Ehrlichial Agents Research Articles

Surveillance of ticks (Acari: Ixodidae) and tick-borne pathogens in Eastern Central Alabama.

Similar to other states in the southeastern United States, human cases of tick-borne diseases in Alabama have risen steadily over the last 2 decades. Nevertheless, limited data have been published on ticks or tick-borne pathogen (TBP) distributions since the 1990s. To better understand the risk of tick and TBP exposure in eastern central Alabama, ticks were sampled repeatedly across 8 sites associated with recreational use during May and June of 2015 to characterize tick density and diversity. Although habitats were similar across sites, tick density varied among locations. Seven species were collected, but 97.7% of 1,310 samples were the lone star tick, Amblyomma americanum (L.), the primary vector of ehrlichial agents and the tick species most commonly linked to alpha-gal syndrome and southern tick-associated rash illness. To investigate pathogen prevalence among sites, we tested A. americanum by a multiplex qPCR assay for 5 bacterial species, including 3 Ehrlichia spp. and 2 Rickettsia spp. None of the specimens tested positive for Panola Mountain Ehrlichia or Rickettsia parkeri. However, causative agents of human ehrlichiosis, Ehrlichia chaffeensis and Ehrlichia ewingii, occurred at half of the locations with, respectively, 0.27% and 0.45% of A. americanum infected on average. In contrast, Rickettsia amblyommatis, a tick endosymbiont suspected to be nonpathogenic, was found in 54.5% of the 1119 A. americanum tested. Despite low infection rates of Ehrlichia spp. in A. americanum, high encounter rates with this species in recreational deciduous woodlands suggest a moderate risk of tick bite and a low-to-moderate risk of TBP exposure in late spring.

Serological Evidence Supporting the Occurrence of Ehrlichia chaffeensis or a Closely Related Species in Brazilian Dogs.

Ehrlichia chaffeensis is a tick-borne bacterium that causes human monocytotropic ehrlichiosis, an emerging life-threatening disease in humans transmitted by Amblyomma americanum. Although most studies have reported bacterial isolations and clinical cases in the United States, their occurrence is not restricted to North America. Some studies in the Southern Cone of South America have molecularly detected a close phylogenetic relative of E. chaffeensis in ticks and wild mammals. Even so, many gaps must be filled to confirm the presence of this agent in the region. To add new data on this issue, we report the first detection of specific anti-E. chaffeensis antibodies in dogs collected from all regions of Brazil. By means of IFA and ELISA with crude and specific antigens of E. chaffeensis, sera from 1134 dogs were analyzed. Serological analyses using ELISA showed nine (0.7%) seropositive dogs, with seven of them exhibiting IFA titers ranging from 160 to 5120. All regions of Brazil had at least one seropositive dog. Our results support the evidence for the occurrence of E. chaffeensis in South America. As dogs have a close relationship with humans, they can be used as an environmental sentinel for these infections because they can act as a bridge to human parasitism or infection with ehrlichial agents.

Molecular detection and genetic characterization of Ehrlichia canis and Ehrlichia sp. in neotropical primates from Brazil

The Anaplasmataceae family includes obligate, arthropod-transmitted intracellular bacteria that can be zoonotic and potentially fatal. Studies focusing on the interaction between neotropical primates and the agents of this family are scarce. The present study aimed to identify agents of the Anaplasmataceae family in the whole blood of free-living and captive neotropical primates in the State of Mato Grosso, Central-West Brazil. Thirty-eight samples of six nonhuman primate (NHP) species were collected in seven municipalities and analysed through polymerase chain reaction (PCR), nucleotide sequencing, and phylogenetic analysis of the dsb, groEL, 16S rRNA, and gltA genes. DNA fragments similar to those of Ehrlichia canis were detected in Sapajus apella and Ehrlichia chaffeensis from Mico melanurus. The sequences generated in this study and homologous sequences retrieved from GenBank® were used for phylogenetic analyses to characterize the Ehrlichial agents detected in NHPs. The agents were then grouped into clades corresponding to different isolates from the NHP species. In addition, an Anaplasma sp. closely related to Anaplasma marginale was identified in two S. apella individuals. These findings shed light on the susceptibility of neotropical NHPs to Anaplasmataceae agents. These bacteria are known to be transmitted by ticks, which can also serve as possible sources of infection for other animals, including humans.

