Results from an indirect fluorescent antibody test using three different strains of Ehrlichia canis

An indirect fluorescent antibody (IFA) test is usually performed to detect antibodies in dogs naturally infected by Ehrlichia canis. In this work, results obtained using three different E. canis strains as antigen (a commercial antigen, the E. canis Oklahoma strain and the E. canis Madrid strain) were compared. One hundred and forty-nine serum samples obtained from dogs living in the centre of Spain were analysed. When qualitative results were evaluated, identical results were detected in 87.2% of samples for the three antigens tested. When comparing antibody titre results, differences between the Madrid strain and the commercial antigen, and between the Madrid and Oklahoma strains were statistically significant (P < 0.0001). No differences were found when comparing the Oklahoma strain with the commercial antigen (P = 0.562). Subtle intra-laboratory variations shown in this study suggest a higher sensitivity of the IFA test when an autochthonous strain is used as antigen.     

Doxycycline clearance of experimentally induced chronic Ehrlichia canis infection in dogs

BACKGROUND: Ineffective clearance of Ehrlichia canis after doxycycline administration has been reported despite the fact that the recommended treatment for canine ehrlichiosis is doxycycline. The effectiveness of doxycycline in clearing E canis infection from the blood and tissues of dogs requires additional evaluation. HYPOTHESIS: Doxycycline (5 mg/kg PO q12h), administered for 4 weeks, will eliminate E canis infection from the blood and tissues of experimentally infected dogs. ANIMALS: Fifteen Walker hound-mixed breed dogs were inoculated subcutaneously with E canis-infected canine histiocytic cells 4 months before doxycycline treatment. METHODS: Four dogs were treated with doxycycline (5 mg/kg PO q12h for 3 weeks), 5 dogs were treated with doxycycline at the same dosage for 4 weeks, and 5 control dogs were not treated. Dexamethasone (0.4 mg/kg i.v.) was given after treatment to precipitate recrudescence of any remaining E canis organisms. Platelet counts, anti-E canis immunofluorescent antibodies, and polymerase chain reaction (PCR) detection of E canis deoxyribonucleic acid (DNA) in blood and tissues were evaluated. RESULTS: E canis DNA was not detected in the blood and tissues of doxycycline-treated dogs after treatment. Platelet counts were within reference intervals, and E canis antibodies decreased. Spontaneous clearance of E canis infection occurred in 2 of 5 control dogs. Three control dogs had E canis DNA detected in blood and tissues, platelet counts remained low or within the reference interval, and E canis antibodies remained high. CONCLUSIONS AND CLINICAL IMPORTANCE: As administered in this study, doxycycline cleared E canis from the blood and tissues of experimentally infected dogs.     

Monitoring C-reactive Protein in Beagle Dogs Experimentally Inoculated with Ehrlichia canis

The concentrations of C-reactive proteins (CRP) in the plasma of five beagle dogs experimentally inoculated with Ehrlichia canis increased markedly. The concentrations began to increase between 4 and 16 days and peaked between 15 and 42 days after inoculation of E. canis. The peak concentrations ranged from 217.8 to 788.8 g/ml (452.6±228.1 SD). After the peak, the concentrations of CRP decreased rapidly. The PCR product of 16S rRNA of E. canis became detectable in the five dogs between 18 and 27 days after inoculation of E. canis. Antibodies to E. canis were detected in plasma from the dogs between 5 and 15 days after inoculation of E. canis. The timings of seroconversion and of the start of the increase in CRP were approximately similar and the high concentrations of CRP in the plasma of the dogs tended to become apparent when the PCR product of 16 S rRNA of E. canis became detectable.     

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.     

Sequence and phylogenetic analysis of the gp200 protein of Ehrlichia canis from dogs in Taiwan

Ehrlichia (E.) canis is a Gram-negative obligate intracellular bacterium responsible for canine monocytic ehrlichiosis. Currently, the genetic diversity of E. canis strains worldwide is poorly defined. In the present study, sequence analysis of the nearly full-length 16S rDNA (1,620 bp) and the complete coding region (4,269 bp) of the gp200 gene, which encodes the largest major immunoreactive protein in E. canis, from 17 Taiwanese samples was conducted. The resultant 16S rDNA sequences were found to be identical to each other and have very high homology (99.4~100%) with previously reported E. canis sequences. Additionally, phylogenetic analysis of gp200 demonstrated that the E. canis Taiwanese genotype was genetically distinct from other reported isolates obtained from the United States, Brazil, and Israel, and that it formed a separate clade. Remarkable variations unique to the Taiwanese genotype were found throughout the deduced amino acid sequence of gp200, including 15 substitutions occurring in two of five known species-specific epitopes. The gp200 amino acid sequences of the Taiwanese genotype bore 94.4~94.6 identities with those of the isolates from the United States and Brazil, and 93.7% homology with that of the Israeli isolate. Taken together, these results suggest that the Taiwanese genotype represents a novel strain of E. canis that has not yet been characterized.     

Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis

An indirect ELISA test was developed for the diagnosis of Brucella canis infection in dogs. A bacterial whole cell extract was used as a solid phase antigen, using B. canis isolated from an infected animal. Sera from culture-positive and healthy negative animals were used as internal reference controls. The cut-off point was determined by a mathematical formula for a statistically valid value, which defined the upper prediction limit, based on the upper tail of the t-distribution of 21 negative control sera readings, for the confidence level of 99.5%. The sensitivity and specificity of the ELISA test were 95% and 91%, respectively. The ELISA test showed a significant concordance index (K=0.84) with the agar gel immunodiffusion test. The reliability of the ELISA for the detection of infected animals was established by a double blind study testing 280 sera provided by serum banks from different diagnostic and research institutions and analyzed by ROC Curve.     

The Pharmacokinetics of a Long-acting Oxytetracycline Formulation in Healthy Dogs and in Dogs Infected with Ehrlichia canis

The pharmacokinetic properties of oxytetracycline were studied following a single injection of a long-acting formulation (20 mg/kg body weight) into the semimembranosus muscle of healthy dogs and of dogs that had been experimentally infected with Ehrlichia canis. The disposition curves of the long-acting oxytetracycline formulation before and after infection were best described by a bi-exponential decline after a first-order absorption. The mean maximum serum concentration (C _max) following infection was significantly lower and the time taken to attain this concentration (t _max) was significantly shorter than that in the healthy dogs. The mean apparent elimination half-life (t _1/2) was significantly increased following infection. The corresponding rate constant () was significantly decreased. The absorption half-life (t _1/2ab) was significantly decreased after infection. The volume of distribution at steady state (V _dss) increased significantly following infection. It was concluded that the pharmacokinetic behaviour of a long-acting oxytetracycline in dogs after intramuscular administration is characterized by a two-compartment model with a slow elimination phase. This could be due to flip-flop kinetics. The febrile reaction in experimental E. canis infection affected some pharmacokinetic parameters of oxytetracycline.     

Host surveys, ixodid tick biology and transmission scenarios as related to the tick-borne pathogen, Ehrlichia canis

The ehrlichioses have been subject to increasing interest from veterinary and public health perspectives, but experimental studies of these diseases and their etiologic agents can be challenging. Ehrlichia canis, the primary etiologic agent of canine monocytic ehrlichiosis, is relatively well characterized and offers unique advantages and opportunities to study interactions between a monocytotropic pathogen and both its vertebrate and invertebrate hosts. Historically, advances in tick-borne disease control strategies have typically followed explication of tick-pathogen-vertebrate interactions, thus it is reasonable to expect novel, more sustainable approaches to control of these diseases as the transmission of their associated infections are investigated at the molecular through ecological levels. Better understanding of the interactions between E. canis and its canine and tick hosts would also elucidate similar interactions for other Ehrlichia species as well as the potential roles of canine sentinels, reservoirs and models of tick-borne zoonoses. This article summarizes natural exposure studies and experimental investigations of E. canis in the context of what is understood about biological vectors of tick-borne Anaplasmataceae.     

Detection of Ehrlichia platys in dogs in Australia

OBJECTIVE: To describe the detection of Ehrlichia platys in free-roaming dogs in Central Australia. PROCEDURE: Blood samples were collected from four dogs and examined for bacterial 16S ribosomal DNA using Polymerase Chain Reaction (PCR)-based assays. The three positive samples obtained were then sequenced and identification of the PCR product carried out. As a result of all three samples being identical to or closely related to part of the 16S rRNA gene of E. platys, blood samples were subsequently obtained from a further 24 dogs. These samples were screened using a PCR-assay to determine the presence of Ehrlichia DNA using genus-specific primers. The positive samples obtained from the screening process were then subjected to a further PCR-assay using E. platys specific primers. RESULTS: Of 28 dogs sampled, Ehrlichia DNA was detected in the blood of 13 dogs. Sequencing of the amplicons obtained indicated a high homology with the 16S rRNA gene for E. platys. When the E. platys-specific PCR was performed for 10 of those dogs, the 678 bp product obtained from the PCR amplification confirmed the identification as part of the 16S rRNA gene of E. platys in all 10 dogs. CONCLUSION: This study reports for the first time Ehrlichia carriage by dogs in Australia. It also indicates the usefulness of the PCR technique in rapidly and accurately identifying diseases that are otherwise difficult to detect. By using universal primers directed against bacterial 16S ribosomal DNA and sequencing analysis, the detection of potentially pathogenic Ehrlichia organisms that had not previously been found in Australia has been made possible.     

