Physical linkage of naturally complexed bacterial outer membrane proteins enhances immunogenicity

The outer membrane proteins (OMPs) of bacterial pathogens are essential for their growth and survival and especially for attachment and invasion of host cells. Since the outer membrane is the interface between the bacterium and the host cell, outer membranes and individual OMPs are targeted for development of vaccines against many bacterial diseases. Whole outer membrane fractions often protect against disease, and this protection cannot be fully reproduced by using individual OMPs. Exactly how the interactions among individual OMPs influence immunity is not well understood. We hypothesized that one OMP rich in T-cell epitopes can act as a carrier for an associated OMP which is poor in T-cell epitopes to generate T-dependent antibody responses, similar to the hapten-carrier effect. Major surface protein 1a (MSP1a) and MSP1b1 occur as naturally complexed OMPs in the Anaplasma marginale outer membrane. Previous studies demonstrated that immunization with the native MSP1 heteromer induced strong immunoglobulin G (IgG) responses to both proteins, but only MSP1a stimulated strong CD4⁺ T-cell responses. Therefore, to test our hypothesis, constructs of CD4⁺ T-cell epitopes from MSP1a linked to MSP1b1 were compared with individually administered MSP1a and MSP1b1 for induction of MSP1b-specific IgG. By linking the T-cell epitopes from MSP1a to MSP1b1, significantly higher IgG titers against MSP1b1 were induced. Understanding how the naturally occurring intermolecular interactions between OMPs influence the immune response may lead to more effective vaccine design.     

Detection of Cattle Naturally Infected with Anaplasma marginale in a Region of Endemicity by Nested PCR and a Competitive Enzyme-Linked Immunosorbent Assay Using Recombinant Major Surface Protein 5

A competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginale is endemic. The true positive and negative A. marginale infection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale. Third, msp5 sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale. The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginale infections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement.     

<Emphasis Type="Italic">Anaplasma marginale</Emphasis> and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis>: Rickettsiales pathogens of veterinary and public health significance

Anaplasma marginale and Anaplasma phagocytophilum are the most important tick-borne bacteria of veterinary and public health significance in the family Anaplasmataceae. The objective of current review is to provide knowledge on ecology and epidemiology of A. phagocytophilum and compare major similarities and differences of A. marginale and A. phagocytophilum. Bovine anaplasmosis is globally distributed tick-borne disease of livestock with great economic importance in cattle industry. A. phagocytophilum, a cosmopolitan zoonotic tick transmitted pathogen of wide mammalian hosts. The infection in domestic animals is generally referred as tick-borne fever. Concurrent infections exist in ticks, domestic and wild animals in same geographic area. All age groups are susceptible, but the prevalence increases with age. Movement of susceptible domestic animals from tick free non-endemic regions to disease endemic regions is the major risk factor of bovine anaplasmosis and tick-borne fever. Recreational activities or any other high-risk tick exposure habits as well as blood transfusion are important risk factors of human granulocytic anaplasmosis. After infection, individuals remain life-long carriers. Clinical anaplasmosis is usually diagnosed upon examination of stained blood smears. Generally, detection of serum antibodies followed by molecular diagnosis is usually recommended. There are problems of sensitivity and cross-reactivity with both the Anaplasma species during serological tests. Tetracyclines are the drugs of choice for treatment and elimination of anaplasmosis in animals and humans. Universal vaccine is not available for either A. marginale or A. phagocytophilum, effective against geographically diverse strains. Major control measures for bovine anaplasmosis and tick-borne fever include rearing of tick-resistant breeds, endemic stability, breeding Anaplasma-free herds, identification of regional vectors, domestic/wild reservoirs and control, habitat modification, biological control, chemotherapy, and vaccinations (anaplasmosis and/or tick vaccination). Minimizing the tick exposure activities, identification and control of reservoirs are important control measures for human granulocytic anaplasmosis.     

