prevalence rates of the three different species of trypanosomes (X - squared =
2.8438, df = 2, p - value = 0.2413). There is just as much chance that an animal infested with
a parasite of the genus Trypanosoma will be infested with T. vivax, T. congolense or T.
theileri as well.

In total, the average prevalence rate of trypanosomosis in agro-ecological areas including each
of the sampled sites or substantially in Benin is estimated at 64% with a confidence interval
between 53.875% and 73.375% at the level of uncertainty of 0.00125. The risks of
trypanosomosis infestation are therefore relatively high in the agro-ecological areas concerned
in Benin.

3.2- Factors influencing prevalence

In contrast to the orther herds,all the sampled cattle from Kpinnou state farm tested positive
for Anaplasma phagocytophilum.Kpinnou State Farm herds being by far the most infested by
Anaplasma phagocytophilum, they are followed by the FEO herds; finally, the herds of agro-
ecological zones II and V including Gogounou township on one hand and Bassila and Kétou
townships on the other have the lowest levels of Anaplasma phagocytophilum infestation.
There is a link between the prevalence of Anaplasma phagocytophilum and the type of herd or
the agro-ecological area considered (p-value = 1.187.10 -6).This particular trend should
normally be linked with the pasture feeding approach and also the humidity and temperature
conditions that are on average 85-90% and 27-28 ° C, allcorresponding to the optimal
conditions for ticks. the case confirms that the transmission of A. phagocytophilum is really
linked to ticks and their presence. However, pasture feeding approach as temperature and
humidity conditions has not been proved to be

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statistically different from one sampled site to another; thus, the only factors of importance
are the type of pasture; opened or closed and also the origin of the animal breed; abroad or
local. Kpinnou and Okpara state farms use closed pasture their herds are relatively separate
from neighbouring farm herds wich is not the case for agroecological II and V sampled sites.
The latters are then more exposed to herds and infestation sources. Unfortunately
agroecological sampled sites are less infested than State farms. Consequently , animal breed

13
origin remain the most important factor; animal exchange and in this case Girolando breed
importation from Brazil to the state farms could have contribute to particular Anaplasma
genus introduction in Benin as Aydin and Bakirci (2007) found in their sutdy. Secondly the
closed breeding approach that make the state farms closed system with least anthropogenic
agricultural actions such as pesticide use could explain the expansion and the increaseof the
parasite genus in the state farms making them permanent infested area.

Regarding the common prevalence rate trend of the other parasites genus, Babesia, Theleria
and Trypanosoma, the fair explanation should be that they were present and well established
in the local parasite genus system in Benin long time ago.

.In regards to factor sex, it appears that none of the parasite genus infestation rate
showsdependency. Gender therefore does not have a significant effect on the infection
rate;there should be an identical susceptibility of both sexes to the parasite genus or its
vectors. These results confirm those of Acapovi-Yao et al., (2009) who demonstrated in their
research that the phenotype, the sex and the age class of the cattle did not influence the
prevalence of trypanosome infestations in Korhogo and Odiénné. . Moreover, analoguous
results concerning Anaplasma phagocytophilum have been found with horses by Leblond et
al., (2005) and Hansen et al., (2010).

. At the opposite, Ingabiré(2009) found higher prevalence rate in females than in males for
trypanosomas genus as Acapovi-Yao and al.(2009) in Koutouba. The only explanation given
for the different prevalence rates in sex is the gentle and impetuous behavior respectively for
female and male when facing vector attacks. The mitigated result related to sex, influencing
or not the prevalence rate should make implement rigorous experiemental design taking into
account sex, age, physiological status, breed and also bredding system all as factors.

3.3- Comparative efficacy of diagnostic methods by microscopy and PCR

The results of parasitic diagnosis by microscopy and PCR reveal a perfect agreement between
the diagnostic methods for Anaplasmosis (Table V). As regards Theileriosis, Babesiosis and
Trypanosomiasis, discrepancies are observed (Table V).

