Immune-Mediated Hemolytic Anemia Associated with Candidatus Mycoplasma Haematoparvum in a Splenectomized Dog in Italy

Abstract This report describes a case of canine hemotropic mycoplasmasosis by Candidatus Mycoplasma haematoparvum in a dog. A five-year-old splenectomized dog was referred to the Veterinary Teaching Hospital of the University of Sassari with clinical symptoms and laboratory findings compatible with immune-mediated hemolytic anemia. Epicellular bacteria were detected in the erythrocytes by microscopic examination of blood smears. PCR and sequencing were positive for Candidatus Mycoplasma haematoparvum. Treatment with doxycycline, prednisolone and blood transfusion was administered. Several studies have described the molecular prevalence of M. hemocanis and Candidatus M. haematoparvum, however there are few clinical reports, especially those describing Candidatus M. haematoparvum infection in dogs, for which only two cases have been reported. To the best of our knowledge this is the first case report of a symptomatic infection caused by Candidatus Mycoplasma haematoparvum in Italy. Hemoplasmosis should be considered as a potential cause of hemolytic anemia in dogs. Following treatment with doxycycline and prednisolone, the clinical signs improved without resolution of infection. This condition was the same at the three-year follow-up.


INTRODUCTION
Canine hemotropic mycoplasmas (hemoplasmas) are cell wall-defi cient bacterial pathogens that attach to the surface of erythrocytes of infected animals. Clinical infections are usually chronic and subclinical in immunocompetent dogs, but may lead to acute signs related to hemolytic anemia following splenectomy, immunosuppression, or concurrent infections [1].

Two different species have been recognized in dogs: Mycoplasma hemocanis (Mhc) and
Candidatus Mycoplasma haematoparvum (CMhp). Several studies have described the molecular prevalence of M. haemocanis and Candidatus M. haematoparvum however there are few clinical reports, especially those describing Candidatus M. haematoparvum infection in dogs, for which only two cases have been reported [2][3].
This report describes the clinical presentation, the use of the correct diagnostic tools and the follow-up of the disease caused by CMhp in a splenectomized dog with immunemediated hemolytic anemia. The aim is to improve the current poor knowledge of canine hemoplasmosis, its pathogenicity, diagnosis, therapy and prognosis.

