Salivary antigens of the cat flea, Ctenocephalides felis felis

The cat flea, Ctenocephalides felis felis, is the major cause of flea bite hypersensitivity (FBH) in dogs and cats, yet little progress has been reported on identifying the antigens responsible. We obtained flea salivary antigens by washing secretions from containers probed by the mouthparts of fleas, and by extracting whole flea salivary glands. Mice were exposed to feeding fleas to generate antibodies to salivary antigens injected in vivo . The sera were tested for antibodies against the salivary antigens described and against a whole flea extract; in indirect ELISA, antibodies to salivary secretions were detected in 60% of the sera from mice exposed to feeding fleas. These sera identified four protein bands at apparent MW 56, 54, 42 and 40 K which corresponded to prominent protein bands in whole salivary gland extracts identified by protein staining after SDS-PAGE. Fixed sections of whole fleas exposed to the antisera showed that only structures within the salivary glands were identified. The salivary secretions and gland extracts are now being used to study immune responses of dogs suffering from FBH.     

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.     

Molecular evidence supports the role of dogs as potential reservoirs for Rickettsia felis

Rickettsia felis causes flea-borne spotted fever in humans worldwide. The cat flea, Ctenocephalides felis, serves as vector and reservoir host for this disease agent. To determine the role of dogs as potential reservoir hosts for spotted fever group rickettsiae, we screened blood from 100 pound dogs in Southeast Queensland by using a highly sensitive genus-specific PCR. Nine of the pound dogs were positive for rickettsial DNA and subsequent molecular sequencing confirmed amplification of R. felis. A high prevalence of R. felis in dogs in our study suggests that dogs may act as an important reservoir host for R. felis and as a potential source of human rickettsial infection.     

Polygenic detection of Rickettsia felis in cat fleas (Ctenocephalides felis) from Israel

The presence of Rickettsia felis, an emerging bacterial pathogen, was investigated in 79 cat flea (Cteno-cephalides felis) pools from Israel (5 to 20 fleas each) by polymerase chain reaction (PCR) and sequencing of 5 different genes. Amplified targets included both metabolic (gltA and fusA) and surface antigen (ompA, ompB, and the 17-kDa antigen) genes. R. felis DNA was detected in 7.6% of the flea pools. Two genotypes similar in their housekeeping gene sequences but markedly different in their surface antigenic genetic milieus were characterized. This is the first detection of this flea-transmitted rickettsia within its vector in Israel and the Middle East. Although no clinical case has been reported in human beings in Israel to date, these findings suggest that this infection is prevalent in Israel.     

First report of the isolation and molecular characterization of Rickettsia amblyommii and Rickettsia felis in Central America

During 2010, 15 adult ticks, identified as Amblyomma cajennense, were collected from horses in Cahuita and Turrialba districts, whereas 7 fleas, identified as Ctenocephalides felis, were collected from a dog in San Jose city, Costa Rica. In the laboratory, three A. cajennense specimens, two from Cahuita and one from Turrialba, were individually processed for rickettsial isolation in cell culture, as was a pool of seven fleas. Rickettsiae were successfully isolated and established in Vero cell culture from the three ticks and from a pool of seven fleas in C6/36 cell culture. The three tick isolates were genotypically identified as Rickettsia amblyommii, and the flea isolate was identified as Rickettsia felis through DNA sequencing of portions of the rickettsial genes gltA, ompA, and ompB of each isolate. In addition, other seven ticks were shown to contain rickettsial DNA. Polymerase chain reaction products of at least two of these ticks were sequenced and also showed to correspond to R. amblyommii. Overall, 66.7% (10/15) of the A. cajennense adult ticks were found to be infected with rickettsiae. This is the first report of a successful isolation in cell culture of R. amblyommii and R. felis from Central America.     

Rickettsia felis and Bartonella henselae in fleas from Lebanon

A total of 155 fleas collected in 2009 in Lebanon from 16 cats (104 Ctenocephalides felis specimens, 1 C. canis specimen) and 2 dogs (50 C. canis specimens) were tested for the presence of Rickettsia spp. and Bartonella spp. using molecular methods, including real-time quantitative polymerase chain reaction (PCR), regular PCR, and sequencing of amplified PCR products. Rickettsia felis, the agent of the emerging flea-borne spotted fever in humans, was identified in 17 (16%) C. felis cat fleas. Bartonella henselae, an agent of cat scratch disease, was identified in three (2.9%) C. felis. Our results emphasize the potential risk of these emerging flea-borne infections in Lebanon.     

