Neorickettsia Helminthoeca Research Articles

Neorickettsia helminthoeca associated lymphoid, enteric, and pulmonary lesions in dogs from Southern Brazil: An immunohistochemical study.

Neorickettsia helminthoeca (NH), the agent of salmon poisoning disease or canine neorickettiosis (CN), is a bacterial endosymbiont of the nematode Nanophyetus salmincola, and infections are spreading among specific fish-eating mammalians. This article describes the pathologic and immunohistochemical findings associated with spontaneous NH-induced infections in dogs from Southern Brazil. The principal pathologic findings were hypertrophy of Peyer patches and lymphadenopathy with lymphocytic proliferation, chronic interstitial pneumonia, and chronic enteritis associated with positive intralesional immunoreactivity to antigens of NH within macrophages and histiocytes. Positive immunoreactivity against canine parvovirus-2 (CPV-2) or/and canine distemper virus was not detected in the evaluated intestinal segments or in the samples from the cerebellum and lungs, respectively, from the dogs evaluated. These findings demonstrated that NH was involved in the enteric, pulmonary, and lymphoid lesions herein described, and provide additional information to confirm the occurrence of this bacterial endosymbiont within this geographical location. It is proposed that chronic pneumonia should be considered as a pathologic manifestation of NH-induced infections. Additionally, our results show that the occurrences of CN seem to be underdiagnosed in Southern Brazil due to the confusion with the incidence of CPV-2.

IMMUNOHISTOCHEMICAL AND MOLECULAR EVIDENCE OF PUTATIVE NEORICKETTSIA INFECTION IN COATIS ( NASUA NASUA) FROM SOUTHERN BRAZIL.

The pathologic, molecular, and immunohistochemical findings associated with Neorickettsia helminthoeca are described in coatis ( Nasua nasua). Tissue sections (small intestine, lungs, kidney, liver, and spleen) of coatis ( n = 3) that died at the Bela Vista Biological Refuge, Foz do Iguaçu, Paraná, southern Brazil were routinely processed from histopathology. Selected formalin-fixed paraffin-embedded (FFPE) tissue sections of the small intestine, lungs, and spleen were used in an immunohistochemical (IHC) assay designed to identify the antigens of N. helminthoeca. Additionally, FFPE tissue sections of the small intestine were used to demonstrate antigens of canine parvovirus-2 (CPV-2) by IHC. Histopathology revealed chronic enteritis in all coatis. Parasitic enteritis was diagnosed in two coatis; one of these contained examples of a trematode within the lumen of the small intestine and the ovum of a trematode encysted in the intestinal mucosa. Other significant pathologic findings included interstitial pneumonia ( n = 2) and pyogranulomatous splenitis ( n = 1). Positive immunolabeling for N. helminthoeca was identified within macrophages of the small intestine and reticuloendothelial cells within the germinal centers of the spleen of all coatis; the intestinal trematode was N. helminthoeca IHC-positive. All pulmonary sections revealed negative immunolabeling for N. helminthoeca. Furthermore, the antigens of CPV-2 were not identified in the intestine of any coati. These findings indicate that these coatis were infected by N. helminthoeca, but since clinical and gross pathological findings were not recorded, it is uncertain if this pathogen produced clinical disease in this canid host; therefore, coatis may be asymptomatic or dead-end hosts for this organism.