Indeed, the precision of microscopy results will only have perfectly matched that of the PCR
in the case of A. phagocytophilum at the Kpinnou State Farm (only samples diagnosed by
both microscopy and PCR), in the differentiation of the two species of Babesia (B. bigemina
and B. bovis) and the three species of Trypanosomes (T. theileri, T. congolense and T. vivax).
Thus, a number of samples diagnosed negative by microscopy for the rest of the parasites (in
addition to A. phagocytophilum) were found to be positive by PCR. This shows that
microscopy displays lower sensitivity than PCR; that is, there were false negatives with the
microscopy. However, their specificities are equivalent since there were no false positives

14
detected by PCR. The low level of sensitivity of the microscopic method could be induced by
a blood load of parasites low enough to be detected and therefore linked to the diagnostic
method. The diagnostic method by PCR appear therefore to be the most reliable.

Anaplasmosis microscopy seems to offer the greatest chance of identification of the parasite
species (minimum chance of 83,16% with an uncertainty of 0,00125) than those directed
towards other parasites; it could be to the single Anaplasma species of interest in the study.
Babesiosis and Trypanosomiasis microscopies come in second position (with respective
minimum chances of 78,18% each), as regardTheileriosis microscopy, it offers the lowest
chances of species identification (63.06%) although there is only one prevalent species of
Theileriosis against three for trypanosomiasis and two for babesiosis in the study.

In addition, the results for anaplasmosis showed 20 positive cases out of 20 diagnosed by
microscopy. This accuracy observed in the present study has shown the ability of microscopy
to be able to detect true positives, giving it a rather remarkable specificity. This could be
explained by the strong parasitaemia observed in the animals sampled (Hansmann et al 2017),

In the case of Theïleriosis, one observes a perfect agreement of the results in 16 cases (16/20)
that is to say 8 true positives and 8 true negatives, and a disagreement in 4 cases (4/20); the
concordant chances of the results of the tests for the detection of Theileriosis by microscopy
and PCR under the conditions of the present experiment vary between 19,1% and 63,8% with
an uncertainty of 0,00125 on each of the extreme values. The discordant results are all
negative by microscopy and positive by PCR; which suggests that we have a proportion of
4/12 cases of false negatives in the negative cases; if 100% reliability were assigned to the
PCR, the chances of obtaining false negatives in the detection of Theileria by microscopy
under the conditions of the present experiment would varyfrom 9.91% to 65.11% at
anuncertainty level of 0,000125. The sensitivity here being established at 0,4 for microscopy
and 0.,6 for PCR with a rather remarkable specificity for the two methods.

With regard to babesiosis, a concordance of results is observed in 16 cases (16/20), ie 15 true

positives and 1 true negative, with disagreement in 4 cases (4/20); the chances of agreement
of the results of the tests for detecting Babesiosis by microscopy and PCR under the
conditions of the present experiment vary between 19.1% and 63.8% with an uncertainty of 0.
00125. Regarding the cases of discrepancies, these are all cases of negative results by
microscopy and positive by PCR, which suggests that there are 4/5 cases of false negatives in
the negative cases; if 100% reliability were assigned to the PCR, the chances of obtaining
false negatives in the detection of Babesiosis by microscopy under the conditions of the
present experiment would varyfrom 28.36% to 99.48% at a level uncertainty of 0.000125. The
sensitivity in this case being set at 0.75 for microscopy and 0.95 for PCR, with a fairly high
specificity for both methods.

