Neorickettsia sp. in coatis (Nasua nasua) in Brazil.

The genus Neorickettsia comprises trematode-associated bacteria that can cause diseases in animals and humans. Despite detection of Neorickettsia antigens in the intestine of coatis kept in captivity in southern Brazil through immunohistochemistry, the molecular identity of the bacteria in South American procyonids remains elusive. The aim of the present study was to investigate the occurrence of Neorickettsia sp. in blood samples from coatis in central-western Brazil. Between March 2018 and January 2019, animals were captured and recaptured in two areas of the Cerrado (Parque Estadual do Prosa, PEP; and Vila da Base Aérea, VBA) located in the city of Campo Grande, state of Mato Grosso do Sul, central-western Brazil. All captures were performed according to convenience. DNA from 97 blood samples was subjected to nested PCR (nPCR) targeting a fragment of the 16S rRNA gene of Neorickettsia sp. Six samples (3.6%; five from VBA and one from PEP) from different coatis were positive in nPCR based on the 16S rRNA. The sequences obtained (~500 bp) showed ˃ 99% similarity to N. risticii. Phylogenetic analysis clustered the sequences detected in the present study in a clade with N. risticii. This is the first molecular detection of Neorickettsia sp. in coatis in Brazil.

Molecular detection of blood-borne agents in vampire bats from Brazil, with the first molecular evidence of Neorickettsia sp. in Desmodus rotundus and Diphylla ecaudata.

Bats (Mammalia, Chiroptera) represent the second largest group of mammals. Due to their ability to fly and adapt and colonize different niches, bats act as reservoirs of several potentially zoonotic pathogens. In this context, the present work aimed to investigate, using molecular techniques, the occurrence of blood-borne agents (Anaplasmataceae, Coxiella burnetii, hemoplasmas, hemosporidians and piroplasmids) in 198 vampire bats sampled in different regions of Brazil and belonging to the species Desmodus rotundus (n = 159), Diphylla ecaudata (n = 31) and Diaemus youngii (n = 8). All vampire bats liver samples were negative in PCR assays for Ehrlichia spp., Anaplasma spp., piroplasmids, hemosporidians and Coxiella burnetii. However, Neorickettsia sp. was detected in liver samples of 1.51% (3/198) through nested PCR based on the 16S rRNA gene in D. rotundus and D. ecaudata. This is the first study to report Neorickettsia sp. in vampire bats. Hemoplasmas were detected in 6.06% (12/198) of the liver samples using a PCR based on the 16S rRNA gene. The two 16S rRNA sequences obtained from hemoplasmas were closely related to sequences previously identified in vampire and non-hematophagous bats from Belize, Peru and Brazil. The genotypic analysis identified a high diversity of bat-associated hemoplasma genotypes from different regions of the world, emphasizing the need for studies on this subject, in order to better understand the mechanisms of co-evolution between this group of bacteria and their vertebrate hosts. The role of neotropical bat-associated Neorickettsia sp. and bats from Brazil in the biological cycle of such agent warrant further investigation.

Detection of Neorickettsia sp. in Oligoryzomys flavescens rodent from a protected urban area in Buenos Aires City (Argentina).

Rodents play an important role in vector-borne pathogens cycle. To detect Anaplasma, Ehrlichia, Neorickettsia, Rickettsia and Borrelia species in rodents from a protected urban area in Buenos Aires City (Argentina) were analyzed 203 organ pools of Mus musculus, Oligoryzomys flavescens, Rattus norvegicus, Deltamys kempi and Scapteromys aquaticus by PCR. Only one O. flavescens (1.2%) was positive by PCR for 16S rRNA fragment for the Anaplasmataceae family and the sequence had 99.7% identity with Neorickettsia risticii. Plus, the sequence obtained for a fragment of the p51 gene for the genus Neorickettsia from positive sample had 95.3-96.1% identity with N. risticii found previously in bats Tadarida brasiliensis from Buenos Aires City. Our study presents the first finding of Neorickettsia in rodents from natural environment, but further studies are necessary about these vector-borne bacteria and the rol of rodents in its epidemiology.

