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•      Confirmation
by Sequencing and phylogenetics.

•      Molecular
Identification of variant species of Babesia  in cattle in Muzaffargarh.

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Tick fever is a key
tick-borne disease caused by protozoan parasite belonging to the genus Babesia.
The prevalence is high in South Africa and East Asia. Currently, peripheral
blood smear examination via microscopy and serological techniques are in use to
diagnose Babesia in blood samples. Morphological similarities between Babesia
,Theileria, Anaplasma and Plasmodium species and low parasitaemia
in case of early and chronic infection impose limitation on traditional methods.
PCR and DNA Sequencing based method may overcome these difficulties and may
help in early and precise diagnosis of B.bovis and B.bigemina
species. Detection of species of B. bovis and B. bigemina will
serve the baseline for vaccine development of babesiosis and better management
of the therapy regime in Pakistan. Keeping in view the importance of babesiosis
the present study is designed to achieve the following objectives.


The first
descriptions of the polymerase chain reaction for confirmatory diagnosis were
reported in 1992 for B. bovis, B. bigemina (Figueroa et al. 2012).
Molecular techniques are highly effective and sensitive for the
detection of parasitic infestations, irrespective of the type of infection and
the presented sample (Quaresma et al. 2009). Amid the variegated techniques available, some methods stand
out for being applicable not only to diagnosis and differentiation of species,
but also to treatment surveiling, and thus have become a propitious tool to
help in clinical diagnosis. Not only being highly specific and sensitive,
molecular assays are potential alternative for direct microscopy also, although
the latter method is still considered the “gold standard” for
identifying parasites.

One can detect up to 1/10000 cells, requiring the analysis of one hundred
fields, the equivalent to 0.5?l of blood. Blood source should be paid special
attention; for species like B.bigemina, B.divergens, or B.gibsoni,
which do not adhere to the vascular endothelium peripheral blood is useful. The
diagnosis of species like B. bovis using this method is feasible if the
blood sample is taken directly from a blood capillary from the ear , compared
with peripheral blood taken from the jugular or caudal veins, since blood in
capillaries contains a higher percentage of infected erythrocytes for these
species. The presence of merozoites inside red blood cells is an indication of
infection but, trophozoites  appear in
different forms and size depending on the species, causing their detection
difficult .

  Parasites inside red
blood cells are observed under a microscope using a 100X objective and a drop
of oil immersion.  Merozoites (paired) of
B.bigemina, B. bovis , and B. divergens measure 2.5-3.5 ,
1.5-2, and 1.5-0.4?m in diameter respectively.

can be detected by conventional method which is cheaper but not sensitive and
specific (Garcia et al. 1993).

Pakistan there is no vaccine against Babesiosis, there are limited choices of
chemotherapy and few low-cost, reliable and fast detection methods. It is
necessary to know the detection and treatment in order to control Babesiosis
and develop a vaccine.

responsible tick for human babesiosis is Ixodes  ricinus (Homer et al.
2000). The predisposing
factors responsible for increase in tick borne infections among humans include
change in climate, low immunity, increased encounter between human and ticks, shift
in community centre and host vector species. Human Babesiosis is mainly
transmitted by ticks but blood infection is also a potential source. Human Babesiosis
may be asymptomatic or fatal. Manifestations vary between different individuals
depending upon the immunological status of the host, age and coinfection with
other pathogens.

first case of human Babesiosis was identified in 1957 in spleenectomized farmer. Up till now, more than 300 cases of human Babesiosis
have been reported but serological survey have shown that actual picture is
quite different (Kjemtrup et
al. 2000). Many species
of Babesia also infecting humans and are emerging as potential zoonosis among
these species infecting humans and causing human Babesiosis include B. divergens
(Qi et al.
2011), B. microti (Gray et al.
2008), B. duncani (Conrad et al.
2006), B. venatorum
(Lempereur et
al. 2011). No human
specific Babesia specie has been documented yet and humans are supposed to be
accidental host (Taboada et
al. 1991). Infectivity, pathogenicity
and virulence of Babesiosis vary among people (Yabsley et
al. 2013). Incubation
period of Babesia parasites ranges between 10 to 14 days and infection
can persist for year or whole life span of animal (Richier et
al. 2006).

of the newly released merozoites transform into non dividing, spherical gamonts
infect new erythrocytes. These gamonts containing cells are ingested by Ixodid
 ticks when feed on infected
vertebrate blood (Mackenstedt et al. 1994). Gamonts start asexual cycle within the tick gut and the
whole process initiate again.