Molecular Detection of Tick-Borne Agents in Cats from Southeastern and Northern Brazil

Even though the epidemiology of tick-borne agents (TBA) in dogs has been extensively investigated around the world, the occurrence, vectors involved, and molecular identity of these agents in cats remains elusive in many regions. Among TBA, Ehrlichia, Anaplasma, Babesia, Cytauxzoon, and Hepatozoon are responsible for diseases with non-specific clinical signs in cats, making essential the use of molecular techniques for accurate diagnosis and proper treatment. The present work aimed to investigate the occurrence and molecular identity of tick-borne agents (Ehrlichia, Anaplasma, Babesia/Theileria, Cytauxzoon, and Hepatozoon) in cats from southeastern (states of São Paulo (SP) and Minas Gerais (MG)) and northern (state of Rondônia (RO)) Brazil. For this purpose, 390 blood samples were collected from domiciled cats in MG (n = 155), SP (n = 151), and RO(n = 84) states, submitted to DNA extraction and PCR assays for Ehrlichia spp. (dsb gene), Anaplasma spp. (rrs gene), piroplasmids (18S rRNA gene), and Hepatozoon spp. (18S rRNA gene), sequencing, and phylogenetic inferences. The overall positivity for Anaplasma spp., Ehrlichia spp., Babesia/Theileria spp., Cytauxzoon spp., and Hepatozoon spp. were 7.4% (12.3% (MG) and 6.6% (SP)), 2% (4.5% (MG) and 0.6% (SP)), 0.7% (0.6% (MG), 0.6% (SP) and 1.2% (RO)), 27.2% (41.9% (MG), 24.5% (SP) and 4.8% (RO), and 0%, respectively. The phylogenetic analysis grouped the obtained sequences with ‘Candidatus Anaplasma amazonensis’, A. platys, B. vogeli, and Cytauxzoon sp. previously detected in wild felids from Brazil. qPCR specific for E. canis based on the dsb gene confirmed the molecular identity of the detected ehrlichial agent. The present study expanded the list and geographical distribution of hemoparasites in cats. ‘Candidatus Anaplasma amazonensis’, recently detected in sloths from northern Brazil, was described for the first time in cats. This is the first report of piroplasmids infecting cats in northern Brazil. Coinfection by Cytauxzoon and other TBA (Ehrlichia, Anaplasma, and B. vogeli) reported in the present study raises the need for veterinary practitioners’ awareness of cats parasitized by multiple TBA.

Serological evidence of Ehrlichia minasensis infection in Brazilian dogs

Ehrlichia spp. are important tick-borne pathogens of animals in Brazil, and Ehrlichia canis is the most prevalent species infecting dogs. Moreover, Ehrlichia minasensis has also recently been identified as a novel ehrlichial agent that infects cattle in Brazil. The objective of this study was to determine whether dogs could be infected by E. minasensis. To investigate this possibility, sera (n = 429) collected from dogs in the Pantanal region were retrospectively analyzed for the presence of antibodies against E. canis and E. minasensis. Canine sera were screened by two isolates of E. canis in indirect immunofluorescence assay (IFA) and the majority (n = 298; 69.4%) had antibodies with endpoint titers ranging from 80 to 327,680. In order to further confirm E. canis-specific antibodies, IFA positive sera were analyzed by ELISA using E. canis-specific peptides (i.e. TRP19 and TRP36 US/BR/CR), which detected E. canis antibodies in 80.2% (239/298) of the dog sera. Fifty-nine (13.7%) samples had detectable antibodies to E. canis by IFA but were negative by E. canis peptide ELISA. These sera were then tested by E. minasensis IFA (Cuiaba strain) as antigen and 67.8% (40/59) were positive (titers ranging from 80 to 20,480). Eleven sera had antibody titers against E. minasensis at least two-fold higher than observed for E. canis and suggests that these dogs were previously infected with E. minasensis. The results of the present study suggest that multiple ehrlichial agents infect dogs in Brazil, which highlights the need to consider different Ehrlichia spp. in Brazilian dogs, particularly in areas where dogs are frequently exposed to multiple tick species. This investigation is the first to provide serologic evidence of E. minasensis infection in dogs from Brazil.

