Maria L. Zambrano

Isolation and Identification of Rickettsia massiliae from Rhipicephalus sanguineus Ticks Collected in Arizona

ABSTRACT Twenty Rhipicephalus sanguineus ticks collected in eastern Arizona were tested by PCR assay to establish their infection rate with spotted fever group rickettsiae. With a nested PCR assay which detects a fragment of the Rickettsia genus-specific 17-kDa antigen gene (htrA), five ticks (25%) were found to contain rickettsial DNA. One rickettsial isolate was obtained from these ticks by inoculating a suspension of a triturated tick into monolayers of Vero E6 monkey kidney cells and XTC-2 clawed toad cells, and its cell culture and genotypic characteristics were determined. Fragments of the 16S rRNA, GltA, rOmpA, rOmpB, and Sca4 genes had 100%, 100%, 99%, 99%, and 99%, respectively, nucleotide similarity to Rickettsia massiliae strain Bar29, previously isolated from R. sanguineus in Catalonia, Spain (L. Beati et al., J. Clin. Microbiol. 34:2688-2694, 1996). The new isolate, AZT80, does not elicit cytotoxic effects in Vero cells and causes a persistent infection in XTC-2 cells. The AZT80 strain is susceptible to doxycycline but resistant to rifampin and erythromycin. Whether R. massiliae AZT80 is pathogenic or infectious for dogs and humans or can cause seroconversion to spotted fever group antigens in the United States is unknown.

Detection and Identification of Rickettsial Agents in Ticks From Domestic Mammals in Eastern Panama

ABSTRACT Several outbreaks of Rocky Mountain spotted fever have occurred in recent years in Colombian communities close to the border with Panama. However, little is known about rickettsiae and rickettsial diseases in eastern Panamanian provinces, the Darien Province and the Kuna Yala, located north of the endemic area in Colombia. In 2007, 289 ticks were collected in several towns from dogs, horses, mules, cows, and pigs. DNA was extracted from 124 Dermacentor nitens, 64 Bhipicephalus sanguineus, 43 Amblyomma ovale, 35 A. cajennense, 10 Boophilus microplus, 4 A. oblongoguttatum, and 9 A. cajennense nymphs. SYBR-Green polymerase chain reaction assays targeting a fragment of the OmpA and 16S rRNA genes were used for detection of DNA of the spotted fever group rickettsiae (SFGR) and Anaplasmataceae (Anaplasma and Ehrlichia), respectively. In total, 37.4% ticks were positive for SFGR, including 20.3% R. sanguineus, 27.9% A. ovale, 25.8% D. nitens, 50% B. microplus, 50% A. oblongoguttatum, and 100% A. cajennense. The presence of Rickettsia amblyommii DNA was confirmed by sequencing in A. cajennense, A. oblongoguttatum, A. ovale, B. microplus, and R. sanguineus. DNA of R. rickettsii was only detected in one D. nitens collected from a horse in Santa Fe, Darien Province. Prevalence of Anaplasmataceae varied from 6.3% in R. sanguineus to 26.5% in A. cajennense. DNA of Ehrlichia chaffensis was found in three D. nitens and three A. cajennense from horses. This is the first study providing molecular characterization and prevalence information on SFGR in ticks from these areas and thus will be helpful for future evaluations of the risk of rickettsial diseases for individuals living in this region.

A Focus of Dogs and Rickettsia massiliae―Infected Rhipicephalus sanguineus in California

A recurrent focus of Rhipicephalus sanguineus infestation was investigated in a suburban area of southern California after reports of suspected Rocky Mountain spotted fever in two dogs on the same property. Abundant quantities of Rh. sanguineus were collected on the property and repeatedly from each dog, and Rickettsia massiliae DNA was detected by polymerase chain reaction (PCR). Whole blood and serum samples from four dogs were tested by using PCR and microimmunofluorescent assay for antibodies against spotted fever group rickettsiae. Serum samples from all four dogs contained antibodies reactive with R. massiliae, R. rhipicephali, R. rickettsii, and 364D Rickettsia but no rickettsial DNA was detected by PCR of blood samples. Serum cross-absorption and Western blot assays implicated R. massiliae as the most likely spotted fever group rickettsiae responsible for seropositivity. To our knowledge, this is the first detection of R. massiliae in ticks in California.

