Molecular detection and characterization of Ehrlichia canis from dogs in Turkey

Seroprevalence of Ehrlichia canis antibodies among dogs in Turkey were previously reported, however, the ehrlichial organism has never been characterized in this region. The current study examined dogs from Ankara with febrile illness for E. canis infection with E. canis-specific PCR. Three of the 12 blood specimens from dogs showing clinical signs compatible with canine ehrlichiosis were found to be positive by PCR using E. canis-specific primers. E. canis detected in one of the blood specimens was designated as Kutahya strain. The representative E. canis strain was characterized by 16S rRNA gene sequencing and Western blot analysis of the plasma sample from the dog infected with E. canis. The 16S rRNA sequence (1,388 bp) of the E. canis Kutahya was identical to that of Ehrlichia ovina from a sheep in Turkey and Venezuelan Dog Ehrlichia (VDE) and was closely related (99.9%) to that of type strain of E. canis, Oklahoma. The plasma of the dog infected with E. canis Kutahya was analyzed by Western blotting using the purified E. canis Oklahoma strain as antigen. The reactive antibody profiles of the dog infected with E. canis Kutahya was found to be similar to those of dogs infected with E. canis Oklahoma and VDE, suggesting the antigenic similarities among these strains. The findings in this study would help for a better understanding of epidemiology of canine ehrlichiosis. This is the first report of molecular detection and characterization of an ehrlichial agent in Turkey.     

Genetic characterization of Anaplasma (Ehrlichia) platys in dogs in Spain

This paper reports the first genetic characterization of Anaplasma (Ehrlichia) platys in Spain from a naturally infected dog. The dog presented clinical signs compatible with canine ehrlichiosis. After DNA extraction and PCR amplification, 16S rRNA gene and citrate synthase gene ( gltA) of this agent were amplified. The GenBank accession number for the nucleotide sequence of the 16S rRNA gene of this strain is AY530806. The A. platys strains registered in France and Japan showed the highest similarity to the 16S rRNA gene sequence obtained from the Spanish strain. In the amplification of the gltA gene, a 1443 bp fragment was obtained, and three nucleotide differences were detected in comparison with other strains sequences. These data confirm the presence of A. platys in a dog showing clinical signs compatible with ehrlichiosis in Spain.     

First confirmed canine case of Ehrlichia canis infection in Japan

An 11-year-old castrated Pekinese dog that had been moved from Indonesia to Japan eight years previously was diagnosed with an Ehrlichia canis infection by haematological characteristics (normocytic anaemia, mild thrombocytopenia and hypergammaglobulinaemia) and serological findings (antibody titre to E canis 1:3,200 or more). The dog did not respond to treatment with tetracycline and died from renal failure. The diagnosis was confirmed postmortem by pathological evaluation and polymerase chain reaction (PCR) followed by sequencing of the 16S rRNA gene. Typical morulae of Ehrlichia were detected in the cytoplasm of macrophages in spleen tissue by immunohistological staining. Ehrlichia-like organisms were also detected in the spleen by electron microscopy. E canis-specific PCR analysis of DNA extracted from the spleen gave a positive signal, and sequence analysis of the fragment revealed that it was identical to part of the 16s rRNA gene of E canis. The dog was the first confirmed clinical case of E canis infection in Japan.     

Serologic and molecular evidence of coinfection with multiple vector-borne pathogens in dogs from Thailand

