Our results indicate that pregnancy changed the clinical aspects of trypanosomiasis, since there was an exacerbation of the infection, manifested by high and persistent parasitemia followed by hyperthermia. Studies evaluating follicular degeneration in goats experimentally infected with T. vivax, using the same dose (1.25 × 105 trypomastigotes) and inoculum from the present study, showed high parasitemia and hyperthermia in the acute phase only, whereas in the chronic phase, parasitemia is either absent or of low intensity. An important feature is that none of the experimental animals died .
Mean values of hematocrit (PCV) showed the typical evolution of trypanosomiasis, with a progressive reduction in the values throughout the infection period. Evaluation of the degree of anemia by hematocrit determination has been frequently used in monitoring the evolution of the disease, since this hematological parameter is most frequently changed in animals experimentally or naturally infected by T. vivax[24–26]. The hematocrit showed a dramatic decrease in infected ewes from this study, reaching 8%. Low hematocrit is associated with more severe stages of trypanosomiasis and may have contributed to the fatal course of disease in Ewes 1 and 3; and to the reduced body weight of newborn lambs and aborted fetuses.
Pathological consequences of the infection in pregnant females appear to be directly related to the gestational period in which it occurs. In the present study, two ewes infected in the first third of pregnancy presented serious infection and died within 65 days of pregnancy (35 dpi). However, the mortality of the ewes infected in the final third of pregnancy did not occur. Two of those ewes aborted, and another delivered a live lamb; but the weak offspring died five days after birth. The negative effect of trypanosomiasis in pregnant females is probably a consequence of the low immunity in this period associated to hematological and biochemical alterations promoted by the hemoparasite, that also increases the metabolic needs of the pregnant female [27–29]. In addition, the ovine placenta starts secreting progesterone around day 55, which is sufficient to maintain pregnancy in most ewes when the corpus luteum is removed . Therefore, the damage to the placenta caused by the protozoa may promote the insufficient placental secretion of progesterone and the consequent interruption of pregnancy.
The evaluation of blood glucose concentration has been applied as an indicator of energy metabolic activity. Thereby, the metabolism of pregnant ewes is characterized by high glucose requirements . The effect of T. vivax on the energetic metabolism in goats promoted an increase of 25% in the energy requirements for maintenance. Hypoglycemia is a common finding in acute trypanosomiasis, and is attributed to energy expenditure caused by hyperthermia and blood glucose consumption by trypanosomes . In this study, hypoglycemia revealed to be an important biochemical change observed in the infected groups. Thus, the combination of increased energy demands in pregnancy associated with a negative energy balance promoted by the infection is incompatible with the maintenance of fetal development in pregnant ewes. In addition, fetal fructose is produced by the placenta from glucose, and comprises about 70-80% of fetal blood sugar. The values of fetal fructose are correlated with the maternal glucose levels, and the maintenance of high concentrations can be seen as an indicator of ideal placental function . Another important metabolic alteration in the infection is the increase in protein catabolism, which is evidenced by the significant decrease of serum total protein and increased urea . A similar trend was observed in this study, in which low serum levels of total protein and high levels of urea were observed in the infected ewes.
Another systemic alteration observed in infected pregnant ewes was the progressive loss of body condition, which was manifested by low body scores achieving the lower limit of 1.5 (Ewe 1 and 3). Whatever the cause, small ruminants with a body condition of 1.5 are nine times more prone to abort compared with those in good body condition . Other systemic manifestations observed in this group, and found in the reviewed literature as capable of promoting abortion, are hyperthermia and anemia [36, 37]. The reduction in serum progesterone is another cause of maternal origin that can trigger abortion. In this study, it was observed that the serum levels of progesterone of infected ewes were significantly lower than those observed in the control group. It is known that the decrease in progesterone may result in disruption of pregnancy and promotes the expulsion of the dead fetus. In female animals, hormonal disorders in trypanosomiasis caused by T. vivax occur due to degeneration of the hypothalamus, pituitary, and gonads; which results in disruption of hormonal secretion, and consequent diminishing plasma concentrations of those hormones, which are fundamental for the reproductive processes, including pregnancy . Another hypothesis for the abortions consists in the occurrence of maternal hypoglycemia and, consequently, fetal hypoglycemia. This event is followed by the successive events of an increase of corticosteroids, estrogen, and prostaglandin, causing luteolysis and abortion .
