Febrile illness associated with Rickettsia conorii infection in dogs from Sicily.

We report serologic and molecular evidence of acute, febrile illness associated with Rickettsia conorii in 3 male Yorkshire terriers from Sicily (Italy).

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Rickettsia conorii, transmitted by Ripicephalus sanguineus, causes Mediterranean spotted fever (MSF) in humans in Mediterranean countries, Sub Saharan Africa and Asia (1). Rickettsia spp. seroprevalence in dogs is high (26%-60%) in disease-endemic regions, and proximity to seroreactive dogs is a risk factor for MSF in humans (2,3). Recent studies reported the detection of Rickettsia DNA in the blood of European dogs (4,5). However, evidence that R. conorii infection causes illness in dogs is lacking (2,3,6). Illness has been associated with R. conorii natural infection in only 2 dogs since human MSF was described in 1932 (6). Moreover, the only clinical signs observed in experimentally infected dogs were pain, erythema, and edema at the injection site; and regional lymphadenopathy (6). We report infection with R. conorii ssp. conorii in 3 acutely ill, febrile Yorkshire terrier dogs, supported by PeR, DNA sequencing, and seroconversion.

The Study

Between May and September 2005, three unrelated intact male Yorkshire terriers with a mean age of 4.3 years from Catania, Sicily, were brought to a local veterinarian; the dogs had the following histories: anorexia and lethargy of 2 days' duration (dog 1); anorexia, lethargy, and intermittent lameness of a few days' duration (dog 2); and intermittent vomiting, anorexia, and lethargy of a few days' duration (dog 3). Despite living mostly indoors, all 3 dogs had a recent history of tick exposure. All dogs had received current vaccination histories and had no history of serious illness. Results of the physical examination and hematologic, biochemical, and serum electrophoresis abnormalities at the time of onset of clinical signs and after 1 month (dogs 2 and 3) and 2 months (dog 1) of follow-up are provided in Table 1. Treatments instituted for all 3 dogs at onset of illness are described in Table 1.

EDTA-blood and serum samples were obtained by the attending veterinarian at the time of clinical assessment (before treatment), then 1 week later and 1 month (dogs 2 and 3) or 2 months later (dog 1). DNA extraction was performed from whole blood samples (5,7). A quantitative PeR (qPCR) for detection of Rickettsia spp., Anaplasma phagocytophilum, Ehrlichia canis, and Leishmania infantum in DNA samples was performed by using a Light Cycler (Roche, Mannheim, Germany). PeR amplification was carried out with Rickettsia (Rr-prim3 5'-GAAACC GAAAGAGAATCTTCCGAT-3' and Rr-prim4 5'-TCC TAGTGTAGAGGTGAAATTCTTA-3' [8]), E. canis, A. phagocytophilum (fragment of 16S rRNA gene), and L. infantum LCSet primers and probes following manufacturer's instructions (TIB Molbiol, Centro Biotecnologie Avanzate, Genova, Italy) (5,7). Conventional Babesia genus PCR was performed (9). Borrelia burgdorferi sensu lato qPCR was performed by a commercial laboratory (www.scanelis.com). PCR results for all infectious agents listed above, with the exception of Rickettsia, were negative in all dogs.

PCRs for Rickettsia that use the outer membrane protein A (ompA) gene to amplify 632 bp (10) and 212 bp (107F 5'-GCTTTATTCACCACCTCAAC-3' and 299R 5'TRATCACCACCGTAAGTAAAT-3') (7) amplicons were performed. For dog 1, a 632-bp amplicon was cloned by using the TOPO TA Cloning (Invitrogen, Carlsbad, CA, USA) and sequenced (GenBank accession nos. DQ518245) (7). For dogs 2 and 3, a 212-bp amplicon was subjected to direct sequencing (accession no. DQ518246, DQ518247) (7). PeR results are summarized in Table 1.