Forty-nine dogs from Thailand were evaluated for serologic evidence of exposure or polymerase chain reaction (PCR) evidence of infection with vectorborne pathogens, including Ehrlichia sp. (Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, and Ehrlichia risticii), Bartonella vinsonii subsp. berkhoffi (Bvb), spotted fever group (SFG) rickettsiae (Rickettsia rickettsii), Typhus group (TG) rickettsiae (Rickettsia canada, Rickettsia prowazekii, and Rickettsia typhi), and Babesia sp. (Babesia canis and Babesia gibsonii). All study dogs had at least 1 of 3 entry criteria: fever, anemia, or thrombocytopenia. By immunofluorescence antibody (IFA) testing, seroreactivity was most prevalent to E chaffeensis (74%) and E canis (71%) antigens, followed by E equi (58%), Bvb (38%), E risticii (38%), R prowazekii (24%), B canis (20%), R rickettsii (12%), R canada (4%), and B gibsonii (4%) antigens. There was 100% concordance between E canis IFA and Western blot immunoassay (WI) for 35 of 35 samples; 2 samples were IFA and WI reactive only to E equi antigens. By PCR amplification, 10 dogs were found to be infected with E canis, 5 with Ehrlichia platys, and 3 with B canis. Sequencing of PCR products was undertaken to compare Ehrlichia strains from Thailand to strains originating from the United States. Partial DNA sequence analysis confirmed infection with E canis and E platys, with identical 16S rRNA sequence alignment to E canis (U26740) and to E platys (M83801), as reported in GenBank. Partial E canis P28.1 and P28.2 amino acid sequences from Thai dogs were divergent from analogous sequences derived from North American E canis (AF082744) strains, suggesting that the Thai dogs were infected with a geographically distinct strain of E canis compared to North American strains. The results of this study indicate that dogs in Thailand have substantial exposure to vectorborne diseases and that coinfection with these pathogens may be common.     

猪附红细胞体16S rRNA基因的序列测定和系统进化分析

从确诊为猪附红细胞体感染的猪场，无菌采集血样，抽提猪附红细胞体基因组DNA，采用真细菌的通用引物进行16S rRNA基因扩增，对扩增产物进行克隆和测序。从3个地理位置不同的猪场均成功地扩增出长度为1469bp的核苷酸序列。系统进化分析表明，3个猪场样品所测序列一致性达99．52％以上，具有相同的基因型，但与国外报道的猪附红细胞体Illinois株同源性为95％，属于同一基因群，但基因型不同；所有种类的附红细胞体和血巴尔通氏体组成同一进化分支，这类血营养菌与支原体科，支原体属的病原最靠近(75％)，而与立克次氏体目的病原较远(70％)。上述研究证实，广东所流行的猪附红细胞体是一种新基因型的猪附红细胞体，建议命名为猪附红细胞体广东株型；为反映进化关系，猪附红细胞体和其它血营养菌应划归于支原体科的支原体属。     

Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada

To identify the tick-borne pathogens in dogs from Grenada, we conducted a serologic survey for Ehrlichia canis in 2004 (104 dogs) and a comprehensive serologic and molecular survey for a variety of tick-borne pathogens in 2006 (73 dogs). In 2004 and 2006, 44 and 32 dogs (42.3% and 43.8%) were seropositive for E. canis, respectively. In 2006, several tick-borne pathogens were identified by serology and PCR. DNA of E. canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, and Bartonella sp. were identified in 18 (24.7%), 14 (19.2%), 5 (7%), 5 (7%), and 1 (1.4%) dogs, respectively. Six (8.2%) dogs were seropositive for Bartonella vinsonii subsp. berkhoffii. All dogs were seronegative and PCR-negative for Rickettsia spp. Coinfection with two or three pathogens was observed in eight dogs. Partial 16S rRNA E. canis and A. platys sequences were identical to sequences in GenBank. Partial 18S rRNA gene sequences from the Grenadian H. canis were identical to each other and had one possible mismatch (ambiguous base) from H. canis detected from Spain and Brazil. Grenadian B. c. vogeli sequences were identical to B. c. vogeli from Brazil and Japan. All of the detected pathogens are transmitted, or suspected to be transmitted, by Rhipicephalus sanguineus. Results of this study indicate that dogs from Grenada are infected with multiple tick-borne pathogens; therefore, tick-borne diseases should be included as differentials for dogs exhibiting thrombocytopenia, leukopenia, fever, or lethargy. One pathogen, E. canis, is also of potential public health significance.     