The second stage is reffered as sporogony. At this stage,
kinetes invade other body organs such as salivary glands, fat bodies, nephrocytes
and ovaries within 2-3 days of tick attachment and initiate sporogony. During
sporogony kinetes start producing sporozoites asexually. Ovarian invasion may
lead to transovarial transmission which can transfer the infection to the next
generation. On the other hand, invasion of the salivary gland may lead to trans
stadial transmission (Telford III et al. 1993). The third stage is also asexual and is referred as
merogony. At this stage parasite invade the erythrocytes of the vertebrate host
via endocytosis and start multiplying into two or sometimes four merozoites (Kakoma et al. 1994). On erythrocytes rupture, the merozoites are released
and target uninfected erythrocytes subsequently multiply. This process continues
till the death of host or the elimination of the merozoites by host immune
system (Uilenberg. 2006).

 A meta analysis of Babesiosis in cattle indicates  overall prevalence as: Babesiosis  18.64% ,B.bovis  18.11% and  B.bigemina  10.03%. In buffaloe prevalence of B.bovis
is 8.63%. A brief account of previous ten years research on
the prevalence of Babesia is described below. In sahiwal region the blood
sample were analysed through microscope using blood smear technique Babesia species
were analysed and positive sample were 7.2%(Atif et al.
2012). In Bahawalpur
region the blood sample were analysed in crossbred cattle through PCR technique
Babesia species were analysed and positive sample were 11% for B.bovis
and 18% for B.bigemina(Chaudhary et al.2010). In Sargodha region the
blood sample were analysed in local cattle breed through thin blood smear
technique .Babesia species were analysed and positive sample were  6.57% for Babesia(Atif et al.2012). In
Bahawalnagar region the blood sample were analysed in Buffaloes and cattle  local breed through thin blood smear  and PCR technique. Babesia species were
analysed and positive sample were  2.7%
for Babesia species and 17.1% for B.bovis(Zulfiqar  et al.2012). In Charsada sawabi region the
blood sample were analysed in (local breed) in which B. bigemina were 11
% ( Ahmad In Peshawar region in the month of May-Nov 2011 Buffaloes
and cattle local breed were analyzed through Blood and PCR samples in which B.
Bigemina was 0.16% and B. bovis was 0.08%(Saad et al. 2015). In
karachi region in the month of Aprail- oct 2011 Buffaloes and local cattle breed
was analyzed through  PCR technique in
which B. Bigemina were 0.03 % and B. bovis was 0.08%(KAKAR. 2015). In Bannu Laki
Marwat( KPK)  region  local breed of cattle B. bigemina and B.
bovis were analyzed through PCR technique in which B. bigemina were
54.6 % and B. bovis was 20.66(Amir et al). Life cycle of Babesia parasite is indirect and complemented by two hosts (Olsen. 1986). Babesia reproduces asexually in tick host and
sexually in vertebrate host. The complete life cycle contain three stages. The
first stage is sexual, characterized by gamonts production (gametogony) which
fuse in tick guts and form gametes. These
gametes fuse to form motile zygote or kinetes. (Mehlhorn et al. 1985).

 Babesiosis is  cosmopolitan in nature  affecting many species of mammals with a major
impact on cattle (Jongejan et
al. 2004). Livestock and
dairy industry play pivotal role in the economy of Pakistan. Livestock sector
contributes 11.6% of national GDP which is approximately 58.55% of the
agriculture value added (Anonymous. 2017).

