Theileria annae (syn. Babesia microti-like) infection in dogs in NW Spain detected using direct and indirect diagnostic techniques: clinical report of 75 cases

Theileria annae (syn. Babesia microti-like) is a recently recognized piroplasm that causes canine piroplasmosis along with other species of the genera Theileria and Babesia [1,2].

Historically, Babesia infection in dogs was identified according to the morphologic appearance of the parasite in the erythrocyte. Based on relative size, these parasites are broadly divided into two groups, large and small piroplasms. Although all large forms reported to date have been ascribed to the genus Babesia, small Babesia spp. and Theileria spp. cannot be distinguished by microscopy and DNA-based molecular techniques are required for an accurate identification [3,4]. Indeed, it is currently unclear if the protozoan T. annae is a member of the genus Theileria or Babesia [2,5]. No evidence was initially presented [2] for extra-erythrocytic infecting stages or for the absence of transovarial transmission in ticks (distinguishing features of Theileria spp.). Morphologically, T. annae resembles small piroplasms such as Babesia gibsoni which, phylogenetically, is considered a “true babesia”. Molecularly, however, it appears to be closer to the genetically-distinct rodent piroplasm B. microti (B. microti group) and only distantly related to “true theilerias” such as Theileria parva [2,6,7].

At present, 12 piroplasm species have been reported in dogs worldwide but some of these have been only detected by molecular techniques [1]. Four species have been described in Europe: Babesia canis, Babesia vogeli, B. gibsoni and T. annae.

Babesia canis is endemic in temperate regions and is the most common species reported in Europe (northern Spain, Portugal, France, central Europe and Eastern Europe). Babesia vogeli and B. gibsoni are widely distributed across both Old and New World continents [8,9]. In Europe, B. vogeli has been described in the Mediterranean basin, whereas B. gibsoni only occasionally appears in Europe [10], mainly as the consequence of introduced infected dogs from endemic areas (Asia, United States and Australia) [8].

Studies in the United States and Australia have indicated that direct dog to dog transmission (in American Pit bull terriers and other fighting dogs) is likely and this could be the main mode of transmission outside Asia for B. gibsoni [11,12].

T. annae was first described in 2000 in a dog from Germany that had travelled to the Pyrenees [2]. Today, the infection is considered endemic in NW Spain (Galicia) [5,13]. Using molecular techniques T. annae has been also detected in Spanish regions outside Galicia, such as Barcelona [14], and in other countries including NW Portugal [15], Croatia [16], US [12] and Sweden [17]. However, in most cases the travel history of the dogs was unknown. Other authors have reported cases of T. annae infecting foxes in Spain [18], Portugal [19], Italy [20], Croatia [21], Canada [22] and the US [23]. Among all piroplasm species reported in Europe, T. annae seems to show the greatest preference for foxes. Thus, T. annae has been detected in red foxes in Spain and Portugal at prevalences from 14% to 69.2%, respectively; while B. canis has been only occasionally identified in these animals [19]. To date, there are no available data regarding clinical impacts on foxes.

The transmission vector of T. annae is presently unknown. Ixodes hexagonus has been proposed as a likely candidate because endemic areas of T. annae infection closely match its distribution range [18,24]. However, T. annae DNA has been observed in both I. hexagonus and I. ricinus, though no data exist to substantiate their competence as vectors for T. annae.

T. annae-infected dogs show severe clinical signs and clinicopathological abnormalities resembling those of other piroplasm infections such as fever, pale mucous membranes or haemoglobinuria [25]. Despite morphologic differences, T. annae is often ascribed by veterinarians to other Babesia spp. mainly B. canis. Currently, there are no diagnostic tools to distinguish between the different piroplasms in routine veterinary practice and their detection by microscopy in red blood cells is still the only method available to practitioners. In laboratory settings, the immunofluorescent antibody test (IFAT) is the most widely used test for a serology diagnosis and is considered highly sensitive and moderately specific to detect chronic infection and subclinical infection in carriers [4,26,27]. The polymerase chain reaction (PCR) is a sensitive and specific diagnostic test widely employed to diagnose canine babesiosis. When PCR is combined with sequencing, species-specific primers/probes, or restriction fragment length polymorphism (RFLP) analysis it can be used to detect infected dogs with low parasitaemia levels and to identify parasites [12,28,29]. However, the literature lacks widespread serological and/or molecular surveys of T. annae infection. Similarly, comparative methodological studies on the available diagnostic procedures are limited.

The present study was designed to examine the clinical picture of T. annae infection in dogs in NW Spain and assess how best to diagnose this largely unknown disease.