Restriction and expansion of Ehrlichia strain diversity

Ehrlichia are tick-borne gram negative, obligately intracellular bacteria. The 16S rRNA gene DNA sequences are highly conserved among strains of each Ehrlichia species. The 28-kDa/Map-1 outer membrane protein genes are highly diversified among strains of Ehrlichia chaffeensis and E. ruminantium, but are highly conserved among E. canis isolates. The diversity of the immunodominant proteins of E. chaffeensis and E. ruminantium in contrast with the conservation of the immunodominant proteins of E. canis suggests that E. chaffeensis and E. ruminantium face more host immune pressure than E. canis or that E. chaffeensis and E. ruminantium evolved earlier than E. canis and have diverged.     

Granulocytic Ehrlichiosis in Dogs from North Carolina and Virginia

Medical records of 3 dogs from North Carolina and 3 dogs from Virginia with ehrlichial morulae in circulating neutrophils were studied retrospectively. Two clinically distinct disease syndromes, including chronic, moderate to severe anemia (n = 3) and polyarthritis (n = 2) were associated with canine granulocytic ehrlichiosis (CGE) in these dogs. One dog was clinically healthy, and abnormalities were not detected during physical examination. Clinical signs were nonspecific and included fever, lethargy, anorexia, vomiting, and diarrhea. The most frequent laboratory abnormalities were normocytic normochromic nonregenerative anemia, moderate thrombocytopenia with large platelets, lymphopenia, and eosinopenia. Considerable variability was found in the serologic responses to Ehrlichia equi, Ehrlichia canis , and Ehrlichia chaffeensis antigens among the 5 dogs for which stored sera were available for indirect fluorescent antibody testing. Polymerase chain reaction amplification and sequencing of portions of the 16S rRNA gene from blood (collected in ethylenediaminetetraacetic acid) of 1 severely anemic dog (dog 3) and 1 polyarthritic dog (dog 4) resulted in DNA sequences nearly identical to the GenBank accessions for Ehrlichia ewingii. The DNA sequence from a 3rd dog (dog 5) was most similar to that of E. canis. Serologic or molecular results support the possibility of E. ewingii, E. equi , and E. canis coinfection or serologic cross-reactivity among canine granulocytic and monocytic Ehrlichia species in dogs from North Carolina and Virginia. Variability in response to tetracycline or doxycycline treatment was noted in these dogs, with more rapid resolution of signs in dogs with polyarthritis. We report the 1st cases of CGE in dogs from North Carolina and Virginia, including recognition of CGE in a healthy dog.     

New advances in molecular epizootiology of canine hematic protozoa from Venezuela, Thailand and Spain

The prevalence of hematozoan infections (Hepatozoon canis and Babesia sp., particularly Babesia canis vogeli) in canids from Venezuela, Thailand and Spain was studied by amplification and sequencing of the 18S rRNA gene. H. canis infections caused simultaneously by two different isolates were confirmed by RFLP analysis in samples from all the geographic regions studied. In Venezuela, blood samples from 134 dogs were surveyed. Babesia infections were found in 2.24% of the dogs. Comparison of sequences of the 18S rRNA gene indicated that protozoan isolates were genetically identical to B. canis vogeli from Japan and Brazil. H. canis infected 44.77 per cent of the dogs. A representative sample of Venezuelan H. canis isolates (21.6% of PCR-positives) was sequenced. Many of them showed 18S rRNA gene sequences identical to H. canis Spain 2, albeit two less frequent genotypes were found in the sample studied. In Thailand, 20 dogs were analyzed. No infections caused by Babesia were diagnosed, whereas 30 per cent of the dogs were positive to hematozoan infection. Two protozoa isolates showing 99.7-100% identity to H. canis Spain 2 were found. In Spain, 250 dogs were studied. B. canis vogeli infected 0.01% of the animals. The sequence of the 18S rRNA gene in Spanish isolates of this protozoa was closely related to those previously deposited in GenBank (> 99% identity). Finally, 20 red foxes were screened for hematozoans employing semi-nested PCR and primers designed to detect Babesia/Theileria. Fifty percent of the foxes were positive to Theileria annae. In addition, it was found that the PCR assay was able as well to detect Hepatozoon infections. Thirty five percent of the foxes were infected with two different H. canis isolates showing 99.8-100% identity to Curupira 1 from Brazil.     