With regard to trypanosomiasis, a perfect agreement is observed in 17 cases (17/20), ie 15

true positives and 2 true negatives, and a disagreement in 3 cases (3/20); the chances of
concordance of the results of the tests for the detection of trypanosomiasis by microscopy and
PCR under the conditions of the present experiment vary between 62.11% and 96.78% with
an uncertainty of 0,00125. The discordant results are all negative by microscopy and positive

15
by PCR; there is therefore a proportion of 3/5 cases of false negatives in the negative cases. If
100% reliability were assigned to PCR, the chances of obtaining false negatives in detecting
trypanosomiasis by microscopy under the conditions of the present experiment would range
from 14.62% to 94.62% at a level uncertainty of 0.000125. The sensitivity is therefore 0.75
for microscopy and 0.9 for PCR. As for specificity, it is also important for both methods.

In the concordant positive cases, the microscopy allowed a good differentiation or

identification of the species. For each of the species, at the level of the positive cases of the
study, the diagnostic methods by microcopy and by PCR give identical results with regard to
the precise identification of the parasite species. The minimum chances of identifying the
precise species of parasite by microscopy vary depending on the parasite (Table VI). These
chances could be equated with the chances of identifying true positives (if the PCR result was
considered to be 100% reliable). But in the present study all the samples positive by
microscopy are also positive by PCR, and therefore true positives, which gives the two
methods an equally important specificity.

Conclusion

A. phagocytophilum, is known as a veterinary pathogen causing disease in ruminants for

several decades, its zoonotic potential is not yet well understood. The ability to cause disease
in humans with potentially serious consequences, the global distribution and the growing
attention to ticks and transmitted diseases have increased the interest and awareness of
veterinarians and other health workers about of this emerging pathogen.

The advent of significant advances in molecular biology in recent years has enabled us to
confirm part of the microscopic analysis of samples by PCR. The study of Anaplasma
phagocytophilum highlights several important elements among wich the effective fisrt
effective prevalence reported in Benin. Its pathogenic role, albeit moderate at the individual
level, can be significant at the herd level. Moreover, the lack of interest shown in it by
practitioners certainly masks a significant impact.

It emerges that the economic losses caused by Anaplasma phagocytophilum deserve a better
awareness of those involved in breeding. The implementation of laboratory tests which must
be simple, economical and reliable is essential. This is ideally the place that PCR should take
in the future to systematize the search for and the precise identification of the bacteria.
Finally, the zoonotic nature of Anaplasma phagocytophilum infection gives it additional
appeal, although it is difficult to predict its incidence in the coming years.

With a view to better taking into account the impact of diseases transmitted by ticks to
livestock, bovine anaplasmosis requires large-scale epidemiological studies in Benin given the
low number of research activities carried out on this disease which could be considered a
major animal or even human health problem. Both for public health reasons and for economic
reasons, it is advisable to fight against the disease of the pasterns. However, this control is
hampered by a lack of knowledge about the epidemiology of A. phagocytophilum and the
quality and duration of the immunity it confers in domestic ruminants.

16
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List of tables
Table I: Number of cattle sampled by site according to their breed or genetic type

Race Borgou Girolando Goudali White Métis

Site Fulani

Athiémé 0 20 0 0 0

Bassila 0 0 6 14 0

23
 Gogounou 15 0 0 05 0

Kétou 11 0 2 7 0

Tchaourou 0 0 0 0 20

Table II: Number of infested cattle in the different sites sampled (Number of heads for
20 cattle / site)

Site

Parasites Athiémé Bassila Gogounou Kétou Tchaourou

Anaplasma 20 8 7 7 13

Babesia 15 8 8 10 13

Theileria 8 5 8 7 9

Trypanosoma 15 12 11 13 13

Table III: Confidence interval of prevalence rates (risk of infection) of Anaplasmosis

Kpinnou farm Okpara farm Area II Area V

(Gogounou) (Bassila Kétou)

Extreme values [83.125 ;100[ [40.875 ; 84.625] [15.575 ; 59.125] [19.125 ;

of the risk of 63.875]

infestation (%)

Table IV: Estimated parameters of the explanatory model of the prevalence rate of
Anaplasma and associated probabilities

