Babesia Microti – Known and Unknown Protists

B. microti is known as the main etiological agent of human babesiosis and there are some case studies for that disease, highlighting the fact that this is an important “emerging tick-borne disease”. However a lot of information about this protist is unclear. The reactions in the liver are noticeable already after the fi rst three weeks of infection. This therefore provides the basis for discussion of the effect of B. microti contact with hepatocytes in vitro and in vivo. This issue is an essential objective of our study. The effect of B. microti on the liver as an organ and the individual hepatocytes is evident. This may be an indirect impact, through the various factors of metabolic pathways. Ultrastructural studies of breeding co-culture of hepatocytes and B. microti has shown, however, that piroplasms B. microti are adhering to the outer surface of the hepatocytes. This contact is associated with destructive changes in hepatocytes both necrotic and apoptotic. Research Article Babesia Microti – Known and Unknown Protists Marta Albertyńska1, Weronika Rupik2*, Mateusz Hermyt2, Hubert Okła3 and Krzysztof Piotr Jasik1* 1Department of Skin Structural Studies, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, Poland 2Department of Animal Histology and Embryology, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, Poland 3Institute of Materials Science, Faculty of Computer Science and Materials Science, University of Silesia in Katowice, Poland Dates: Received: 29 December, 2016; Accepted: 1 February, 2017; Published: 2 February, 2017 *Corresponding author: Weronika Rupik, Assistant Professor, Department of Animal Histology and Embryology, University of Silesia in Katowice, 9 Bankowa Street, 40-007, Katowice, Poland, E-mail: Krzysztof Jasik, Associate professor, Head of Department, Department of Skin Structural Studies, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, 3 Kasztanowa Street, 41-200, Sosnowiec, Poland, E-mail: https://www.peertechz.com


Introduction
Babesia microti is a species of the phylum Apicomplexa (Piroplasmida) and invades red blood cells of vertebrates. B. microti is known to be a cosmopolitan species, but the main areas of its occurrence are the United States and European countries such as: France, England, Ireland, Germany, Spain, Switzerland, Russia and Poland [1,2]. In the US, these piroplasm have been reported mainly in the northeastern and midwestern States [3,4].
The life cycle of B. microti is associated with the metagenesis and runs with two hosts: intermediate -vertebrate, and the defi nitive -the tick. Protozoan parasites reproduce asexually in the intermediate hosts, while sexual reproduction occurs in the defi nitive host [5][6][7].
The main intermediate hosts and reservoirs of these protists are rodents (Apodemus fl avicolii, Clethrionmys glaveolus, Sorex araneas). B. microti may be optionally prevalent human parasite.
Typical fi nal hosts and vectors of B. microti in Europe are Ixodes ricinus and I. trianguliceps [8], and in North America -I. scapularis [9]. Despite B. microti is known as the main etiological factor of the human babesiosis and there are the case reports pertaining to this disease which indicated the fact that it is "Emerging tick-borne disease" [10] there are still many inconsistencies and unsolved issues. This issues are associated with the taxonomy of this pathogen, its pathogenesis and the correlation between the pathogen and the host. Commonly used name of B. microti is often negated and these piroplasms alternatively are called Theileria microti [11]. This is explained by the fact that in the course of the asexual reproduction in the intermediate host, piroplasms invade not only erythrocytes but also the lymphocytes, which is contrary to members of genus Babesia [11].
However the genome of B. microti has been sequenced.
These analysis shows that this species does not belong to none of the established genera -Babesia and Theileria -but to a separate genus [12].
A typical life cycle of Babesia (Theileria) microti presented in the literature [13] includes merogony and forming the pre-gametocytes in intermediate hosts, and gamogony and sporogony in defi nitive hosts ( Figure 1). Babesiosis was fi rst described in 1888 by Victor Babeş, in cattle with symptoms of fever and haemoglobinuria. After 9 years, it was found that the etiological agent of this disease is transmitted by ticks. Human babesiosis was fi rst found in 1957 Citation: Albertyńska  In tropical and subtropical climate babesiosis occurs in Africa, Asia (China, Taiwan, and India) and Australia [16].

Pathogenesis of human babesiosis
Clinical signs of human babesiosis appear between 1 and 4 weeks from man's prick by a tick infected with B. microti or between 9 weeks and 6 months in the case of babesiosis after blood transfusion.
Sporozoites penetrating into the interior of the cell cause the physical, chemical and structural changes of cell membrane.
Inside the erythrocytes sporozoites are divided into 2-4 merozoites, and formed in this way merozoites leave the red blood cell, causing its damage and degradation.
The effects of merogony are hemolytic anemia, thrombocytopenia, increased number of reticulocytes and low hematocrit.
Physical and chemical changes of membranes of vascular endothelial cells, thrombocytes and red blood cells are associated with occlusion of the small blood vessels and the formation of blockages. In the course of babesiosis, the characteristic signs include splenomegaly, hepatomegaly and dysfunction of the liver, kidneys and central nervous system [6,16,17].
During the course of the disease activation of the complement system and production of pro-infl ammatory cytokines occurs, which results in emerging fl u-like symptoms. It also causes stimulatation of suppressor T cells (stimulation of primary) and B cells (stimulating secondary). Blood chemistry shows increase in liver enzymes, such as transaminases, alkaline phosphatase, lactate dehydrogenase and free bilirubin [17].
The clinical course babesiosis in humans may be asymptomatic, mild or severe. Statistically at 25% of adults and 50% of children, babesiosis occurs asymptomatic and sometimes it ends with a self-healing or goes into latent phase [18].
However, babesiosis can be deadly, especially for the elderly or immunocompromised [18,19].
B. microti as a representative of the Apicomplexa is usually compared with Theileria spp. and Plasmodium spp. The effect of these comparisons draws attention to the lack of tropism Babesia spp. to lymphocytes, in contrast to Theileria spp. and the inability to penetrate piroplasm of the genus Babesia to hepatocytes, in contrast to the representatives of the Plasmodium spp., which in the fi rst period of invasion penetrate to the liver cells. However, analysing of the etiology of babesiosis in rats draws attention to the fact that piroplasms induce signifi cant changes in hepatocytes [20]. The fi rst reactions in the liver are noticeable already after the fi rst three weeks of infection. This therefore provides the basis for discussion of the effect of B. microti contact with hepatocytes in vitro and in vivo. This issue is an essential objective of the study.