The relationship between the degree of thrombocytopenia and infection with Ehrlichia canis in an endemic area

Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis. In order to evaluate platelet counts as a screening test for E. canis in an endemic area, 217 whole blood samples from dogs were divided into three groups: 71 non-thrombocytopenic samples (group A, platelet counts greater than 200 000/mL) and 146 thrombocytopenic samples (less than 200 000/mL). The thrombocytopenic group was further divided into 62 with platelet counts between 100 000-200 000/mL (Group B) and 84 samples with less than 100 000 platelets/mL (Group C). All samples were examined for the presence of a segment of the Ehrlichia canis 16S rRNA gene using a nested polymerase chain reaction. Sixty-seven of the 217 samples (30.9%) were positive for the presence of the E. canis 16S rRNA gene; 53 (63.1%) of the group C samples and 13 (21%) of group B. Only one (1.4%) of the non-thrombocytopenic samples (Group A) was positive. These data support the concept that platelet counts may be a good screening test for canine monocytic ehrlichiosis, and that the magnitude of thrombocytopenia may increase the reliability of diagnosis.     

Sequence and phylogenetic analysis of the 16S rRNA gene of Ehrlichia canis strains in dogs with clinical monocytic ehrlichiosis

The purpose of this study was to characterize, at the molecular level, the Ehrlichia canis strains involved in naturally occurring canine monocytic ehrlichiosis (CME) in Greece, and to investigate if any sequence diversity exists between the 16S rRNA genes of those involved in the mild non-myelosuppressive or the severe myelosuppressive form of CME. To this end, amplification of the ehrlichial 16S rRNA gene was attempted by nested polymerase chain reaction (PCR) assays in bone marrow (BM) aspirates from 20 dogs tentatively diagnosed as having non-myelosuppressive (n=10, group A) or myelosuppressive (n=10, group B) CME. PCR assay using E. canis-specific primers revealed that 15 BM samples, including all group A and 5 group B dogs, were positive. Using universal PCR primers, a nearly full-length 16S rRNA gene could be amplified from 13 BM samples, including 9 group A and 4 group B dogs. The 16S rDNA analysis based on secondary structure revealed that all sequences of the Greek strains were identical to each other and indicated 100% identity among some American (Venezuelan and Brazilian), European (Greek), Middle Eastern (Turkish) and Asiatic (Thailand) strains. The results of this study suggest that the E. canis strains involved in the non-myelosuppressive and myelosuppressive forms of CME in Greece share an identical 16S rRNA genotype.     

红腹锦鸡大肠杆菌病的细菌学诊断

无菌采集病死红腹锦鸡肝脏、脾脏和心血,接种多种培养基分离细菌。以生化试验和小鼠攻毒试验,鉴定分离细菌及其致病性。采用PCR方法扩增分离细菌16S rRNA,测定核苷酸序列,与GenBank中大肠杆菌相应基因序列进行分析比较,绘制系统进化树。结果表明,分离细菌为大肠杆菌,可以致小鼠死亡。其16S rRNA基因序列与GenBank登录的大肠杆菌相应基因同源性超过99%。由此证明,该红腹锦鸡死于大肠杆菌病。     

一例雏鸡铜绿假单胞菌的分离鉴定及其16SrRNA基因序列分析

为对死亡雏鸡进行病因诊断,通过大体剖检、细菌分离、生化鉴定,证实为铜绿假单胞菌感染。测定了该分离株的16SrRNA基因序列,并与GenBank中收录的序列比较,结果发现所分离的铜绿假单胞茵及参考株的16SrDNA基因序列极其保守,相似性达99％～100％;与大肠杆菌、沙门氏菌、巴氏杆菌的相似性差异约为9％;与鸭疫里默氏...     

