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.     

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.     

Detection of Ehrlichia canis by polymerase chain reaction in dogs from Portugal

Antibodies against Ehrlichia canis, the cause of canine monocytic ehrlichiosis, have been reported previously in clinically ill and stray dogs from Portugal. In this study, the 16S rRNA gene of E. canis was detected by the polymerase chain reaction (PCR) in 12/55 (22%) of dogs with suspected tick-borne disease in the Algarve region in Portugal.     

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.     

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.     

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.     

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.     

A preliminary investigation of Ehrlichia species in ticks, humans, dogs, and capybaras from Brazil

A molecular epidemiologic investigation in two Brazilian states (Rondônia and São Paulo) was undertaken to determine if Ehrlichia species responsible for human and animal ehrlichioses in North America could be found in Brazilian vectors, potential natural mammalian reservoirs and febrile human patients with a tick bite history. Samples, including 376 ticks comprising 9 Amblyomma species, 29 capybara (Hydrochaeris hydrochaeris) spleens, 5 canine blood, and 75 human blood samples from febrile patients with history of tick bites were tested by a real-time PCR assay targeting a fragment of the Ehrlichia dsb gene. Ehrlichia DNA was not detected in any tick, capybara or human samples. In contrast, 4 out of 5 dogs contained Ehrlichia canis DNA in their blood, which were sequenced, representing the first report of E. canis infecting dogs in the Amazon region of Brazil. Further studies are needed to evaluate the presence of other agents of human and animal ehrlichioses in Brazil.     

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 and molecular characterization of a Brazilian strain of Borrelia anserina, the agent of fowl spirochaetosis

Borrelia anserina the agent of fowl spirochaetosis, has a worldwide distribution, where it is transmitted by Argas spp. ticks. The present study reports the first molecular characterization and in vitro isolation of an avian spirochaete strain from Brazil, presumably identified as B. anserina originated from naturally infected Argas miniatus ticks. DNA fragments of the rrs and flab genes were amplified by PCR and sequenced to determine phylogenetic similarities. The resulting sequences were 99.8% (483 of 484) and 98.7% (754 of 764) similar to GenBank corresponding sequences of B. anserina rrs and flaB genes, respectively. By neighbor-joining phylogenetic analysis, the flaB sequence of the Brazilian strain clustered in a monophyletic group with the sequence of B. anserina under 100% bootstrap support. The isolate was successfully isolated in BSK medium, with seven passages performed. The spirochaete strain isolated in the present study was genetically identified as B. anserina labeled as strain PL.     

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.     

Hepatozoon canis infecting dogs in the State of Espírito Santo, southeastern Brazil

From May 2007 to March 2008, blood samples were collected from 92 healthy dogs living in 21 households (17 farms in rural area, and 4 homes in urban area) in 6 counties of the State of Espírito Santo, southeastern Brazil. In addition, ticks were collected from these dogs. A mean of 4.4 ± 3.0 dogs (range: 1–12) were sampled per household; 78 and 14 dogs were from rural and urban areas, respectively. Polymerase chain reaction (PCR) designed to amplify fragments of the 18S rDNA gene of Babesia spp or Hepatozoon spp revealed amplicons of the expected size in 20 (21.7%) dogs for Babesia, and 54 (58.7%) dogs for Hepatozoon. All Babesia-positive dogs were also Hepatozoon-positive. Among the 21 households, 15 (71.4%) from 3 counties had at least one PCR-positive dog, including 13 farms (rural area) and 2 homes (urban area). A total of 40 PCR products from the Hepatozoon-PCR, and 19 products from the Babesia-PCR were submitted to DNA sequencing. All generated sequences from Hepatozoon-PCR were identical to each other, and to corresponding 18S rDNA sequences of H. canis in GenBank. Surprisingly, all generated sequences from the Babesia PCR were also identical to corresponding 18S rDNA sequences of H. canis in GenBank. Dogs from 10 rural and 2 urban households were found infested by Rhipicephalus sanguineus ticks. Immature of Amblyomma cajennense ticks were found in dogs from only 4 rural households (also infested by R. sanguineus). All but one household with R. sanguineus-infested dogs had at least one Hepatozoon-infected dog. Statistical analysis showed that the presence of ticks (i.e. R. sanguineus) infesting dogs in the households was significantly (P < 0.05) associated with at least one PCR-positive dog. There was no significant association (P > 0.05) between PCR-positive dogs and urban or rural households. Canine hepatozoonosis caused by H. canis is a high frequent infection in Espírito Santo, Brazil, where it is possibly vectored by R. sanguineus. Since all infected dogs were found apparently healthy, the pathogenicity of H. canis for dogs in Espírito Santo is yet to be elucidated.     