In this study, T. annae infection was assessed by PCR, LM and IFAT on blood and serum samples obtained from dogs in NW Spain suspected of having piroplasmosis.

Microscopy examination is the easiest and most accessible diagnostic test, requiring a well prepared and suitably stained blood smear together with a trained observer. Our results indicate the good specificity (95.56%) and moderate sensitivity (76%) of this procedure as well as its moderate agreement with PCR. However, it should be noted that we assume that most of the dogs examined here were in the clinical phase of T. annae infection when the visual detection of piroplasms is easier than in animals with low parasitaemia levels due to chronic disease [1]. In effect, LM has been described as less sensitive to detect chronic and sub-clinical piroplasmosis in carrier dogs [40].

We considered the molecular approach as the gold standard method for the diagnosis of small piroplasm infections. Accordingly, using the molecular diagnostic test, a larger number of positive dogs for T. annae infection were detected (62.5%). Other PCR assays used to diagnose B. gibsoni infections have shown a high specificity and sensitivity [29,41]. In the latter studies, PCR was able to detect the parasite both at an earlier stage of infection than IFAT or LM and in the late stages of infection, when parasitaemia levels are low and Giemsa-stained thin blood smears return negative results [41]. Nevertheless, false negative PCR results have been reported in chronic babesiosis, attributed to parasite elimination from the circulating blood by the host [40]. This could determine that in the long term (up until 420 days post-infection) an infection might only be revealed (retrospectively) by serology [42].

In agreement with a previous report [43], we observed moderate agreement between our IFAT and PCR results. In contrast, discrepancies were reported by Kubelová et al. [44] for these techniques in endemic areas of canine piroplasmosis.

Serological cross-reactions between T. annae and B. canis were produced in two dogs. Cross reactions have been also reported between B. gibsoni and B. canis by other authors [45]. Most of the present dogs testing seropositive for T. annae (n = 59) showed low antibody titres (1/64 or 1/128). Such titres reflect an early stage of infection. For B. canis, it has been described that the first detectable IgG antibodies usually appear 2–3 weeks after infection [46,47]. In addition, low antibody titres could be indicative of past infection or exposure and not necessarily of present infection. A serological diagnosis is therefore a more reliable method for the detection of hidden or past infections (i.e. chronically infected carrier dogs) though acute infections may not be accurately diagnosed if this technique is performed alone. This issue, however, needs confirmation owing to a lack of serological data. It has recently been argued that the diagnosis of infection by a vector-borne pathogen in dogs can be improved by running serological and PCR based tests in parallel [48]. However, we observed no benefits of the use of both techniques over that of PCR alone. Serology is unable to distinguish between B. canis, T. annae and B. gibsoni infection, and blood smears cannot distinguish between T. annae and B. gibsoni. Indeed, the latter technique is also unable to discriminate B. canis which, though considered a large Babesia, can appear as having pleomorphic intermediate-sized intraerythrocyte stages.

In the current study, PCR and sequencing using universal primer sets specific for piroplasmida enabled the detection of a larger number of animals harbouring T. annae, compared to the other techniques (Figure 1). However, our study failed to clarify the taxonomy of T. annae such that more work is needed to resolve this well recognized systematics conundrum [7].

The possibility of co-infections should also be considered. In the north of Spain, summers are warm, winters cool and rainfall is evenly distributed all year round. Accordingly to that, Rhipicephalus sanguineus and Phlebotomus perniciosus, vectors frequently reported in Spain, are uncommon in this region and the prevalences of L. infantum and E. canis are lower than in the rest of Spain. The seroprevalence of L. infantum recorded in our study (10.8%) was higher than previously reported for NW Spain (3.7% and 4.1%) [49,50]. This discrepancy could be attributed to the sick population selected for our study rather than a cross-sectional population. An exemption might be the Orense province in NW Spain, where a prevalence of 35.6% has been observed, similar to that reported in endemic areas of Spain [50]. This prevalence was attributed by the authors to the bioclimatic characteristics of this geographical area. Moreover, in this latter region, the presence of the sandfly, P. perniciosus was also detected. Only one dog from Ourense, which tested negative for L. infantum, was enrolled in this study. Twelve of the dogs examined here showed coinfection with piroplasms (T. annae, B. canis or B. gibsoni) and L. infantum. The findings are similar to those in a previous study that showed high co-infection rate of L. infantum in the Babesia positive dogs from Portugal [9]. No E. canis antibodies were detected in our study, coinciding with the low seroprevalence (1.4%) reported for this infection in NW Spain [30].