Epidemiological survey of Anaplasma platys and Ehrlichia canis using ticks collected from dogs in Japan

Detection of Anaplasma platys and Ehrlichia canis in ticks recovered from dogs in Japan was attempted using a species-specific nested PCR based on the 16S rRNA gene. A total of 1211 ticks recovered from 1211 dogs from all over Japan were examined for A. platys and E. canis. Four tick samples from Fukushima, Miyazaki and Kagoshima Prefectures recovered from four different dogs showed a positive reaction for A. platys. Although the four dogs did not show any clinical signs and no blood examination data were available, it is possible that A. platys has already been spread widely in Japan. No positive reactions were observed in any ticks examined for E. canis.     

Prevalence of Ehrlichia canis infection in thrombocytopenic dogs from Rio de Janeiro, Brazil

BACKGROUND: Infection with Ehrlichia canis causes a highly variable, multisystemic disease in dogs. Nevertheless, many clinicians in Rio de Janeiro, Brazil, use the presence of only thrombocytopenia to make a presumptive diagnosis of E canis infection. OBJECTIVE: The objective of this study was to determine the prevalence of E canis in thrombocytopenic dogs from Rio de Janeiro, Brazil, using polymerase chain reaction (PCR). METHODS: Following DNA extraction of whole blood samples from 226 dogs, PCR assays were done using primers for rickettsial DNA (including Ehrlichia spp, Anaplasma platys and A phagocytophilum) and using E canis-specific primers (16S rRNA gene). Dogs were grouped as thrombocytopenic and nonthrombocytopenic based on platelet counts. The null hypothesis that there was no difference in the prevalence of E canis in these groups was rejected at P<.05. RESULTS: Thirty-six (32.1%) of the thrombocytopenic dogs and 4 (3.5%) of the nonthrombocytopenic dogs were positive for rickettsial gene sequences (P<.0001). Further, 30 (26.8%) of thrombocytopenic dogs and 4 (3.5%) nonthrombocytopenic dogs were positive for E canis-specific gene sequences (P<.0001). CONCLUSIONS: Although the prevalence of E canis infection was higher in thrombocytopenic dogs, less than one third of these dogs had demonstrable E canis infection. Thus, thrombocytopenia is not specific for the detection of E canis infection and should not be used solely to establish a diagnosis of canine ehrlichiosis, even in a geographic area with relatively high disease prevalence.     

Molecular survey of Ehrlichia infection in ticks from animals in Yamaguchi Prefecture, Japan

A total of 82 ticks collected from wild animals and dogs in Yamaguchi Prefecture, Japan were examined for Ehrlichia infection by using polymerase chain reaction (PCR) primers that amplify DNA of most members of the genus Ehrlichia. A DNA sample from an Ixodes ovatus nymph from a bear in Yamaguchi Prefecture, Japan, was positive in the screening PCR. Subsequent PCR using two sets of primers yielded a 1431 bp segment of the 16S rRNA gene and the sequence was very similar to those of E. chaffeensis and E. muris, and a strain variant of a recently described Ehrlichia species isolated from I. ovatus in other prefectures of Japan.     

Detection of Ehrlichia canis by PCR in different tissues obtained during necropsy from dogs surveyed for naturally occurring canine monocytic ehrlichiosis

A molecular study for the detection of Ehrlichia canis was carried out on tissues obtained at necropsy from randomly selected dogs with the intention of investigating naturally-occurring canine ehrlichiosis. The tissues evaluated for the presence of E. canis included lymph nodes, spleen, liver, bone marrow, and blood. Eight of the 18 dogs included were found to be positive for E. canis by polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene. Two dogs were positive for Anaplasma platys of which one dog was co-infected with E. canis and A. platys. Blood (5/8) and lymph nodes (5/8) were the tissues found to yield the highest number of positive E. canis PCR results with 7/8 dogs positive in the blood or lymph node. E. canis and A. platys DNA could be amplified by PCR when tissue samples were obtained 72h after the time of death.     