Salmon poisoning disease in grizzly bears with population recovery implications

ABSTRACTWildlife restoration programs depend on having animals that have evolved to handle the constraints, including disease, posed by the new environment. Grizzly bears (Ursus arctos horribilis) once thrived while feeding on salmon (Onchorhynchus spp.) and other foods in California, Oregon, Washington, and Idaho, USA. Unlike Canadian and Alaskan salmon, salmon in lower latitudes often carry an encysted fluke (Nanophyetus salmincola) containing an endosymbiont (Neorickettsia spp.) that can produce a deadly disease in ursids and canids called salmon poisoning disease (SPD). Current recovery plans for grizzly bears in the North Cascades of Washington and the mountains of central Idaho, where infected salmon currently occur, call for using bears from several interior populations. We hypothesized that such bears with no history of salmon consumption will be sensitive to SPD. To test that idea, we fed a limited amount of fresh Chinook salmon (O. tshawytscha) harvested from rivers draining the east and west sides of the Cascades to 11 captive grizzly bears in which both parents were from populations with no history of SPD. We detected N. salmincola ova in the feces, and 8 bears exhibited typical SPD symptoms, which included decreased or no appetite, lethargy, vomiting, and diarrhea. We identified N. helminthoeca and Stellantchasmus falcatus (SF) agent (Neorickettsia sp.) in the salmon, but only SF agent occurred in the sick bears. Stellantchasmus falcatus agent is a relatively new arrival in the Pacific Northwest. Because of the severity of the disease and the potential for mortality without treatment, additional studies should be conducted to determine the distribution and prevalence of SF agent in salmon and kokanee (O. nerka) in the recovery area, develop a serum antibody test that could be used to determine exposure of bears to SF agent, and use that test to determine if grizzly bears or American black bears (U. americanus) currently living in the Pacific Northwest have been infected and recovered. If either of the latter have occurred, SPD may not prevent the successful restoration of grizzly bears where SF agent‐infected salmon will be consumed. © 2018 The Wildlife Society.

Metagenomic survey of bacteria associated with the invasive ladybird Harmonia axyridis (Coleoptera: Coccinellidae)

The Asian ladybird Harmonia axyridis is an invasive insect in Europe and the Americas and is a great threat to the environment in invaded areas. The situation is exacerbated by the fact that non native species are resistant to many groups of parasites that attack native insects. However, very little is known about the complex microbial community associated with this insect. This study based on sequencing 16S rRNA genes in extracted metagenomic DNA is the first research on the bacterial flora associated with H. axyridis. Lady beetles were collected during hibernation from wind turbines in Poland. A mean ± SD of 114 ± 35 species of bacteria were identified. The dominant phyla of bacteria recorded associated with H. axyridis were Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. Representatives of these phyla are common in the environment, e.g. in the soil, and are often identified as the dominant bacteria associated with arthropods. We also identified animal pathogenic bacteria, such as Burkholderia, Rhodococcus, Chlamydiae and Anaplasmataceae spp. (Neorickettsia helminthoeca and Ehrlichia ovina). We also identified Wolbachia pipientis in a single beetle. This bacterium is a causative agent of reproductive alterations in arthropods. These results support the enemy release hypothesis in the case of this ladybird invasion. Pathogenic bacteria were recorded in only a few samples. Moreover, male-killing bacteria such as Spiroplasma spp., Wolbachia spp. and Rickettsia spp. were only recorded in single insects so they cannot be responsible for the observed alterations in the sex-ratio of the ladybird population studied.

Analysis of complete genome sequence and major surface antigens of Neorickettsia helminthoeca, causative agent of salmon poisoning disease.

Summary Neorickettsia helminthoeca, a type species of the genus Neorickettsia, is an endosymbiont of digenetic trematodes of veterinary importance. Upon ingestion of salmonid fish parasitized with infected trematodes, canids develop salmon poisoning disease (SPD), an acute febrile illness that is particularly severe and often fatal in dogs without adequate treatment. We determined and analysed the complete genome sequence of N. helminthoeca: a single small circular chromosome of 884 232 bp encoding 774 potential proteins. N. helminthoeca is unable to synthesize lipopolysaccharides and most amino acids, but is capable of synthesizing vitamins, cofactors, nucleotides and bacterioferritin. N. helminthoeca is, however, distinct from majority of the family Anaplasmataceae to which it belongs, as it encodes nearly all enzymes required for peptidoglycan biosynthesis, suggesting its structural hardiness and inflammatory potential. Using sera from dogs that were experimentally infected by feeding with parasitized fish or naturally infected in southern California, Western blot analysis revealed that among five predicted N. helminthoeca outer membrane proteins, P51 and strain‐variable surface antigen were uniformly recognized. Our finding will help understanding pathogenesis, prevalence of N. helminthoeca infection among trematodes, canids and potentially other animals in nature to develop effective SPD diagnostic and preventive measures. Recent progresses in large‐scale genome sequencing have been uncovering broad distribution of Neorickettsia spp., the comparative genomics will facilitate understanding of biology and the natural history of these elusive environmental bacteria.

