Rabies vaccination for dogs is an active immunization used to prevent rabies. Adverse effects of rabies vaccination have been reported such as acute hypersensitivity (fever, anaphylaxis, ataxia, etc.), local signs (pain, edema, pruritus, etc.), neurologic disorders, encephalitis, and autoimmune diseases like immune-mediated thrombocytopenia (IMT) or immune- mediated hemolytic anemia (IMHA) (9,11,18). In dogs, cases of IMT (5,25) or IMHA (5,14,27) related to vaccination have been reported, but cases of Evans’ syndrome related to rabies vaccination have not been reported.

Evans’ syndrome is a rare immune-mediated disease in dogs defined by the combination of IMT and IMHA (7). The incidence rate of Evans’ syndrome in dogs has been estimated to be 0.01% (17). In humans, several cases of Evans’ syndrome associated with vaccination have been reported, including after vaccination for influenza and hepatitis B (15,26). There have been no reports of Evans’ syndrome caused by rabies vaccination in humans or dogs. IMT is a common autoimmune disease in dogs caused by the formation of antibodies to platelets, and it is caused by infection, inflammatory diseases, neoplasia, drugs, and vaccines (7,11,13,16,22,25,28,33). In rare cases, sequential or concurrent IMHA occurs in dogs with IMT (23). To diagnose Evans’ syndrome, all other causes must be excluded, and direct Coomb’s positive hemolytic anemia, thrombocytopenia, and spherocytosis must be present simultaneously (6,8,12). The treatment for Evans’ syndrome includes immunosuppressive drugs, anticoagulation, and blood transfusion (19). The first-line immunosuppressive drugs in dogs are glucocorticoids, such as prednisolone and dexamethasone, and it has been reported that cyclosporine, mycophenolate mofetil (MMF), azathioprine, vincristine, chlorambucil, leflunomide, and human immunoglobulin (hIVIG) are available for treatment (25,29).

This case shows that serious adverse effects due to rabies vaccination can occur and should be monitored more carefully. In these cases, a combination of high-dose corticosteroids and MMF may be a successful treatment option.

A 11-year-old neutered male Maltese dog was referred to the hospital with an acute hypersensitivity reaction at a vaccine injection site. The patient received a rabies vaccine (Rabisin^®, Boehringer Ingelheim International GmbH, Germany) subcutaneously on right scapular lesion. After one hour, purpura with edema was observed at injection site. On a complete blood count (CBC) conducted at a local animal hospital confirmed severe thrombocytopenia (14 K/μL; reference range 148-484 K/μL), then the patient was referred.

On anamnesis, the patient had been vaccinated annually, but no specific symptoms had appeared before. Also, the patient had no history of immune-medicated diseases. Physical examination confirmed purpura (3.7 × 4.4 × 0.6 cm) with edema and heat in the scapular area (Fig. 1A). No lesions were identified except for in this area. On a CBC, severe thrombocytopenia (0 K/μL; reference range 148-484 K/μL) was noted (Fig. 2). Serum chemistry, blood electrolyte, and coagulation analyses (prothrombin time, activated partial thromboplastin time, and D-dimer levels) were performed, showing no remarkable findings. On a peripheral blood smear, thrombocytopenia was observed, but other blood cell abnormalities and parasites were not observed. Abdominal radiographs revealed no remarkable findings. The patient was hospitalized and administered vitamin K (1 mg/kg SC BID; vitamin K1 inj, Daihan Pharm, Seoul, Korea) and desmopressin (0.1 mg/dog PO BID; Desmin tab, Dongkook, Seoul, Korea) as antihemorrhagic agents. In addition, chlorpheniramine (0.5 mg/kg SC; Chlorpheniramine maleate inj, Huons, Gyeonggi, Korea) and methylprednisolone sodium succinate (MPSS, 4 mg/kg IV; Predi-Sol inj, Reyon Pharm, Seoul, Korea) were administered intravenously to reduce the hypersensitivity reaction. During the hospitalization period, the purpura lesion gradually began spreading to the surrounding area such as right forelimb, neck, and thorax. On a CBC, a mild regenerative anemia (hematocrit 31.7%; reference range, 37.3-61.7%) (reticulocyte index (RI), 1.18) developed, and the thrombocytopenia persisted (1 K/μL; reference range, 148-484 K/μL) (Fig. 2). Autoagglutination was observed on a saline agglutination test (SAT) (Fig. 3A), and spherocytes were observed on a peripheral blood smear (Fig. 3B), but direct antiglobulin test (DAT, Coomb’s tests) was negative. Other infectious diseases caused by parasites (e.g., Anaplasma spp., Babesia spp., and Theileria spp.) and bacteria (e.g., Ehrlichia spp., Borrelia spp., Bartonella spp., Rickettsia spp., and Leptospira spp.) were ruled out using a SNAP 4Dx Plus Test (IDEXX Laboratories, Inc., Westbrook, ME) and real-time PCR (Pobanilab, Gyeonggi, Korea). Based on several tests results, secondary IMT and IMHA associated with rabies vaccination were considered; therefore, vaccine-associated Evans’ syndrome was tentatively diagnosed. Then, prednisolone (2 mg/kg PO BID; Solondo^® tab, Yuhan, Seoul, Korea) and MMF (10 mg/kg PO BID; My-Rept Cap, CKD Pharm, Seoul, Korea) were added as immunosuppressive drugs to the treatment course.

Figure 1. Photographs of the dog. A firm raised area of purpura (3.7 × 4.4 × 0.6 cm) with heat was confirmed at the vaccination site at the first visit (A). Four weeks later, no lesions were identified on physical examination (B).