Detection of Ehrlichia sp. in Amblyomma sculptum parasitizing horses from Brazilian Pantanal wetland

Bacteria of the genus Ehrlichia are transmitted by ticks and also are an important cause of infection in wild and domestic mammals. Infection with Ehrlichia spp. has been reported in horses, especially in the USA, Nicaragua and Brazil. In this study, we report the parasitism by Amblyomma sculptum, Rhipicephalus microplus and Dermacentor nitens ticks in horses from a ranch located in south Pantanal wetland. Molecular and serological analyzes to determine infection by Ehrlichia spp. in horses and their respective ticks were carried out. A total of 12 horses were submitted to blood collection to investigate antibodies by indirect immunofluorescence assay (IFA) using Ehrlichia canis crude antigens and to be tested by polymerase chain reaction (PCR) in order to amplify fragments of the 16S rRNA, dsb, groEL and sodB gene of Ehrlichia spp. A total of 164 tick specimens were removed from horses, stored in isopropanol and later identified as D. nitens, A. sculptum and R. microplus. DNA from ticks were extracted and subjected to the same PCR assays to detect Ehrlichia spp. Anti-Ehrlichia spp. antibodies were detected in five/12 (41.7 %) horses by IFA, with antibody titers ranging from 40 to 160. All horse DNA samples were negative for the 16S rRNA, dsb, groEL and sodB of Ehrlichia spp. One A. sculptum female was positive to all target genes of Ehrlichia. This tick was parasitizing an Ehrlichia-seropositive horse with antibody titer of 80. Nucleotide sequences of 16S rRNA, dsb, groEL and sodB genes showed close relationship with different strains of Ehrlichia detected in wild mammals, Amblyomma ticks and horses from Brazil and Argentina. Detection of anti-Ehrlichia sp. antibodies suggests that horses have been exposed to an ehrlichial agent in the Pantanal. Future studies on Ehrlichia infection should be carried out to better elucidate and to bring new information about equine ehrlichiosis, since these animals are important hosts of ticks in the Brazilian Pantanal wetlands.

Tick-borne pathogens in dogs, wild small mammals and their ectoparasites in the semi-arid Caatinga biome, northeastern Brazil

Caatinga is a biome exclusive to the semiarid zone of Brazil, where studies on ticks and tick-borne diseases are scarce. Herein, we investigated the occurrence of Rickettsia, Ehrlichia, and Coxiella in wild mammals, domestic dogs and their ectoparasites using molecular and serological techniques. During 2014–2016, blood samples and ectoparasites were collected from 70 small mammals (51 rodents, 18 marsupials, 1 wild canid) and 147 domestic dogs in three areas of the Caatinga. Through serological analyses of domestic dogs of the three areas, 8 to 11 % were seropositive for Rickettsia rickettsii, 9 to 37 % for Rickettsia amblyommatis, 61 to 75 % for Ehrlichia canis, and 0–5% for Coxiella burnetii. All wild mammals were seronegative for Rickettsia spp. and C. burnetii, except for one rodent (Wiedomys pyrrhorhinos) and one marsupial (Didelphis albiventris) that were seroreactive to C. burnetii, one wild canid (Cerdocyon thous) for R. amblyommatis, and two Rattus rattus for Rickettsia spp. Through PCR targeting DNA of Rickettsia, Ehrlichia or Coxiella, all blood samples were negative, except for the presence of Ehrlichia canis DNA in 8.8 % of the domestic dogs, and a recently reported novel agent, Ehrlichia sp. strain Natal, in one marsupial (Gracilinanus agilis). A total of 222 ticks, 84 fleas, and six lice were collected. Ticks were mostly Rhipicephalus sanguineus sensu lato, some Ixodes loricatus, Ornithodoros rietcorreai, Haemaphysalis sp., and Amblyomma spp.; fleas were Ctenocephalides felis felis, Pulex sp. and Polygenis (Polygenis) bohlsi jordani; and lice were Polyplax sp. and Gyropus sp. Through molecular detection of microorganisms, 9% of C. felis felis contained Rickettsia felis, 20 % of A. auricularium contained R. amblyommatis and 13 % of A. parvum contained ‘Candidatus Rickettsia andeanae’, whereas Ehrlichia canis DNA was detected in at least 6% of the R. sanguineus s.l. from one area. We report a variety of ectoparasites infesting small mammals and domestic dogs in the Caatinga biome, where these ectoparasites probably act as vectors of rickettsiae, ehrlichial agents (E. canis and Ehrlichia sp. strain Natal) and C. burnetii. Our results highlight to the potential risks of human infection by these tick-borne agents in the Caatinga biome.