Nanophyetus salmincola, vector of the salmon poisoning disease agent Neorickettsia helminthoeca, harbors a second pathogenic Neorickettsia species

The trematode Nanophyetus salmincola is known as the carrier of Neorickettsia helminthoeca, an obligate intracellular endosymbiotic bacterium that causes salmon poisoning disease (SPD), a fatal disease of dogs. The bacteria are maintained through the complex life cycle of N. salmincola that involves snails Juga plicifera as the first intermediate host, salmonid fishes as the second intermediate host and fish-eating mammals as definitive hosts. N. salmincola was also found to harbor a second species of Neorickettsia that causes the Elokomin fluke fever disease (EFF) which has clinical signs similar to SPD in bears, but only low grade illness in dogs. The EFF agent has not been sequenced. In this study we identified N. salmincola as the vector of yet additional species of Neorickettsia known as Stellanchasmus falcatu (SF) agent using DNA sequencing.

Salmon Poisoning Disease in Dogs: 29 Cases

Salmon poisoning disease (SPD) is a trematode-borne disease of dogs caused by Neorickettsia helminthoeca. To determine risk factors and spatial epidemiology of SPD in dogs from northern California; to describe the clinicopathologic, microbiologic, and imaging findings of SPD in these dogs; and to evaluate treatments and outcomes for SPD. Twenty-nine dogs with SPD based on the finding of trematode ova in the feces, or organisms consistent with N. helminthoeca in specimens submitted for microscopic examination. Information regarding signalment, fish exposure, clinical signs, diagnostic evaluation, treatments, and outcomes was obtained for each dog. Archived lymph node aspirates and histopathology specimens were subjected to polymerase chain reaction (PCR) testing for Neorickettsia spp. Labrador Retrievers and intact male dogs were overrepresented. Exposure locations were often distant from the dogs' residence. Some dogs had neurologic signs, including twitching and seizures. Dogs lacking peripheral lymphadenomegaly had abdominal lymphadenomegaly on ultrasound examination. A combination of centrifugation fecal flotation and sedimentation had greatest sensitivity for finding fluke ova. N. helminthoeca DNA was amplified by PCR from 4/10 dogs. Penicillins, cephalosporins, and chloramphenicol did not appear to be effective treatments. Mortality rate was 4/29 (14%). SPD should be suspected in dogs with inappetence, gastrointestinal, or neurologic signs, with or without fever or peripheral lymphadenomegaly in the appropriate geographical setting. Diagnosis is facilitated by a combination of fecal sedimentation and centrifugal flotation, abdominal ultrasonography, and PCR-based assays on lymphoid tissue. The treatment of choice is tetracycline antimicrobials.

Veterinary issues related to bears (Ursidae)

A wide variety of infectious and non‐infectious diseases has been described in bears. Some viral (e.g. canine distemper, infectious canine hepatitis), bacterial (e.g. salmon poisoning) and parasitic diseases (particularly skin mites and ascarid infections) are of concern. Non‐infectious conditions, such as dental disease, degenerative joint disease and neoplasia, are very important in the management of captive bears. Appropriate anaesthesia is essential in both veterinary and biological interventions with bears.

Neorickettsia helminthoeca and salmon poisoning disease: A review

Neorickettsia helminthoeca is an obligate intra-cytoplasmic bacterium that causes salmon poisoning disease (SPD), an acute, febrile, fatal disease of dogs. The complex life-cycle of this pathogen involves stages in an intestinal fluke (Nanophyetus salmincola), a river snail (Oxytrema silicula), in fish, and in fish-eating mammals. This complexity has created confusion with respect to the various bacterial and parasitic infections associated with the disease and its significance in dogs in specific geographical locations has likely to have previously been under-estimated. This paper addresses the history, taxonomy, microbiology of N. helminthoeca and summarises the pathogenesis, clinical signs and pathological features associated with infection. Furthermore, the biological cycles, treatment, control, and both public and veterinary health impacts associated with this pathogen and the intestinal fluke N. salmincola are discussed.