Canine babesiosis caused by Babesia canis vogeli in rural areas of the State of Minas Gerais, Brazil and factors associated with its seroprevalence

This epidemiological survey on canine babesiosis was carried out in three distinct rural regions (Lavras, Belo Horizonte and Nanuque) of the State of Minas Gerais, Brazil. Ticks and blood samples were collected during a dry season (Lavras, n = 92; Belo Horizonte, n = 50; Nanuque, n = 102) and the subsequent rainy season (Lavras, n = 71; Belo Horizonte, n = 28; Nanuque, n = 66) from dogs living on farms. Plasma samples were analyzed by the indirect fluorescent antibody test for detection of anti-Babesia canis vogeli antibodies. DNA was extracted from blood of serologically positive dogs and molecular characterization of Babesia species was performed. Rhipicephalus sanguineus, Amblyomma cajennense and Boophilus microplus were the tick species identified in all regions. In Lavras, in addition to those tick species, A. tigrinum and A. ovale were also identified. The most prevalent tick species was A. cajennense (35.3%), followed by R. sanguineus (19%) and B. microplus (4.0%). Dogs living in Nanuque region were more heavily infested with ticks than dogs living in Belo Horizonte and Lavras regions. The overall frequency of anti-B. c. vogeli antibodies in the canine population in rural areas of Minas Gerais was 28.7%, with prevalence rates of 49.0% in Nanuque, 34.0% in Belo Horizonte and 3.3% in Lavras. The age of the animals and tick infestation were associated with seroprevalence of B. c. vogeli. The sequence analysis showed that B. c. vogeli was the only Babesia species present in all three regions. This study showed different rates of prevalence and incidence of canine babesiosis among the three rural regions sampled in Minas Gerais State. The results point to the importance of canine babesiosis in rural areas and to the need for further studies related to its transmission and maintenance in nature.     

Molecular characterization and genogrouping of VP1 of aquatic birnavirus GC1 isolated from rockfish Sebastes schlegeli in Korea

The cDNA nucleotide sequence of genome segment B encoding the VP1 protein was determined for the aquatic birnavirus GC1 isolated from the rockfish Sebastes schlegeli in Korea. The VP1 protein of GC1 contains a 2,538 bp open reading frame, which encodes a protein comprising 846 amino acid residues that has a predicted MW of 94 kDa. The sequence contains 6 potential Asn-X-Ser/Thr motifs. Eight potential Ser phosphorylation sites and 1 potential Tyr phophorylation site were also identified. GC1 contains the Leu-Lys-Asn (LKN) motif instead of the typical Gly-Asp-Asp (GDD) motif found in other aquatic birnaviruses. We also identified the GLPYIGKT motif, the putative GTP-binding site at amino acid position 248. In total, the VP1 regions of 22 birnavirus strains were compared for analyzing the genetic relationship among the family Birnaviridae. Based on the deduced amino acid sequences, GC1 was observed to be more closely related to the infectious pancreatic necrosis virus (IPNV) from the USA, Japan, and Korea than the IPNV from Europe. Further, aquatic birnaviruses containing GC1 and IPNV have genogroups that are distinct from those in the genus Avibirnaviruses and Entomo-birnaviruses. The birnavirusstrains were clustered into 5 genogroups based on their amino acid sequences. The marine aquatic birnaviruses (MABVs) containing GC1 were included in the MABV genogroup; the IPNV strains isolated from Korea, Japan, and the USA were included in genogroup 1 and the IPNV strains isolated primarily from Europe were included in genogroup 2. Avibirnaviruses and entomobirnaviruses were included in genogroup 3 and 4, respectively.     

