Shell-vial culture and real-time PCR applied to Rickettsia typhi and Rickettsia felis detection.

Murine typhus is a zoonosis transmitted by fleas, whose etiological agent is Rickettsia typhi. Rickettsia felis infection can produces similar symptoms. Both are intracellular microorganisms. Therefore, their diagnosis is difficult and their infections can be misdiagnosed. Early diagnosis prevents severity and inappropriate treatment regimens. Serology can't be applied during the early stages of infection because it requires seroconversion. Shell-vial (SV) culture assay is a powerful tool to detect Rickettsia. The aim of the study was to optimize SV using a real-time PCR as monitoring method. Moreover, the study analyzes which antibiotics are useful to isolate these microorganisms from fleas avoiding contamination by other bacteria. For the first purpose, SVs were inoculated with each microorganism. They were incubated at different temperatures and monitored by real-time PCR and classical methods (Gimenez staining and indirect immunofluorescence assay). R. typhi grew at all temperatures. R. felis grew at 28 and 32 °C. Real-time PCR was more sensitive than classical methods and it detected microorganisms much earlier. Besides, the assay sensitivity was improved by increasing the number of SV. For the second purpose, microorganisms and fleas were incubated and monitored in different concentrations of antibiotics. Gentamicin, sufamethoxazole, trimethoprim were useful for R. typhi isolation. Gentamicin, streptomycin, penicillin, and amphotericin B were useful for R. felis isolation. Finally, the optimized conditions were used to isolate R. felis from fleas collected at a veterinary clinic. R. felis was isolated at 28 and 32 °C. However, successful establishment of cultures were not possible probably due to sub-optimal conditions of samples.

The role of cats in the eco-epidemiology of spotted fever group diseases.

BACKGROUND: Mediterranean Spotted Fever (MSF), whose etiological agent is R. conorii, is one of the oldest described vector-borne infectious diseases. Although it is endemic in the Mediterranean area, clinical cases have also been reported in other regions. R. massiliae-Bar29 is related to MSF cases. This strain is distributed worldwide. R. conorii and R. massiliae-Bar29 are transmitted by ticks. Dogs are considered the sentinel of R. conorii infection. Cats could also be involved in their transmission. Rickettsia felis, etiological agent of Flea-borne spotted fever, is mainly transmitted by the cat flea, Ctenocephalides felis. Up to now, the role of cats in its transmission is not entirely elucidated. The aim of the study is to analyze the infection in cats by these microorganisms. METHODS: The study was undertaken in Northeastern Spain. Twenty municipalities of seven regions participated in the study. 212 cats (pets and stray cats) were analyzed. Variables surveyed were: date of collection, age, sex, municipality, source, living place, outdoor activities, health status, type of disease, contact with other animals, and ectoparasite infestation. Sera were evaluated by indirect immunofluorescence antibody assay (IFA). Molecular detection (real-time PCR and sequencing) and cultures were performed on blood samples. RESULTS: There were 59 (27.8%) cats seroreactive to one or more microorganisms. Considering cross-reactions, the seroprevalences were 15.6%-19.5% (R. massiliae-Bar29), 1.9%-6.2% (R. conorii), and 5.2%-7.5% (R. felis). A weak association was observed between SFG seropositivity and tick infestation. Ticks found on seropositive cats were Rhipicephalus pusillus, R. sanguineus and R. turanicus. DNA of Rickettsia was detected in 23 cats. 21 of them could be sequenced. Sequences obtained were identical to those sequences of SFG rickettsiae similar to R. conorii and R. massiliae. No amplification of R. felis was obtained. CONCLUSIONS: Cats can be infected by SFG rickettsiae and produce antibodies against them. Cats may play a role in the transmission cycle of R. conorii and R. massiliae-Bar29, although the role in the R. felis cycle needs further analysis.

Molecular detection of Rickettsia typhi in cats and fleas.