Material and Methods
The research was conducted in both in vivo and in vitro.

In vivo analyses (light microscopy observations)
The animals were earlier infected by reference strain culture of B. microti (ATCC 30221). Infection was caused by intraperitoneal (i.p.) injection of 0.5 mL protozoan culture in the six-week-old rats. The animals were housed in standardized conditions: temperature aprox. 20-22 o C, humidity -50-60% and lighting cycles -12 hours of light and 12 hours of darkness. The breeding was conducted in cages with pine sawdust which was autoclaved. The animals had unlimited access to water and fodder. Control material was livers collected from 2 healthy rats. During the experimental steps, 0.5 mL sodium chloride physiological solution for intravenous injection (Inj. Natrii Chlorati Isotonica Polpharma, 9 mg/mL; Polpharma, Starogard Gdanski, Poland) was injected intraperitoneal in the rats. Control rats were housed in same conditions as animals infected with B. microti.
Detection of parasitemia in blood smears was performed using commercially available Histology FISH Accessory Kit (see below).
Three week after inoculation, the animals were euthanized by inhalation anesthesia with isofl urane (Forane® -Baxter, Deerfi eld, IL, USA). During post-mortem examinations, liver samples were collected for histological analysis.
Detection of B. microti genetic material in liver specimens and blood smears was performed using commercially available

In vitro analyses (light and transmission electron microscopy observations)
In this study AML 12 line of normal hepatocytes (ATCC CRL-2254) was infected by reference strain culture of B. microti (ATCC 30221).

Results
The presence of B. microti DNA was demonstrated in blood cells using FISH method (Figure 2).
The spot signals of fl uorescence were also observed in the samples of infected rat's liver (Figure 3).  hepatocytes culture has shown the contact between the hepatocytes and piroplasms B. microti. The presence of piroplasms inside cultured cells is ambiguous and questionable. Above the free surface of hepatocytes monolayer there are many leftover membranes after erythrocytes from the inoculum. In some places, oval structures containing the cell nucleus with a diameter of approx. 1-3 μm, adhere to the outer membrane of cultured hepatocytes ( Figure 6). In the cross sections by a layer of infected hepatocytes there are visible also the places in which the layer is discontinuous and the cells in these places are destroyed. Images of the destruction of cells are heterogeneous, as they can be subtle and the small fragments of individual cells that have been disintegrated -apoptotic bodies -can be noticed (Figure 7). In others fi elds of observation there are large spaces with vacuolization of fragments of the cells. In the latter case, the degenerative changes are more extensive

Discussion
According to the literature Babesia microti penetrates directly into erythrocytes without the stages of initial proliferation at other organs [5,21].This feature is related to the specifi city of the impact of the membrane of red blood cells with a particular species of piroplasm [22,23].
The most common complications of severe babesiosis include acute respiratory failure, congestive heart failure, liver and renal failure and splenic rupture [24,25]. Our studies confi rm the previously described hepatic tissue changes in rats infected long-term with Babesia microti [20].   The intense apoptotic processes in hepatocytes cultured with B. microti we confi rmed with use of the TUNEL method, however, the mechanism of induction of this phenomenon requires further analysis.
The interesting thing is that the processes of apoptosis in hepatocytes are clearly hampered by Plasmodium spp. However at late stages, upon completion of schizogony, a novel form of host cell death is triggered. It displays some hallmarks of apoptosis, but also marked differences, such as the failure of the infected hepatocytes to activate caspases and to display phosphatidylserine on its surface of the host cell [32].
The infl uence of protozoan parasites on death of the host cells has been shown in many publications. It was noted that the impact can be differentiated both in terms of stage on which it occurs as well as the fi nal effect, i.e., whether the inhibition or stimulation of the cell death process takes place [33,34]. However it is characteristic, that the modulation of the cell death process involves mainly cells that are engaged in the life cycle of the given species [34].
Thus remains unanswered question, what is the meaning of hepatocytes for the Babesia microti development.

Summary
Babesia microti is an intraerythrocytic parasite, however, the impact of this species on the host organism is multilateral.
Although it is not clearly demonstrated the ability to infect by B. microti other cells than erythrocytes and lymphocytes, possibly, however, the impact of these piroplasm in different organs is very strong and diverse.
The effect of B. microti on the liver as an organ and the individual hepatocytes is evident. This may be an indirect impact, through the various factors of metabolic pathways.
Ultrastructural studies of breeding co-culture of hepatocytes and B. microti has shown, however, that piroplasms B. microti are adhere to the outer surface of the hepatocytes. This contact is associated with destructive changes in hepatocytes both necrotic and apoptotic.