Direct identification of Ehrlichia canis by a novel polymerase chain reaction method and molecular analysis of the citrate synthase (gltA) gene from various Italian strains.

Fourteen blood samples collected from dogs that were seropositive for Ehrlichia canis were examined for the presence of the citrate synthase gene using a highly specific and sensitive novel polymerase chain reaction assay. The assay detected E. canis DNA in 3 dogs. The complete nucleotide sequence of the citrate synthase gene was determined in 2 of the test-positive samples, and represents the first sequence of the gene to be derived from Italian isolates. The sequence data displayed high identity (99.2%) between the geographically separated Italian samples and the Oklahoma strain of E. canis. The high-sequence conservation revealed by molecular analysis confirmed the usefulness of the citrate synthase gene as a target for detection of E. canis.     

First isolation and molecular characterization of Ehrlichia canis in Costa Rica, Central America

The present study investigated Ehrlichia species in blood samples from dogs suspected of clinical ehrlichiosis, using molecular and isolation techniques in cell culture. From a total of 310 canine blood samples analyzed by 16S rRNA nested PCR, 148 (47.7%) were positive for Ehrlichia canis. DNA from Ehrlichia chaffeensis or Ehrlichia ewingii was not detected in any sample using species-specific primers in separated reactions. Leukocytes from five PCR-positive dogs were inoculated into DH82 cells; successful isolation of E. canis was obtained in four samples. Partial sequence of the dsb gene of eight canine blood samples (including the five samples for in vitro isolation) was obtained by PCR and their analyses through BLAST showed 100% of identity with the corresponding sequence of E. canis in GenBank. This study represents the first molecular diagnosis, isolation, and molecular characterization of E. canis in dogs from Costa Rica.     

The pCS20 PCR assay for Ehrlichia ruminantium does not cross-react with the novel deer ehrlichial agent found in white-tailed deer in the United States of America

White-tailed deer are susceptible to heartwater (Ehrlichia [Cowdria] ruminantium infection) and are likely to suffer high mortality if the disease spreads to the United States. It is vital, therefore, to validate a highly specific and sensitive detection method for E. ruminantium infection that can be reliably used in testing white-tailed deer, which are reservoirs of antigenically or genetically related agents such as Ehrlichia chaffeensis, Anaplasma (Ehrlichia) phagocytophilum (HGE agent) and Ehrlichia ewingii. Recently, a novel but as yet unnamed ehrlichial species, the white-tailed deer ehrlichia (WTDE), has been discovered in deer populations in the United States. Although the significance of WTDE as a pathogen is unknown at present, it can be distinguished from other Ehrlichia spp. based on 16S rRNA gene sequence analysis. In this study it was differentiated from E. ruminantium by the use of the pCS20 PCR assay which has high specificity and sensitivity for the detection of E. ruminantium. This assay did not amplify DNA from the WTDE DNA samples isolated from deer resident in Florida, Georgia and Missouri, but amplified the specific 279 bp fragment from E. ruminantium DNA. The specificity of the pCS20 PCR assay for E. ruminantium was confirmed by Southern hybridization. Similarly, the 16S PCR primers (nested) that amplify a specific 405-412 bp fragment from the WTDE DNA samples, did not amplify any product from E. ruminantium DNA. This result demonstrates that it would be possible to differentiate between E. ruminantium and the novel WTDE agent found in white tailed deer by applying the two respective PCR assays followed by Southern hybridizations. Since the pCS20 PCR assay also does not amplify any DNA products from E. chaffeensis or Ehrlichia canis DNA, it is therefore the method of choice for the detection of E. ruminantium in these deer and other animal hosts.