A putative novel strain of Ehrlichia infecting Amblyomma tigrinum associated with Pampas fox (Lycalopex gymnocercus) in Esteros del Iberá ecoregion, Argentina

The current work evaluated road-killed Pampas foxes (Lycalopex gymnocercus) and their ticks for the presence of vector-borne agents in the ecoregion Esteros del Iberá in northeastern Argentina. Spleen, lung and blood samples and Amblyomma tigrinum adult ticks collected from the foxes were tested by polymerase chain reaction (PCR) assays targeting bacteria of the genera Ehrlichia, Anaplasma, and Rickettsia. All foxes tested were negative for the three genera, but evidence of Ehrlichia and Rickettsia infection was detected in the ticks. One A. tigrinum (out of 12 tested) was infected by an ehrlichial agent, here named Ehrlichia sp. strain Iberá, related to ehrlichial agents recently detected in platypuses in Tasmania (Ornithorhynchus anatinus) and in voles (Myodes rutilus and Myodes rufocanus) and shrews (Sorex araneus) in the Russian Far East. Regarding Rickettsia, all A. tigrinum ticks (100%) were infected by ´Candidatus Rickettsia andeanae´, a member of the spotted fever group rickettsia of unknown pathogenicity. Further research is necessary to unveil the ecology of Ehrlichia sp. strain Iberá as well as its zoonotic relevance. The species of the genus Ehrlichia are known to be pathogenic to mammals, including humans and domestic animals, thus the presence of this ehrlichial agent in A. tigrinum is a potential risk for veterinary and public health, as the adults of A. tigrinum are common parasites of dogs in rural and peri-urban environments, and humans are also frequently bitten by this tick species.

Isolation and molecular detection of Ehrlichia species from ticks in western, central, and eastern Japan

Ehrlichiosis is a tick-borne bacterial disease caused by pathogens of the Ehrlichia genus. Although human ehrlichiosis has not been reported in Japan, Ehrlichia spp., which are closely related to Ehrlichia chaffeensis, were detected in several species of ixodid ticks. In this study, the presence of Ehrlichia spp. in ticks in Japan was studied by using isolation and molecular detection methods. In total, 1237 ticks were collected from vegetation in western, central, and eastern parts of Japan. The ticks were tested for detection of ehrlichial DNA with a nested polymerase chain reaction and/or isolation by inoculation of mice with the homogenate. Ehrlichial DNA was detected in 29 of these ticks. The ehrlichial DNAs, groEL and 16S rRNA genes, detected in Ixodes turdus showed a high similarity to those of E. chaffeensis with 94.7% and 99.2% identity, respectively. Ehrlichia sp. HF and Candidatus Neoehrlichia mikurensis were also detected in I. ovatus. Furthermore, Ehrlichia sp. HF was isolated from laboratory mice that were intraperitoneal inoculated with I. ovatus tick homogenate. Some ehrlichial agents detected in Ixodes ticks might be a previously unknown Ehrlichia species. In this study, Candidatus N. mikurensis was detected in I. ovatus ticks. Because I. ovatus is distributed widely and cases of its tick bite in humans are ubiquitously reported in Japan, there is a potential for ehrlichiosis to be endemic to Japan, necessitating intensive surveillance of this infectious disease.

