An atypical Toxoplasma gondii genotype in a rural Brazilian dog co-infected with Leishmania ( Viannia ) braziliensis

Toxoplasmosis and leishmaniasis are two worldwide zoonoses caused by the protozoan parasites Toxoplasma gondii and Leishmania spp., respectively. This report describes the clinical and laboratorial fi ndings of a co-infection with both parasites in a 4-year-old female dog suspected of ehrlichiosis that presented anemia, thrombocytopenia, hypoalbuminemia, hyperglobulinemia, tachyzoite-like structures to the lung imprints, and polymerase chain reaction (PCR) results positive for T. gondii (kidney, lung, and liver) and Leishmania spp. Co-infection with Toxoplasma gondii and Leishmania braziliensis was confi rmed by sequencing; restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) confi rmed an atypical T. gondii genotype circulating in dogs that has been reported to cause human congenital toxoplasmosis.

Toxoplasmosis is a worldwide anthropozoonosis caused by a coccidian protozoan, Toxoplasma gondii.Felids are the defi nitive hosts, while most warm-blooded animals are intermediate hosts (1) .Dogs are considered sentinels for human infection; they are infected by ingesting raw or poorly cooked meat containing tissue cysts and by ingesting sporulated oocysts present in the contaminated feces of cats (2) (3) (4) .
Leishmaniasis is a zoonotic disease caused by Leishmania spp., and manifested in South America as American visceral leishmaniasis (AVL, Leishmania infantum chagasi) and American tegumentary leishmaniasis (ATL, mainly Leishmania braziliensis) (1) .L. braziliensis can cause visceral disease as a consequence of host immunosuppression, and is considered an opportunistic disease transmitted by phlebotomine bites after blood feeding from infected dogs, the main urban reservoir.
Hence, we report a T. gondii and L. braziliensis co-infection in a Brazilian dog suspected to have an Ehrlichia canis infection, and an atypical T. gondii genotype circulating in dogs.