Ultrastructural study of the infection process of Rickettsia conorii in the salivary glands of the vector tick Rhipicephalus sanguineus

This work was designed to study the infection process of Rickettsia conorii in the salivary glands of experimentally infected Rhipicephalus sanguineus ticks. One hundred six uninfected engorged nymphs were intracelomically inoculated with approximately 2 x 10(3) plaque-forming units of a rickettsial suspension. After the molt, unfed and fed adults were dissected, and the salivary glands were extracted and processed for transmission electron microscopy observation. Three different uninfected control groups were used for (1) evaluating the impact of the inoculation procedure, (2) establishing the feeding period of infected ticks, and (3) ultrastructural characterization of the salivary glands. Overall, 75.5% (80 of 106) of the nymphs inoculated with rickettsiae died during the molt or soon after hatching into adult instars; 50% (12 of 24) of the remaining infected adults showed severe malformations compromising their viability. In apparently healthy specimens, time of engorgement was longer. The contrast with the negative control groups was statistically significant, suggesting that R. conorii exerts a strong negative effect on the vector ticks. The ultrastructural study showed that in the salivary glands of infected ticks, rickettsial growth occurs preferentially in central, peripheral, and interstitial acini cells.     

Molecular detection and characterization of spotted fever group rickettsiae in Taiwan

Rickettsioses are emerging infectious diseases caused by rickettsiae in association with arthropods. We report the detection of spotted fever group rickettsiae (SFGR) in Taiwan using molecular methods. Phylogenetic analyses of the 17-kd protein and citrate synthase (gltA) genes showed that SFGR TwKM01 detected in Rhipicephalus haemaphysaloides ticks was most similar to Rickettsia rhipicephali. Three TwKM01 isolates were obtained from three individual R. haemaphysaloides ticks. Small, intracellular, coccobacillary bacteria were found in infected L929 cells using immunofluorescence antibody testing and transmission electron microscopy. Two other SFGRs, TwKM02 and TwKM03, identified in Leptotrombidium chigger mites, were closely related to R. australis and R. felis URRWXCal(2), respectively. The TwKM03 strain was also detected in Ixodes granulatus ticks and widely distributed in Hualien, Kinmen, and Lienchiang counties in Taiwan. The endonucleases MaeII and HhaI selected for restriction fragment length polymorphism analysis of the gltA and 17-kd polymerase chain reaction products, respectively, were useful for genotyping Rickettsia species TwKM01, TwKM02, TwKM03, and other SFGRs. Although their infectivity and pathogenicity for vertebrates are unknown, the finding of SFGRs raises the possibility that bacteria other than Orientia tsutsugamushi, Coxiella burnetii, and R. typhi may be involved in rickettsial diseases in Taiwan.     

Rickettsia typhi IN RODENTS AND R. felis IN FLEAS IN YUCATáN AS A POSSIBLE CAUSAL AGENT OF UNDEFINED FEBRILE CASES

Rickettsia typhi is the causal agent of murine typhus; a worldwide zoonotic and vector-borne infectious disease, commonly associated with the presence of domestic and wild rodents. Human cases of murine typhus in the state of Yucatán are frequent. However, there is no evidence of the presence of Rickettsia typhi in mammals or vectors in Yucatán. The presence of Rickettsia in rodents and their ectoparasites was evaluated in a small municipality of Yucatán using the conventional polymerase chain reaction technique and sequencing. The study only identified the presence of Rickettsia typhi in blood samples obtained from Rattus rattus and it reported, for the first time, the presence of R. felis in the flea Polygenis odiosus collected from Ototylomys phyllotis rodent. Additionally, Rickettsia felis was detected in the ectoparasite Ctenocephalides felis fleas parasitizing the wild rodent Peromyscus yucatanicus. This study’s results contributed to a better knowledge of Rickettsia epidemiology in Yucatán.     

Molecular detection of Rickettsia felis, Rickettsia typhi and two genotypes closely related to Bartonella elizabethae

A total of 56 fleas were collected from mice, rats, and one hedgehog in national parks of mainland Portugal and the Madeira Island. All fleas were tested for the presence of bacteria of the genera Rickettsia and Bartonella using PCR assays. In fleas from mainland Portugal, we detected Rickettsia felis in one Archaeopsylla erinacei maura flea and in one Ctenophtalmus sp. In five Leptopsylla segnis fleas taken from rats in the Madeira Island, we identified Rickettsia typhi. In addition, in four fleas from the genera Ornithophaga and Stenoponia collect from mice and a rat in mainland Portugal, we detected the presence of two new Bartonella genotypes closely related to Bartonella elizabethae. Our findings emphasize the potential risk of flea-transmitted infections in mainland Portugal and the Madeira archipelago, and extend our knowledge of the potential flea vectors of human pathogens.     