Serologic Cross-Reactivity between Anaplasma marginale and Anaplasma phagocytophilum

In the context of a serosurvey conducted on the Anaplasma marginale prevalence in Swiss cattle, we suspected that a serological cross-reactivity between A. marginale and A. phagocytophilum might exist. In the present study we demonstrate that cattle, sheep and horses experimentally infected with A. phagocytophilum not only develop antibodies to A. phagocytophilum (detected by immunofluorescent-antibody assay) but also to A. marginale (detected by a competitive enzyme-linked immunosorbent assay). Conversely, calves experimentally infected with A. marginale also developed antibodies to A. phagocytophilum using the same serological tests. The identity of 63% determined in silico within a 209-amino-acid sequence of major surface protein 5 of an isolate of A. marginale and one of A. phagocytophilum supported the observed immunological cross-reactivity. These observations have important consequences for the serotesting of both, A. marginale and A. phagocytophilum infection of several animal species. In view of these new findings, tests that have been considered specific for either infection must be interpreted carefully.     

Anaplasma marginale Superinfection Attributable to Pathogen Strains with Distinct Genomic Backgrounds

Strain superinfection occurs when a second pathogen strain infects a host already infected with a primary strain. The selective pressures that drive strain divergence, which underlies superinfection, and allow penetration of a new strain into a host population are critical knowledge gaps relevant to shifts in infectious disease epidemiology. In regions of endemicity with a high prevalence of infection, broad population immunity develops against Anaplasma marginale, a highly antigenically variant rickettsial pathogen, and creates strong selective pressure for emergence of and superinfection with strains that differ in their Msp2 variant repertoires. The strains may emerge either by msp2 locus duplication and allelic divergence on an existing genomic background or by introduction of a strain with a different msp2 allelic repertoire on a distinct genomic background. To answer this question, we developed a multilocus typing assay based on high-throughput sequencing of non-msp2 target loci to distinguish among strains with different genomic backgrounds. The technical error level was statistically defined based on the percentage of perfect sequence matches of clones of each target locus and validated using experimental single strains and strain pairs. Testing of A. marginale-positive samples from tropical regions where A. marginale infection is endemic identified individual infections that contained unique alleles for all five targeted loci. The data revealed a highly significant difference in the number of strains per animal in the tropical regions compared to infections in temperate regions and strongly supported the hypothesis that transmission of genomically distinct A. marginale strains predominates in high-prevalence areas of endemicity.     

Detection of Anaplasma marginale in Hyalomma asiaticum ticks by PCR assay

A polymerase chain reaction (PCR) assay was performed in this study to amplify the major surface protein 5 (msp5) gene from the genomic DNA of Anaplasma marginale in Hyalomma asiaticum ticks by species-specific primers. Sequence analysis showed that the msp5 gene was 643 bases long and that the PCR products from the samples had an identical sequence (JX507127). Moreover, the BLAST showed that the sequence was identical to the msp5 sequences of A. marginale and most closely related to the A. marginale msp5 gene (AB704328) and the Liangdang strain of the A. marginale msp5 gene (EF546443) with similarity of 99 % (differing only by two bases). An epidemiological survey was performed in several dairy farms: a total of 68 ticks were collected from 49 cattle. As a result, 14 of the 49 (28.57 %) blood smears stained with Wright–Giemsa and 22 of the 68 (32.35 %) ticks examined by PCR assay exhibited A. marginale infection. The results of the PCR assay were mostly consistent with the results of the microscopic examination. A number of results were negative in blood smear but positive in PCR, which is important for the early diagnosis of anaplasmosis.     

Persistence of Anaplasma ovis Infection and Conservation of the msp-2 and msp-3 Multigene Families within the Genus Anaplasma

Goats which have recovered from acute Anaplasma ovis infection remain seropositive, although infected erythrocytes cannot be detected by microscopic examination. Persistence of A. ovis 17 to 21 months following experimental infection was demonstrated by PCR detection of the msp-5 gene. Quantitative analysis of persistent rickettsemia over time showed that all levels were below the limit of microscopic detection and ranged from a low of 10² organisms/ml to peaks of 10⁶ organisms/ml. Two patterns of persistent rickettsemia were observed: the first was characterized by cyclic fluctuations at 6- to 9-week intervals, similar to the pattern described for A. marginale-infected cattle, while in the second pattern, repetitive cycles did not occur and the rickettsemia levels were relatively constant. The msp-2 and msp-3 multigene families, which provide the genetic capacity for outer membrane protein antigenic variation during persistent A. marginale rickettsemia, were identified in the A. ovis genome by Southern blot analysis, and expression of an MSP-2 homologue was confirmed by using immunoblots.     