CASE REPORT
A mixed breed female dog weighing 15.3kg and approximately 4 years of age from the rural area of Botucatu, SP, Brazil (22°53'08"S; 48°26'42"W) was brought to the Veterinary Hospital, School of Veterinary Medicine and Animal Science [Faculdade de Medicina Veterinária e Zootecnia (FMVZ)], São Paulo State University [Universidade Estadual Paulista (UNESP)] in May 2013.The animal had a previous history of fever and apathy starting 1 year prior.During that time, the animal was examined at a private veterinary clinic, clinically diagnosed, and treated for ehrlichiosis.One year later, the animal presented hyporexia, diarrhea with mucous, tachypnea, a reactive left submandibular lymph node, rectal temperature of 39.9°C, and was suspected to have bronchopneumonia.
Blood samples were collected for a complete blood cell count, serum biochemical analyses, and testing for T. gondii, Neospora caninum, and Leishmania spp.antibodies by the indirect fl uorescent antibody test (IFAT) method.The IFAT was assayed with antigens produced in-house (tachyzoites of T. gondii, RH strain; tachyzoites of N. caninum, NC-1 strain; In addition, the animal presented pulmonary fi elds with diffuse opacifi cation on a thoracic radiographic image with bronchial, interstitial, and alveolar patterns, and enlargement of the bronchial wall, suggesting bronchopneumonia.The heart presented a discrete hogback with respect to the right atrium.Three days later, the previous fi ndings had worsened with accentuated diffuse opacifi cation.The heart limits of the right atrium and pulmonary trunk were still curved.After 3 days, impairment was observed with a mixed pattern that tended to be micronodular in structure, suggestive of a fungal infection. An erythrogram demonstrated normocytic normochromic anemia [mean corpuscular volume (MCV) = 70.5%;mean corpuscular hemoglobin concentration (MCHC) = 33.1%)and thrombocytopenia (28,000 platelets/µL -1 ) (Table 1); lymphopenia (290 cells/µL) and eosinopenia (0.0 cells/µL) were observed on a leukogram; and hypoalbuminemia (1.5g/dL) and hyperglobulinemia (5.1g/dL) were found in the biochemical analyses (5) .Rouleaux was still observed.Chloramphenicol sodium succinate (50mg/kg three times a day; Ariston, São Paulo, SP, Brazil) was used to treat the ehrlichiosis and bronchopneumonia.Furosemide (4mg/kg twice a day; Sanofi , São Paulo, SP, Brazil), bromhexine (3mg/kg once a day, Boehringer-Ingelheim, São Paulo, SP, Brazil), and oxygen therapy were used for pulmonary complications; fl uid therapy with lactated Ringer's solution plus glucose for dehydration; and a multivitamin (5mL twice a day, Clusivol ® ; Wyeth Consumer, São Paulo, SP, Brazil) to stimulate the appetite.The animal died 10 days later, and pneumonia associated with renal and hepatic lesions was confi rmed by necropsy.Lung, kidney, and liver samples were fi nely chopped, imprinted in slides, and stained with Giemsa; they showed negative results for Ehrlichia spp., but presented tachyzoite-like structures in the lung tissue.
The samples were tested for T. gondii (6) , N. caninum (7) , E. canis (8) , and Leishmania spp.deoxyribonucleic acid (DNA) (9) by the PCR (Table 2).Positive results for T. gondii were observed in the lung, kidney, and liver samples; Leishmania spp. was found in the lung and liver.All samples yielded negative results for E. canis, N. caninum, and L. infantum chagasi.
Samples positive for Leishmania spp.were typed by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) using one marker for species identifi cation and the restriction enzyme HaeIII (9) , and showed L. braziliensis.In the same way, samples positive for T. gondii were also typed by RFLP-PCR using 11 markers for genotype identifi cation (10) .All three tissue samples were confi rmed to contain T. gondii by genotyping, and demonstrated an atypical genotype (Table 3) reported in the scientifi c literature to cause human congenital toxoplasmosis, TgCTBr5 (4) .All amplifi cations were performed in a MasterCycler ep gradient (Eppendorf, USA).Sequencing of the purified PCR products (Tg18s58F and Tg18s348R for T. gondii; LITSR and L5.8S for Leishmania spp.) was performed, and the results searched in the National Center for Biotechnology Information (NCBI) GenBank database using the Basic Local Alignment Search Tool (BLAST).All T. gondii     (11) In this study, a dog undergoing routine treatment at the FMVZ-UNESP Veterinary Hospital presented a T. gondii and L. braziliensis co-infection.This type of co-infection has already been reported in Brazil in 17/66 (25.8%) cats by molecular methods (11) , but was caused by L. infantum chagasi, not L. braziliensis.Both parasites have been reported to be opportunistic.Moretti et al. (2) reported a case of a dog coinfected with T. gondii, distemper virus, and ehrlichiosis.Immunosuppressive factors decrease host immunity and cause the rupture of pre-existing cysts, reactivating latent infections (1) (3) (2) .In this case, even with a negative result for E. canis, ehrlichiosis was not ruled out because the animal could have been a carrier.Additionally, an atypical T. gondii genotype was identifi ed from the Brazilian dog; curiously, the same genotype has been isolated only in Brazil from cats, chickens, capybaras, sheep, rabbits, and mice, and was recently reported by Carneiro et al. (4) to cause human congenital toxoplasmosis in the State of Minas Gerais, Brazil, but with no clinical signs.The authors reported that it was confi rmed as an avirulent genotype, among others, by mouse bioassay.The present study emphasizes the occurrence of this genotype and disease with pulmonary complications probably caused by the opportunistic multiplication of T. gondii as a consequence of immunosuppression.Even under these conditions, an avirulent parasite can multiply, infect other hosts, and cause disease.The fi ndings observed by Moretti et al. (2) were similar, but they observed a virulent type I genotype (SAG2 locus) rather than the type III genotype (SAG2 and alt-SAG2 loci) found in this study.This report emphasizes the importance of T. gondii, the circulation of the same genotypes in animals and humans, and supports dogs as perfect sentinels for this infection and genotype, as observed in other species.
The hematological, biochemical, and imaging analyses suggested a T. gondii and Leishmania spp.co-infection with an additional immunosuppression factor causing a visceral form of the disease by L. braziliensis.L. braziliensis causes ATL in humans and dogs; however, both here and in the scientifi c literature, it has been reported to be an opportunistic parasite in immunosuppressed individuals (12) .
The low titer for the T. gondii antibody fi ndings associated with the hematological, biochemical, and imaging results suggests a possible acute infection with a low parasite load producing a low humoral immune response dependent on a cellular response.Alternatively, it could suggest a reactivation of a chronic T. gondii infection caused by any immunosuppressive factor, for example, ehrlichiosis (2) ; the same was observed for leishmaniasis.Immunosuppression caused by an immunosuppressive factor changed the cutaneous form of leishmaniasis caused by L. braziliensis into a visceral pattern, as observed by Hernandez et al. (12) .The positive result from N. caninum antibody testing suggests an old infection with a low parasite load.
Thus, this report demonstrates a T. gondii and L. braziliensis co-infection in a dog presumed to have ehrlichiosis and a cutaneous Leishmania species causing a visceral form of the disease, as well as the circulation of the same T. gondii atypical genotype in dog and human populations, causing congenital toxoplasmosis in humans and domestic animals.These fi ndings highlight the importance of molecular techniques as epidemiological and diagnostic tools and adjuncts to standard tests to determine the causative agent(s), and supports dogs as the perfect sentinel model for this infection.