Figure 2. The patient’s hematocrit and platelet count during the therapeutic monitoring period. After the diagnosis of Evans’ syndrome, immunosuppressive drugs were included in the treatment. Four weeks after drug administration, the patient’s platelet count and hematocrit recovered to normal levels.

Figure 3. Saline agglutination test (SAT) and peripheral blood smear cytology. Before drug administration, autoagglutination positivity (A) and spherocytes were confirmed (B, red arrow). After immunosuppressive therapy, autoagglutination (C) and spherocytes (D) were not identified. The patient’s thrombocytopenia also improved after treatment (B, D).

After six days, new lesions were not observed, and the previous lesions improved. On a CBC, the thrombocytopenia (25 K/μL; reference range, 148-484 K/μL) was mildly improved, but the regenerative anemia had worsened (hematocrit, 28.5%; reference range, 37.3-61.7%) (RI 1.6) (Fig. 2). Furthermore, a corticosteroid-induced stress leukogram was identified on a CBC, and elevated liver enzymes observed on a serum chemistry analysis. On the SAT, autoagglutination was negative (Fig. 3C), but spherocytes were still present on a peripheral blood smear. On anamnesis, the patient’s stool color was normal; therefore, corticosteroid-induced intestinal bleeding was excluded. It was judged that the improvement in the thrombocytopenia was due to drug administration; thus, the anemia was evaluated one week later.

After one-week, the regenerative anemia (hematocrit, 35.2%; reference range, 37.3-61.7%) (RI 2.0) was improved, and a platelet count at the lower limit of normal (157 K/μL; reference range. 148-484 K/μL) was identified on a CBC (Fig. 2). The dose of prednisolone (1 mg/kg PO BID) was tapered. Four weeks after medication administration, the patient’s hematocrit (43.5%; reference range, 37.3-61.7%) and platelet count (459 K/μL; reference range, 148-484 K/μL) recovered to normal levels (Fig. 2). In addition, spherocytes were not observed on a peripheral blood smear (Fig. 3D). The dose of prednisolone was tapered over 40 days, and MMF was administered for four months and then discontinued. A physical examination, CBC, and serum chemistry analysis were performed, showing no remarkable findings (Figs. 1B, 2). These values were maintained for six months without recurrence of related symptoms.

This is the first case report of rabies vaccine-associated Evans’ syndrome in dog. This case shows that serious adverse effects due to rabies vaccination can occur, and management with high-dose corticosteroids and MMF was successful.

Vaccines facilitate the development of immunity to a certain disease by stimulating the body’s immune system to develop acquired immunity (20). It has been reported that the mechanism of action of vaccines can lead to immune-mediated diseases (2,5,9-11,16,18,23,24,28). The mechanisms that lead to immune-mediated disease are as follows: First, vaccines affect the immune system, leading to the production of abnormal antibodies that attack the body’s own cells such as red blood cells (RBCs) and platelets (25). Second, vaccine components may attach to RBC or platelet membranes and induce an immune response (5). Third, vaccination may affect the immune system; then, normal cells are destroyed by a hyperactive immune system (6).

Among the glucocorticoids, MPSS, which is a fast-acting and intravascular injectable agent, was applied at the beginning of treatment. In this case, a combination of prednisolone and MMF was used for treatment.

MMF has a main mechanism of action of suppressing immunity by a lymphocyte-selective toxicity effect through depletion of guanosine and deoxyguanosine nucleotides (29). Other mechanisms include suppress of dendritic cell maturation and reduction of monocyte migration to the inflamed site (29). Several cases of MMF treatment in canine immune-mediated diseases such as IMT, IMHA, pemphigus, and acquired myasthenia gravis have been reported (1,4,21,30,31). MMF was applied to treatment by referring to the following studies.

In one study comparing a group of dogs with IMHA treated with corticosteroids and MMF and another treated with other immunosuppressive drugs (cyclosporine, azathioprine, and hIVIG), there was no significant difference in the survival rate, but fewer adverse effects occurred in the MMF group (30). In addition, one study comparing the survival period, adverse effects, and cost of MPSS/corticosteroid and cyclosporin/corticosteroids treatment in dogs with primary IMT have been reported (3). As a result, the MPSS/corticosteroids group had fewer adverse effect and less treatment cost (3). Furthermore, another study has reported that use of MMF with corticosteroids in dogs with pemphigus can be reduce the adverse effects caused by corticosteroids (21).

The mortality rates of IMT and IMHA in dogs are reported to be 28% to 70% and 10% to 30%, respectively (17). The prognosis of Evans’ syndrome is worse than that of IMT or IMHA alone (17). However, the exact prognosis of Evans’ syndrome in dogs has not yet been reported.

This case report has limitations. First, in this case, antiplatelet antibodies were not measured. Several techniques have been reported to measure the serum levels of anti-platelet antibodies such as platelet factor 3 (PF-3) and radioactive immunoglobulin in humans (5). We could not find an institution that measures anti-platelet antibodies in dogs in Korea; thus, this test was not performed in this patient. Second, to diagnose Evans’ syndrome, DAT-positive hemolytic anemia should be confirmed, but in this case, DAT was negative (6,8,12). DAT is not very sensitive or specific diagnostic method for IMHA, as only 60-70% of dog IMHA cases have been confirmed positive (32). The causes of DAT false positives include lack of antibodies in RBCs, test performance temperature, inappropriate antigen: antibody ratio, and low-efficiency antibody elution in RBCs during washing were reported (32).

This is the first case report of rabies vaccine-associated Evans’ syndrome in dog. This case shows that serious adverse effects due to rabies vaccination can occur, and management with high-dose corticosteroids and MMF was successful.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2023R1A2C1005348).

The authors have no conflicting interests.