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New findings on members of the family Anaplasmataceae of veterinary importance.

Members of the family Anaplasmataceae are obligate intracellular Gram-negative bacteria that naturally infect a variety of wild and domestic animal species, the spillover of which may lead to zoonosis. I discuss new findings on members of the family Anaplasmataceae of veterinary importance and therefore, I will describe the recent findings on Neorickettsia risticii in the trematode and related Neorickettsia species. I also will review the recent progress on Aegyptianella pullorum and other Aegyptianella sp., "Candidatus Neoehrlichia mikurensis" and Anaplasma phagocytophilum strains in various hosts. The whole genome sequences of two important veterinary pathogens-Anaplasma marginale, the bovine anaplasmosis agent, and Ehrlichia (formerly Cowdria) ruminantium, the agent of heartwater of ruminants-have been published. Taken together, these advances in research of the family Anaplasmataceae in the veterinary field provide us with insights into the evolution, reservoir, and transmission of these organisms in nature and their pathogenesis in natural and accidental hosts. It is through this work that surveillance, diagnosis, preventive measures, and treatment of ehrlichioses of both animals and humans can be improved.

Anaplasmataceae↗

Ultrastructure and phylogenetic analysis of 'Candidatus Neoehrlichia mikurensis' in the family Anaplasmataceae, isolated from wild rats and found in Ixodes ovatus ticks.

A novel bacterium that infects laboratory rats was isolated from wild Rattus norvegicus rats in Japan. Transmission electron microscopy of the spleen tissue revealed small cocci surrounded by an inner membrane and a thin, rippled outer membrane in a membrane-bound inclusion within the cytoplasm of endothelial cells. Phylogenetic analysis of the 16S rRNA gene sequence of the bacterium found in R. norvegicus rats and Ixodes ovatus ticks in Japan revealed that the organism represents a novel clade in the family Anaplasmataceae, which includes the Schotti variant found in Ixodes ricinus ticks in the Netherlands and the Ehrlichia-like Rattus strain found in R. norvegicus rats from China. The novel clade was confirmed by phylogenetic analysis of groESL sequences found in R. norvegicus rats and Ixodes ovatus ticks in Japan. No serological cross-reactivity was detected between this bacterium and members of the genera Anaplasma, Ehrlichia or Neorickettsia in the family Anaplasmataceae. It is proposed that this new cluster of bacteria should be designated 'Candidatus Neoehrlichia mikurensis'.

Anaplasmataceae↗

Differentiation of Anaplasmataceae through partial groEL gene analysis.

The nucleotide sequences (287 bp) of the partial groEL gene from 14 reference strains of Anaplasmataceae were determined and compared. A partial groEL gene is useful for the identification and characterization of Anaplasmataceae, in spite of its short nucleotide sequences.

Anaplasmataceae↗

Description of Aegyptianella botuliformis n. sp. (Rickettsiales: Anaplasmataceae) from the helmeted guineafowl, Numida meleagris.

Aegyptianella botuliformis n. sp. (Rickettsiales: Anaplasmataceae) isolated from helmeted guineafowls Numida meleagris from the Kruger National Park is described. The rickettsia occurs within a membrane-bound vacuole in the cytoplasm of erythrocytes with up to 8 organisms in a mature inclusion. The initial body resembles that of Aegyptianella pullorum. The tightly packed, sausage-shaped intermediate forms are a distinctive morphological feature, seen as irregular, pleomorphic forms under light microscopy. While more larvae and nymphs of Amblyomma hebraeum and Amblyomma marmoreaum were found on the birds than larvae of an Argas sp., it is believed that the latter are the vectors of A. botuliformis n. sp. In addition to the Kruger National Park, positive blood smears were obtained from guineafowls at other localities in the Transvaal.

Anaplasmataceae↗

Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.

Anaplasma↗

Mechanisms to create a safe haven by members of the family Anaplasmataceae.

