Impact of Babesia microti on the initiation and course of pregnancy in murine model of vertically transmitted infection

Genus Babesia groups tick-transmitted protozoa causing babesiosis, a malaria-like disease. Vertical transmission of Babesia spp. was reported in mammals, however, the exact timing and mechanisms involved in this mode of transmission are not currently known. In this experimental study we evaluated: 1) the reproductive success, and success of vertical transmission of Babesia microti in mice mated in acute and chronic phases of the infection and in pregnant mice infected during early and advanced pregnancy; 2) possible influence of the pregnancy on the course of parasite infection (parasitaemia) in females; and 3) pathological changes in females and their embryos induced by infection. Blood smears and PCR targeting the 550 bp 18S rRNA gene fragment were used for the detection of B. microti. Histopathological examination was performed on collected tissues. Successful development of pregnancy was recorded only in females in the chronic phase of infection. The success of vertical transmission of B. microti in this group was 63% (71/112). In females mated in the acute phase of infection or on the 4th day of pregnancy, no evidence for pregnancy development were observed. In the group infected on the 12th day of pregnancy, numerous complications including pregnancy loss and stillbirth were recorded. During the acute phase of infection, parasitaemia was lower in pregnant females in comparison to infected, non-pregnant control females. Acute B. microti infection prevents pregnancy initiation and development of pregnancy at a very early stage, and causes severe complication in BALB/c mice in the second and third trimester of pregnancy. Chronic B. microti infection has no negative impact on the initiation and development of pregnancy, but resulted with congenital infections. Further study is required to determine to what extent maternal antibabesial immune responses and potential placental accumulation of parasites contribute to compromised pregnancy in the murine model of congenital Babesia infection. Author summary The mouse is the most common mammalian model for studying human parasitic diseases, including malaria, toxoplasmosis, Chagas disease, and babesiosis. Babesiosis is an emerging intraerythrocytic infection caused by protozoal parasites, mostly Babesia microti. Our previous work in murine model proved that vertical transmission of Babesia microti, is a third way - after tick-bite and blood/organ transfusion - to acquire babesiosis. In this study we focused on investigating how the infection influences the course of pregnancy. We were interested in how variations in infection acquisition time and infection phase influence the reproductive success of mice and vertical transmission of parasites. We expected that the infection causes severe pathological changes in the organs of infected females and their offspring. Results obtained in this study have shown that vertical transmission of B. microti was only possible in chronically infected mice, in which health status and reproductive success were not compromised by the infection. Acute infection made successful reproduction impossible, however, the infection had no significant effect on the histopathological condition of tissues. We hope that these insights into B. microti vertical transmission will lead to the better understanding of congenital babesiosis.

* at the end of the experiment on an autopsy date; ** in 3 females 17 uterine scars were found, 127 suggesting embryos implantation, but no further development; NA-not applicable.

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Babesia microti strain 129 The female mice were infected with the B. microti King's 67 strain, which originated 130 from field voles in the Oxford area, United Kingdom [28]. The blood is passaged from 131 infected to naive mice by intraperitoneal injections [7,17]. Mice are infected with 5 × 10 6 132 infected red blood cells (iRBCs) in the volume of 0.2 ml. This method has been successfully 133 used in experimental studies on Babesia and other blood parasites worldwide [7,17,29,30]. Study design 143 The first set of experiments involved females infected with B. microti before mating.

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Females were assigned to two experimental groups: 145 In Group A, six females in the appropriate phase of the reproductive cycle were joined with 146 males on the 7 th day post infection with B. microti (acute phase). Group A were autopsied on the 12 th day of the expected pregnancy. As their weight did not 168 change since fertilisation, and USG examination was unable to detect any developing 169 embryos, females from this group were autopsied to check the development of pregnancy.

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Females from Group D were autopsied between the 14 th and 18 th day of pregnancy (2 females 176 on 14 th , 6 females on 16 th , 6 females on 18 th ), and 6 females on the 1 st day postpartum. were obtained with the mean litter size 6.8±1.0 (Tables 1-2). USG monitoring has shown no malformations in embryos or pregnancy termination in any of the females from this group. All 266 pups were in good health condition following delivery until the day of autopsy (    postpartum. From embryos, organs were isolated on the 12 th (n = 12 from Group B), 14 th (n = 390 8 from Group B and n = 12 from Group D), 16 th (n = 22 and n = 36, respectively), and 18 th (n 391 = 29 and n = 21, respectively) day of pregnancy.

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DNA of Babesia was found in all types of tested tissues (Table 3). Hearts, lungs, spleen, and 393 placenta presented the highest B. microti positive rate. Higher positive rates in tissues of the 394 liver, spleen, kidneys, and placentas was noted in offspring from Group D in comparison to 395 Group B (Table 3).

Histopathological changes in female BALB/c mice 397
The material for histopathological examination was collected from selected females in 398 experimental and control groups. A variety of histopathological changes (lesions) were found 399 in organs of females in experimental and control groups (Table 5). There was no significant  Blood parameters 420 We present the comparison of mean blood parameters for 6 females from each group (Fig 4).

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As females from Groups A and C presented no evidence of pregnancy, mean counts of RBC 422 and platelets from these groups were compared to the counts obtained from Group NPI (non-423 pregnant, uninfected females) (Fig 4ab). RBC count was the lowest in Group A, and slightly 424 higher in Group C. In both experimental groups, mean RBC counts were significantly lower 425 than in the Control Group NPI (t=-17.92, df=10, p<0.001; t= -17.36, df=10, p<0.001, 426 respectively). Platelet count was the lowest in Group C, and slightly higher in Group A. In 427 both groups, platelet counts were significantly lower in comparison to the Control Group NPI 428 (t= -3.69, df=10, p<0.05; t= -3.36, df=10, p<0.05, respectively).

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Mean blood counts of females in Groups B and D were compared to the counts from 430 Group UI (pregnant, uninfected females) (Fig 4cd). The mean RBC count (but not the platelet  showed minor increases of parasitaemia, but it was not recorded in this study [17].

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In protozoan infections, parasitaemia levels in pregnant women/females seems to be 506 an important factor contributing to occurrence of vertical transmission, e.g. congenital