Neorickettsia helminthoeca in Brazilian dogs: a cytopathological, histopathological and immunohistochemical study

Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD,USAINTRODUCTIONNeorickettsia helminthoeca is an intracellular bacte-rium of reticuloendothelial cells of dogs thatcauses salmon poisoning disease (SPD), whichwas considered endemic only to specific geo-2graphical locations of North America [1–3].However, 20 mature dogs demonstrated lesionsconsistent with this infection in Southern Braziland intracytoplasmic and intralesional neorickett-sial bodies were identified within reticuloendo-thelial cells by Giemsa stain [4]. Additionally, bymolecular biology methods, it was shown that themesenteric lymph node of one dog containedDNA that was similar to N. helminthoeca as knownin the USA [5].This article describes the cytological, histopath-ological and immunohistochemical findings asso-ciated with N. helminthoeca in additional casesfrom Brazil.MATERIALS AND METHODS

Detection of new Anaplasmataceae in the digestive tract of fish from southeast Asia

The bacteria of the genus Neorickettsia are strict intracellular bacteria of the Anaplasmataceae family. They are transmitted to their definitive host (humans and animals) by ingestion of raw fish, snails or aquatic insects that are parasitised by infected trematodes. Among them, three species (N. sennetsu, N. helminthoeca and N. risticii) are known to cause diseases in mammals (humans and animals) [1] but two have not yet been associated with the disease (SF agent, and Ehrlichia species trout isolate). About 50 million people in the world are infested by food-borne trematodes, predominantly in southeast Asia. Uncooked fish and shellfish consumption is the principal mode of transmission. Fish-borne trematodes in humans are increasing in endemic areas, and are emerging in Europe and America [2]. Globalisation, international travels and new cooking fashions such as eating raw fish ‘Sushi’ and ‘Sashimi’ are likely to lead to new emerging infections caused by Neorickettsia. We studied here the presence of Neorickettsia in fish and fish-based ingredients that are usually consumed uncooked in southeast Asia. A total of 126 fish and 19 ingredients made from uncooked fish were studied. Between January and February 2007, 57 frozen fish and seven ingredients imported from Thailand and Vietnam were bought in the Asian market in Marseille. In March 2007, 69 other fish preserved in 70% ethanol and 12 supplementary ingredients were collected in Cambodia. After dissection of defrosted fish, stomach and intestine were collected for direct examination for parasite by binocular microscope, and then processed for PCR analysis, culture and histology. Found worms were kept in the 70% ethanol for morphological and molecular identification of the 18S gene by using primers P6.F and 18sr2R [3]. Following DNA extraction of the fish digestive tract, DNA of Anaplasmataceae was detected by rrS PCR using primers: Ehr16SD and Ehr16SR [1]. We completed rrS sequences found by using specific primers (Ncs-614F: 5¢-TTG-GTG-TAG-GGG-TGAAAT-CC, Ncs-606R: 5¢-CCC-CTA-CAC-CAA-AAATTC-CA) and non-specific primers (NcsMA-14F: 5¢-GGC-AGA-CGG-GTG-CGT-AAC, Ncs-1321R: 5¢-GAC-GGG-CAG-TGT-GTA-CAA-GA) newly designed respectively by alignment of rrS sequences of three new Anaplasmataceae with Neorickettsia sequences available in GenBank and by alignment of rrS sequence of all Anaplasmataceae available in GenBank. DNA fragment of the citrate synthase gene (gltA) of Anaplasmataceae was identified by using F1 and R1b as previously described [1]. Negative controls consisted of DNA extracted from human colon, uninfected fish and sterile water. DNA of A. phagocytophilum was used as positive control of rrS PCR and N. sennetsu for gltA PCR. We designed new primers CG-442F (5¢-GGC-CCATAC-CCC-ARN-AAT-G) and CG-877R (5¢-GCDAGT-TTT-TGT-CAR-GTN-GA) to confirm the species of positive fishes. The sequence diversity of the rrS gene was used to define a new species when it was at least 3% between bacterial species. The new genus was definite when rrs gene sequence similarities were lower than 97% [4]. We identified 35 worms in six Channa striata imported into Marseille from Thailand. Sequence analysis of the 18S gene showed that all worms were belonging to Neoechinorhynchus pseudemydis species. Three new Anaplasmataceae were detected in three fish by rrS PCR by using Ehr16SD and Ehr16SR primers (Table 1). After completion of the rrS sequences, the Anaplasmataceae from the stomach of Channa striata fish imported from Thailand was identified as a new genus with a Corresponding author and reprint requests: P. Brouqui, URMITE CNRS ⁄ IRD UMR 6236, IFR 48, Universite de la Mediterranee, Faculte de Medecine, 27 Boulevard Jean Moulin, 13385 Marseille cedex 5, France E-mail: philippe.brouqui@univmed.fr