Ticks, rickettsial and erlichial infection in small mammals from Atlantic forest remnants in northeastern Brazil

We evaluated infection by Rickettsia spp. and Ehrlichia spp in small mammals and their ticks from two Atlantic forest conservation areas in the state of Rio Grande do Norte, northeastern Brazil. A total of 39 small mammals were captured during 2012–2013, encompassing 33 marsupials (29 Didelphis albiventris, four Monodelphis domestica), three Cricetidae rodents (two Necromys lasiurus, one Rattus rattus), one Caviomorpha rodent (Thrichomys apereoides) and two armadillos (Euphractus sexcinctus). The ticks Amblyomma auricularium, Ixodes loricatus, and Ornithodoros mimon were collected from D. albiventris, whereas only A. auricularium was collected from armadillos. Through immunofluorescence assay with Rickettsia spp. antigens, 6/28 (21%) D. albiventris and the single R. rattus specimen reacted to at least one rickettsial antigen, with highest seroprevalence and endpoint titers to Rickettsia amblyommatis. A total of 150 ticks (126 A. auricularium, nine I. loricatus, 15 O. mimon) was tested for rickettsial infection by PCR, which detected only R. amblyommatis in most of the A. auricularium ticks. Lung and spleen samples were collected from small mammals (two N. lasiurus, six D. albiventris, three M. domestica, one T. apereoides, one R. rattus) and were tested by PCR for Anaplasmataceae agents. The spleen from one D. albiventris contained a new ehrlichial agent, here named as Ehrlichia sp. strain Natal. Phylogenetic analysis inferred from the dsb gene of Ehrlichia spp. indicates that this novel agent is potentially a new species. Future studies should monitor the possible role of rickettsial and/or ehrlichial microorganisms as agents of emerging diseases in these degraded areas of Atlantic forest, just as has occurred with other agents in degraded areas of this biome in southeastern Brazil.

A novel ehrlichial agent detected in tick in French Polynesia

Ticks are hematophageous arthropods that are known to host and transmit miscellaneous pathogens including zoonotic bacteria. The aim of this study was to investigate the presence of tick-associated microorganisms in Tahiti, French Polynesia with molecular tools. A total of 658 ticks from two species including Rhipicephalus sanguineus s.l. and Rh. annulatus were collected with forceps on dogs and cattle respectively, or with a flag on pasture in several locations of Tahiti in 2013. Two Rickettsia belonging to the spotted fever group different from R. conorii and R. massiliae were detected by qPCR in two Rh. sanguineus s.l. ticks, but sequencing failed. A Rh. annulatus tick was found positive for a new ehrlichial agent characterized by amplification and sequencing of fragments of the Anaplasmataceae 23S and Ehrlichia 16S genes. Phylogenetic analyses based on the 23S and 16S sequences reveals that this bacterium is a new genotype, genetically close to Ehrlichia minasensis, a recently described Ehrlichia sp. close to Ehrlichia canis.

A novel Ehrlichia genotype strain distinguished by the TRP36 gene naturally infects cattle in Brazil and causes clinical manifestations associated with ehrlichiosis