Members of the family Anaplasmataceae are obligatory intracellular bacteria with unique host cell specificities. Depending on each bacterial species, granulocytes, platelets, endothelial cells, monocytes, macrophages, red blood cells, and cells of invertebrates are specifically infected. This unique host cell specificity has been the major hurdle to overcome in order to cultivate this group of bacteria. Because these bacteria cannot survive outside host cells, once released from a host cell, they need to rapidly induce signals for their own internalization into another host cell unique to each species. How these bacteria enter and continue to survive and replicate within the host milieu, then exit the host cell is largely unknown. Recently, however, unique strategies employed by some of these bacteria for successful parasitism of mammalian leukocytes have begun to be uncovered. When these bacteria interact with host cells, signals are transduced both inside the host cells and inside the bacteria. These signals disable the alarm system, as well as microbicidal mechanisms, of the leukocytes and condition the host cells to accept these intruders to share space and nutrient resources. Signals transduced inside the bacteria allow them to finely tune their metabolism and physiology in the new host cell environment and to disguise themselves as "insiders" so that their sojourn does not upset the host cell physiology until they have sufficiently multiplied. This paper discusses our recent findings on these topics.

Adaptation, Physiological↗

Prevalence of Borrelia burgdorferi sensu lato and Anaplasmataceae members in Ixodes ricinus ticks in Alsace, a focus of Lyme borreliosis endemicity in France.

Due to the high Lyme borreliosis incidence in Alsace, in northeastern France, we investigated in 2003-2004 three cantons in this region in order to determine the density of Ixodes ricinus ticks infected by Borrelia burgdorferi sensu lato and Anaplasmataceae. The peak density of nymphs infected by B. burgdorferi sensu lato at Munster and Guebwiller, where the disease incidence was high, was among the highest reported in Europe (105 and 114 per 100 m(2), respectively). In contrast, the peak density of infected nymphs was low in the canton of Dannemarie (5/100 m(2)), where the disease incidence was low. The two main species detected in ticks were Borrelia afzelii, more frequent in nymphs, and Borrelia garinii, more frequent in adult ticks. The rates of tick infection by Anaplasma phagocytophilum were 0.4% and 1.2% in nymphs and adults, respectively.

Anaplasmataceae↗

Aegyptianella ranarum sp. n. (Rickettsiales, Anaplasmataceae): ultrastructure and prevalence in frogs from Ontario.

Aegyptianella ranarum sp. n. (Rickettsiales, Anaplasmataceae) was recorded from bullfrogs (Rana catesbeiana Shaw), green frogs (Rana clamitans Latreille) and mink frogs (Rana septentrionalis Baird) from five sites in southern Ontario. The rickettsia occurs within membrane-bound vacuoles in the cytoplasm of erythrocytes with up to 120 organisms in mature inclusions. The pattern of replication of A. ranarum in host erythrocytes and its prevalence over a 3-yr period in frogs from Algonquin Park, Ontario are discussed.

Anaplasmataceae Infections↗

Adhesion of outer membrane proteins containing tandem repeats of Anaplasma and Ehrlichia species (Rickettsiales: Anaplasmataceae) to tick cells.

Infection of cells by tick-borne rickettsiae appears to be mediated by outer membrane proteins that allow pathogens to adhere to host cells. Major surface protein (MSP) 1a of Anaplasma marginale, the type species for the genus Anaplasma, was shown previously to be an adhesin for tick cells. The A. marginale MSP1a has a variable number of tandem 28 or 29 amino acid repeats located in the amino terminal region of the protein that contains an adhesion domain that is necessary and sufficient for infection of tick cells. The MSP1a studies demonstrated the importance of combining structural and functional characteristics for identification of adhesive proteins. In the present study other outer membrane proteins containing tandem repeats were selected from organisms of the family Anaplasmataceae and studied for their adhesive properties to tick cells. The adhesive properties and protein characteristics were then analyzed in order to provide a predictor of the adhesion function of proteins identified from genome sequences. Proteins selected included the A. marginale MSP1a, A. phagocytophilum 100 and 130 kDa, Ehrlichia chaffeensis 120 kDa, E. canis 140 kDa and E. ruminantium "mucin", which were all cloned and expressed in Escherichia coli and then tested as adhesins for cultured IDE8 cells. Of the proteins studied, the A. marginale MSP1a and the E. ruminantium "mucin" were found to be adhesins for tick cells. Although all of these recombinant outer membrane proteins were glycosylated, the A. marginale MSP1a and E. ruminantium "mucin" adhesins shared a common feature of having a high Ser/Thr content in the tandem repeats. The results reported herein provide new information on the role of E. ruminantium "mucin" as an adhesin for tick cells and also suggest a role of glycans in adhesin molecules.