Salmon poisoning disease in two Malayan sun bears

2 captive sun bears (Helarctos malayanus) were evaluated because of acute onset of vomiting, mucoid diarrhea, lethargy, and anorexia 1 week after eating live trout from a northern California reservoir. In 1 of the bears, a CBC and serum biochemical analyses revealed mild anemia, mild eosinophilia, moderate lymphopenia, moderate hypoalbuminemia, and high serum G-glutamyltransferase activity. Ultrasonographic examination of the same bear revealed ascites and mesenteric lymphadenopathy. Histologic examination of gastrointestinal tract biopsy specimens revealed moderate to severe lymphoplasmacytic and eosinophilic gastritis, enteritis, and colitis. Ova of Nanophyetus salmincola, the trematode vector of Neorickettsia helminthoeca (a rickettsial organism that causes salmon poisoning disease), were detected in fecal samples from both bears. The bears were treated with oxytetracycline, doxycycline, praziquantel, and famotidine. Within 1 week after initiation of treatment, the appetite and fecal consistency of each bear were considered normal. Fecal ova shedding began 4 days after onset of clinical signs and ceased 9 days later. Salmon poisoning disease can be rapidly fatal in untreated animals, but if diagnosed early and treated appropriately, full recovery can be achieved. Domestic dogs and captive exotic bears are highly susceptible to clinical disease after ingestion of trematode-infected fish. Salmon poisoning disease may develop outside the geographic range in which the causative organism is endemic as a result of the transplantation of infected fish for sport fishing; veterinarians practicing in areas where infected fish may be transplanted should be aware of appropriate diagnostic and treatment protocols.

Neorickettsia helminthoecain Dog, Brazil

<i>Neorickettsia helminthoeca</i>in Dog, Brazil

Lymph node aspirate from a California wine‐country dog

A 4-year-old, male Golden Retriever was presented to the Veterinary Medical Teaching Hospital at the University of California-Davis with a history of lethargy, inappetance, and vomiting. The patient had generalized lymphadenomegaly, marked thrombocytopenia, mild anemia, and moderate hypoalbuminemia. Moderate to marked histiocytic inflammation and lymphocytic-plasmacytic reactivity of the mesenteric, left popliteal, and right mandibular lymph nodes were diagnosed cytologically. Many macrophages contained granular to amorphous material of a uniform blue color, occasionally in morula formation, suggestive of rickettsial organisms. Exposure to raw trout was subsequently documented, leading to a presumptive diagnosis of salmon poisoning disease (SPD). The patient responded quickly to doxycycline therapy for the causative agent of SPD (Neorickettsia helminthoeca). SPD should be considered as a differential diagnosis for a canine patient with clinical signs of vomiting, diarrhea, lethargy, and lymphadenomegaly; laboratory findings of thrombocytopenia and hypoalbuminemia; and potential exposure to raw fish from an endemic area. The cytologic finding of rickettsial inclusions within lymph node macrophages is reportedly seen within a majority of SPD cases and can be valuable in supporting a clinical suspicion of SPD, as it was in this case.