The demonstration of parasite DNA by PCR in the placenta, blood, and tissue from aborted fetuses and stillbirths suggest that the protozoan traverses the maternal bloodstream. Thus, the infection invades the pregnant uterus, causing placental damage, and spreads into the blood and fetal tissues. Our histopathological results showed pericarditis, hepatitis, and encephalitis in the fetuses, therefore classifying these abortions as of infectious origin .
The abortion or birth of weak lambs may also be explained by placental insufficiency. Reproductive failure due to chorionic epithelial damage may occur due to inadequate nutrition or fetal oxygenation. In such cases, the fetus suffers anoxia, releasing the adrenocorticotrophic hormone with the subsequent release of fetal cortisol, which stimulates the production of estrogen and prostaglandin F2α by the placenta. This event results in luteolysis, with a consequent decrease in progesterone . The presence of the DNA of T. vivax in the placenta associated with its damage suggests that the parasite has an important role in the pathogenesis of placental dysfunction and abortion.
The detection of T. vivax in the placenta, amniotic fluid, fetal blood, and tissues is an unprecedented event, and confirms the first diagnosis of the transplacental transmission through detection of the parasite DNA by PCR in ewes. Although little investigated, transplacental transmission was confirmed for the first time in 1972 by the finding of large numbers of trypanosomes in the blood four hours after the birth of lambs from experimentally inoculated ewes with T. vivax in the final trimester of gestation . Later, there was the confirmation of the transplacental transmission of T. vivax in cattle by the detection of high parasitemia in a calf born from an infected cow in the same period of pregnancy . In Venezuela, congenital transmission was also observed in a calf, in which parasitemia and anti-T. vivax antibodies were detected by indirect immunofluorescence . Recently, in Brazil, the probable transplacental transmission was recognized in four three-day-old calves with high parasitemia and born from chronically infected cows . These authors attribute the high prevalence of trypanosomiasis by T. vivax in Northeastern Brazil to the transplacental transmission, as the regional climate (hot and dry) does not favor the development of host insects during most of the year.
In naturally infected flocks and herds, the transplacental transmission of the parasite is not well known and is underestimated by farmers and practitioners. The published cases of placental transmission by T. vivax do not describe lesions and parasitism of the placenta, and show no morphological evidence of the infection in the fetus. In this study, the occurrence of transplacental transmission in ewes was based on the identification of pathological lesions, suggesting protozoan infection, and also by the detection of the parasite’s DNA in the placenta, amniotic fluid, blood, and tissues from aborted fetuses and recently dead neonates. The transplacental transmission of T. vivax is unquestionable, as well as its effects on fetuses and newborns, which may occur in the initial or final third of gestation. It is likely that this type of transmission contributes to the survival of the parasite and the spread of infection in herds, and is also associated with cases of abortion, premature births, low birth weight, and perinatal mortality. These facts are often reported during the outbreaks of T. vivax infection in the Brazilian semiarid region [15, 18, 20].
Our experiment confirms the importance of T. vivax as a causative agent of abortion and perinatal mortality, as previously demonstrated in the outbreaks of infection in cattle, ewes, and goats in the Brazilian semiarid region [14, 19, 20, 44]. Our data suggest that T. vivax is able to cause abortions or perinatal mortality in different ways. In fact, the reproductive disturbances caused by the parasite may be multifactorial, since maternal systemic effects and injury of the placenta and fetus were observed.