To the best of our knowledge, this is the first report of molecular identification of the B. microti-like piroplasm both in dogs from Portugal and in dogs suspected of clinical piroplasmosis outside of Spain.
The designation B. microti-like piroplasm has been used in the present description, as genetic analyses revealed that T. annae is more closely related to B. microti, a rodent piroplasm that causes babesiosis in humans, than to other Theileria spp. [8, 39, 40]. Furthermore, no evidence has been presented for pre-erythrocytic stages of this infection in leukocytes, which is a characteristic of the theilerial life cycle . The name "Spanish isolate" also used to describe this pathogen  comes from the first description of this causative agent, which was made from a dog with piroplasmosis (originated from a Babesia-free area in Germany) that had travelled to northeastern Spain .
The B. microti-like piroplasm can cause severe disease and mortality in dogs and is endemic in Galicia, northwestern Spain [31, 39], which northerly borders the area where the three dogs from the present report lived in Portugal. According to the published information, the B. microti-like piroplasm had been molecularly sequenced from dogs with clinical babesiosis only from Spain and mostly from Galicia [31, 39, 42]. Besides, there are reports of infection in a few clinically healthy dogs: one from Tarragona, northeastern Spain , another one from Croatia  and an additional one from Mississipi . DNA of the B. microti-like piroplasm has also been detected in foxes from central and northern Spain [44–46], eastern Canada, North Carolina in the USA , Croatia ; in cats from Portugal  and Italy ; in a donkey from northern Spain ; in roe deer from Italy ; in feral raccoons from Japan ; in Ixodes ricinus and Rhipicephalus sanguineus ticks from Italy [54, 55]; and in I. ricinus and Ixodes hexagonus from northern Spain . To date, this piroplasm species has not been reported in Africa or Australia.
Ixodes hexagonus is the main candidate vector of the B. microti-like piroplasm in Galicia . This tick species has also been found in northern Portugal [57, 58], but the endemic nature of the small piroplasm in this area cannot be ascertained. In the present report, the bitch and the male dog were most probably infected during the 10-day period they spend in Vigo (Galicia), although the possibility that the infection originated in Portugal should not be excluded. Both dogs became clinically suspected two months after returning from Vigo. This fact suggests a longer incubation period for the disease caused by the B. microti-like piroplasm in comparison with the 4 to 21 days described for other canine babesial infections . However, the pup never left Portugal and could have been infected by vertical transmission. The transplacental transmission of B. gibsoni has been experimentally demonstrated in a bitch that delivered a litter of one stillborn and four live pups . These four pups died from congenital babesiosis between 14 and 39 days post-birth.
In horses, once an animal is infected with T. equi it remains a lifelong carrier, since anti-theilerial drugs do not completely eliminate the parasite . Infected mares can transmit T. equi piroplasms across the placenta and this might result in abortion or neonatal piroplasmosis. Colostral antibodies to T. equi may suppress parasitaemia in newborn foals thereby reducing the incidence of clinical neonatal equine piroplasmosis, which could control parasitaemia during the foals' early months of life . In the present report, a more than 2-month time interval between possible infection in utero and clinical disease might be explained by a similar protection conferred on the pup by maternal colostrum. Nevertheless, and although it had no detectable ticks, the possibility that the pup was infected after birth by a tick vector cannot be ruled out.
A severe regenerative haemolytic anemia and moderate to severe thrombocytopenia are common findings among dogs infected with the B. microti-like piroplasm [60, 61]. In the present report, the three dogs had regenerative anaemia, based on reticulocyte count (performed only for the bitch), presence of polychromasia, anisocytosis and nucleated RBC. The mechanisms related with severe hemolytic anemia may be more dependent on the host immune response than on the direct destruction of RBC by the piroplasm . The bitch and pup also had confirmed severe thrombocytopenia, while platelet count could not be assessed in the male dog. Mechanisms of local or systemic intravascular coagulopathy, immune-mediated destruction or splenic sequestration may be implicated in severe thrombocytopenias. On the other hand, the almost constant presence of macroplatelets in blood smears is associated with a bone marrow regenerative response to platelet consumption, as well as sequestration or destruction .
The bitch had normal leukocyte counts, while the male dog presented leukocytosis and the pup had leukopenia. Camacho-García  describes that half the dogs with piroplasmosis due to the B. microti-like piroplasm had normal leukocyte counts. Nevertheless, the values may range between leukopenia and leukocytosis, with the latter reaching extreme counts compatible with a leukemoid response in immune-mediated hemolytic anemia . Many cases also develop serum biochemistry abnormalities compatible with a glomerular component of renal failure . In a study describing 58 infected dogs, 36% were azotemic at the time of diagnosis and 22% died with azotemia being the main cause of mortality . In the present report, serum creatinine and urea were assessed only for the bitch and found to be within their normal range.
According to Camacho-García , animals infected with the B. microti-like piroplasm had a syndrome clinically more severe than those infected with B. canis, treatment with imidocarb dipropionate was less effective and evolution towards renal failure more frequent. At the time the pup died from parvoviral enteritis, one month after diagnosis of babesial infection, it had apparently recovered from the clinical disease caused by the small piroplasm.
The definitive diagnosis of babesiosis for the male dog was achieved only after the PCR and sequencing results. Indeed, in dogs clinically suspected of babesiosis, microscopy may lack sensitivity due to low parasitaemia . Sensitive molecular detection and species identification are important for the selection of the appropriate therapy and for prognosis, as well as for the screening of subclinical infections and blood donors .