Experimental inoculation of beagle dogs with Ehrlichia species detected from Ixodes ovatus

Three beagle dogs were inoculated with mice spleen/liver homogenate infected with Ehrlichia species detected from Ixodes ovatus (EIO) and one dog was used as a control. All three infected dogs did not show clinical signs of disease except for mild pyrexia throughout the 41-day study period. Splenomegaly was observed from Day 7 post-inoculation (p.i.) in two of the dogs. Hematological and biochemical abnormalities included mild thrombocytopenia, hypoproteinaemia, hypoalbuminaemia and increased C-reactive protein values. One of the dogs' splenic aspirate sample was PCR-positive for Ehrlichia Day 7 p.i. and another dogs' blood and bone marrow aspirate sample was PCR-positive Day 41 p.i. Sequence analysis of the PCR products showed 100% homology with the 16SrRNA partial gene sequence of Ehrlichia sp. HF565. Antibody titers to EIO were observed in all three experimentally infected dogs starting from the first week p.i. and cross-reactivity with Ehrlichia canis was detectable in one of the dogs starting Day 7 p.i. These data suggest that infection of dogs with EIO is possible, though is probably of low pathogenic importance. Cross-reactivity of EIO infected dog serum with E. canis raises the likelihood of false E. canis seropositive dogs.     

Isolation,in vitro propagation,genetic analysis,and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlândia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlândia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlândia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlândia and São Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.     

Molecular characterization of Babesia canis canis isolates from naturally infected dogs in Poland

Babesia canis has generally been considered the only large Babesia to infect dogs. In this study, we used PCR to detect and characterize B. canis canis isolated from naturally infected dogs in Poland by amplifying and sequencing a portion of the 18S ribosomal RNA (rRNA) gene. Venous blood samples were collected from 76 Babesia-symptomatic dogs. A 559-bp fragment of the B. canis canis 18S rRNA gene was amplified by PCR. The PCR products were then digested with HincII restriction enzyme, and isolates were classified according to whether they were cut (group A) or not (group B) by this endonuclease. Sequencing of the PCR products from the isolates led to the identification of seven sequence variants (four in group A, and three in group B). Sequences were compared with GenBank sequences, and alignments showed that all B. canis canis isolates from Europe may be classified into groups A or B as defined in our study.     

Diagnosis of canine monocytotropic ehrlichiosis (Ehrlichia canis): An overview

Canine monocytotropic ehrlichiosis (CME), caused by the rickettsia Ehrlichia canis, an important canine disease with a worldwide distribution. Diagnosis of the disease can be challenging due to its different phases and multiple clinical manifestations. CME should be suspected when a compatible history (living in or traveling to an endemic region, previous tick exposure), typical clinical signs and characteristic hematological and biochemical abnormalities are present. Traditional diagnostic techniques including hematology, cytology, serology and isolation are valuable diagnostic tools for CME, however a definitive diagnosis of E. canis infection requires molecular techniques. This article reviews the current literature covering the diagnosis of infection caused by E. canis.     

Allele-specific polymerase chain reaction diagnostic test for the functional MDR1 polymorphism in dogs

The major multidrug transporter P-glycoprotein (Pgp) contributes to the barrier function of several tissues and organs, including the brain. In a subpopulation of Collies and seven further dog breeds, a 4 base pair deletion has been described in the Pgp-encoding MDR1 gene. This deletion results in the absence of a functional form of Pgp and loss of its protective function. Severe intoxication with the Pgp substrate ivermectin has been attributed to the genetically determined lack of Pgp. An allele-specific polymerase chain reaction (PCR)-based screening method has been developed to detect the mutant allele and to determine if a dog is homozygous or heterozygous for the mutation. Based on this validation, the allele-specific PCR proved to be a robust, reproducible and specific tool, allowing rapid determination of the MDR1 genotype of dogs of at risk breeds using blood samples or buccal swabs.     