Prevalence of Theileria equi, Babesia caballi, and Anaplasma phagocytophilum in horses from the north of Portugal

Piroplasmid protozoa Theileria equi and Babesia caballi and zoonotic rickettsial bacterium Anaplasma phagocytophilum are important agents of equine vector-borne diseases (EVBD). This study aimed at investigating the prevalence of infections with or exposure to these pathogens in horses from the north of Portugal. Blood was randomly collected from 162 horses, living in 72 different stables, to prepare Giemsa-stained slide smears. Additionally, plasma samples were tested for antibodies to T. equi and B. caballi by two competitive enzyme-linked immunosorbent assays and to A. phagocytophilum by an indirect fluorescence antibody test. Five horses were positive to T. equi by microscopy (3.1 %), three to B. caballi (1.9 %), and none to A. phagocytophilum with no horse simultaneously positive for the two piroplasms. Clinically suspect animals had a significantly higher positivity to T. equi by microscopy in comparison with the nonsuspect ones (21.4 vs. 1.4 %). Twenty-nine horses were seropositive to T. equi (17.9 %), 18 to B. caballi (11.1 %), and 21 to A. phagocytophilum (13.0 %). Combined serology and microscopy positive results to T. equi and B. caballi were 19.1 and 11.7 %, respectively, with 33.3 % of the horses found positive to at least one agent. Forty horses were positive to single agents and 14 to more than one agent. An outdoor or mixed outdoor/indoor type of housing was found to be a risk factor for the combined positivity to T. equi. Infections with T. equi, B. caballi, and A. phagocytophilum are endemic in the north of Portugal. In addition to the treatment of positive horses, preventive measures should be put in practice to reduce exposure to and infection with agents of EVBD.     

Reduction of tick infections with Anaplasma marginale and A. phagocytophilum by targeting the tick protective antigen subolesin

Subolesin was recently shown by both gene silencing and immunization with the recombinant protein to protect against tick infestations, and to cause reduced tick survival and degeneration of gut and salivary gland tissues. In this research, we extended these studies by testing whether targeting subolesin by RNAi or vaccination interfered with the ability of ticks to become infected with two tick-borne pathogens, Anaplasma marginale which causes bovine anaplasmosis and Anaplasma phagocytophilum, the causative agent of human granulocytin anaplasmosis. For the A. marginale studies, Dermacentor variabilis males were injected with subolesin dsRNA or saline and then were allowed to feed on cattle with ascending rickettsemias, while for the A. phagocytophilum studies, mice were immunized with the recombinant subolesin protein, infected with the pathogen and then infested with larval Ixodes scapularis. Tick infections were determined by quantitative polymerase chain reaction of gut and salivary gland tissues. In both experimental approaches, tick infections were significantly reduced. These results suggest that subolesin appears to be a candidate vaccine antigen that may contribute to control of multiple tick species and the reduction of tick-borne pathogens.     

Infection Exclusion of the Rickettsial Pathogen Anaplasma marginale in the Tick Vector Dermacentor variabilis

Anaplasma marginale is a tick-borne, rickettsial cattle pathogen that is endemic in several areas of the United States. Recent studies (J. de la Fuente, J. C. Garcia-Garcia, E. F. Blouin, J. T. Saliki, and K. M. Kocan, Clin. Diagn. Lab. Immunol. 9:658-668, 2002) demonstrated that infection of cultured tick cells and bovine erythrocytes with one genotype of A. marginale excluded infection with other genotypes, a phenomenon referred to as infection exclusion. The present study was undertaken to confirm the phenomenon of infection exclusion of A. marginale genotypes in a tick vector, Dermacentor variabilis. Only one genotype of A. marginale (Virginia isolate) was detected by PCR in ticks that fed first on a calf infected with a Virginia isolate and second on a calf infected with an Oklahoma isolate. These studies demonstrate that infection exclusion of A. marginale genotypes also occurs in naturally infected ticks, as well as in cattle and cultured tick cells, and results in establishment of only one genotype per tick.     