Real-Time PCR Differential Detection of Neorickettsia findlayensis and N. risticii in Cases of Potomac Horse Fever.

Potomac horse fever (PHF) is an acute and potentially fatal enterotyphlocolitis of horses with clinical signs that include anorexia, fever, diarrhea, and laminitis. Its incidence is increasing despite a commercially available vaccine. PHF is caused by Neorickettsia risticii, and the recently rediscovered and classified N. findlayensis. PHF diagnosis is currently accomplished using serology or nested PCR. However, both methods cannot distinguish the two Neorickettsia species that cause PHF. Further, the current N. risticii real-time PCR test fails to detect N. findlayensis. Thus, in this study, two Neorickettsia species-specific real-time PCR assays based on Neorickettsia ssa2 and a Neorickettsia genus-specific real-time PCR assay based on Neorickettsia 16S rRNA gene were developed. The ssa2 real-time PCR tests differentiated N. findlayensis from N. risticii in the field samples for which infection with either species had been verified using multiple other molecular tests and culture isolation, and the 16S rRNA gene real-time PCR detected both Neorickettsia species in the samples. These tests were applied to new field culture isolates from three Canadian provinces (Alberta, Quebec, Ontario) and Ohio as well as archival DNA samples from suspected PHF cases to estimate the prevalence of N. findlayensis in different geographic regions. The results suggest that N. findlayensis frequently causes PHF in horses in Alberta and Quebec. The development of these tests will allow rapid, sensitive, and specific diagnosis of horses presenting with clinical signs of PHF. These tests will also enable rapid and targeted treatment and help develop broad-spectrum vaccines for PHF.

Anaplasmataceae closely related to Ehrlichia chaffeensis and Neorickettsia helminthoeca from birds in Central Europe, Hungary.

Increasing amount of data attest that (in the context of vector-borne infections) birds are not only important as hosts of blood-sucking arthropod vectors, but also as reservoirs of vector-borne pathogens. From 2015 to 2019 cadavers of 100 birds (from 45 species, nine orders) were collected in Hungary, and their organs were screened for DNA from a broad range of vector-borne bacteria with PCR and sequencing. Molecular analyses revealed the presence of Anaplasmataceae, and sequencing identified bacteria closely related to Neorickettsia helminthoeca and Ehrlichia chaffeensis in a Eurasian teal (Anas crecca) and a song thrush (Turdus philomelos), respectively. All samples were PCR negative for rickettsiae, borreliae, Francisella and Coxiella spp., as well as for piroplasms. To our knowledge, this is the first report of a Neorickettsia and an Ehrlichia sp., which belong to the phylogenetic groups of N. helminthoeca and E. chaffeensis, respectively, from Europe. The potential presence of these two vector-borne bacteria needs to be taken into account during future studies on the eco-epidemiology of Anaplasmataceae in Europe.

A Novel Neorickettsial Infection in 3 Dogs in the Pacific Northwest.

The genus Neorickettsia includes obligate, intracellular bacteria responsible for diseases including Potomac horse fever caused by Neorickettsia risticii and salmon poisoning disease (SPD) caused by Neorickettsia helminthoeca. The Stellanchasmus falcatus (SF) agent is a member of this genus previously associated only with mild clinical signs in dogs. Between 2013 and 2016, 3 dogs in Washington State (USA) presented with disease suggestive of SPD, but N. helminthoeca was not detected by molecular techniques. Clinical signs included depression, anorexia, and diarrhea. Cytologic examination of aspirates supported a diagnosis of granulomatous lymphadenitis with organisms suggestive of Neorickettsia. Dogs either died or were humanely euthanized due to poor response to therapy. Necropsy findings included lymphadenomegaly and hepatomegaly. Histopathology identified granulomatous and lymphoplasmacytic splenitis, lymphadenitis, enteritis, and hepatitis with extensive necrosis. Neorickettsia DNA was detected using genus-specific primers and direct sequencing showed 100% sequence identity to the SF agent in all 3 dogs. This is the first clinicopathologic description of severe disease in dogs attributed to the SF agent. These findings may suggest the emergence of a novel neorickettsial disease in the Pacific Northwest.

