Molecular survey and genetic identification of Anaplasma species in goats from central and southern China

Anaplasma species are obligate intracellular rickettsial pathogens that impact the health of humans and animals. Few studies have been carried out on Anaplasma infections in central and southern China. This study was conducted to determine the coinfection rates of Anaplasma ovis, A. bovis, and A. phagocytophilum from 262 field blood samples of goats in these regions. The average prevalences of single infection of A. ovis, A. bovis, and A. phagocytophilum were 15.3, 16.0, and 6.1%, respectively. Coinfection of A. ovis and A. bovis was dominant, with an infection rate of 27.1%. Coinfection of A. ovis and A. phagocytophilum was 1.9% and that of A. bovis and A. phagocytophilum was 4.2%. Three-pathogen coinfection was found in three of four investigated provinces with a prevalence between 0 and 5.3%. The accuracy of the PCR results was corroborated by sequencing. Analysis of the 16S rRNA gene sequences of A. bovis and A. phagocytophilum confirmed the presence of these pathogens at the investigated sites and indicated the possible genetic diversity of A. phagocytophilum. Field blood inoculation of experimental animals led to successful identification and observation of the morphological shapes of A. bovis in the infected monocytes of sheep. Phylogenetic study with msp4 sequences of A. ovis indicated that the A. ovis genotypes from sheep in the north differed from the genotypes of goats in the investigated sites.     

Cloning and modeling of CD8 beta in the amphibian ambystoma Mexicanum. Evolutionary conserved structures for interactions with major histocompatibility complex (MHC) class I molecules

Bovine anaplasmosis is a rickettsial disease of world-wide economic importance caused by Anaplasma marginale. Several major surface proteins with conserved gene sequences have been examined as potential candidates for vaccines and/or diagnostic assays. Major surface protein 1 (MSP1) is composed of polypeptides MSP1a and MSP1b. MSP1a is expressed from the single copy gene msp1 alpha and MSP1b is expressed by members of the msp1 beta multigene family. In order to determine if the msp1 genes are conserved, primers specific for msp1 alpha, msp1 beta(1), and msp1 beta(2) genes were synthesized and used to amplify msp1 sequences of A. marginale from tick cell cultures, from cattle during acute and chronic infections and from salivary glands of Dermacentor variabilis. Protein sequences of MSP1a, MSP1b(1) and MSP1b(2) were conserved during the life cycle of the parasite. No amino acid changes were observed in MSP1a. However, small variations were observed in the MSP1b(1) and MSP1b(2) protein sequences, which could be attributed to recombination, selection for sub-populations of A. marginale in the vertebrate host and/or PCR errors. Several isolate-specific sequences were also observed. Based on the information obtained in this study, the MSP1 protein appears to be fairly well conserved and a potential vaccine candidate.     

Structural basis for segmental gene conversion in generation of Anaplasma marginale outer membrane protein variants

Bacterial pathogens in the genus Anaplasma generate surface coat variants by gene conversion of chromosomal pseudogenes into single-expression sites. These pseudogenes encode unique surface-exposed hypervariable regions flanked by conserved domains, which are identical to the expression site flanking domains. In addition, Anaplasma marginale generates variants by recombination of oligonucleotide segments derived from the pseudogenes into the existing expression site copy, resulting in a combinatorial increase in variant diversity. Using the A. marginale genome sequence to track the origin of sequences recombined into the msp2 expression site, we demonstrated that the complexity of the expressed msp2 increases during infection, reflecting a shift from recombination of the complete hypervariable region of a given pseudogene to complex mosaics with segments derived from hypervariable regions of different pseudogenes. Examination of the complete set of 1183 variants with segmental changes revealed that 99% could be explained by one of the recombination sites occurring in the conserved flanking domains and the other within the hypervariable region. Consequently, we propose an 'anchoring' model for segmental gene conversion whereby the conserved flanking sequences tightly align and anchor the expression site sequence to the pseudogene. Associated with the recombination sites were deletions, insertions and substitutions; however, these are a relatively minor contribution to variant generation as these occurred in less than 2% of the variants. Importantly, the anchoring model, which can account for more variants than a strict segmental sequence identity mechanism, is consistent with the number of msp2 variants predicted and empirically identified during persistent infection.     

Susceptibility of two Rocky Mountain bighorn sheep to experimental infection with Anaplasma ovis.