BACKGROUND: Rickettsiatyphi is the etiological agent of murine typhus (MT), a disease transmitted by two cycles: rat-flea-rat, and peridomestic cycle. Murine typhus is often misdiagnosed and underreported. A correct diagnosis is important because MT can cause severe illness and death. Our previous seroprevalence results pointed to presence of human R. typhi infection in our region; however, no clinical case has been reported. Although cats have been related to MT, no naturally infected cat has been described. The aim of the study is to confirm the existence of R. typhi in our location analyzing its presence in cats and fleas. METHODOLOGY/PRINCIPAL FINDINGS: 221 cats and 80 fleas were collected from Veterinary clinics, shelters, and the street (2001-2009). Variables surveyed were: date of collection, age, sex, municipality, living place, outdoor activities, demographic area, healthy status, contact with animals, and ectoparasite infestation. IgG against R. typhi were evaluated by indirect immunofluorescence assay. Molecular detection in cats and fleas was performed by real-time PCR. Cultures were performed in those cats with positive molecular detection. Statistical analysis was carried out using SPSS. A p < 0.05 was considered significant. Thirty-five (15.8%) cats were seropositive. There were no significant associations among seropositivity and any variables. R. typhi was detected in 5 blood and 2 cultures. High titres and molecular detection were observed in stray cats and pets, as well as in spring and winter. All fleas were Ctenocephalides felis. R. typhi was detected in 44 fleas (55%), from shelters and pets. Co-infection with R. felis was observed. CONCLUSIONS: Although no clinical case has been described in this area, the presence of R. typhi in cats and fleas is demonstrated. Moreover, a considerable percentage of those animals lived in households. To our knowledge, this is the first time R. typhi is detected in naturally infected cats.

The role of dogs in the eco-epidemiology of Rickettsia typhi, etiological agent of Murine typhus.

Rickettsia typhi, etiological agent of Murine typhus (MT), is transmitted to humans from an animal reservoir through two cycles: a classic rat-flea-rat cycle, and a peridomestic animal cycle. There are not many studies concerning which animals are involved in the peridomestic cycle, and most of them are focused on cats. The aim of this study was to determine the presence of R. typhi in dogs, not only by serological methods but also by direct methods such as culture and molecular detection. Two hundred and one dog blood samples were collected from Veterinary clinics, kennels, and shelters in Northeastern Spain (2006-2008). Age, sex, municipality, living place, healthy status, contact with animals, and ectoparasite infestations were surveyed. IgG was measured by IFA. Titers ≥ 1/64 were considered positive. Cultures were carried out using samples of dogs with titers ≥ 1/128. The molecular detection was performed by real-time PCR. Nine dogs (4.5%) were positive according to IFA (5: 1/64; 3: 1/128; 1: 1/512). There were no significant differences in the rates of antibodies related to any of the variables. Rickettsial DNA was detected in two cultures. Sequences obtained were identical to those of R. typhi. The results show direct and indirect evidences of the presence of R. typhi infection in dogs.

Prevalence of Bartonella henselae in cats in Catalonia, Spain.

Bartonella henselae, an emerging pathogen bacterium, is the main causative agent of the cat scratch disease. While the first clinical descriptions were associated with immunosupressed patients, it is now more frequently observed in patients with normal immune status (endocarditis and bacteremia). Cats were found to be the only known reservoir of B. henselae. In this paper, we report the results obtained in the first study made to investigate the prevalence of B. henselae bacteremia and antibodies in domestic cats in Catalonia, Spain. Serum samples from 115 cats were tested for antibodies to B. henselae by immunofluorescent antibody testing, and 29.6% had a titer >or= 1:64. Seven B. henselae strains were isolated using standard culture techniques and amplification by a polymerase chain reaction and subsequent sequencing was performed on the intergenic spacer region between the 16 and 23S ribosomal RNA genes. Of all factors concerning the studied bacteremia rate (age, sex, habitat, presence of antibodies, contact with animals, parasites), only the presence of antibodies to B. henselae was statistically significant.