Consensus sequences were aligned [(BIOEDIT version 7.0 (ClustalW)] with known sequences in GenBank using the basic local alignment search tool (BLAST; available from http://www.ncbi.nlm.nih.gov/BLAST/). The sequence obtained from all 3 dogs was 100% homologous to a portion of the complete genome sequence corresponding to the ompA gene from R. conorii (Malish 7, accession no. AE008674).

Immunofluorescent assays to detect antibodies to R. rickettsii, R. conorii, B. burgdorferi sensu stricto, E. canis, Babesia canis, A. phagocytophilum, L. infantum, Bartonella henselae, and B. vinsonii ssp. berkhoffi antigens were performed (3,7). Results are presented in Table 2.

Conclusions

Clinicopathologic abnormalities detected in these dogs at initial examination, including acute onset of fever, lethargy, thrombocytopenia, anemia, mildly increased liver enzyme activities and hypoalbuminemia, were very similar to abnormalities associated with spotted fever group (SFG) rickettsioses in dogs and humans (1). In addition, R. conorii DNA was amplified in all dogs during the acute illness. Further evidence for R. conorii infection as a cause of the associated clinical signs was provided by the subsequent failure to detect DNA in dogs 1 and 2, 1 week after treatment with doxycycline and the rapid resolution of clinical signs 2 days after initiating doxycycline therapy. Clinical signs in dog 3 resolved in 4 days, while the dog was receiving ceftriaxone, which has no known anti-rickettsial efficacy (1). Spontaneous immune clearance of R. conorii likely accounted for the resolution of clinical signs in dog 3 (6).

The 4-fold increase in IgG antibody titers in dogs 2 and 3 supports seroconversion, which is consistent with an acute R. conorii infection (11). Additionally, the initially high IgM titer in dog 1 after the onset of illness compared with a much lower IgM titer after 65 days is also supportive of an acute infection and is consistent with observations of human serologic test results (1). IgM titers rise rapidly and then disappear by day 35 and 80 in dogs experimentally infected with R. conorii and R. rickettsii, respectively (6,11). However, high R. rickettsii IgM titers are detected in dogs that do not seroconvert, based upon IgG antibodies (11). Thus, the presence of IgM supports but does not prove acute SFG infection in dogs.

Coinfection with A. phagocytophilum or B. burgdorferi could have contributed to clinical signs observed in dog 1. This dog had a low serum A. phagocytophilum titer 7 days after initial examination and also seroconverted to B. burgdorferi. A. phagocytophilum causes an acute febrile illness in dogs and humans, similar to the findings described here (12). B. burgdorferi does not cause clinical signs in dogs until 60-150 days after experimental infection (13); therefore, despite seroconversion, the acute clinical signs in dog 1 were not likely to have been related to B. burgdorferi infection. Moreover, PCR amplification of DNA from organisms other than R. conorii was not found in any dog.

All dogs were intact, male, genetically unrelated Yorkshire terriers. Although an increased risk for Rocky Mountain spotted fever has not been reported in Yorkshire terriers, purebred dogs infected with R. rickettsii appear to be more prone to clinical illness (14). Notably, this breed seems to be at increased risk for Babesia canis infection (15). Male dogs and men may be at increased risk for infection and may develop more severe illness with R. rickettsii and R. conorii (1,14), and male dogs are more likely to be R. conorii seroreactive (3). It has been suggested that more severe illness may develop in English springer spaniels with suspected phosphofructokinase deficiency and persons with glucose 6-phosphate dehydrogenase deficiency when infected with R. rickettsii and R. conorii (1,14). Although inherited immunodeficiencies have not been reported in Yorkshire terriers, and all dogs were previously healthy, an inherited metabolic or immunologic defect cannot be ruled out because specific testing was not performed.

Although a metabolic or immunologic defect may be necessary for illness to develop in dogs of various breeds after R. conorii infection, other potential explanations can be made for the discrepancy between high R. conorii seroprevalence rates among healthy dogs and lack of reports of clinical illness. The high R. conorii seroprevalence in healthy dog populations suggests that exposure to SFG rickettsiae is common. However, the acute, nonspecific, and potentially self-limiting nature of R. conorii infection, combined with a low index of suspicion by regional veterinarians and a historical lack of specific diagnostic techniques, may have precluded the prior association of clinical signs with R. conorii infection in dogs. Further evidence should be gathered regarding the extent to which R. conorii causes clinical disease in dogs.