In North America, the role of wild ruminants in the epidemiology of anaplasmosis has not been clearly defined. Such information is particularly meager in regard to bighorn sheep. We report the susceptibility of two Rocky Mountain bighorn sheep (Ovis canadensis canadensis) to experimental infection with a well characterized field isolate of Anaplasma ovis obtained from domestic sheep in Idaho. Both bighorn sheep developed infection resulting in severe clinical disease, with relatively high parasitemias, icterus and anemia. One animal required tetracycline therapy and responded well to treatment, while the other recovered uneventfully without treatment. Both bighorn sheep were spleen-intact, a condition that in A. ovis-exposed domestic sheep typically is associated with mild infection. The results indicate that bighorn sheep may be adversely affected if exposed to the organism in nature.     

Experimental anaplasmosis in mule deer: persistence of infection of Anaplasma marginale and susceptibility to A. ovis

An experimental Anaplasma marginale infection was induced in a splenectomized mule deer (Odocoileus hemionus hemionus) which persisted subclinically at least 376 days as detected by subinoculation into susceptible cattle. Anaplasma ovis was experimentally transmitted from sheep to a splenectomized and a spleen-intact mule deer, and back to sheep. The pathogenesis in deer was very similar to that seen in sheep using ovine blood inoculations.     

First evidence for a restriction-modification system in Leptospira sp

Anaplasma marginale genomic DNA was tested for the presence of repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC)-like sequences in order to evaluate the genetic diversity of multiple A. marginale isolates. A. marginale isolates were obtained from cattle of six different states of Brazil, from the US and an Anaplasma centrale strain was obtained from Uruguay. Patterns obtained from A. marginale isolates varied from 14 to 17 fragments by REP-polymerase chain reaction (PCR) and 6 to 14 fragments by ERIC-PCR. All A. marginale isolates presented a 0.75-kb fragment by REP and two common fragments (0.38 and 1.0 kb) by ERIC-PCR. These two fragments were not detectable in A. centrale. Both methods produced similar patterns (80%) among A. marginale isolates obtained from the same region, although some isolates within regions shared less similarity. Isolates from Parana and Pernambuco, were differentiated by these methods. The study demonstrates the presence of ERIC and REP-like elements in A. marginale isolates and shows that A. marginale isolates and strains can be differentiated by these methods.     

The hypervariable region of Anaplasma marginale major surface protein 2 (MSP2) contains multiple immunodominant CD4+ T lymphocyte epitopes that elicit variant-specific proliferative and IFN-gamma responses in MSP2 vaccinates

Major surface protein 2 (MSP2) is an immunodominant outer membrane protein of Anaplasma marginale and Anaplasma phagocytophilum pathogens that cause bovine anaplasmosis and human granulocytic ehrlichiosis, respectively. MSP2 has a central hypervariable region (HVR) flanked by highly conserved amino and carboxyl termini. During A. marginale infection, dynamic and extensive amino acid sequence variation in MSP2 occurs through recombination of msp2 pseudogenes into the msp2 expression site, followed by sequential segmental gene conversions to generate additional variants. We hypothesized that MSP2 variation leads to significant changes in Th cell recognition of epitopes in the HVR. T cell epitopes were mapped using T cells from native MSP2-immunized cattle and overlapping peptides spanning the most abundant of five different MSP2 HVRs in the immunogen. Several epitopes elicited potent effector/memory Th cell proliferative and IFN-gamma responses, including those in three discreet blocks of sequence that undergo segmental gene conversion. Th cell clones specific for an epitope in the block 1 region of the predominant MSP2 variant type failed to respond to naturally occurring variants. However, some of these variants were recognized by oligoclonal T cell lines from MSP2 vaccinates, indicating that the variant sequences contain immunogenic CD4(+) T cell epitopes. In competition/antagonism assays, the nonstimulatory variants were not inhibitory for CD4(+) T cells specific for the agonist peptide. Dynamic amino acid sequence variation in MSP2 results in escape from recognition by some effector/memory MSP2-specific Th cells. Antigenic variation in MSP2 Th cell and B cell epitopes may contribute to immune evasion that allows long-term persistence of A. marginale in the mammalian reservoir.     

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paran , Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post-immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.     