Suspected Cases of Neorickettsia‐Like Organisms in Brazilian Dogs

Preliminary findings of gross and histopathological lesions consistent with salmon poisoning disease in 10 dogs from southern Brazil are described. Lesions were restricted to the spleen, lymph nodes, and intestinal lymphoid tissues. Grossly, there was marked hyperplasia of mesenteric lymph nodes and Peyer's patches. Microscopic alterations were characterized by diffuse hyperplasia of intestinal lymphoid tissues and Peyer's patches. Intracytoplasmic organisms consistent with Neorickettsia helminthoeca were demonstrated by Giemsa stain in reticuloendothelial cells of the intestine, spleen, Peyer's patches, and lymph nodes. We have named this organism Neorickettsia helminthoeca-like because of its marked similarity with the agent described in the United States.

Analysis of p51, groESL, and the major antigen P51 in various species of Neorickettsia, an obligatory intracellular bacterium that infects trematodes and mammals.

The p51 gene that encodes the major antigenic 51-kDa protein in Neorickettsia risticii was identified in strains of Neorickettsia sennetsu and the Stellantchasmus falcatus agent but not in Neorickettsia helminthoeca, suggesting that p51-based diagnosis would be useful to distinguish among them. groESL sequencing results delineated the phylogenic relationships among Neorickettsia spp.

Citrate synthase gene sequence: a new tool for phylogenetic analysis and identification of Ehrlichia.

The sequence of the citrate synthase gene (gltA) of 13 ehrlichial species (Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia muris, an Ehrlichia species recently detected from Ixodes ovatus, Cowdria ruminantium, Ehrlichia phagocytophila, Ehrlichia equi, the human granulocytic ehrlichiosis [HGE] agent, Anaplasma marginale, Anaplasma centrale, Ehrlichia sennetsu, Ehrlichia risticii, and Neorickettsia helminthoeca) have been determined by degenerate PCR and the Genome Walker method. The ehrlichial gltA genes are 1,197 bp (E. sennetsu and E. risticii) to 1,254 bp (A. marginale and A. centrale) long, and GC contents of the gene vary from 30.5% (Ehrlichia sp. detected from I. ovatus) to 51.0% (A. centrale). The percent identities of the gltA nucleotide sequences among ehrlichial species were 49.7% (E. risticii versus A. centrale) to 99.8% (HGE agent versus E. equi). The percent identities of deduced amino acid sequences were 44.4% (E. sennetsu versus E. muris) to 99.5% (HGE agent versus E. equi), whereas the homology range of 16S rRNA genes was 83.5% (E. risticii versus the Ehrlichia sp. detected from I. ovatus) to 99.9% (HGE agent, E. equi, and E. phagocytophila). The architecture of the phylogenetic trees constructed by gltA nucleotide sequences or amino acid sequences was similar to that derived from the 16S rRNA gene sequences but showed more-significant bootstrap values. Based upon the alignment analysis of the ehrlichial gltA sequences, two sets of primers were designed to amplify tick-borne Ehrlichia and Neorickettsia genogroup Ehrlichia (N. helminthoeca, E. sennetsu, and E. risticii), respectively. Tick-borne Ehrlichia species were specifically identified by restriction fragment length polymorphism (RFLP) patterns of AcsI and XhoI with the exception of E. muris and the very closely related ehrlichia derived from I. ovatus for which sequence analysis of the PCR product is needed. Similarly, Neorickettsia genogroup Ehrlichia species were specifically identified by RFLP patterns of RcaI digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.

Molecular detection of an Ehrlichia-like agent in rainbow trout ( Oncorhynchus mykiss) from Northern California

Ehrlichia DNA was identified by nested PCR in rainbow trout ( Oncorhynchus mykiss) collected from a creek in northern California where Potomac horse fever is endemic. Ehrlichia DNA was found in tissues from several organs including the gills, heart, spleen, liver, kidneys and intestine of trout and from three different adult digenetic trematodes ( Deropegus sp., Crepidostomum sp., Creptotrema sp.) parasitizing the gallbladder and/or the intestine of the trout. Sequencing of PCR-amplified DNA from the 16S rRNA gene indicated that the source organism was most closely related to the sequences of E. risticii (level of sequence similarity 96.0%), the SF agent (95.9%), E. sennetsu (95.8%), and Neorickettsia helminthoeca (95.3%). The data suggest that trout and parasitic trematodes may be involved in the epidemiology of an Ehrlichia-like agent belonging to the E. sennetsu genogroup. Whether the fish agent infects horses, dogs, or human beings, and whether it causes disease, remain to be determined.