Isolation and Molecular Analysis of a Novel Neorickettsia Species That Causes Potomac Horse Fever.

Potomac horse fever (PHF), a severe and frequently fatal febrile diarrheal disease, has been known to be caused only by Neorickettsia risticii, an endosymbiont of digenean trematodes. Here, we report the cell culture isolation of a new Neorickettsia species found in two locations in eastern Ontario, Canada, in 2016 and 2017 (in addition to 10 variable strains of N. risticii) from N. risticii PCR-negative horses with clinical signs of PHF. Gene sequences of 16S rRNA and the major surface antigen P51 of this new Neorickettsia species were distinct from those of all previously characterized N. risticii strains and Neorickettsia species, except for those from an uncharacterized Neorickettsia species culture isolate from a horse with PHF in northern Ohio in 1991. The new Neorickettsia species nonetheless had the characteristic intramolecular repeats within strain-specific antigen 3 (Ssa3), which were found in all sequenced Ssa3s of N. risticii strains. Experimental inoculation of two naive ponies with the new Neorickettsia species produced severe and subclinical PHF, respectively, and the bacteria were reisolated from both of them, fulfilling Koch's postulates. Serological assay titers against the new Neorickettsia species were higher than those against N. risticii Whole-genome sequence analysis of the new Neorickettsia species revealed unique features of this bacterium compared with N. risticii We propose to classify this new bacterium as Neorickettsia finleia sp. nov. This finding will improve the laboratory diagnosis of and vaccine for PHF, environmental risk assessment of PHF, and understanding of PHF pathogenesis and Neorickettsia biology in general.IMPORTANCE Despite the detection of Neorickettsia species DNA sequences in various trematode species and their hosts, only three Neorickettsia species have been cell culture isolated and whole-genome sequenced and are known to infect mammals and/or cause disease. The molecular mechanisms that enable the obligatory intracellular bacterium Neorickettsia to colonize trematodes and to horizontally transmit from trematodes to mammals, as well as the virulence factors associated with specific mammalian hosts, are unknown. Potomac horse fever (PHF) is a severe and acute systemic infectious disease of horses, with clinical signs that include diarrhea. Neorickettsia risticii is the only known bacterial species that causes PHF. Ingestion of insects harboring N. risticii-infected trematodes by horses leads to PHF. Our discovery of a new Neorickettsia species that causes PHF and whole-genome sequence analysis of this bacterium will improve laboratory diagnosis and vaccine development for PHF and will contribute to our understanding of Neorickettsia ecology, pathogenesis, and biology.

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.

Assessing bat droppings and predatory bird pellets for vector-borne bacteria: molecular evidence of bat-associated Neorickettsia sp. in Europe.

In Europe, several species of bats, owls and kestrels exemplify highly urbanised, flying vertebrates, which may get close to humans or domestic animals. Bat droppings and bird pellets may have epidemiological, as well as diagnostic significance from the point of view of pathogens. In this work 221 bat faecal and 118 bird pellet samples were screened for a broad range of vector-borne bacteria using PCR-based methods. Rickettsia DNA was detected in 13 bat faecal DNA extracts, including the sequence of a rickettsial insect endosymbiont, a novel Rickettsia genotype and Rickettsia helvetica. Faecal samples of the pond bat (Myotis dasycneme) were positive for a Neorickettsia sp. and for haemoplasmas of the haemofelis group. In addition, two bird pellets (collected from a Long-eared Owl, Asio otus, and from a Common Kestrel, Falco tinnunculus) contained the DNA of a Rickettsia sp. and Anaplasma phagocytophilum, respectively. In both of these bird pellets the bones of Microtus arvalis were identified. All samples were negative for Borrelia burgdorferi s.l., Francisella tularensis, Coxiella burnetii and Chlamydiales. In conclusion, bats were shown to pass rickettsia and haemoplasma DNA in their faeces. Molecular evidence is provided for the presence of Neorickettsia sp. in bat faeces in Europe. In the evaluated regions bat faeces and owl/kestrel pellets do not appear to pose epidemiological risk from the point of view of F. tularensis, C. burnetii and Chlamydiales. Testing of bird pellets may provide an alternative approach to trapping for assessing the local occurrence of vector-borne bacteria in small mammals.