Texas fever or Babesiosis is
caused by protozoan of the genus Babesia which is transmitted by tick and this
malady is characterized by anemia (hemolytic) and fever (104-1050F),
with often hemoglobinuria and death (Ristic. 1981). The acute form
of Babesiosis is characterised by general findings such as pyrexia, weakness,
lymphadenopathy, depression and malaise.



The antigens of parasite in the tissues
of host can not be diagnosed directly by immunological method. Now a days, nucleic acid identification and
amplification is proved to be the most sensitive and reliable method for the
identification of Babesia. Intraerythrocytic presence
of parasite in host cells is indication of the acute stage of the disease.
However,  chronically infected animals ,
this method is not effective and other, more sophisticated techniques should be
exercised. PCR assay for detection  and its uniqueness of high sensitivity and
speci?city  for babesiosis have been proved (Oliveira-Sequeira et al. 2005).
PCR has been accepted 100% sensitive for Babesia parasite detection (Schaaschmidt
et al. 2006). Apposite gene marker is required for molecular detection. The
frequently used markers for Babesia diagnosis are 18S rRNA, Beta-tubulin
protein and Heat Shock Protein(HSP-70)(Caccio et al. 2000). Virulence, prognosis
and response to medicines in case of bovine babesiosis is  species related.

Babesiosis can be detected by microscopy which is considered to be
a gold standard but it lacks sensitivity(Bashiruddin et al. 1999).
Babesiosis can be detected via Immunoflorescent Assay Technique (IFAT) and
Enzyme linked Immunosorbent Assay (ELISA) in carrier animals but suffer  drawbacks of cross-reaction between B.bigemina
and B.bovis and cannot differentiate between past and present infection (Choopa. 2016). However,
presently many  molecular techniques have
been developed that can detect and differentiate various species of Babesia in
carrier and infected animals (Choopa. 2016).

point prevalence of Babesiosis in Buffalo is 36.05%, Cattle 9.97%, Sheep 13.92%,
Goat 10.99, Equine 50.56% and Camel is 8.33% respectively(Sahib et al.2015). The cattle tick Rhipicephalus
microplus is
a competent vector of B.  bovis, B.  bigemina,
and A. marginale, which
cause tick fever in Pakistan and the rest of the world(Karim et al. 2017). Hyalomma
species are known vectors of T.

and Tick borne diseases cause substantial economic losses in different
livestock species by reducing productivity and fertility, and sometimes causing
deaths. Tick borne diseases and tick cause an
estimated US$ 12.9 to 18.6 billion loss and an annual shortfall of nearly three
billion pieces of hide in cattle(Karim et al.
2017). The  economic loss of production globally by ticks
are about 13-19 billion per year (De Castro et al. 1993). Out of 12 billion cattle
over 500 million cattle are at potential risk of being infected by Babesiosis (Dantas-Torres et
al. 2012) . Recently, studies in Australia and India
have also estimated annual losses at $26 million and $499 million , respectively (Bhat et al. 2015).

Babesiosis is one of the
most important tick transmitted diseases worldwide. Babesiosis is
caused by the protozoan parasites. The Genus Babesia include more than one
hundred species, widespread in nature and infecting a large range of cultivated
and wild cattle (National Academies of Sciences et al.
2016). Babesiosis has worldwide
importance due to  distribution of large
range of host tick vectors. This ubiquitous parasite primarily infects the host
erythrocytes.  The second most important
haemoparasite of mammalians next to trypanosomes is Babesia(Barandika et al. 2016). B. bovis and B.
bigemina belong to the genus Babesia. B.bigemina is the most pervasive specie infecting cattle
in hot and mild-hot regions of the world amid a variety of species.  The livestock sector bear huge losses which
are attributed by cattle mortality, ill-thrift, loss of milk and meat
production, draft power, cost on control measures, and also through its impact
on international cattle trade in Pakistan (Bhat et al. 2015).

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