A novel Ehrlichia genotype most closely related to E. canis was reported in North American cattle in 2010, and a similar agent was subsequently identified in the hemolymph of Brazilian Rhipicephalus (Boophilus) microplus ticks and isolated in 2012. The purpose of this study was to determine whether this or other novel ehrlichial agents naturally infect Brazilian cattle. Using PCR targeting the genus-conserved dsb gene, DNA from this novel ehrlichial agent in Brazilian cattle was detected. Attempts to isolate the organism in vitro were performed using DH82 cells, but morulae and ehrlichial DNA could only be detected for approximately one month. In order to further molecularly characterize the organism, PCR was performed using primers specific for multiple E. canis genes (dsb, rrs, and trp36). Sequence obtained from the conserved rrs and dsb genes demonstrated that the organism was 99–100% identical to the novel Ehrlichia genotypes previously reported in North American cattle (rrs gene) and Brazilian ticks (rrs and dsb genes). However, analysis of the trp36 gene revealed substantial strain diversity between these Ehrlichia genotypes strains, including divergent tandem repeat sequences. In order to obtain preliminary information on the potential pathogenicity of this ehrlichial agent and clinical course of infection, a calf was experimentally infected. The calf showed clinical signs of ehrlichiosis, including fever, depression, lethargy, thrombocytopenia, and morulae were observed in peripheral blood monocytes. This study reports a previously unrecognized disease-causing Ehrlichia sp. in Brazilian cattle that is consistent with the genotype previously described in North America cattle and ticks from Brazil. Hence, it is likely that this is the organism previously identified as Ehrlichia bovis in Brazil in 1982. Furthermore, we have concluded that strains of these Ehrlichia genotypes can be molecularly distinguished by the trp36 gene, which has been widely utilized to define E. canis strain diversity.

Diagnostic Evaluation of <i> Ehrlichia</i> <i>canis</i> Human Infections

Ehrlichiosis is an infection of the group of Vector Borne Diseases (VBD), caused by different echrlichia species (spp). Ehrlichiae are primarily recognized as causers of animal diseases, later as human diseases—Humane Monocytic (HME) and Humane Granulocytic (HGE) ehrlichiosis. The dogs, cats and rodents have natural reservoirs of ehrlichial agents. Ehcrlichiae are spread to human’s trough, the bites of infected ticks, sometimes resulting from contact with animals, possible to the transfusion. In period from 2008 to 2013, we analyzed 250 patients suspect of VBD. On examination, there were included clinical, laboratory and epidemiological methods. The confirmation of Ehrlichia canis was in serum of 64 patients, thanks to Indirect Immune fluorescent Assay (IIF). The other laboratory and serological methods (Elisa, IIF, and Western blood) are utilized for differential diagnosis, and detection of co-infective forms of diseases. Ehrlichia canis infection has been reported in humans as a cause of serious illnesses characterized by non specific symptoms: fever, headache, musculoskeletal pain, enlarged liver, neurological, hematological dysfunction. Co-infections with E. canis due to causers of different VBD have been documented in humans. In 53 cases, we detected co-infective participation of E. canis with Rickettsia conorii in 24 cases, Coxiella burneti in 6, and Borrelia burgdorferi in 23 cases. The preferred drug for both human ehrlichiosis is doxycycline, except children to 8 years, pregnant females. Ehrlichiosis is a difficult infectious disease to diagnose and prognose because of dominant non specific symptoms and co-infections.

NovelEhrlichiaandHepatozoonAgents Infecting the Crab-Eating Fox (Cerdocyon thous) in Southeastern Brazil

This study evaluated infection by vector-borne agents in 58 crab-eating fox (Cerdocyon thous L.) that were road-killed in an Atlantic rainforest reserve in the state of Espírito Santo, southeastern Brazil. Spleen, lung, or blood samples collected from the foxes were tested in the laboratory by a battery of polymerase chain reaction (PCR) assays targeting bacteria of the genera Rickettsia, Borrelia, Coxiella, Anaplasma, and Ehrlichia; and protozoa of the genera Babesia, Hepatozoon, and Leishmania. Of the targeted organisms, evidence of infection in the foxes was detected for Ehrlichia and Hepatozoon organisms only. Overall, six (10.3%) foxes were infected by an ehrlichial agent closely related to an ehrlichial agent recently detected in free-ranging Jaguars [(Panthera onca (L.)] in central-western Brazil, and to Ehrlichia ruminantium. For Hepatozoon, 28 (48.3%) foxes were infected by an agent closely related to Hepatozoon sp. Curupira 2 and H. americanum; and one (1.7%) fox was infected by an organism closely related to reptile-associated Hepatozoon agents. Finally, 11 (19.0%) foxes were found infested by Amblyomma cajennense (F.) nymphs, which were all PCR negative for the range of vector-borne agents cited above. Because the haplotypes found in free-ranging foxes are genetically closely related to pathogens of great veterinary importance, namely E. ruminantium and H. americanum, it is highly desirable to know if these novel organisms have any important role as agents of diseases in domestic animals and wildlife in Brazil.