Antigenic variation of Anaplasma marginale msp2 occurs by combinatorial gene conversion

The rickettsial pathogen Anaplasma marginale establishes lifelong persistent infection in the mammalian reservoir host, during which time immune escape variants continually arise in part because of variation in the expressed copy of the immunodominant outer membrane protein MSP2. A key question is how the small 1.2 Mb A. marginale genome generates sufficient variants to allow long-term persistence in an immunocompetent reservoir host. The recombination of whole pseudogenes into the single msp2 expression site has been previously identified as one method of generating variants, but is inadequate to generate the number of variants required for persistent infection. In the present study, we demonstrate that recombination of a whole pseudogene is followed by a second level of variation in which small segments of pseudogenes recombine into the expression site by gene conversion. Evidence for four short sequential changes in the hypervariable region of msp2 coupled with the identification of nine pseudogenes from a single strain of A. marginale provides for a combinatorial number of possible expressed MSP2 variants sufficient for lifelong persistence.     

Sharing of Pasteurella spp. between free-ranging bighorn sheep and feral goats

Pasteurella spp. were isolated from feral goats and free-ranging bighorn sheep (Ovis canadensis canadensis) in the Hells Canyon National Recreation Area bordering Idaho, Oregon, and Washington (USA). Biovariant 1 Pasteurella haemolytica organisms were isolated from one goat and one of two bighorn sheep found in close association. Both isolates produced leukotoxin and had identical electrophoretic patterns of DNA fragments following cutting with restriction endonuclease HaeIII. Similarly Pasteurella multocida multocida a isolates cultured from the goat and one of the bighorn sheep had D type capsules, serotype 4 somatic antigens, produced dermonecrotoxin and had identical HaeIII electrophoretic profiles. A biovariant U(beta) P.haemolytica strain isolated from two other feral goats, not known to have been closely associated with bighorn sheep, did not produce leukotoxin but had biochemical utilization and HaeIII electrophoretic profiles identical to those of isolates from bighorn sheep. It was concluded that identical Pasteurella strains were shared by the goats and bighorn sheep. Although the direction of transmission could not be established, evidence suggests transmission of strains from goats to bighorn sheep. Goats may serve as a reservoir of Pasteurella strains that may be virulent in bighorn sheep; therefore, goats in bighorn sheep habitat should be managed to prevent contact with bighorn sheep. Bighorn sheep which have nose-to-nose contact with goats should be removed from the habitat.     

Experimental anaplasmosis in American bison: persistence of infections of Anaplasma marginale and non-susceptibility to A. ovis

Blood collected 314 and 496 days after experimentally infecting splenectomized and spleen-intact American bison (Bison bison) with Anaplasma marginale was infective for splenectomized bovine steers. The pathogenesis was identical to that seen in bovine studies using bovine blood inoculations. A splenectomized bison remained normal clinically, hematologically and serologically for 10 mo after repeated inoculation of ovine blood infected with A. ovis.     

Analysis of the Anaplasma marginale major surface protein 1 complex protein composition by tandem mass spectrometry

The protective major surface protein 1 (MSP1) complex of Anaplasma marginale is a heteromer of MSP1a and MSP1b, encoded by a multigene family. The msp1beta sequences were highly conserved throughout infection. However, liquid chromatography-tandem mass spectrometry analysis identified only a single MSP1b protein, MSP1b1, within the MSP1 complex.     

A novel clinical syndrome and detection of Anaplasma ovis in Mongolian reindeer (Rangifer tarandus)

The Tsaatan (or Dhuka) peoples of northern-western Mongolia are one of the few remaining reindeer-herding cultural groups in the world. Recently a disease condition that involves sudden death of reindeer and cases involving fever, lethargy, and pale mucous membranes has been reported. Examination of blood smears collected in the 2005 field season resulted in the identification of intra-erythrocytic inclusions resembling Anaplasma spp. in smears from clinically sick animals. Using universal polymerase chain reaction (PCR) primers for the amplification of the 60 kDa chaperonin gene (cpn60, also known as hsp60 or groEL), we detected sequences corresponding to Anaplasma ovis in reindeer blood samples. Species-specific PCR primers for A. ovis were designed and validated and used to screen blood samples from Mongolian reindeer. Screening of 66 blood samples collected in the 2006 field season resulted in the detection of A. ovis in 80% of the samples. Our results indicate a high prevalence of A. ovis in the Tsaatan reindeer herds and an association with clinical disease that is likely to be anaplasmosis. To our knowledge this is the first report of natural A. ovis infection in reindeer.     

