Anaplasmosis in two dogs in France and molecular and phylogenetic characterization of Anaplasma phagocytophilum

Two dogs in France were diagnosed with Anaplasma phagocytophilum infection by real-time PCR. The most remarkable hematologic and biochemical findings were severe thrombocytopenia, mild neutrophilia, morulae in neutrophils, and increased serum concentration of the α2-globulin fraction detected by agarose gel electrophoresis of serum proteins. Using sequencing of the partial 16S rRNA and ankA genes, molecular characterization of the A. phagocytophilum strains showed that the organisms from both dogs were identical to the European strains isolated from horses and people. Based on phylogenetic analysis, the ankA gene was more discriminating than the 16S rRNA gene in distinguishing the majority of European and American strains of A. phagocytophilum infecting people and animals. Three isolates of A. phagocytophilum, 1 from Spain (cow) and 2 from Norway (sheep and deer), were external to the European and American clades.     

Molecular Evidence for Persistence of Anaplasma phagocytophilum in the Absence of Clinical Abnormalities in Horses after Recovery from Acute Experimental Infection

Background: Anaplasma phagocytophilum infects several mammalian species, and can persist in sheep, dogs, and calves. However, whether this organism persists in horses or induces long-term clinical abnormalities is not known.
Objectives: To evaluate whether A. phagocytophilum can persist in horses and to document clinical findings for 3 months after complete recovery from acute disease.
Animals: Five clinically normal adult horses that had recovered spontaneously from experimentally induced acute disease caused by a Swedish equine isolate of A. phagocytophilum .
Methods: Horses were monitored for up to 129 days post inoculation (PI) by daily clinical examination and at least alternate day blood sampling for evidence of A. phagocytophilum on polymerase chain reaction (PCR) and blood smears. All horses were euthanized and underwent postmortem examination.
Results: All horses were periodically PCR positive after recovery from acute infection. Before day 66 PI 2 horses were persistently PCR negative whereas 3 horses were intermittently PCR positive. Subsequently, 4 of 5 horses were intermittently PCR positive, particularly after stress mimicking interventions. One animal was positive immediately before postmortem examination. Clinical abnormalities related to persistence of anaplasma were not observed. No specific changes were found at postmortem examination, and all sampled tissues from all horses were negative on PCR for A. phagocytophilum .
Conclusions and Clinical Importance: Infection with A. phagocytophilum can persist in the horse for at least 129 days. However, the continued presence of the organism is not associated with detectable clinical or pathological abnormalities.     

Molecular detection and characterization of Anaplasma phagocytophilum strains

The zoonotic rickettsial pathogen Anaplasma phagocytophilum has a broad geographic distribution and a high degree of biological and clinical diversity. To investigate the genetic diversity of A. phagocytophilum strains in the Baltic region and Norway, three species of Ixodidae ticks were examined for A. phagocytophilum infection, and two genes of the pathogen genome were analyzed. Analysis of partial 16S rRNA and partial major surface protein (msp4) gene sequences was accomplished through nested PCR and sequence analysis. Strains identified in this study were compared with those originating from other European countries and the United States. Seven 16S rRNA gene variants and fifteen msp4 gene variants of A. phagocytophilum were detected. Nine sequences had unique nucleotide polymorphisms and therefore differed from other A. phagocytophilum sequences previously submitted to GenBank. The present study represents the first molecular characterization of A. phagocytophum strains circulating in Lithuania and describes the strains detected in Ixodes persulcatus ticks in Latvia and Estonia. This is also the first report describing A. phagocytophilum strains isolated from Dermacentor reticulatus ticks.     

Molecular analyses of a potentially novel Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer (Cervus nippon yesoensis) in Japan

An Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer in Hokkaido, Japan was molecularly analyzed using 16S rRNA, citrate synthase (gltA), and heat-shock operon (groEL) gene sequences. Genome walking was performed to determine its complete gltA and groEL sequences (1233 bp and 1650 bp, respectively). Percent identities to the closest A. phagocytophilum sequences from the US and European strains were 98.6-98.8%, 76.5%, and 80.3-80.8% for 16S rRNA, gltA, and groEL genes, respectively. For deduced amino acid sequences, percent identities to the closest A. phagocytophilum sequences were 66.7% and 97.6% for gltA and groEL genes, respectively. Phylogenetic analyses revealed divergence from any known A. phagocytophilum strain. The lower identities and the divergent phylogenetic position of the Anaplasma sp. detected from sika deer in Japan with established A. phagocytophilum strains provide evidence of its potential novelty.     