Sero-prevalence and risk indicators for canine ehrlichiosis in three rural areas of Brazil

Ehrlichia canis has a worldwide geographic distribution, occurring particularly in tropical and subtropical areas. In Brazil, the main vector in urban areas is believed to be the brown dog tick Rhipicephalus sanguineus, but little is known about the occurrence, transmission and other epidemiological aspects of canine ehrlichiosis in rural areas, where Amblyomma ticks are found more frequently than R. sanguineus. A sero-prevalence study of canine ehrlichiosis was carried out in three distinct rural regions of the State of Minas Gerais, Brazil. Serum samples were collected from 226 dogs living on farms in Lavras (n=85), Belo Horizonte (n=45), and Nanuque (n=96) and were analyzed by an indirect fluorescent antibody test for the detection of anti-Ehrlichia canis antibodies. Age, breed, sex, presence of ticks and packed cell volume were also recorded. There were 65.6% positive dogs in Nanuque, 37.8% in Belo Horizonte, and 24.7% in Lavras. Animals living in Nanuque were 4.6 times more likely to be serologically positive than dogs living in the other two regions and antibody titres were considerable higher in this area. Male dogs, dogs >5 years of age, those infested with ticks, and mongrels all showed higher rates of positivity. The results point to the importance of canine ehrlichiosis in rural areas and indicate the need for further studies on natural transmission and maintenance of the disease.     

应用PCR技术检测鹅细小病毒

根据鹅细小病毒GPVH1株结构蛋白VP1与VP3编码基因非重叠核苷酸序列设计了一对引物GPR1 /GPR2 ,用这对引物对感染器官内的GPR和接种鹅胚增殖的GPV进行PCR扩增 ,其结果引物GRP1 /GPR2能特异性地扩增GPVVP1 VP3区段核苷酸序列 ,扩增出与设计核苷酸片段大小相符的 0 6kb的序列 ,而对照的鹅副粘病毒cDNA及鸭瘟病毒 (DPR)DNA对照组均出现阴性结果。     

PCR制备地高辛标记的探针检测禽流感病毒核酸

用聚合酶链反应（PCR）技术,制备了广东禽流感无致病力分离株A/goose/China/24/96(H7N3）核蛋白基因片段（NPc）的地高辛标记的cDNA探针。建立并优化了检测禽流感病毒核酸的探针杂交法,探针杂交法能鉴别出非免疫鸡胚和SPF鸡胚尿囊液中的病毒,攻毒后第3天的SPF和非免疫鸡泄殖腔拭子中AIV的最大检出率为1/10,对临床样品中的AIV的最大检出率为1/7,而直接HA和HI法及AGP试验检不出临床样品的AIV。该探针具有较好的特异性和敏感性,为从分子水平探讨AIV的发病机理、临床早期快速诊断提供了新的研究手段。     

Detection of an Ehrlichia bovis-like Organism in Cultured Buffalo Monocytes

Monocytes from a buffalo were cultured in RPMI 1640 medium following separation of plasma by the erythrocyte sedimentation technique and subsequent separation of mononuclear cells by density gradient centrifugation. Growth of an organism considered to be Ehrlichia bovis was noticed in the cultured monocytes after 10 days. The inclusions were considered to be those of E. bovis from their morphology, staining characteristics and growth characteristics in culture, and by indirect immunofluorescence examination with an anti-E. canis serum. The utility of peripheral blood monocyte cultures opens the possibility of diagnosing the carrier status of ehrlichiosis in animals.     

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.     

Detection of neoplastic lymphocytes in peripheral blood of dogs with lymphoma by polymerase chain reaction for antigen receptor gene rearrangement

BACKGROUND: Uniquely rearranged immunoglobulin and T-cell receptor gene sequences can be amplified and electrophoretically separated by size to detect a clonal population of lymphocytes. OBJECTIVE: The purpose of this study was to determine whether the polymerase chain reaction (PCR) detects neoplastic (clonal) lymphocytes more frequently than do microscopic methods. METHODS: We identified neoplastic lymphocytes in peripheral blood by both routine and standardized microscopic examination of blood smears and by PCR amplification of blood-derived DNA and compared the 3 methods for frequency of detection of leukemic involvement. For standardized microscopic examination (200 leukocytes counted on Wright-Giemsa-stained blood smears), samples were categorized as negative (1% prolymphocytes, no lymphoblasts), or positive (>/=1 lymphoblast). A PCR-amplified sample was positive if 1 or 2 discrete bands were seen on the gel, or negative if no bands, a smear, or a faint ladder was seen. RESULTS: Using PCR, neoplastic lymphocytes were detected in peripheral blood 2.5 times more frequently than with routine or standardized microscopic evaluation. Eighty-three percent of samples negative by microscopy were positive by PCR. CONCLUSION: PCR is more sensitive than microscopy for the detection of clonal lymphocytes in peripheral blood. The results of this study also suggest that neoplastic lymphocytes circulate in peripheral blood at a higher frequency than previously reported. PCR may be useful for detecting or phenotyping lymphoma, monitoring response to therapy, identifying recurrence, and screening breeds at risk.