Experimental infections of Anaplasma ovis in pronghorn antelope

Anaplasma ovis was experimentally transmitted from sheep to pronghorn antelope (Antilocapra americana) and back to sheep. Anaplasma ovis was recovered in splenectomized sheep, from two of three spleen-intact pronghorns following their inoculation with blood from known A. ovis carrier sheep. These two pronghorns exhibited a 0.5% or higher A. ovis parasitemia within 48 days after exposure, and an anaplasmosis-positive serological response 91 days after exposure. Clinical signs of illness were not observed. Blood from the infected pronghorns produced disease in four splenectomized sheep.     

Evolution of antigen variation in the tick-borne pathogen Anaplasma phagocytophilum

Anaplasma phagocytophilum is an obligately intracellular tick-transmitted bacterial pathogen of humans and other animals. During the course of infection, A. phagocytophilum utilizes gene conversion to shuffle ～100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which codes for the major surface antigen and major surface protein 2 (MSP2). The role and extent of msp2(p44) recombination, particularly in hosts that only experience acute infections, is not clear. In the present study, we explored patterns of recombination and expression of the msp2(p44) gene of A. phagocytophilum in a serially infected mouse model. Even though the bacterium was passed rapidly among mice, minimizing the opportunities for the host to develop adaptive immunity, we detected the emergence of 34 unique msp2(p44) expression cassette variants. The expression of msp2(p44) pseudogenes did not follow a consistent pattern among different groups of mice, although some pseudogenes were expressed more frequently than others. In addition, among 263 expressed pseudogenes, 3 mosaic sequences each consisting of 2 different pseudogenes were identified. Population genetic analysis showed that genetic diversity and subpopulation differentiation tended to increase over time until stationarity was reached but that the variance that was observed in allele (expressed pseudogene) frequency could occur by drift alone only if a high variance in bacterial reproduction could be assumed. These findings suggest that evolutionary forces influencing antigen variation in A. phagocytophilum may comprise random genetic drift as well as some innate but apparently nonpurifying selection prior to the strong frequency-dependent selection that occurs cyclically after hosts develop strong adaptive immunity.     

Immunologic responses of domestic and bighorn sheep to a multivalent Pasteurella haemolytica vaccine

The efficacy of a Pasteurella haemolytica vaccine (serotypes A1, A2, and T10) to induce humoral antibodies and alter colonization of the upper respiratory tract by related P. haemolytica spp. strains was evaluated in 10 bighorn (Ovis canadensis canadensis) and 10 domestic (Ovis aries) sheep. Sheep of each species were divided into five pairs based on age and history of respiratory disease. One sheep in each pair was vaccinated twice 2 wk apart with 2 ml of vaccine (VAC group) and the remaining animals (NV group) were injected with 2 ml of sterile saline. Mild, transient lameness was the only observed adverse effect. Blood sera from the sheep were tested for agglutinating antibodies against whole cells of A1, A2, and T10 and for leukotoxin neutralizing antibodies. Antibody titers were expressed as the reciprocal log2 of the highest reactive dilutions. Domestic sheep > 1-yr-old and two bighorn sheep with a history of A1 infection had higher titers throughout the study against A1 cells than domestic sheep < 1-yr-old and bighorns without a history of A1 infection. Both domestic and bighorn sheep had log2 titers of 8 to 12 against A2 cells and 6 to 12 against T10 cells during this time. Bighorn sheep in the VAC group had 2 to 32 fold titer increases for A1 cells by 2 wk post-vaccination (PV) compared to 0 to 2 fold increases in VAC domestic sheep. Two to 16 and 0 to 8 fold increases in antibodies titers to A2 and T10 cells, respectively, were detected in sera of both VAC groups. Sera of bighorn sheep with a history of respiratory disease and all domestic sheep had log2 leukotoxin neutralizing antibody titers of 4 to 14 in contrast to < or = 2 in sera of bighorn sheep without a history of respiratory disease. Neutralizing antibody titers of two bighorns without a history of respiratory disease in the VAC group increased from log2 0 to 5 in one and from 0 to 9 in the other 2 wk PV. Antibody increases in these animals were no longer evident at 16 wk PV while titers of animals with histories of disease remained relatively stable. The types and numbers of Pasteurella spp. isolated from nasal and pharyngeal swabs varied throughout the study without conclusive evidence of suppression of colonization. Although the animals were not experimentally challenged to determine the efficacy of the vaccine, one VAC and one NV bighorn sheep died following introduction of an A2 P. haemolytica strain when leukotoxin neutralizing antibodies had returned to pre-vaccination levels. This vaccine appeared to be safe for use in bighorn sheep and stimulated moderate but transient increases in antibody levels which should provide some protection against naturally occurring disease. A vaccine which would induce production of high and maintained antibodies against multiple strains of P. haemolytica would be valuable for use in bighorn sheep maintained in captivity or when captured for relocation.     