Bartonella henselae infection in domestic cat and dog fleas

We studied on the infection of domestic cat and dog fleas with Bartonella henselae by polymerase chain reaction (PCR). A total of 62 fleas (36 Ctenocephalidis felis from cats, 24 C. felis from dogs and 2 Ctenocephalidis canis from dogs), stored in 70% ethanol, were analyzed by PCR for B. henselae specific DNA. Of the 62 fleas, C. felis from cats and dogs were positive for B. henselae specific DNA in 12 of the 36 (33.3%) and in 5 of the 24 (20.8%), respectively, and C. canis from dogs was positive in 2 of the 2 (100%). Our results demonstrated that pet fleas were infected with B. henselae, and suggest that flea transmission of B. henselae between cats or dogs may occur, and direct transmission of B. henselae from pet fleas to human may cause cat scratch disease.     

Ectoparasites and associated pathogens of free-roaming and captive animals in zoos of South Carolina

A survey of ectoparasites and their associated pathogens was conducted in two South Carolina zoos, from 2004 to 2007. Dead, wild birds and mammals, as well as captive animals examined during routine veterinary checks constituted the study populations. Ectoparasites were tested for species of Anaplasma, Bartonella, Coxiella burnetii, Ehrlichia, Rickettsia, and Trypanosoma. Forty-six species of ectoparasites were collected from 133 free-roaming and captive hosts and their associated nesting and bedding materials. Six vector-borne pathogens were detected molecularly in the ectoparasites, including Anaplasma phagocytophilum in the tick Ixodes dentatus Marx from an eastern cottontail rabbit, Bartonella clarridgeiae in the cat flea Ctenocephalides felis (Bouché) from a Virginia opossum, Bartonella sp. Oh6 in the squirrel flea Orchopeas howardi (Baker) from an eastern grey squirrel, Bartonella sp. T7498 in the sucking louse Neohaematopinus sciuri Jancke from a squirrel, Rickettsia sp. Rf2125 in C. felis from a zookeeper and a grizzly bear, and Rickettsiales sp. Ib 2006 in Ixodes brunneus Koch from an American crow. While the pathology of some of these pathogens is poorly known, Anaplasma phagocytophilum (causative agent of human granulocytic anaplasmosis) and Bartonella clarridgeiae (causative agent of a disease similar to cat-scratch disease) can infect humans. Ectoparasites and their pathogens, especially those originating from free-roaming animals, present a potential threat to captive animals and humans.     

Urban focus of Rickettsia typhi and Rickettsia felis in Los Angeles, California

Classic murine typhus, caused by Rickettsia typhi, is endemic in the continental United States in areas of Texas and southern California. We conducted an environmental investigation in an urban area of Los Angeles identified as the probable exposure site for a case of murine typhus. Four Rattus norvegicus heavily infested with Xenopsylla cheopis (average 32.5 fleas per animal, range 20-42) were trapped, and fleas, blood, and tissues were collected. DNAs from all specimens were tested for R. typhi and Rickettsia felis using a TaqMan assay targeting the rickettsial citrate synthase gene. Although rickettsiemia was not detected, DNA of R. felis was detected in at least one tissue from each rat. Tissues from 3 rats were also positive for R. typhi DNA. R. typhi and R. felis DNAs were detected in fleas collected from each animal with average minimal infection rates of 10% and 32.3%, respectively. Although R. typhi still circulates in urban Los Angeles in the classic Oriental flea-rat cycle, R. felis is more prevalent, even in this association.     