Mounting evidence for an ehrlichial involvement in systemic lupus erythematosus

H agents, well studied in veterinary medicine, were first suggested as a human pathogen in an article in Nature New Biology in 1972, and there is growing evidence of their involvement in systemic lupus erythematosus (SLE). Over the last four decades a small body of published and unpublished research has reported the following: Ehrlichial antigens identified in the glomeruli of patients with lupus nephritis; Ehrlichia-like intra-erythrocytic inclusions in patients with SLE and endosomal structures suggestive of Ehrlichia in the marrow of patients with SLE. Furthermore, compassionate antibiotic treatment of very ill patients using an antibiotic known to be effective against Ehrlichial agents was conducted, with strongly positive results including changes in observed intra erythrocytic bacterial structures. Self-reactive antibodies are characteristic of SLE and Ehrlichia have been shown to produce errors in transcription in parasitized cell division especially in proliferating cells of the marrow. If Ehrlichia invade the immune system through activity against bone marrow cells (especially stem cells or other precursors of mature B and/or T cells) and interfere with their construction, function and elimination before release to the circulation, the body may be unable to eliminate them in a normal fashion and the self-reactive antibodies would be unleashed on the tissues of the infected patient. Molecular studies using the genetic sequences of the Ehrlichia identified in a human patient with another possibly related illness of the immune system should be undertaken to see if it can be positively identified in the marrow and blood of lupus patients. If so, a compelling alternative explanation of the disease complex of SLE would be proved.

Detection and Identification ofEhrlichiaSpecies inRhipicephalus(Boophilus)microplusTicks in Cattle from Xiamen, China

A total of 289 Rhipicephalus (Boophilus) microplus Canestrini (Acari: Ixodidae) collected from cattle in Xiamen city of Fujian province, southeastern China, were tested for ehrlichial agents by nested polymerase chain reaction assay. Twenty-seven (9.3%) ticks from seven cattle were found positive for Ehrlichia. The polymerase chain reaction products from positive samples were sequenced and found identical with each other. The 1458-bp nucleotide sequence of ehrlichial 16S rRNA gene was obtained and showed the greatest levels of sequence similarity to 16S rRNA gene sequences of the Ehrlichia spp. detected in ticks near Thai-Myanmar-Vietnam border and that in Tibet, China, differing only 2 and 4 bp, respectively. Sequence comparison of the 16S rRNA gene with the members of the genus Ehrlichia reveals that this ehrlichial agent differed from the sequences of E. chaffeensis in ticks from southeastern China by 17 bp, showing close relation to E. chaffeensis. This was the first report on ehrlichial pathogens in R. (Boophilus) microplus from cattle in southeastern China. The spatial distance between Fujian province and Tibet is more, whereas the ehrlichial agents from both regions are highly homologous. The veterinary and public health significances of the agent deserve further investigation.

Detection of the New Ehrlichia Species Closely Related to Ehrlichia ewingii from Haemaphysalis longicornis in Yonaguni Island, Okinawa, Japan

We collected a total of 206 Haemaphysalis longicornis ticks by flagging in pastures in Yonaguni Island, Okinawa, Japan, in April 2008. Four of the 206 tick DNA samples tested were positive in a polymerase chain reaction (PCR) screening for the 16SrRNA gene of Anaplasmataceae. Partial sequences of 4 PCR products were identical to each other. Longer sequences of the 16SrRNA gene were successfully determined in 2 of the 4 tick samples, and the obtained 1,392 bp and 1,300 bp sequences revealed high similarity to the 16SrRNA gene sequences of the validated Ehrlichia species, including Ehrlichia ewingii, E. chaffeensis, and E. canis (98.3-98.6%). We also sequenced 1,304 bp of the groEL gene from the 2 tick samples, and found that these had the highest similarity to sequences of E. ewingii (94.0-94.4%) in the validated ehrlichial species. Based on the 16SrRNA and groEL gene sequences, the ehrlichial agents detected in this study were similar to the Ehrlichia species detected in Asia and may compose a new Ehrlichia species with other Ehrlichia species detected in Asia.