With respect to the clinical picture, the most common clinical signs observed in the dogs included in our study were weakness, pale mucous membranes, haemoglobinuria, tachycardia, hyperthermia, tachypnea, and hepatosplenomegaly. These signs consistent with those reported previously for T. annae [25] and Babesia spp. infection [51] were to be expected because many were responsible for a clinical suspicion of piroplasmosis and were thus criteria for inclusion in our study. We observed orangey faeces in 12.5% of the infected dogs, probably due to high levels of excreted bilirubin.

Severe regenerative anaemia and thrombocytopenia were the main haematological findings observed, consistent with prior reports [13]. Regenerative anaemia was typically macrocytic/hypochromic with increased numbers of reticulocytes observed that were relatively larger than mature red blood cells. Reticulocytes are hypochromic because they have not completed haemoglobin synthesis. These haematological abnormalities have been also described by others in T. annae infected dogs in NW Spain [25].

The red blood cell counts, haematocrits and haemoglobin levels recorded in the present animals with suspected piroplasmosis are in agreement with those reported for 62 T. annae -infected dogs examined in 2003, 90% of which showed values lower than 4.46 x 10⁶ erythrocytes/μL, 10.52 g haemoglobin/dL and 31.04% haematocrit [13]. Leukocyte abnormalities have been inconsistently observed in dogs with piroplasmosis [25]. Total leukocyte counts were greater than 17 x10³ cells/μL in 25% of the animals examined here, and there was a trend towards neutrophilia and eosinopenia. This could be a consequence of the severe stress associated with this illness, in line with observations by other authors [13].

We only detected a few cases of azotaemia (9.8%) despite others observing its high prevalence in this disease (36%) [13,52] and suggesting its strong correlation with the likelihood of death within the first week of diagnosis [52]. High ALP activity was observed in 43.8% of the infected dogs despite reports of liver disease only in other types of piroplasmosis [53].

Our epidemiological results are in agreement with observations by García et al. [13], who mentioned that the age distribution of their study population reflected a greater risk of infection in younger animals [13]. Frequencies of infection by Babesia species in endemic areas have been described as inversely proportional to animal age. The correlation observed here, between an animal weight under 22 kg or a hunting type dog and a greater likelihood of T. annae infection, could reflect the fact that hunting dogs are usually fairly light. A large number of the T. annae infected dogs included in our study lived in rural areas and were infested by ticks. Other authors have reported a greater risk of T. annae infection in hunting dogs or dogs infested by ticks [25,54]. As also noted for other Babesia species [54,55], we observed no correlation between T. annae infection and sex or breed. García et al. [13] reported that autumn and winter were the periods when most cases of T. annae were observed. However, we observed no significant correlation between season and T. annae infection.

By PCR and sequencing, we detected three positive cases of B. gibsoni infection among the 120 dogs with suspected piroplasmosis. However, we have insufficient data to confirm the autochthonous nature of these cases. B. gibsoni has been sporadically reported in dogs travelling to endemic areas [10,14] and more epidemiological studies are needed to evaluate its presence in Spain. In contrast, B. canis has been often identified in dogs in northern Spain and Portugal [56] and both its diagnosis and clinical management by veterinarians in these regions are effective [8]. Despite not being included in our study, among dogs diagnosed in the participating clinics as having piroplasmosis caused by B. canis, 12 cases proved PCR positive. This could reflect the high prevalence of B. canis in NW Spain and its similar clinical signs to T. annae infection. Moreover, the pleomorphic nature of B. canis hinders its microscopy identification and a molecular confirmation is often required on which to base selection of the best treatment option. One of the two PCR-confirmed cases in Asturias (outside Galicia) had never left that area, suggesting that T. annae infection could be spreading to neighbouring regions. This idea requires confirmation in future studies.

Currently, microscopy detection of the parasite is the most simple and rapid diagnostic method, provided it is performed by a specialized technician. This procedure shows a moderate sensitivity for the pathogen in the acute infection stage when IFAT-determined antibody titres are low. However, sensitivity increases when IFAT and LM are used together. PCR is able to detect a larger number of positive cases and confirm the species involved. For an accurate diagnosis, we would recommend an integrative approach based on epidemiological evidence, the clinical picture, LM and/or IFAT, and confirmation of the infecting species by a molecular method. We propose that the decision tree in Figure 1 may be useful for clinically managing T. annae infection in endemic regions or in dogs travelling to an endemic area. With regards to the possibility that this small piroplasm could spread across northern Spain, we fear that bordering areas of Galicia with similar climate conditions could be already affected. This concern determines a need for larger epidemiological surveys in which molecular and serological methods are used to detect dogs with chronic or subclinical infection. The data emerging from such studies will serve to more reliably establish the current prevalence of T. annae infection in northern mainland Spain. Finally, our study does not clarify the systematics of T. annae such that we recommend more work in this area.