Anaplasma phagocytophilum infection in a dog: identifying the causative agent using PCR

A diagnosis of Anaplasma phagocytophilum infection was confirmed in a two-year-old male golden retriever displaying few clinical and haematological abnormalities. This was achieved by demonstrating ehrlichial organisms in circulating neutrophils, by indirect immunofluorescence assay using A phagocytophilum as an antigen, and by detecting DNA specific for the 16S rRNA gene of granulocytic Anaplasma by PCR. After treatment with doxycycline for 10 days the dog showed improvement and the laboratory values returned to normal.     

Broad range 16S rRNA gene PCR compared to bacterial culture to confirm presumed synovial infection in horses

The objectives of the present study were to evaluate the accuracy of broad range 16S rRNA gene PCR compared to bacterial culture for the detection of synovial infection in horses. The study included 57 synovial fluid samples from horses with presumed synovial infection and a control group consisting of 31 synovial fluid samples originating from clinically normal horses and horses with aseptic synovial inflammation. All samples were analysed by 16S PCR with reverse line blot (RLB) hybridisation. Synovial fluid samples were cultured using conventional agar plate methods (APM) and/or blood culture medium (BCM). The results of the study showed a superior detection rate (89.5%) for 16S PCR with RLB. Bacterial culture had lower sensitivity, but highly acceptable detection rates (77.6%) were observed using BCM. APM had very low sensitivity (37.8%) and infection was never detected by plate isolation without positive incubation in BCM. The highest sensitivity (91.8%) for the detection of synovial infection was achieved when the results of incubation in BCM and 16S PCR were combined. For all the tests, the specificity was higher than 90%.     

Identification of Anaplasma spp. rickettsia isolated from horses from clinical disease cases in Poland

This study was aimed at determining the cause of the diseases in five horses exhibiting symptoms of fever, joint oedema and ataxia and thrombocytopenia. The PCR technique revealed the presence in the blood of 16S RNA Anaplasma/Ehrlichia spp. genetic material. DNA amplification with primers EHR 521 and EHR 747 gave a product with a size of 247 bp. The sequence of the PCR product obtained showed a 97.6-99.6% similarity with a sequence of a fragment of 16S RNA Anaplasma phagocytophilum, gene number EU 090186 from GenBank. Intravenous administration of oxytetracycline at a dose of 8 mg/kg of body mass for 7 days resulted in a gradual recovery.     

Seroprevalence of Anaplasma phagocytophilum among healthy dogs and horses in Israel

The presence of reacting antibodies to Anaplasma phagocytophilum has previously been demonstrated in Israel, both in humans and the golden jackal (Canis aureus syriacus). This study was undertaken to determine the seroprevalence of A. phagocytophilum antibodies in two additional potential hosts, domestic dogs and horses in order to investigate the possibility of exposure to the organism in Israel. Of 195 dogs tested, 9% were seroreactive with A. phagocytophilum antigen and 30% were seroreactive to Ehrlichia canis. Twenty-nine percent of the dogs seropositive for E. canis were also reactive to A. phagocytophilum. Two dogs had immunofluorescence antibody (IFA) antibody titres for A. phagocytophilum greater than E. canis. The equine serological survey (n = 300) revealed no seroreactive horses. The results presented in this study suggest that dogs in Israel could have been accidentally exposed to A. phagocytophilum, for example by ticks carried on migrating birds, however, the possibility of cross-reaction with E. canis should also be considered. In spite of the high prevalence of ticks on horses in Israel during the summer months, no evidence for exposure to A. phagocytophilum was apparent.     

Detection and quantitation of Ehrlichia risticii genomic DNA in infected horses and snails by real-time PCR

A real-time quantitative PCR using the TaqMan fluorogenic detection system (TaqMan PCR) was established for identification of Ehrlichia risticii, the agent of Potomac horse fever (PHF). The TaqMan PCR identified an 85 base pair section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for eight tested E. risticii strains. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene of E. risticii. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The TaqMan PCR was evaluated on horses with infectious colitis and on freshwater stream snails collected from regions with a history of PHF. E. risticii could be detected in 22 of 153 (14.4%) horses with infectious colitis and in 25 of 234 (10.7%) snails in the TaqMan PCR. The same results were obtained in the conventional nested PCR. The Ehrlichia-load was in the range of 10,000-9,000,000 and 35,000-680, 000,000 Ehrlichia equivalents per microg leukocyte DNA and snail DNA, respectively.     