Serologic evidence of equine granulocytic anaplasmosis in horses from central West Brazil

Ehrlichiosis is a zoonotic disease caused by gram-negative and intracellular obligatory bacterial organisms. Equine Granulocytic Anaplasmosis - EGA (formerly Equine Granulocytic Ehrlichiosis, EGE) is a seasonal disease, normally self-limited in horses. There are few reports in Brazil about this ehrlichial agent, as well as its natural vectors. Nowadays, veterinarians are considering the suspicion of EGA in horses with suggestive symptoms of ehrlichiosis and which do not respond to piroplasmosis treatment. The aim of the present study was to identify horses exposed to the agent A. phagocytophilum by serological and molecular techniques. Twenty equine blood and serum samples from the central West region of Brazil were evaluated by microscopic examination of buffy coat smear, enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody test (IFAT) and nested polymerase chain reaction (nPCR). Additionally, the serodiagnosis of Theileria equi by IFA and ELISA were carried out, as well as molecular diagnosis by nPCR. Thirteen (65%) serum samples were positive for A. phagocytophilum by ELISA, but none of them were positive by buffy-coat smear examination or nPCR. Antibodies IgG anti-T. equi were detected in 18 (90%) and 17 (85%) horses by IFA and ELISA, respectively and the agent was detected in 9 (45%) animals by nPCR. Our data may be considered as important information to understanding the occurrence of EGA and equine piroplasmosis in central West Brazil.

Predominance of Ehrlichia chaffeensis in Rhipicephalus sanguineus ticks from kennel-confined dogs in Limbe, Cameroon

Rhipicephalus sanguineus ticks (n = 63) collected from five dogs (two adults and three puppies) housed in a kennel were screened for Ehrlichial agents (Ehrlichia canis, E. chaffeensis, and E. ewingii) using a species-specific multicolor real-time TaqMan PCR amplification of the disulphide bond formation protein (dsb) gene. Ehrlichia chaffeensis DNA was detected in 33 (56%) ticks, E. canis DNA was detected in four (6%) ticks, and one tick was coinfected. The E. chaffeensis and E. canis nucleotide sequences of the amplified dsb gene (374 bp) obtained from the Cameroonian R. sanguineus ticks were identical to the North American genotypes.

Detection and Identification of Bacterial Agents inIxodes persulcatusSchulze Ticks from the North Western Region of Russia

Ixodes persulcatus Schultze ticks are traditionally associated with transmission of Lyme disease, babesiosis, and tick-borne encephalitis. Here we compared the prevalence of infection with Borrelia burgdorferi, and rickettsial and ehrlichial agents in I. persulcatus ticks collected in different locations of the North Western administrative region of Russia. Altogether, 27.7% of ticks were infected with at least one organism, while the DNA of two or more bacteria was found in 11.8% of ticks tested. The highest average prevalence of Anaplasmataceae (20.8%) was detected in ticks from Arkhangel'sk province, while the prevalence in ticks from Novgorod province and St. Petersburg, respectively, was 7.3% and 12.2%. Only Ehrlichia muris DNA was identified by DNA sequencing. In comparison, the prevalence of B. burdorferi DNA was 16.6%, 5.8%, and 24.5% in the respective locations. The 382-bp amplicon of gltA from Candidatus Rickettsia tarasevichiae was detected in 2.75% and 1.6%, respectively, of ticks from Arkhangel'sk and Novgorod provinces, extending further west and north the area where this rickettsia is known to be present. DNA of the rickettsia-like endosymbiont Montezuma was primarily associated with female ticks, 8-28% of which were infected. Since I. persulcatus is so commonly infected with multiple agents that may cause human diseases, exposure to these ticks poses significant risk to human health in this region.