Acknowledgments

We thank Silvia Beccaro, Julie Bradley, and Matthew Poore for serologic testing, Claudia Zampieron for molecular testing, and Barbara Hegarty for helpful discussions.

Dr Solano-Gallego is a veterinarian at the Private Veterinary Hospital and Laboratory San Marco (Padua, Italy). Her primary research interests include the study of vectorborne zoonotic diseases of dogs and cats.

References

(1.) Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin Microbiol Rev. 2005;18:719-56.

(2.) Segura-Porta F, Diestre-Ortin G, Ortuno-Romero A, Sanfeliu-Sala I, Font-Creus B, Munoz-Espin T, et al. Prevalence of antibodies to spotted fever group rickettsiae in human beings and dogs from an endemic area of Mediterranean spotted fever in Catalonia, Spain. Eur J Epidemiol. 1998;14:395-8.

(3.) Solano-Gallego LJ, Osso M, Hegarty B, Breitschwerdt E. A serological study of exposure to arthropod-borne pathogens in dogs from northeastern Spain. Vet Res. 2006;37:231-44.

(4.) Estrada-Pena A, Venzal Bianchi J. Efficacy of four anti-tick chemicals to break the transmission of Rickettsia conorii to dogs. In: Abstracts of the Fourth International Conference on Rickettsiae and Rickettsial Diseases, Logrono, Spain; 2005 Jun 18-21. P-100.

(5.) Solano-Gallego L, Razia L, Trotta T, Furlanello T, Caldin M. Molecular survey of Ehrlichia canis, Anaplasma phagocytophilum and Rickettsia spp. from blood of dogs living in Italy. In: Abstracts of the Fourth International Conference on Rickettsiae and Rickettsial Diseases, Logrono, Spain; 2005 Jun 18-21. P-107.

(6.) Kelly PJ, Matthewman LA, Mason PR, Courtney S, Katsande C, Rukwava J. Experimental infection of dogs with a Zimbabwean strain of Rickettsia conorii. J Trop Med Hyg. 1992;95:322-6.

(7.) Kidd LB. Evaluation of a PCR assay for detection of spotted fever group Rickettsia in dog blood. North Carolina State University; 2006 [PhD dssertation].

(8.) Breitschwerdt EB, Papich MG, Hegarty BC, Gilger B, Hancock SI, Davidson MG. Efficacy of doxycycline, azithromycin, or trovafloxacin for treatment of experimental Rocky Mountain spotted fever in dogs. Antimicrob Agents Chemother. 1999;43:813-21.

(9.) Carret C, Walas F, Carcy B, Grande N, Precigout E, Moubri K, et al. Babesia canis canis, Babesia canis vogeli, Babesia canis rossi: differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal RNA genes. J Eukaryot Microbiol. 1999;46:298-303.

(10.) Roux V, Fournier PE, Raoult D. Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein rOmpA. J Clin Microbiol. 1996;34:2058-65.

(11) Breitschwerdt EB, Levy MG, Davidson MG, Walker DH, Burgdorfer W, Curtis BC, et al. Kinetics of IgM and IgG responses to experimental and naturally acquired Rickettsia rickettsii infection in dogs. Am J Vet Res. 1990;51:1312-6.

(12.) Neer TM, Breitscbwerdt EB, Greene RT, Lappin MR. Consensus statement on ehrlichial disease of small animals from the infectious disease study group of the ACVIM. American College of Veterinary Internal Medicine. J Vet Intern Med. 2002;16:309-15.

(13.) Grcene CE, Straubinger RK. Borreliosis. In: Green C, ed. Infectious diseases of the dog and cat. 3rd ed. Philadelphia: Saunders, Elsevier; 2006. p. 417-34.