Genotypic characterization of Staphylococcus aureus isolated from bovines, humans, and food in Indonesia

The present study determined the genetic relationships between 41 Staphyloccocus (S.) aureus isolates from bovines, humans, and food using a single enzyme amplified fragment length polymorphism (AFLP) technique. We evaluated the prevalence of staphylococcal enterotoxin (SE) genes and other virulence gene determinants by PCR. The identification of S. aureus was based on culturing and biochemical tests, and by amplifying a specific section of the 23S rRNA gene. PCR amplification of the SE genes (sea, seb, sec, see, seg, seh, and sei) singly or in combination was observed. Most isolates of bovine origin harbored hla (84%) and cap5 (74%), while most isolates from humans harbored hla (73%), cap8 (91%), and fnbA (100%). Strains from food sources were positive for hla (100%), cap5 (100%), and cap8 (64%) unlike isolates from humans or bovines. A single enzyme AFLP analysis revealed a correlation between AFLP clusters of some strains and the source of the isolates The genotypic results of the present study might help to better understand the distribution of prevalent S. aureus clones among humans, bovines, and food and will help control S. aureus infections in Indonesia.     

Cloning, expression, and characterization of the MSP1a and MSP1b recombinant proteins from PR1 Anaplasma marginale strain, Brazil

The Anaplasma marginale is a bacterium that has obligate intraerythrocytic multiplication in cattle causing important economic loss. The A. marginale major surface protein 1 (MSP1) complex, heterodimer composed of MSP1a and MSP1b, has been identified as adhesins for bovine erythrocytes. The objectives of this study were to sequences the msp1β gene and produce and characterize recombinant MSP1a and MSP1b from a Brazilian strain of A. marginale, PR1. The msp1α and msp1β genes from the PR1 strain were cloned and expressed in E. coli BL21 Star using the vectors pET102 and pET101/D-TOPO. Antibodies were produced against the recombinant proteins and were shown to react with rMSP1a and rMSP1b demonstrating a molecular mass of 70 kDa to 105 kDa and 100 kDa, respectively for these proteins. Bovine erythrocytes were agglutinated by BL21/rMSP1a and BL21/rMSP1b and, this agglutination was inhibited by the presence of the IgY anti-rMSP1a, confirming the adhesion function of these proteins. Additionally, using the IgY anti-rMSP1a and rMSP1b in a IFI, the presence of rMSP1a and rMSP1b was confirmed on the outer membrane of the recombinant E. coli BL21. Our results show that the msp1β gene from the PR1 strain has both the conserved region and contain the defined polymorphism regions previously described for other strains of A. marginale. The results from this study confirm adhesive functions for rMSP1a and rMSP1b from PR1 strain in bovine erythrocytes invasion.     

Isolation of an atypical Leptospira strain assigned to the Sejroe serogroup from a water buffalo in Brazil