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

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.

Ultrastructure and localization of Neorickettsia in adult digenean trematodes provides novel insights into helminth-endobacteria interaction.

BACKGROUND: Neorickettsia are a group of intracellular α proteobacteria transmitted by digeneans (Platyhelminthes, Trematoda). These endobacteria can also infect vertebrate hosts of the helminths and cause serious diseases in animals and humans. Neorickettsia have been isolated from infected animals and maintained in cell cultures, and their morphology in mammalian cells has been described. However, limited information is available on the morphology and localization of Neorickettsia in the trematode host. METHODS: We used a Neorickettsia-infected strain of the model trematode Plagiorchis elegans to infect Syrian Golden hamsters to produce adult worms. Ultrastructure of Neorickettsia was assessed by transmission electron microscopy of high pressure freezing/freeze substitution fixed specimens. A Neorickettsia surface protein from P. elegans (PeNsp-3) was cloned and antibodies against the recombinant protein were used to localize Neorickettsia by immunohistochemistry. RESULTS: Ultrastructural analysis revealed moderate numbers of pleomorphic endobacteria with a median size of 600 × 400 nm and characteristic double membranes in various tissue types. Endobacteria showed tubular membrane invaginations and secretion of polymorphic vesicles. Endobacteria were unevenly localized as single cells, or less frequently as small morula-like clusters in the ovary, Mehlis' gland, vitelline follicles, uterus, intrauterine eggs, testis, cirrus-sac, tegument, intestine and the oral and ventral sucker. Examination of hamster small intestine infected with P. elegans showed many endobacteria at the host-parasite interface such as the oral and ventral sucker, the tegument and the excretory pore. CONCLUSIONS: We conclude that adult P. elegans trematodes carry Neorickettsia endobacteria in varying numbers in many tissue types that support vertical transmission, trematode to trematode transmission via seminal fluid, and possibly horizontal transmission from trematodes to vertebrate hosts. These means appear to be novel mechanisms of pathogen transmission by endoparasitic worms.

Real-time PCR detection and phylogenetic relationships of Neorickettsia spp. in digeneans from Egypt, Philippines, Thailand, Vietnam and the United States.

Neorickettsia (Rickettsiales, Anaplasmataceae) is a genus of obligate intracellular bacterial endosymbionts of digeneans (Platyhelminthes, Digenea). Some Neorickettsia are able to invade cells of the digenean's vertebrate host and are known to cause diseases of domestic animals, wildlife, and humans. In this study we report the results of screening digenean samples for Neorickettsia collected from bats in Egypt and Mindoro Island, Philippines, snails and fishes from Thailand, and fishes from Vietnam and the USA. Neorickettsia were detected using a real-time PCR protocol targeting a 152bp fragment of the heat shock protein coding gene, GroEL, and verified with nested PCR and sequencing of a 1853bp long region of the GroESL operon and a 1371bp long region of 16S rRNA. Eight unique genotypes of Neorickettsia were obtained from digenean samples. Neorickettsia sp. 8 obtained from Lecithodendrium sp. from Egypt; Neorickettsia sp. 9 and 10 obtained from two species of Paralecithodendrium from Mindoro, Philippines; Neorickettsia sp. 11 from Lecithodendrium sp. and Neorickettsia sp. 4 (previously identified from Saccocoelioides lizae, from China) from Thailand; Neorickettsia sp. 12 from Dicrogaster sp. Florida, USA; Neorickettsia sp. 13 and SF agent from Vietnam. Sequence comparison and phylogenetic analysis demonstrated that the forms, provisionally named Neorickettsia sp. 8-13, represent new genotypes. We have for the first time detected Neorickettsia in a digenean from Egypt (and the African continent as a whole), the Philippines, Thailand and Vietnam based on PCR and sequencing evidence. Our findings suggest that further surveys from the African continent, SE Asia, and island countries are likely to reveal new Neorickettsia lineages as well as new digenean host associations.