Identification of a haemomycoplasma species in anemic reindeer (Rangifer tarandus)

During an 18-mo period (May 2002-November 2003), 10 animals in a herd of 19 reindeer (Rangifer tarandus) at the National Animal Disease Center (NADC) experienced episodes of anemia. Affected animals had histories of weight loss, unthriftiness, occasionally edema of dependent parts and moderate anemia characterized by microcytosis or macrocytosis, hypochromasia, schistocytosis, keratocytosis, acanthocytosis, and dacryocytosis. Numerous basophilic punctate to ring-shaped bodies, measuring less than 1.0 microm, were found on the surface of red blood cells and were often observed encircling the outer margins of the cells. Based on cytologic findings, DNA preparations from selected affected animals in the NADC herd and one animal from a private herd experiencing similar episodes of anemia were assayed by polymerase chain reaction (PCR) for the presence of hemotropic bacteria using primers targeting the 16S rRNA genes of Mycoplasma (Eperythrozoon) suis, Mycoplasma (Haemobartonella) haemofelis, Anaplasma marginale, Anaplasma spp., and Ehrlichia spp. Amplification products were detected from four of the affected animals using primers specific for the 16S rRNA gene of M. haemofelis and Mycoplasma haemocanis. Product from one of the animals was sequenced and internal primers were designed from the resulting sequence to perform a nested PCR assay. Samples from 10 reindeer were positive using the nested PCR reaction and products from seven animals were sequenced; BLAST searches and phylogenetic analysis were performed on the resulting sequences. Sequence data from six animals revealed homology to an organism most closely related to Mycoplasma ovis, Mycoplasma wenyonii, and Mycoplasma haemolamae; sequence from a single animal was most closely related to M. haemofelis and M. haemocanis. This represents the first identification of a haemomycoplasma species in reindeer. Although several animals were also infected with abomasal nematodes, the presence of this newly described haemomycoplasma may have contributed to the anemic syndrome.     

Microbial diversity in bighorn sheep revealed by culture-independent methods

We investigated the effectiveness of culture-independent molecular methods for determining host-associated microbial diversity in bighorn sheep (Ovis canadensis). Results from bacterial culture attempts have been the primary source of information on host-associated bacteria, but studies have shown that culture-based results significantly underestimate bacterial diversity in biological samples. To test the effectiveness of culture-independent methods, we extracted DNA from nasal and oropharyngeal swab samples collected from bighorn sheep in four different populations. From these samples, we amplified, cloned, and sequenced small subunit (16S) ribosomal DNA (rDNA) to identify the scope of microbial diversity in bighorn respiratory tracts. Phylogenetic analysis of these rDNA gene sequences revealed organismal diversity an order of magnitude higher than was determined by culture methods. Pasteurellaceae bacteria were the most diverse phylogenetic group in live bighorn sheep, and members of bacterial genera often associated with respiratory disease were found in all the samples. Culture-independent methods were also able to directly detect leukotoxin (lktA) gene sequences in swab and lung tissue samples. Overall, our results show the power of culture-independent molecular methods for identifying microbial diversity in bighorn sheep and the potential for these methods to detect the presence of virulence genes in biological samples.     

Stability and tick transmission phenotype of gfp-transformed Anaplasma marginale through a complete in vivo infection cycle

We tested the stability and tick transmission phenotype of transformed Anaplasma marginale through a complete in vivo infection cycle. Similar to the wild type, the gfp-transformed A. marginale strain established infection in cattle, a natural reservoir host, and persisted in immune competent animals. The tick infection rates for the transformed A. marginale and the wild type were the same. However, there were significantly lower levels of the transformed A. marginale than of the wild type in the tick. Despite the lower levels of replication, ticks transmitted the transformant. Transformants can serve as valuable tools to dissect the molecular requirements of tick colonization and pathogen transmission.