(14.) Greene CE, Breitschwerdt EB. Rocky Mountain spotted fever, murine typhuslike disease, rickettsialpox, typhus, and Q fever. In: Greene C, editor. Infectious diseases of the dog and cat. 3rd ed. Philadelphia: Saunders, Elsevier; 2006. p. 232-45.

(15.) Martinod S, Laurent N, Moreau Y. Resistance and immunity of dogs against Babesia canis in an endemic area. Vet Parasitol. 1986;19:245-54.

Laia Solano-Gallego, * Linda Kidd, ([dagger]) Michele Trotta, * Marco Di Marco, ([double dagger]) Marco Caldin, * Tommaso Furlanello, * and Edward Breitschwerdt ([dagger])

* Clinica e Laboratorio Veterinario Privato "San Marco," Padova, Italy; ([dagger]) North Carolina State University, Raleigh, North Carolina, USA; and ([double dagger]) Clinica Veterinaria, Catania, Italy

Address for correspondence: Laia Solano-Gallego, Clinica e Laboratorio Veterinario Privato "San Marco," Via Sorio 114/c 35141, Padova, Italy; email: [e-mail]

Table 1. Clinical and laboratory data at the time of initial
and follow-up examinations in 3 dogs with serologic and molecular
evidence of natural Rickettsia conorii infection *

Date of
evaluation,
2005                       Physical examination abnormalities

Dog 1
  May 31                  Fever (40.1[degrees]C), tachycardia,
  ([double dagger])   mildly enlarged right popliteal lymph node,
  ([section])           blepharitis, hunched posture, stiff gait

  Jun 8                             No abnormalities

  Aug 8                             No abnormalities

Dog 2
  Sep 19              Fever (41[degrees]C), ptyalism, joint pain,
  ([double dagger])            lameness in right rear limb
  ([section])

  Sep 28                            No abnormalities

  Oct 25                            No abnormalities

Dog 3
  Sep 17                 Fever (41[degrees]C), abdominal pain,
  ([section]) #         dehydration, peripheral lymphadenomegaly
                        (popliteal and prescapular lymph nodes),
                                     conjunctivitis

  Sep 23                            No abnormalities
  ([double dagger])

  Oct 25                            No abnormalities

Date of                        Hematologic, biochemical,
evaluation,                    and serum electrophoresis
2005                            abnormalities ([dagger])

Dog 1
  May 31               Left shift neutrophilia (segmented 11,700;
  ([double dagger])     bands: 468) and thrombocytopenia (112).
  ([section])             [up arrow] ALT (112), hypoproteinemia
                      (5.0) and hypoalbuminemia (40.4). [up arrow]
                       [[alpha].sub.2-] (16.5) and [beta].sub.1-]
                             globulins (13.9) ([paragraph])

  Jun 8                      Mature neutrophilia (16,400),
                        hyperglobulinemia (4.3); [up arrow] CRP
                       (2.31); [up arrow] GGT (10.4); [up arrow]
                       [[alpha].sub.2-] (18.5), [gamma]-globulins
                             (23.3); hypoalbuminemia (40.9)

  Aug 8                             No abnormalities

Dog 2
  Sep 19               Microcytic-hyperchromic anemia (MCHC 63.7;
  ([double dagger])      MCV 56; Hct 25.3); mature neutrophilia
  ([section])                    (11,680) ([paragraph])

  Sep 28                Hyperglobulinemia (4.3); [up arrow] CRP
                      (0.64); [up arrow] [[alpha].sub.2-] (17.5),
                      and [gamma]-globulin (24.6); hypoalbuminemia
                                         (41.1)

  Oct 25                            No abnormalities

Dog 3
  Sep 17              Thrombocytopenia (69), hypoproteinemia (5.3);
  ([section]) #            hypoalbuminemia (28.6); [up arrow]
                         [[alpha].sub.2-] (14.8), 131- (13.0),
                        [beta].sub.2-] (15.6), [gamma]-globulins
                                  (23.0) ([paragraph])