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.

Detection of the bacterial endosymbiont Neorickettsia in a New Zealand digenean.

Neorickettsia are endosymbiotic bacteria that infect digeneans (Trematoda). These bacteria are of interest worldwide because of their ability to move from the parasite to its host, where they can cause serious diseases of humans and animals. While several disease-forming species of Neorickettsia have been well studied, and numerous Neorickettsia types have been identified in regions such as North America and parts of Asia, records from other locations are sparse. To date, there have been no reports of Neorickettsia from New Zealand. We screened ten species of digeneans infecting seven native gastropod species (both marine and freshwater) found near Dunedin, New Zealand, for the presence of neorickettsial infections. A >1300 bp long section of 16S rRNA belonging to a Neorickettsia bacterium was isolated from opecoelid digeneans of two individuals of the mudflat topshell snail Diloma subrostrata. These sequences represent the first evidence of neorickettsial infection in native New Zealand animals and are also the first Neorickettsia found in digeneans of the family Opecoelidae.

The numbers game: quantitative analysis of Neorickettsia sp. propagation through complex life cycle of its digenean host using real-time qPCR.

Bacteria of the genus Neorickettsia are obligate intracellular endosymbionts of parasitic flukes (Digenea) and are passed through the entire complex life cycle of the parasite by vertical transmission. Several species of Neorickettsia are known to cause diseases in domestic animals, wildlife, and humans. Quantitative data on the transmission of the bacteria through the digenean life cycle is almost completely lacking. This study quantified for the first time the abundance of Neorickettsia within multiple stages of the life cycle of the digenean Plagiorchis elegans. Snails Lymnaea stagnalis collected from a pond in North Dakota were screened for the presence of digenean cercariae, which were subsequently tested for the presence of Neorickettsia. Three L. stagnalis were found shedding P. elegans cercariae infected with Neorickettsia. These snails were used to initiate three separate laboratory life cycles and obtain all life cycle stages for bacterial quantification. A quantitative real-time PCR assay targeting the GroEL gene was developed to enumerate Neorickettsia sp. within different stages of the digenean life cycle. The number of bacteria significantly increased throughout all stages, from eggs to adults. The two largest increases in number of bacteria occurred during the period from eggs to cercariae and from 6-day metacercariae to 48-h juvenile worms. These two periods seem to be the most important for Neorickettsia propagation through the complex digenean life cycle and maturation in the definitive host.

Germs within Worms: Localization of Neorickettsia sp. within Life Cycle Stages of the Digenean Plagiorchis elegans.

Neorickettsia spp. are bacterial endosymbionts of parasitic flukes (Digenea) that also have the potential to infect and cause disease (e.g., Sennetsu fever) in the vertebrate hosts of the fluke. One of the largest gaps in our knowledge of Neorickettsia biology is the very limited information available regarding the localization of the bacterial endosymbiont within its digenean host. In this study, we used indirect immunofluorescence microscopy to visualize Neorickettsia sp. within several life cycle stages of the digenean Plagiorchis elegans Individual sporocysts, cercariae, metacercariae, and adults of P. elegans naturally infected with Neorickettsia sp. were obtained from our laboratory-maintained life cycle, embedded, sectioned, and prepared for indirect immunofluorescence microscopy using anti-Neorickettsia risticiihorse serum as the primary antibody. Neorickettsiasp. was found within the tegument of sporocysts, throughout cercarial embryos (germ balls) and fully formed cercariae (within the sporocysts), throughout metacercariae, and within the tegument, parenchyma, vitellaria, uteri, testes, cirrus sacs, and eggs of adults. Interestingly, Neorickettsia sp. was not found within the ovarian tissue. This suggests that vertical transmission of Neorickettsia within adult digeneans occurs via the incorporation of infected vitelline cells into the egg rather than direct infection of the ooplasm of the oocyte, as has been described for other bacterial endosymbionts of invertebrates (e.g.,Rickettsia and Wolbachia).