Characterization of the SF Agent, an Ehrlichia sp. Isolated from the Fluke Stellantchasmus falcatus, by 16S rRNA Base Sequence, Serological, and Morphological Analyses

The organism designated the SF agent was originally isolated in Japan in 1962 from Stellantchasmus falcatus metacercaria parasitic on gray mullet fish. The SF agent resembles members of the genus Ehrlichia morphologically and exhibits weak antigenic cross-reactivity with Ehrlichia sennetsu. This organism causes mild clinical signs in dogs, but severe splenomegaly and lymphadenopathy in mice. This suggests that the SF agent may be similar to either Neorickettsia helminthoeca, an intracellular parasite of a fluke and the cause of salmon poisoning disease in dogs, or E. sennetsu, the causative agent of human sennetsu ehrlichiosis in Japan and Malaysia. In order to determine the phylogenetic relationship between the SF agent and other ehrlichial species, the 16S rRNA gene was amplified by the PCR and sequenced. The SF agent sequence was most closely related to the sequences of Ehrlichia risticii (level of sequence similarity, 99.1%), the causative agent of Potomac horse fever, and E. sennetsu (level of sequence similarity, 98.7%). The next most similar sequence was that of N. helminthoeca, but the level of sequence similarity was only 93.7%. E. sennetsu, E. risticii, the SF agent, and N. helminthoeca formed a distinct cluster that was separated from all other ehrlichial species. As determined by immunofluorescence labeling, antiserum against the SF agent cross-reacted strongly with E. sennetsu, E. risticii, and N. helminthoeca. When three genetically distinct ehrlichial isolates obtained from horses with Potomac horse fever were compared with the SF agent, we found that the SF agent was most closely related to Ohio isolate 081, followed by IllinoisT (T = type strain) and a Kentucky isolate. We observed strong antigenic cross-reactivities and similarities in Western blot (immunoblot) reaction profiles when we compared the SF agent, E. risticii, and E. sennetsu; however, weaker antigenic cross-reactivity was observed when the SF agent and N. helminthoeca were compared. Our results indicate that the SF agent is antigenically more closely related to E. risticii and E. sennetsu than to N. helminthoeca. The biological and antigenic characteristics and the 16S rRNA sequence data suggest that the SF agent is a new species that belongs to the genus Ehrlichia.

Cytopathic effect, plaque formation, and lysis of Ehrlichia chaffeensis grown on continuous cell lines.

Ehrlichiae are strict intracellular bacterial pathogens that parasitize leukocytes or other blood cells. Only six agents of the tribe Ehrlichieae, namely, Cowdria ruminantium, Neorickettsia helminthoeca, Ehrlichia risticii, Ehrlichia sennetsu, Ehrlichia canis, and Ehrlichia chaffeensis, have been adapted to growth in continuous cell lines. E. chaffeensis, the agent of human ehrlichiosis, has been cultured only in a cell line of canine origin. We adapted purified cell-free E. chaffeensis for growth in human embryonic lung (HEL) fibroblasts (HEL 299), green monkey kidney cells (Vero), and a human cervical epithelioid carcinoma (HeLa) cell line. We observed a cytopathic effect with both Vero cells and HEL cells and plaque formation with cellular lysis when infected Vero cells were cultured in agar. Human fibroblasts are already commonly used for the isolation of viruses, coexiellae, and rickettsiae. Furthermore, the capability of these cells to support the growth of ehrlichiae suggests that they may be useful for primary isolation of ehrlichiae as well. The cytopathic effect produced in Vero or HEL cells offers a very helpful indicator of the infection. Plaque formation in Vero cells is a new phenomenon not yet reported for ehrlichiae and will allow the titration of inocula and clonal purification of this bacterium.