Rickettsia felis and Bartonella spp. in fleas from cats in Albania

Fleas can serve as vectors for bacterial pathogens like Bartonella and Rickettsia species, which have been isolated worldwide. However, the knowledge of the epidemiology of vector-borne diseases in general and thus on flea-borne diseases in Albania is limited. Therefore, from 78 free-roaming cats in Tirana, Albania, fleas (371 Ctenocephalides felis and 5 Ctenocephalides canis) were collected to examine them for the presence of Rickettsia and Bartonella species. Ten of the 371 C. felis (2.7%) were positive for Rickettsia felis, and 24 (6.5%) for Bartonella spp. (B. henselae and B. clarridgeiae). In total, fleas from 15 cats (19.2%) were positive for either one or the other of the pathogens. The results of this study provided evidence for the presence of R. felis (causing flea-borne spotted fever) and Bartonella spp. (causing cat scratch disease) in Albania. Thus, these infectious diseases should be considered as differential diagnoses when febrile symptoms are presented, especially after contact with cats or their fleas.     

Rickettsia, Ehrlichia, Anaplasma, and Bartonella in ticks and fleas from dogs and cats in Bangkok

Flea and tick specimens (5-10 fleas or ticks) on dogs and cats from various sites in Bangkok were tested by polymerase chain reaction and DNA sequencing to detect DNA of bacteria Rickettsia (gltA and 17?kDa genes), Anaplasmataceae (16S rRNA gene), and Bartonella (pap31 and its genes). We confirmed that Rickettsia sp. related to Rickettsia felis was detected in 66 of 98 (67.4%) flea specimens from dogs, whereas 8 Bartonella henselae and 2 Bartonella clarridgeiae were detected in 10 of 54 (18.5%) flea specimens from cats. Further, this work provides the first evidence of 10 Ehrlichia canis (3.3%), 7 Anaplasma platys (2.3%), and 2 Wolbachia spp. (0.66%) in 304 Rhipicephalus sanguineus tick specimens in Thailand.     

Phylogenetic analysis of the rompB genes of Rickettsia felis and Rickettsia prowazekii European-human and North American flying-squirrel strains

The rickettsial outer membrane protein B (rompB) gene encodes the major surface antigens of Rickettsia species. We undertook sequencing and molecular analysis of the rompB gene of Rickettsia felis and a comparison with its homologs in spotted fever group (SFG) and typhus group (TG) rickettsiae, including the complete sequences of two North American flying squirrel strains and two European human strains of Rickettsia prowazekii. We sequenced 5,226 base pairs (bp) of the R. felis rompB, encoding a protein of 1,654 amino acids. We also sequenced 5,015 bp of rompB of the flying squirrel strains, encoding a protein of 1,643 amino acids. Analysis of the R. felis rompB gene sequence showed 10-13% divergence from SFG rickettsiae and 18% divergence from the TG rickettsiae. The rompB of all sequenced strains of R. prowazekii showed an overall similarity of 99.7-99.9%.     

Bartonella clarridgeiae, B. henselae and Rickettsia felis in fleas from Morocco

A total of 554 fleas were collected in the Moroccan Casablanca and Tiznit regions from domesticated animals and ruminants between August 2007 and October 2008 and were tested for the presence of Rickettsia spp. and Bartonella spp. using molecular methods. For the first time in Morocco, we found Rickettsia felis, the agent of flea-borne spotted fever in Ctenocephalides felis; B. henselae, an agent of cat scratch disease; and Bartonella clarridgeiae, a cat pathogen and potentially a human pathogen.     

First detection of Rickettsia felis in Ctenocephalides felis fleas parasitizing rats in Cyprus

Rickettsia felis was identified by polymerase chain reaction amplification and DNA sequencing analysis in Ctenocephalides felis fleas parasitizing rats in Cyprus. Murine typhus caused by R. typhi was believed to be the only flea-transmitted rickettsiosis on the island. This is the first report of this pathogen in southeastern Europe.     

吸血期蓖麻硬蜱雌虫体内周身性莱姆病螺旋体生长动态的组织学研究

为查明吸血期蓖麻硬蜱雌虫体内周身性莱姆病螺旋体生长动态,用直接免疫荧光试验、银染色组织学及电子显微镜技术,对饥饿及吸血期蓖麻硬蜱雌虫脑、唾液腺和卵巢组织中莱姆病螺旋体的分布和数量进行了观察。结果显示,饥饿蟀组织中螺旋体数量极为有限,吸血期蜱组织中螺旋体的密度和数量随吸血时间的延长而快速增长,至开始吸血后4或5天,脑、唾液腺和卵巢组织中螺旋体的数量都已达到无法计数的程度;本研究首次对周身性莱姆病螺旋体的分裂增殖提出了假设。螺旋体在吸血期蜱唾液腺的大量检出,为莱姆病螺旋体经由蜱唾液分泌而传播给脊椎动物宿主的假设提供了新的证据。