Detection of bacterial DNA in synovial fluid from horses with infectious synovitis

Standard culturing techniques are often unrewarding in confirming diagnosis of synovial infection in the equine patient. Several human studies report the use of sensitive polymerase chain reaction (PCR) techniques for the detection of bacterial involvement in acute synovitis. However, successful extraction of bacterial DNA directly from clinical samples from horses without prior culture has not been reported yet. The goal of this study was to develop a sensitive and reliable method for molecular detection and identification of bacterial species in synovial fluid from horses with infectious synovitis. Synovial fluid samples from 6 horses with culture confirmed synovial infection were used for broad range 16S rRNA gene PCR. Synovial aspirates of 2 healthy horses were used as negative controls. Following extraction and purification of synovial fluid DNA, all samples were processed by touchdown PCR. Amplicons were detected by reverse line blot hybridisation and visualised with chemiluminescence. Pathogen-specific detection of 16S rRNA gene sequences was successful in all 6 synovial fluid samples. No bacterial DNA was detected in the aspirates from the negative control horses using touchdown PCR followed by 25 additional cycles of amplification. The identity of the pathogens was confirmed by DNA sequencing of the amplicons. It can be concluded that broad range 16S rRNA gene PCR followed by reverse line blot hybridisation is a promising technique for detection of bacterial DNA in synovial fluid samples. Further research should aim at the detection of bacterial DNA in synovial fluid samples suspected of infection but having negative culture results. When the 16S PCR proves to be reliable and more sensitive than standard culturing techniques, it may become a powerful tool in the diagnosis of synovial infection.     

Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus)

Anaplasmataceae organisms comprise a group of obligate intracellular gram-negative, tick-borne bacteria that can infect both animals and humans. In the present work we investigate the presence of Ehrlichia, Anaplasma, and Neorickettsia species in blood samples from Brazilian marsh deer (Blastocerus dichotomus), using both molecular and serologic techniques. Blood was collected from 143 deer captured along floodplains of the Paraná River, near the Porto Primavera hydroelectric power plant. Before and after flooding, marsh deer were captured for a wide range research program under the financial support of S?o Paulo State Energy Company (CESP), between 1998 and 2001. Samples were divided into four groups according to time and location of capture and named MS01 (n=99), MS02 (n=18) (Mato Grosso do Sul, before and after flooding, respectively), PX (n=9; Peixe River, after flooding), and AGUA (n=17; Aguapeí River, after flooding). The seroprevalences for Ehrlichia chaffeensis and Anaplasma phagocytophilum were 76.76% and 20.2% in MS01, 88.88% and 5.55% in MS02, 88.88% and 22.22% in PX, and 94.12% and 5.88% in AGUA, respectively. Sixty-one animals (42.65% of the total population) were PCR-positive for E. chaffeensis PCR (100.0% identity based on 16S rRNA, dsb, and groESL genes). Seventy deer (48.95% of the total population) were PCR-positive for Anaplasma spp. (99.0% of identity with A. platys, and in the same clade as A. phagocytophilum, A. bovis, and A. platys based on 16S rRNA phylogenetic analysis). Our results demonstrate that Brazilian marsh deer are exposed to E. chaffeensis and Anaplasma spp. and may act as reservoirs for these rickettsial agents, playing a role in disease transmission to humans and other animals.     

A survey for piroplasmids in horses and Bactrian camels in North-Eastern Mongolia

Equine piroplasmosis caused by Babesia caballi and Theileria equi is widespread in Asia. The presence of these haemozoans in Mongolia was previously confirmed in domestic as well as in reintroduced Przewalski horses in which they cause significant pathology. The data on occurrence of piroplasms from Bactrian camels in Asia is lacking. A total of 192 horses, 70 Bactrian camels, and additional 16 shepherd dogs from the Hentiy province were included in our study. No clinical signs typical for piroplasmid infection were observed during the field survey. Microscopic examination revealed the presence of T. equi in blood smears from 67% of examined horses, with camels and dogs being negative. A two step PCR approach was used to detect piroplasms in peripheral blood. In the first "catch all" PCR reaction, amplification of the 496 bp-long fragment of the SSU rRNA gene enabled the detection of Babesia and Theileria spp. Second round multiplex PCR reaction used for species discrimination allowed the amplification of T. equi- and B. caballi-specific 340 bp and 650 bp-long regions of the SSU rRNA, respectively. This assay detected T. equi in 92.7% of horses, while the infections with B. caballi and dual infections were rare. In both PCR setups, camels and dogs were negative indicating that in the studied region, these hosts do not share piroplasms with horses.     