Prevalence of selected rickettsial infections in cats in Southern Germany.

Prevalence of Anaplasma, Ehrlichia, Neorickettsia, and Wolbachia DNA in blood of 479 cats collected in different veterinary clinics in Southern Germany was determined using a previously published conventional PCR using 16S-23S intergenic spacer primers (5' CTG GGG ACT ACG GTC GCA AGA C 3' - forward; 5' CTC CAG TTT ATC ACT GGA AGT T 3' - reverse). Purified amplicons were sequenced to confirm genus and species. Associations between rickettsial infections, and feline immunodeficiency virus (FIV), as well as feline leukemia virus (FeLV) status were evaluated. Rickettsial prevalence was 0.4% (2/479; CI: 0.01-1.62%). In the two infected cats, Anaplasma phagocytophilum DNA was amplified. These cats came from different environment and had outdoor access. Both were ill with many of their problems likely related to other diseases. However, one cat had neutrophilia with left shift and the other thrombocytopenia potentially caused by their A. phagocytophilum infection. There was no significant difference in the FIV and FeLV status between A. phagocytophilum-negative and -positive cats. A. phagocytophilum can cause infection in cats in Southern Germany, and appropriate tick control is recommended.

Laboratory maintenance of the bacterial endosymbiont, Neorickettsia sp., through the life cycle of a digenean, Plagiorchis elegans.

The Digenea (Platyhelminthes: Trematoda) are a diverse and complex group of internal metazoan parasites. These parasites can serve as hosts to obligate intracellular bacteria belonging to the genus Neorickettsia (Family: Anaplasmataceae). Neorickettsiae persist within all stages of the fluke life cycle and thus are maintained through vertical transmission. However, the low prevalence of Neorickettsia in nature limits study of their transmission biology at different steps of digenean life cycles. To resolve this dilemma, we have developed for the first time a laboratory model allowing to maintain Neorickettsia sp. through the whole life cycle of a digenean, Plagiorchis elegans. The laboratory life cycle of P. elegans consists of a snail first intermediate host, Lymnaea stagnalis, an aquatic arthropod second intermediate host, Culex pipiens (mosquito larva), and a vertebrate definitive host, Mesocricetus auratus (Syrian hamster). This paper focuses on the development of the laboratory life cycle, as well as outlines its potential uses in studying the transmission biology of Neorickettsia and its evolutionary relationship within its digenean host.

DHR-ICMR Guidelines for diagnosis & management of Rickettsial diseases in India.

Rickettsial diseases, caused by a variety of obligate intracellular, gram-negative bacteria from the genera Rickettsia, Orientia, Ehrlichia, Neorickettsia, Neoehrlichia, and Anaplasma, belonging to the Alphaproteobacteria, are considered some of the most covert emerging and re-emerging diseases and are being increasingly recognized. Among the major groups of rickettsioses, commonly reported diseases in India are scrub typhus, murine flea-borne typhus, Indian tick typhus and Q fever. Rickettsial infections are generally incapacitating and difficult to diagnose; untreated cases have case fatality rates as high as 30-45 per cent with multiple organ dysfunction, if not promptly diagnosed and appropriately treated. The vast variability and non-specific presentation of this infection have often made it difficult to diagnose clinically. Prompt antibiotic therapy shortens the course of the disease, lowers the risk of complications and in turn reduces morbidity and mortality due to rickettsial diseases. There is a distinct need for physicians and health care workers at all levels of care in India to be aware of the clinical features, available diagnostic tests and their interpretation, and the therapy of these infections. Therefore, a Task Force was constituted by the Indian Council of Medical Research (ICMR) to formulate guidelines for diagnosis and management of rickettsial diseases. These guidelines include presenting manifestations, case definition, laboratory criteria (specific and supportive investigations) and treatment.