Anaplasma↗

Anaplasma marginale (Rickettsiales: Anaplasmataceae): recent advances in defining host-pathogen adaptations of a tick-borne rickettsia.

The tick-borne intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) develops persistent infections in cattle and tick hosts. While erythrocytes appear to be the only site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks and transmission occurs via salivary glands during feeding. Many geographic isolates occur that vary in genotype, antigenic composition, morphology and infectivity for ticks. In this chapter we review recent research on the host-vector-pathogen interactions of A. marginale. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells. The MSP1a protein, which is an adhesin for bovine erythrocytes and tick cells, is differentially regulated and affects infection and transmission of A. marginale by Dermacentor spp. ticks. MSP2 undergoes antigenic variation and selection in cattle and ticks, and contributes to the maintenance of persistent infections. Phylogenetic studies of A. marginale geographic isolates using msp4 and msp1alpha provide information about the biogeography and evolution of A. marginale: msp1alpha genotypes evolve under positive selection pressure. Isolates of A. marginale are maintained by independent transmission events and a mechanism of infection exclusion in cattle and ticks allows for only the infection of one isolate per animal. Prospects for development of control strategies by use of pathogen and tick-derived antigens are discussed. The A. marginale/vector/host studies described herein could serve as a model for research on other tick-borne rickettsiae.

Anaplasma marginale↗

Isolation and identification of Aegyptianella pullorum (Rickettsiales, Anaplasmataceae) in wild turkeys from North America.

Isodiagnosis of blood from Rio Grande wild turkeys from southern Texas revealed a small, intraerythrocytic rickettsia, Aegyptianella pullorum Carpano, 1928, in 24 of 300 samples. Identification of this first isolate from North America was made using both light and transmission electron microscopy. It is suggested that the translocation of wild turkeys from Texas to other states could spread this pathogen to both wild birds and domestic poultry.

Anaplasmataceae↗

Identification of Anaplasmataceae (Haemobartonella) antigen and antibodies in systemic lupus erythematosus.

Free antigen related to Anaplasma marginale (AM) (Haemobartonella) was demonstrated in the glomeruli of one patient with lupus nephritis. Indirect fluorescent antibodies against this rickettsia were demonstrated in all of 22 lupus sera tested, with titers ranging from 1:20 to 1:1280. Geometric mean titer (GMT) was 116. Fifty-eight percent of 102 controls did not react to AM by indirect fluorescent antibody technique, and GMT of all controls was 10.7 Immunofluorescence was eliminated by neutralization and blocking techniques.

Adult↗

Blood parasites of sheep in the Netherlands. I. Anaplasma mesaeterum sp.n. (Rickettsiales, Anaplasmataceae).

On two occasions an anaplasm was isolated from sheep on the Dutch island of Ameland. The organism proved to be highly pathogenic for splenectomised sheep; a non-splenectomised animal recovered spontaneously after the packed cell volume had decreased by 40%. Treatment with oxytetracycline was effective. Its pathogenicity for goats appeared to be low, and the organism was apparently not infective to splenectomised cattle. This anaplasm differs from Anaplasma ovis in that less than 30% of the organisms are marginally situated in the red cell, as against over 70% in A. ovis; cross-immunity with A. ovis was incomplete and the latter appeared to be far more pathogenic to goats than the Dutch anaplasm, for which the name Anaplasma mesaeterum sp.n. is proposed. Its ultrastructure is similar to that of A. marginale and A. ovis. The vector is either Ixodes ricinus or Haemaphysalis punctata. Its practical importance remains to be ascertained.

Anaplasma↗

Development of Anaplasma ovis (Rickettsiales: Anaplasmataceae) in male Dermacentor andersoni (Acari: Ixodidae) transferred from infected to susceptible sheep.