  Sep 23                Lymphocytosis (5,594); hyperglobulinemia
  ([double dagger])   (4.1); [up arrow] CRP (2.7); [up arrow] BUN
                       (75); [up arrow] [[alpha].sub.2]-globulin
                             (19.3); hypoalbuminemia (44.9)

  Oct 25                           [up arrow] BUN (54)

                      Rickettsia
Date of                (16S and
evaluation,           ompA gene)
2005                      PCR

Dog 1
  May 31               Positive
  ([double dagger])
  ([section])

  Jun 8                Negative

  Aug 8                Negative

Dog 2
  Sep 19               Positive
  ([double dagger])
  ([section])

  Sep 28               Negative

  Oct 25               Negative

Dog 3
  Sep 17               Positive
  ([section]) #

  Sep 23               Negative
  ([double dagger])

  Oct 25               Negative

* ALT, alanine aminotransferase; CRP, C-reactive protein; GGT,
[gamma]-glutamyl transferase; MCHC, mean cell hemoglobin
concentration; MCV, mean cell volume; Hct, hematocrit; BUN,
blood urea nitrogen; [up arrow], increase.

([dagger]) Reference interval: Hct 38.6%-54.5%; MCV 61-72 fL;
MCHC 34-38 g/dL; segmented neutrophils 3,800-8,800/[micro]L; bands
neutrophils 0-300/[micro]L; lymphocytes 1,300-4,100/[micro]L;
platelets 160-440 x [10.sup.3]/[micro]L); total protein 5.5-7.5
g/[micro]L; globulins 2.6-4.0 g/dL; albumin 53%-65%;
[[alpha].sub.2]-globulins 8.0%-14.0%; [[beta].sub.1]-globulins
2.0%-5.0%; [[beta].sub.2]-globulins 3.0%-9.0 %; [gamma]-globulins
6.0%-15.0%); BUN 18-43 mg/dL; CRP 0.0-0.15 mg/dL; ALT 15-65 IU/L;
GGT 2.0-0.0 IU/L.

([double dagger])Treatment with doxycycline (10 mg/kg/once a day
by mouth/1 month) was started.

([paragraph]) CRP was not measured

([section]) Intravenous fluids were administered.

(#) Treatment with ceftriaxone (30 mg/kg/twice a day intravenously/
for 5 days) was started.

Table 2. Reciprocal IFA titers for the 3 dogs with clinical
and molecular evidence of natural Rickettsia conorii infection *

Dog   Date, 2005          R. conorii            R. rickettsii
no.   (days after
       clinical        IgM         IgG         IgM         IgG
        signs)

1      Jun 8 (7)     1:1,280    1:20,480     1:1,280    1:20,480
1     Aug 3 (65)      1:80        1:320       1:80        1:320
2     Sep 19 (0)      1:640       1:80        1:640       1:80
2     Sep 28 (9)     11,280     1:40,960      1:640     1:20,480
2     Oct 25 (37)     1:640     1:10,240      1:640      1:5,120
3     Sep 17 (0)     1:1,280      1:80       1:2,560       Neg
3     Sep 23 (6)    1:10,240    1:10,240    1:10,240     1:5,120
3     Oct 25 (39)    1:2,560     1:5,120     1:5,120     1:5,120

Dog   Date, 2005       Anaplasma       Borrelia
no.   (days after   phagocytophilum   burgdorferi
       clinical
        signs)            IgM             IgG

1      Jun 8 (7)         1:160            Neg
1     Aug 3 (65)          Neg            1:160
2     Sep 19 (0)          Neg             Neg
2     Sep 28 (9)          Neg             Neg
2     Oct 25 (37)         Neg             Neg
3     Sep 17 (0)          Neg             Neg
3     Sep 23 (6)          Neg             Neg
3     Oct 25 (39)         Neg             Neg

* IFA, immunofluorescence assay; Ig, immunoglobulin; Neg, negative.

([dagger]) All dogs were seronegative for L. infanturn, E. canis,
B. canis, B. henselae, and B. vinsonii ssp. berkhoffi at all time
points