Molecular Typing of Novel Rickettsia rickettsii Isolates from Arizona

Abstract:  Seven isolates of Rickettsia rickettsii were obtained from a skin biopsy, two whole‐blood specimens, and from Rhipicephalus sanguineus ticks from eastern Arizona. Molecular typing of seven isolates of R. rickettsii and DNA samples from two other Rh. sanguineus ticks infected with R. rickettsii was conducted by PCR and DNA sequencing of rompA and 12 variable‐number tandem repeat regions (VNTRs). All DNA specimens from Arizona were identical to each other and to reference human and Dermacentor andersoni isolates of R. rickettsii from Montana in their rOmpA gene sequences and 10 VNTRs. Two of the twelve VNTRs had differences in the number of repeat sequences in isolates from Arizona compared to those from Montana, thus conferring the novelty of the Rh. sanguineus‐associated R. rickettsii

Rickettsia felis in cat fleas, Ctenocephalides felis parasitizing opossums, San Bernardino County, California

Los Angeles and Orange Counties are known endemic areas for murine typhus in California; however, no recent reports of flea‐borne rickettsioses are known from adjacent San Bernardino County. Sixty‐five opossums (Didelphis virginiana) were trapped in the suburban residential and industrial zones of the southwestern part of San Bernardino County in 2007. Sixty out of 65 opossums were infested with fleas, primarily cat fleas, Ctenocephalides felis (Bouché, 1835). The flea minimum infection rate with Rickettsia felis was 13.3% in pooled samples and the prevalence was 23.7% in single fleas, with two gltA genotypes detected. In spite of historic records of murine typhus in this area, no evidence for circulation of R. typhi in fleas was found during the present study. Factors contributing to the absence of R. typhi in these cat fleas in contrast to its presence in cat fleas from Orange and Los Angeles Counties are unknown and need to be investigated further in San Bernardino County.

Rickettsia felis in Ctenocephalides felis from Guatemala and Costa Rica

Rickettsia felis is an emerging human pathogen associated primarily with the cat flea Ctenocephalides felis. In this study, we investigated the presence of Rickettsia felis in C. felis from Guatemala and Costa Rica. Ctenocephalides felis were collected directly from dogs and cats, and analyzed by polymerase chain reaction for Rickettsia-specific fragments of 17-kDa protein, OmpA, and citrate synthase genes. Rickettsia DNA was detected in 64% (55 of 86) and 58% (47 of 81) of flea pools in Guatemala and Costa Rica, respectively. Sequencing of gltA fragments identified R. felis genotype URRWXCal(2) in samples from both countries, and genotype Rf2125 in Costa Rica. This is the first report of R. felis in Guatemala and of genotype Rf2125 in Costa Rica. The extensive presence of this pathogen in countries of Central America stresses the need for increased awareness and diagnosis.

Rickettsia Species Isolated from Dermacentor occidentalis (Acari: Ixodidae) from California

Abstract The Pacific Coast tick (Dermacentor occidentalis Marx, 1892) is one of the most widely distributed and frequently encountered tick species in California. This tick is the primary vector of an unclassified spotted fever group rickettsial pathogen, designated currently as Rickettsia 364D, the etiologic agent of a recently recognized tick-borne rickettsiosis known as Pacific Coast tick fever. Despite intensified interest in this pathogen, important questions remain regarding its taxonomic status and possible variations in genotype among different strains that could influence its pathogenicity. Only the extensively passaged prototypical isolate (strain 364-D) is widely available to rickettsiologists and public health scientists worldwide. To achieve a larger, more geographically diverse, and contemporary collection of strains, 1,060 questing adult D. occidentalis ticks were collected from 18 sites across six counties in northern and southern California in 2016 and 2017. Fourteen ticks (1.3%) yielded DNA of Rickettsia 364D and from these, 10 unique isolates from Lake and Orange counties were obtained. Additionally, Rickettsia rhipicephali was detected in 108 (10.2%) ticks, from which eight isolates were obtained, and Rickettsia bellii in six (0.6%), from which three isolates were obtained. The panel of recently acquired, low-passage strains of Rickettsia 364D derived from this study could enhance opportunities for investigators to accurately determine the taxonomic standing of this agent and to develop specific diagnostic assays for detecting infections with Rickettsia 364D in ticks and humans.