Genetic diversity of Streptococcus equi subsp. zooepidemicus isolated from horses

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic and zoonotic pathogen of horses. In this study, genetic intraspecies variability of SEZ obtained mainly from respiratory and genital samples of horses was investigated by analysis of the 16S–23S rRNA intergenic spacer region (ISR) and of the 16S rRNA gene. 16S–23S ISR rRNA type A1 was predominant, although a high rate of multiple products (30.5%) was obtained. Phylogenetic analysis of the 16S rRNA gene detected three genogroups (I, II and III). 16S rRNA variable regions V1 and V2 are the most important regions for evaluating SEZ intraspecies variability, but at least V1-V5 regions should be considered to avoid mistakes. Analysis of all 16S rRNA sequences available in databases assigned human SEZ to groups I and III but not to group II. These results show a high genetic variability in SEZ collected from different specimens of horses from various regions of Italy.     

Variant -and individual dependent nature of persistent Anaplasma phagocytophilum infection

Background

Anaplasma phagocytophilum is the causative agent of tick-borne fever in ruminants and human granulocytotropic anaplasmosis (HGA). The bacterium is able to survive for several months in immune-competent sheep by modifying important cellular and humoral defence mechanisms. Little is known about how different strains of A. phagocytophilum propagate in their natural hosts during persistent infection.

Methods

Two groups of five lambs were infected with each of two 16S rRNA gene variants of A. phagocytophilum, i.e. 16S variant 1 which is identical to GenBank no {"type":"entrez-nucleotide","attrs":{"text":"M73220","term_id":"148293","term_text":"M73220"}}M73220 and 16S variant 2 which is identical to GenBank no {"type":"entrez-nucleotide","attrs":{"text":"AF336220","term_id":"13325085","term_text":"AF336220"}}AF336220, respectively. The lambs were infected intravenously and followed by blood sampling for six months. A. phagocytophilum infection in the peripheral blood was detected by absolute quantitative real-time PCR.

Results

Both 16S rRNA gene variants of A. phagocytophilum established persistent infection for at least six months and showed cyclic bacteraemias, but variant 1 introduced more frequent periods of bacteraemia and higher number of organisms than 16S rRNA gene variant 2 in the peripheral blood.

Conclusion

Organisms were available from blood more or less constantly during the persistent infection and there were individual differences in cyclic activity of A. phagocytophilum in the infected animals. Two 16S rRNA gene variants of A. phagocytophilum show differences in cyclic activity during persistent infection in lambs.     

A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses

: A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.     

Acute clinical, hematologic, serologic, and polymerase chain reaction findings in horses experimentally infected with a European strain of Anaplasma phagocytophilum

Six horses were experimentally infected by administration of horse blood containing a Swedish strain of Anaplasma phagocytophilum. The polymerase chain reaction (PCR) signal was consistently detected 2-3 days before appearance of clinical signs and persisted 4-9 days beyond abatement of clinical signs, whereas diagnostic inclusion bodies were 1st noted on average 2.6 +/- 1.5 (SD) days after onset of fever. Clinical signs and hematologic changes were largely indistinguishable from those previously reported for diseases caused by A phagocytophilum (formerly Ehrlichia equi--"Californian agent") and the human-derived human granulocytic ehrlichiosis agent. Horses 1st demonstrated antibody response 12-16 days after inoculation, 2 cases of which were still febrile, and serotiters rapidly peaked within 3-7 days of clinical illness. One horse died during the acute stage of disease, but initial clinical signs and hematologic changes were similar to those of other infected horses. This report shows that, despite minor genetic differences, a European equine-derived strain of A. phagocytophilum may be similar in pathogenicity to the Californian agent. The PCR used holds promise to widen the diagnostic window and would also be diagnostic during the initial days of clinical disease when inclusions in neutrophils in blood smears are not yet apparent.     

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Ticks are known or suspected vectors for a wide range of bacterial pathogens. One of the first steps for tick-borne risk assessment is the detection of these pathogens in their vectors. In the present study, a broad-range PCR amplification of the eubacterial gene encoding the 16S rRNA gene combined with Temporal Temperature Gradient gel Electrophoresis (TTGE) was evaluated as a method allowing the one-step detection of bacterial pathogen DNA in ticks. Firstly, DNA extracts from bacteria known to be tick-borne pathogens, i.e., Borrelia burgdorferi lato sensu, Anaplasma phagocytophilum, Spotted Fever Group (SFG) Rickettsia spp., were used to establish a TTGE pathogen DNA reference marker. Secondly, we used broad-range PCR-TTGE to detect the presence of DNA from these three pathogens in 55 DNA extracts from pools of 10 nymphal Ixodes ricinus ticks, which have been previously shown to carry DNA from at least one of those bacteria by specific PCR. Among the 20 B. burgdorferi specific-PCR samples, 15 (75%) were also found to be positive using PCR-TTGE. Sixteen of the seventeen (94%) Rickettsia spp. PCR-specific samples were positive using PCR-TTGE detection and all PCR-specific positive extracts (11/11, 100%) for A. phagocytophilum were also positive using PCR-TTGE. Moreover, we identified unexpected bacterial sequences that were not related to any of the three pathogens such as a sequence related to Spiroplasma sp. Thus, broad-range PCR-TTGE allowed the single step detection of DNA from up to 3 pathogens in the same co-infected samples as well as detection of DNA from unexpected bacteria.     

Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus

Strangles is a contagious equine disease caused by Streptococcus equi subsp. equi. In this study, clinical strains of S. equi (n=24) and Streptococcus equi subsp. zooepidemicus (n=24) were genetically characterized by sequencing of the 16S rRNA and sodA genes in order to devise a real-time PCR system that can detect S. equi and S. zooepidemicus and distinguish between them. Sequencing demonstrated that all S. equi strains had the same 16S rRNA sequence, whereas S. zooepidemicus strains could be divided into subgroups. One of these (n=12 strains) had 16S rRNA sequences almost identical with the S. equi strains. Interestingly, four of the strains biochemically identified as S. zooepidemicus were found by sequencing of the 16S rRNA gene to have a sequence homologous with Streptococcus equi subsp. ruminatorum. However, they did not have the colony appearance or the biochemical characteristics of the type strain of S. ruminatorum. Classification of S. ruminatorum may thus not be determined solely by 16S rRNA sequencing. Sequencing of the sodA gene demonstrated that all S. equi strains had an identical sequence. For the S. zooepidemicus strains minor differences were found between the sodA sequences. The developed real-time PCR, based on the sodA and seeI genes was compared with conventional culturing on 103 cultured samples from horses with suspected strangles or other upper respiratory disease. The real-time PCR system was found to be more sensitive than conventional cultivation as two additional field isolates of S. equi and four of S. zooepidemicus were detected.     

Identification and characterization of Streptococcus agalactiae isolated from horses.

Seven group B streptococcal cultures isolated from three horses reacted with group B-specific antiserum, were CAMP positive, pigmented and showed the typical biochemical properties of Streptococcus agalactiae. The identification could be confirmed by PCR amplification of the 16S rRNA gene and a subsequent RsaI restriction pattern typical for S. agalactiae. In addition, the isolates were identified by amplification of species specific parts of the 16S rRNA gene, the 16S-23S rRNA intergenic spacer region and by amplification of the CAMP-factor (cfb) gene. Six isolates could be classified as serotype III/Rib, one isolate as serotype Ia/cbeta. The occurrence of the protein antigens Rib and cbeta could be confirmed by PCR amplification of the respective genes. The six isolates of serotype III/Rib were hyaluronidase negative, had a hylB gene with a size of 4.6 kb and an insertion element IS1548 of 0.98 kb. The isolate of serotype Ia/cbeta was hyaluronidase positive, had a hylB gene with a size of 3.3 kb and no insertion element IS1548. In addition, all seven isolates had the insertion element ISSag2 and the gene lmb encoding the laminin binding surface protein Lmb and the gene scpB encoding C5a peptidase. According to the present results the group B streptococci isolated from horses showed characteristics of human isolates of this species.     

Anaplasma phagocytophilum in Danish sheep: confirmation by DNA sequencing

Background

The presence of Anaplasma phagocytophilum, an Ixodes ricinus transmitted bacterium, was investigated in two flocks of Danish grazing lambs. Direct PCR detection was performed on DNA extracted from blood and serum with subsequent confirmation by DNA sequencing.

Methods

31 samples obtained from clinically normal lambs in 2000 from Fussingø, Jutland and 12 samples from ten lambs and two ewes from a clinical outbreak at Feddet, Zealand in 2006 were included in the study. Some of the animals from Feddet had shown clinical signs of polyarthritis and general unthriftiness prior to sampling. DNA extraction was optimized from blood and serum and detection achieved by a 16S rRNA targeted PCR with verification of the product by DNA sequencing.

Results

Five DNA extracts were found positive by PCR, including two samples from 2000 and three from 2006. For both series of samples the product was verified as A. phagocytophilum by DNA sequencing.

Conclusions

A. phagocytophilum was detected by molecular methods for the first time in Danish grazing lambs during the two seasons investigated (2000 and 2006).