CASE PRESENTATION
A fi ve-year-old female Beagle dog, living in Sardinia (Italy), was presented to the Veterinary Teaching Hospital of the University of Sassari. The anamnesis reported depression, asthenia and anorexia for three days. Two months prior to the illness, the dog had been splenectomized due to a perforating wound of the abdomen caused by a wild boar. Vaccinations had been performed regularly up to the second year of life and prophylaxis against ecto-and endoparasites was not constant. Physical examination revealed pale mucous membranes, tachycardia, tachypnea, depression, increased capillary refi ll time, and massive fl ea infestation.
The results of the cell blood count and serum biochemical profi le included severe macrocytic hypochromic anemia with a marked reticulocytosis, infl ammatory leukocytosis characterized by an increase in band neutrophils and lymphocytosis, mild hypoalbuminemia, and increased renal parameters (Table 1). Blood smear examination revealed signs of immune-mediated hemolytic anemia: microagglutination, hypochromia, anisocytosis, polychromasia, Howell-Jolly bodies, leptocytes, nucleated red blood cells (nRBC), and signs of infl ammatory response: band neutrophils and activated lymphocytes. The presence of nRBC was also related to the splenectomized condition. Observation of the blood smear with a 100X objective highlighted the presence of parasites on the surface of red blood cells, probably belonging to the genus Mycoplasma. These appeared as basophilic, round or rod-shaped structures organized individually or in chains ( Figure 1). The presence of IgG-IgM anti-erythrocyte antibodies, detected by cytofl uorimetry, and a positive Coombs test confi rmed the immune-mediated hemolytic anemia. Abdominal and thoracic ultrasound examination did not show clinically signifi cant abnormalities. Serologic tests (IFAT) against Leishmania infantum, Ehrlichia canis, and Anaplasma phagocytophilum were negative. DNA was extracted from a blood sample and used in two single PCRs for amplifi cation of a 1429 bp and 232 bp fragment of the 16S rRNA and RNase P gene specifi c for Mycoplasma spp. The sequenced amplicons, deposited in the GenBank under access numbers MH094850 and MH090015, were closely related to Candidatus Mycoplasma haematoparvum (99% identity with 100% coverage).     Using the ClustalW option of BioEdit [4], a fragment of 1297 bp of the 16S rRNA sequence and the 187 pb sequence of the rnpB gene determined in this study were aligned with the corresponding region of 38 other mammalian hemotropic mycoplasmas and 12 other canine and feline hemotropic mycoplasmas, respectively, available in the GenBank. Phylogenetic analysis using the neighbor-joining [5] method was conducted with MEGA 6 software [6]. Internal branches of the trees were statistically supported by bootstrapping with 1,000 iterations [7] (Figures 2 and 3). The dog was treated with doxycycline (10 mg/kg orally every 24 hours for fi ve months) and prednisolone (2 mg/kg orally every 24 hours for one month). In order to stabilize the patient, the dog was immediately subjected to blood typing (DEA1.1+), crossmatching and transfusion with 20 ml/kg of compatible whole blood and fl uid therapy with Ringer lactate solution. Following the initiation of therapy, the dog improved clinically, and renal parameters returned to the normal range on the assumption of a pre-renal condition. On day 60, hematological values were normalized except for thrombocytosis, as a consequence of the splenectomy, and reticulocytosis which was decreased compared to the fi rst day. The persistent reticulocytosis was explained by a compensated anemia, a condition described in immunomediated hemolysis [8]. The microorganisms were no longer detected on blood smears. However, the PCR for Mycoplasma spp, performed 30 and 60 days after diagnosis, was still positive. The therapy was thus prolonged for another three months, after which the patient was still PCR positive, but without symptoms. It was decided to interrupt the antibiotic therapy and to monitor the dog. In the following months the physical examination and blood count were similar to the time of discharge. Three years after the diagnosis of mycoplasmosis, the owner reported that the dog was fi ne and had no symptoms.
Although several studies on the molecular prevalence of Mhc and CMhp have been described, reports about clinical signs are rare, especially regarding CMhp, for which only two cases have been reported. The fi rst clinical case was described by Sykes et al. in 2004 in a splenectomized dog with hematic alterations caused by a T-cell lymphoproliferative disease in California [2]. Ten years later Sharifi yazdi et al. reported a case of fever, regenerative anemia, leukocytosis, thrombocytosis, and urinary bilirubin, associated with CMhp in a non-splenectomized dog in southern Iran [3].
Phylogenetic analysis of 16S rRNA and the rnpB sequence confi rmed that our isolate clustered within the CMhp group and was more distantly related to Mhc, similarly to fi ndings by Sykes and Sharifi yazdi [2][3]. However, it is not possible to clinically compare our case and Sykes' report because the clinical signs of his patient were caused by the lymphoproliferative disease and the dog was consequently euthanized.
The laboratory alterations caused by the immune-mediated hemolytic anemia in our dog were similar to the fi ndings observed by Sharifi yazdi, although the latter was not splenectomized. Our case is similar to infections due to M. haemocanis described by other authors: splenectomized patient, hemolytic anemia, progression to a subclinical form, and persistence of PCR positivity after treatment with doxycycline [9][10][11][12].
To the best of our knowledge, our study is the fi rst report of a symptomatic infection with Candidatus Mycoplasma haematoparvum in Italy. Two previous studies have been reported on the distribution of canine hemotropic mycoplasma infections in Italy. Novacco et al. (2010) reported a prevalence of 5.8% of CMhp infection in randomly selected dogs from central and southern Italy without clinical signs which were clearly attributable to canine hemoplasma infection. An interesting aspect underlined by these authors is the higher CMhp load found in the splenectomized dog [13]. Ravagnan et al. (2017) found a lower prevalence of CMhp in dogs from northern Italy (1.4% positivity in dogs from shelters in Padua and Treviso) [14].
Doxycycline is the drug of choice in mycoplasmosis therapy (5-10 mg/kg orally every 24 hours for 21 days) [9,15], however data are not available on its effi cacy in CMhp therapy. In our case, doxycycline for fi ve months improved clinical signs with excellent results, however it was interrupted without complete clearance of mycoplasma infection. Despite this, the dog had no recurrence of the disease at the three-year follow-up. This is in line with fi ndings by other authors in a dog with Mhc. The resolution of the clinical disease in dogs treated with a long-term antibiotic therapy could be due to the development of an effective immune response by the dog, which often remains a chronic carrier after clinical signs have resolved [12].
Our patient's clinical recovery implies a good prognosis, but not in terms of resolving the infection. This is a typical report characterized by the transition from an acute form with immune-mediated hemolytic anemia to a subclinical/chronic condition in which the presence of the parasite is not accompanied by any clinical manifestation . In fact, the fl uctuating fi nding of some laboratory alterations, such as piastrinocytosis and nucleated red blood cells, are related to the splenectomy condition.
In our study we confi rmed the higher sensitivity of PCR compared to a blood smear. PCR, or quantitative PCR, is the gold standard for the detection of canine hemoplasmas, especially in chronic or subclinical forms or in a follow up, during which there are fewer bacteria [16]. Most non-splenectomized infected dogs without evidence of disease do not have a suffi cient number of organisms present in the blood to be recognized during routine blood smear examination [11]. PCR should be used to monitor the exacerbation of chronic infection in dogs undergoing splenectomy or immunosuppressive therapy, and for screening blood donor dogs [9].