Coefficients Estimators Standard z value Pr(>|z|)

deviation

Intercept 0.09247 0.49804 0.186 0.85271

FACthelthel 0.49809 0.62698 0.794 0.42695

24
 FACbabesNbabes 1.18968 0.83867 1.419 0.15604

FACtryptryp 1.41735 0.61497 2.305 0.02118*

FACthelthel:FACbabesNbabes 2.5101 0.93414 2.687 0.00721**

FACbabesNbabes:FACtryptry 1.48385 0.94716 1.567 0.11720

Intercept, FACthelthel, FACbabesNbabes, FACtryptryp, FACthelthel:FACbabesNbabes

FACbabesNbabes:FACtryptryp

Finally, the minimum explanatory model retained is:

z ~ FACtryp + FACthel:FACbabeswhere z represents the prevalence of Anaplasma.

Table V: Proportions of discordant and concordant results for microscopy and PCR
diagnoses (Number of cases / 20)

Anaplasmosis Theileriosis Babesiosis Trypanosomiasis

Matched positive 20 8 15 15

cases

Matched negative 0 8 1 2

cases

Discordant cases 0 4 4 3

Table VI: Estimates of the minimum chances of accurately identifying the species of
parasites by microscopy in the event of a positive test

Anaplasmosis Theileriosis Babesiosis Trypanosomiasis

Minimum chances

(%) at an 83.16 63.06 78.18 78.18

uncertainty level of

25
 0.00125

List of figures

Figure 1 : Map representing the study sites

26
 CA factor map CA factor map

1.5
Tchaourou Gogounou
Bassila
0.5

Metis_Gir Borgou
ZebuPeuhl
Goudali Ketou
Goudali
Bassila

1.0
0.0

Dim 3 (17.82%)
Dim 2 (41.07%)

-0.5

ZebuPeuhl

0.5
-1.0

Tchaourou Athieme

0.0
Metis_Gir Girolando
-1.5

Ketou

-0.5
-2.0

Athieme
Girolando Gogounou

-1.0
Borgou
-2.5

-2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 -2.0 -1.5 -1.0 -0.5 0.0 0.5

Dim 1 (41.07%) Dim 1 (41.07%)

Fi

gure 2: Projection of breeds or genetic types of cattle and agroecological areas in a factorial plane

CA factor map
CA factor map
0.2

0.2

Bassila
0.1

0.1

Trypanosoma
Gogounou
Dim 2 (16.29%)

Dim 3 (3.43%)

Ketou
Anaplasma Bassila Theileria
Athieme Babesia
0.0

Trypanosoma
0.0

Anaplasma Athieme
Tchaourou
Ketou
Tchaourou Babesia
Gogounou
-0.1

-0.1

Theileria
-0.2
-0.2

-0.2 -0.1 0.0 0.1 0.2 -0.2 -0.1 0.0 0.1 0.2

Dim 1 (80.29%) Dim 1 (80.29%)

Figure 3 : Projection of the different kinds of parasites and agro-ecological areas in a factorial plane

27
 100

non infeste
infeste
80
Frequences (%)

60
40
20
0

Athieme Bassila Gogounou Ketou Tchaourou

Communes

Figure 4 : Prevalence of Anaplasma phagocytophilum according to agro-ecological areas

70

infeste
non infeste
60
Frequences(%)

50
40
30
20
10
0

Athieme Bassila Gogounou Ketou Tchaourou

Communes

Figure 5 : Prevalence of Theileria mutans according to agro-ecological areas

28
 non infeste
70

infeste
60
50
Frequences (%)

40
30
20
10
0

Athieme Bassila Gogounou Ketou Tchaourou

Communes

Figure 6 : Prevalence of parasites of the genus Babesia according to agro-ecological areas

70

infeste
non infeste
60
Frequences(%)

50
40
30
20
10
0

Athieme Bassila Gogounou Ketou Tchaourou

Communes

Figure 7 : Prevalence of parasites of the genus Trypanosoma according to agro-ecological areas

29