Novel Rickettsia Species Infecting Dogs, United States

In 2018 and 2019, spotted fever was suspected in 3 dogs in 3 US states. The dogs had fever and hematological abnormalities; blood samples were Rickettsia seroreactive. Identical Rickettsia DNA sequences were amplified from the samples. Multilocus phylogenetic analysis showed the dogs were infected with a novel Rickettsia species related to human Rickettsia pathogens.


The Cases
On May 15, 2018, a 10-year-old male neutered mixed breed dog (case 1) from Tennessee was examined by a veterinarian for lethargy and hyporexia. The owner reported removing a tick (species unknown) within the previous 2 weeks. On physical examination, the dog had fever (39.8°C) and possible hepatomegaly.
Radiographic imaging results were unremarkable. Thrombocytopenia was the only abnormality noted on complete blood count (CBC). Serum biochemistry panel (SBP) abnormalities included hyperglobulinemia, increased serum alkaline phosphatase activity, hypoglycemia, and hyponatremia ( Table 1). Results of urine dipstick and sediment examination were unremarkable. The dog's samples were R. rickettsii seroreactive and PCR positive for Rickettsia (Table 2). Clinical abnormalities resolved after treatment with doxycycline, and the dog remained healthy during the 1-year follow-up period.
On May 8, 2019, a 9-year-old male neutered Boston terrier (case 2) from Illinois was examined by a veterinarian for lethargy, difficulty walking, and painful elbows. Clinical signs developed 3 days after returning from a tick-infested area in Arkansas. Abnormalities noted on physical examination included fever (40.1°C), dehydration, joint effusion, elbow pain, and shifting leg lameness. Thrombocytopenia and mild leukocytosis were the only CBC abnormalities (Table 1). SBP abnormalities included hypoalbuminemia, increased alanine amino transferase activity, alkaline phosphatase activity, hypercholesterolemia, and hypocalcemia (Table 1). Mild microalbuminuria was noted. Neutrophilic inflammation was documented by synovial fluid cytology in the right and left stifle joints, right tarsus, and left elbow joint. The left carpus contained moderate, chronic inflammation with very rare extracellular cocci; however, culture resulted in no bacterial growth. The dog experienced cardiorespiratory arrest during sedated arthrocentesis but recovered after CPR and sedative reversal. Thoracic radiographs were unremarkable. The dog's samples were R. rickettsii seroreactive and PCR-positive for Rickettsia and convalescent titers demonstrated 4-fold seroconversion ( Table 2). Most clinical abnormalities resolved after administration of doxycycline to treat rickettsiosis, prednisone to treat potential immune-mediated component, omeprazole to prevent gastric ulcers, and metronidazole to treat In 2018 and 2019, spotted fever was suspected in 3 dogs in 3 US states. The dogs had fever and hematological abnormalities; blood samples were Rickettsia seroreactive. Identical Rickettsia DNA sequences were amplified from the samples. Multilocus phylogenetic analysis showed the dogs were infected with a novel Rickettsia species related to human Rickettsia pathogens. assumed dysbiosis. All SBP changes resolved within 5 months of treatment and the dog remained healthy during the 5-month follow-up.
On August 28, 2019, a 9-year-old male neutered terrier mixed-breed (case 3) from Oklahoma was examined by a veterinarian for lethargy, hyporexia, and polydipsia. Physical examination revealed fever (39.8°C) and palpable abdominal tenderness. CBC abnormalities included a normocytic normochromic anemia and thrombocytopenia. SBP abnormalities included hypoproteinemia, hypocalcemia, and mild azotemia. A protein-losing nephropathy (PLN) was documented by urine dipstick and protein/creatinine ratio. Blood samples were R. rickettsii seroreactive and Rickettsia PCR positive, and convalescent titers demonstrated 4-fold seroconversion ( Table 2).  The year before, in August 2018, the dog described in case 3 was examined by a veterinarian for lethargy after tick attachment. At that time, fever (39.7°C), anemia, thrombocytopenia, hyperbilirubinemia, and hypoproteinemia were documented. IFA serology tests performed by 1 diagnostic laboratory showed samples were R. rickettsii seroreactive (1:320) but seronegative for Anplasma spp., Borrelia burdorferi, and Ehrlichia spp. by SNAP 4Dx Plus (IDEXX Laboratories, https://www.idexx.com). Doxycycline and immunosuppressive doses of prednisone were administered concurrently for RMSF and potential immune-mediated disease. Clinical and hematologic abnormalities resolved, and treatment was transitioned from prednisone to cyclosporine due to adverse side effects. Cyclosporine was discontinued in January 2019 and serial monthly CBCs remained normal through March 2019. When rechecked on August 9, 2019, for joint pain, hematocrit and platelet count were normal, but hypoproteinemia, hypoalbuminemia, and hypocalcemia were detected. By August 30, 2019, the dog's anemia and thrombocytopenia worsened, despite treatment with doxycycline and prednisone. Marked abdominal effusion was documented by abdominal ultrasound, without evidence of an intra-abdominal mass. Prednisone and mycophenolate were administered for presumptive immune-mediated thrombocytopenia, and within 3 weeks, the platelet count normalized and titers increased by 4-fold. Despite medical therapy for PLN, nephrotic syndrome developed, and the dog was euthanized.
We obtained identical Rickettsia DNA gene sequences from each dog's blood specimen. We confirmed novel Rickettsia sp. by PCR targeting 3 genes (gltA, htrA, and ompA) and 2 intergenic spacer re- Figure. Multilocus phylogenetic tree of Rickettsia spp. obtained from a dog with Rocky Mountain spotted fever-type symptoms in 2019 (bold) compared with reference sequences. We noted 3 dogs with RSMF symptoms. Rickettsia DNA were identical among all 3 cases; however, complete sequences from all 5 regions were obtained only from case 3, which we used to represent the novel Rickettsia species strain 2019-CO-FNY. We used 2,576 nucleotides concatenated from regions within 3 genes (gltA, htrA, and ompA) and 2 intergenic spacer regions (23S-5S and mmpA-purC). We used the maximum-likelihood method and Tamura-Nei model (6,7) optimized for branch length, topology, and substitution rate to assemble the tree by using the PhyML 3.3.20180621 plugin in Geneious Prime 11.0.0+7 (https:// www.geneious.com). Numbers at nodes indicate bootstrap percentages obtained from 1,000 resamplings. Numbers in parentheses are GenBank accession numbers. The tree is drawn to scale. Scale bar indicated the number of nucleotide substitutions per site.
gions (23S-5S and mmpA-purC) ( Table 2). Rickettsia amplicons were 100% identical among the 3 dogs. We amplified a larger region of the ompA and gltA genes by using 3 different quantitative PCRs from case 3. We submitted sequences from this dog's serum samples to GenBank (accession nos. MT050445-8 and MT066187). We also used the Rickettsia sequences from case 3 to generate a phylogenetic tree (Table 2) based on concatenated novel Rickettsia sp. DNA sequences and reference Rickettsia spp. We generated the phylogenetic tree by using the maximum-likelihood method based on the Tamura-Nei model (Figure) (6,7). Multilocus phylogenetic analysis placed the novel Rickettsia sp. in a clade among SFG Rickettsia between the human pathogens R. heilongjiangensis and R. massiliae. We attempted cell culture isolation of the Rickettsia sp. from whole blood but were unsuccessful (Appendix, https://wwwnc.cdc.gov/EID/ article/26/12/20-0272-App1.pdf).