The isolation of leptospires from buffaloes worldwide is still limited to a few strains. Thus, the aim of this study was to describe the first Leptospira isolate from buffalo urine, assigned to the Sejroe serogroup, which does not belong to the Wolffi subgroup, traditionally isolated in Brazil. A total of 244 urine samples of water buffaloes (Bubalus bubalis) raised in the Brazilian Amazon were subjected to bacteriological culturing and polymerase chain reaction (PCR) for the detection of leptospires. The obtained isolate was characterized by serogrouping using polyclonal antibodies, partial DNA sequencing, Hardjo-Bovis-specific PCR, multiple-locus variable-number tandem repeat analysis (MLVA/VNTR) and experimental infection in hamsters. PCR was performed on the urine samples; 11/244 were positive (4.5 %) for Leptospira, and only one isolate was recovered (0.4 %). Regarding characterization, the isolate was assigned to the Sejroe serogroup with high titers (12,800) for the Saxkoebing and Sejroe serovar antisera. The isolate was negative for Hardjo-Bovis-specific PCR, and the species Leptospira borgpetersenii was identified by DNA sequencing. The MLVA results showed that the VNTR profile of the isolate was 1−2-5, compatible with that of serovars Sejroe/Istrica. In the experimental infection in hamsters, the animals did not develop clinical signs, and no macroscopic lesions were observed on the organs at necropsy; however, the strain was detected in the kidneys, uterus, and testicles of the animals. The isolate described herein highlights infection by Sejroe strains that may be overlooked in buffaloes and that may be different from those normally isolated and used in serological studies.     

Intraoperative Bleeding in Dogs from Grenada Seroreactive to Anaplasma platys and Ehrlichia canis

Background

Frequent exposure of Grenadian dogs to Rhipicephalus sanguineus results in Anaplasma platys, and Ehrlichia canis seroreactivity. During elective surgeries, substantial intraoperative hemorrhage occurs in some seroreactive dogs.

Objectives

To assess hemostatic parameters and bleeding tendencies as well as prevalence of PCR positivity in apparently healthy A. platys and E. canis seroreactive and seronegative free‐roaming dogs from Grenada.

Animals

Forty‐seven elective surgery dogs allocated to 4 groups: Seronegative control (n = 12), A. platys (n = 10), E. canis (n = 14) and A. platys, and E. canis (n = 11) seroreactive.

Methods

Preoperatively, hemostasis was assessed by platelet count, prothrombin time, activated partial thromboplastin time, and buccal mucosal bleeding time. Intra‐ and postoperative bleeding scores were subjectively assigned. Blood, spleen, bone marrow, and lymph node aspirates were tested by PCR.

Results

Bleeding scores in dogs coseroreactive for A. platys and E. canis were higher (P = .015) than those of seronegative dogs. A. platys DNA was amplified from 7/21 (33%) A. platys seroreactive dogs and from 1 E. canis seroreactive dog; E. canis DNA was amplified from 21/25 (84%) E. canis seroreactive dogs. E. canis DNA was amplified most often from blood, whereas A. platys DNA was amplified most often from bone marrow.

Conclusions and Clinical Importance

Apparently healthy, free‐roaming dogs coseropositive for A. platys and E. canis may have increased intraoperative bleeding tendencies despite normal hemostatic parameters. Future investigations should explore the potential for vascular injury as a cause for bleeding in these dogs. Improved tick control is needed for dogs in Grenada.     

Distribution,quantitative load and characterization of Salmonella associated with swine farms in upper-northern Thailand

This study was conducted to analyze the prevalence and quantitative loads of Salmonella spp. on pig farms in Chiang Mai, Lamphun, Thailand to assess loading levels before slaughtering. The serotype diversity, antimicrobial-resistance pattern and pulse-field type of Salmonella spp. were also characterized to assess the dynamic propagation of the pathogen. The Salmonella-positive prevalence was 246/805 (30.56%), and the quantitative loads varied from 1.48~4.04 Log₁₀MPN/g, with a mean ± standard deviation of 2.11 ± 0.57. AMP/S/TE (ampicillin/streptomycin/tetracycline) was the highest frequency antimicrobial resistance pattern found in this study. In addition, Salmonella Rissen was the primary serotype in this region. PFGE results indicated the occurrence of infection by cross contamination among pig farms. Our study showed that pork is easily contaminated with this pathogen. Farm control programs must be based on strict biosecurity and hygienic measures, which could further reduce the contamination pressure at slaughterhouses or retail shops.