The development of Anaplasma ovis was studied in Dermacentor andersoni males transferred from infected to susceptible sheep. Laboratory-reared male D. andersoni were allowed to feed for 6 d on a sheep with ascending A. ovis parasitemia. The ticks were removed and held at room temperature in a humidity chamber for 6 d, after which they were allowed to feed on five susceptible sheep for 1, 3, 5, 7, or 9 d. Gut and salivary glands were collected from ticks during the 21-d experiment and examined with light and electron microscopy. Anaplasmosis developed in all susceptible sheep. Colonies of A. ovis were first observed in midgut epithelial cells on the 3rd d ticks fed on the infected sheep, and infection persisted in gut cells throughout the experiment. The first colonies contained one large organism that subsequently gave rise to many reticulated ones, which became electron dense over time. After ticks were transferred to susceptible sheep and began the second feeding, individual A. ovis organisms were found from days 3-9 in muscle cells on the hemocoel side of the gut basement membrane. However, colonies did not develop in these cells, and the host cells did not hypertrophy as did cells similarly infected with A. marginale. A final site of development of A. ovis was in salivary glands. Individual organisms were first seen in acinar cells on the first day that ticks fed on the second calves, and salivary gland infections persisted throughout the 9-d feeding period. Colonies of A. ovis developed in salivary gland acinar cells and organisms within these colonies were initially electron lucent but became electron dense. Multiple colonies often were observed within salivary gland cells and often contained organisms in various stages of development.

Anaplasma↗

Persistence of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in male Dermacentor andersoni (Acari: Ixodidae) transferred successively from infected to susceptible calves.

The persistence of Anaplasma marginale Theiler in male Dermacentor andersoni Stiles ticks exposed to the organism as adults was studied as the ticks were successively transferred to five susceptible calves. All calves fed upon by these ticks rapidly developed clinical anaplasmosis; incubation periods of infection ranged from 19 to 26 d and did not change significantly with successive feedings. Development of A. marginale in tick midgut and salivary glands was followed daily during tick feeding (total, 35 d) with light microscopy and DNA hybridization. With microscopy, A. marginale colonies persisted in midgut cells throughout the experiment. Large colonies were observed in gut muscle cells on days 8 through 35 and were the predominant infected cell type during this part of feeding. Colonies were seen in salivary gland acini from day 2 throughout the 35-d experiment. The DNA probe confirmed the presence of Anaplasma DNA in midgut and salivary glands throughout the experiment. Quantitative estimates of infection intensity in tissues of individual ticks approximated 10(7) initial body equivalents, confirming heavy infections. A marginale in midgut tissues decreased with feeding time, whereas the estimated number of organisms in salivary glands remained constant. These data demonstrate that D. andersoni males are efficient vectors of A. marginale and may be potential reservoirs of infection for ruminants for extended periods.

Anaplasma↗

Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in hemolymph of Dermacentor andersoni (Acari: Ixodidae) with the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect Anaplasma marginale in hemolymph collected from live Dermacentor andersoni Stiles ticks. Hemolymph was collected from severed legs of male and female ticks exposed to A. marginale as either nymphs or adults. Heat treatment was found to be the optimum method of hemolymph preparation for PCR. Hemolymph samples were collected and pooled from adult ticks exposed as nymphs on days 0-10 of feeding on a susceptible calf. For male and female ticks exposed as adults, samples were collected as ticks fed 7 d on an infected calf, while being held 9 d between feedings, and during a second feeding of 10 d (or to repletion) when they transmitted the parasite. Hemolymph samples were collected from uninfected ticks at the same times to serve as controls. Anaplasma marginale DNA was amplified with primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAGCAGCAGCAAGACCTTCA-3'), which flank a 409-bp fragment of the A. marginale Florida isolate msp1 beta gene. Infected tick hemolymph was PCR-positive for A. marginale at all collection times, including unfed adults infected as nymphs and previously unexposed adults that fed on infected calves for only 1 d. The PCR-based assay of tick hemolymph proved to be a sensitive method for identification of infected ticks, potentially without killing them; it would be well suited for identification of laboratory- or field-infected ticks that could then be used for further studies. The primers used in this assay were also found specific when tested with species of 18 different genera, and universal for 7 A. marginale isolates from diverse geographical areas of the United States.

Anaplasma↗