Conclusions
We report similar illnesses among 3 dogs from different US states associated with tick exposures occurring in summer months. All 3 cases demonstrated fever, lethargy, and thrombocytopenia, abnormalities commonly associated with RMSF. Case 1 had a typical acute onset fever and rapidly responded to treatment with doxycycline; case 2 had a neutrophilic polyarthritis, which has been associated with RMSF in dogs (8). Case 3 was examined for acute onset febrile illness 1 year before the novel Rickettsia sp. infection was documented; Rickettsia IFA seroreactivity was documented on both occasions. This dog likely had an unidentified, concurrent disease process that contributed to PLN.
The cases were geographically distributed among 4 states; the dogs resided in Illinois, Oklahoma, and Tennessee, but the dog from Illinois had traveled to a tick-infested area of Arkansas. The tick species were not identified, but ticks common to these states include Amblyomma americanum, Dermacentor variabilis, and Rhipicephalus sanguineus sensu lato, all of which are known to transmit Rickettsia (3). Haemophysalis longicornis, an invasive tick species recently confirmed in the United States, including in Tennessee and Arkansas, should be considered a potential vector for Rickettsia spp. (9,10).
Based on serologic cross-reactivity, presence of ompA, and phylogenetic tree analysis, the new Rickettsia sp. is an SFG Rickettsia, phylogenetically related to human pathogenic R. heilongjiangensis and R. massiliae, with only 95% identity to each (11,12). Thus, we report a previously unknown and unique Rickettsia sp. with clinical significance for dogs and potentially humans. Because this novel Rickettsia cross-reacts with R. rickettsia on IFA, it could be underdiagnosed and more geographically widespread. Studies aimed at identifying the tick vector, potential animal reservoirs, and prevalence are ongoing. These 3 canine rickettsioses cases underscore the value of dogs as sentinels for emerging tickborne pathogens (13,14).