Anaplasma phagocytophilum Inhibits Apoptosis and Promotes Cytoskeleton Rearrangement for Infection of Tick Cells

Anaplasma phagocytophilum causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects gene expression in both the vertebrate host and the tick vector, Ixodes scapularis. Here, we identified new genes, including spectrin alpha chain or alpha-fodrin (CG8) and voltage-dependent anion-selective channel or mitochondrial porin (T2), that are involved in A. phagocytophilum infection/multiplication and the tick cell response to infection. The pathogen downregulated the expression of CG8 in tick salivary glands and T2 in both the gut and salivary glands to inhibit apoptosis as a mechanism to subvert host cell defenses and increase infection. In the gut, the tick response to infection through CG8 upregulation was used by the pathogen to increase infection due to the cytoskeleton rearrangement that is required for pathogen infection. These results increase our understanding of the role of tick genes during A. phagocytophilum infection and multiplication and demonstrate that the pathogen uses similar strategies to establish infection in both vertebrate and invertebrate hosts.     

Serotyping Isolates of Anaplasma phagocytophilum by Using Monoclonal Antibodies

Ten mouse monoclonal antibodies (MAbs) that react with Anaplasma phagocytophilum (the human granulocytic ehrlichiosis agent) Webster isolates were developed. Seven different isolates of A. phagocytophilum were subtyped with these MAbs. Western blot analysis revealed that these MAbs reacted mainly with 41- to 46-kDa Msp2 proteins. Six MAbs reacted with all isolates. Four other MAbs reacted with human isolates from Wisconsin, but not with human isolates from New York or with animal isolates. Three different serotypes were identified. These features may lead to the development of other specific MAbs in order to provide tools for antigenic characterization of human isolates of A. phagocytophilum.     

Anaplasma phagocytophilum in central and western Wisconsin: a molecular survey

Anaplasma phagocytophilum is an obligate intracellular bacterium that is transmitted to humans through the bite of Ixodes spp. ticks, and causes a febrile disease known as human granulocytic anaplasmosis. The presence of A. phagocytophilum in Wisconsin white-tailed deer blood and in deer ticks was assessed using PCR and DNA sequencing. Sampling sites in the western part of the state (Buffalo County) and central region (Waushara, Waupaca, and Green Lake counties) were used. In Buffalo County, 5.6% of deer and 8.9% of ticks were infected. At Hartman Creek State Park (Waupaca County), 11.5% of ticks were infected, while the observed prevalence in deer from counties to the south of the park (Waushara and Green Lake) reached 19–26%. Based on 16S rRNA sequences, A. phagocytophilum strains associated and not associated with human infections were identified. Furthermore, two novel A. phagocytophilum variants were found in deer blood samples. Transmission of Lyme disease has been documented in both the Western and Central regions we sampled, and the presence of A. phagocytophilum in naturally occurring tick populations could present an additional risk of disease to humans that enter tick habitats.     

Seroprevalence of tick-borne diseases among cattle in the Sudan

This study was carried out to determine the prevalence of Theileria annulata, Theileria mutans, Babesia bigemina, and Anaplasma marginale antibodies among cattle in the Sudan. A total of 600 serum samples were collected from indigenous (zebu) and crossbred cattle (zebu × Friesian) of both sex and different age groups. Indirect enzyme-linked immunosorbent assay was used to assess antibodies against tick-borne diseases in apparently healthy cattle. The overall prevalence rates of T. annulata, T. mutans, B. bigemina, and A. marginale antibodies were found to be 30.8%, 6.1%, 10.7%, and 38.9%, respectively. The highest seroprevalence of T. annulata was reported in Atbara and El Damer, Northern Sudan. There were no significant associations for the seroprevalence of all tick-borne diseases reported among different age groups. Although there were no significant differences between the two breeds of cattle examined for T. annulata, T. mutans, and B. bigemina antibodies, there was a significant difference for prevalence of antibodies against A. marginale, with highest percentages of antibodies in indigenous cattle. Six different combinations of mixed infection were detected. This is the first report in which antibodies against A. marginale among cattle in Northern Sudan is reported. The findings imply that antibodies to tick-borne infections are widely distributed in the region. The need for further investigations using more advanced techniques is recommended.     

A Novel High-Resolution Melt PCR Assay Discriminates Anaplasma phagocytophilum and “Candidatus Neoehrlichia mikurensis”

“Candidatus Neoehrlichia mikurensis” (Anaplasmataceae) is an emerging pathogen transmitted by Ixodes ticks. Conventional PCR and the newly developed high-resolution melt PCR were used to detect and discriminate “Candidatus Neoehrlichia mikurensis” and Anaplasma phagocytophilum. Both bacterial species were frequently found in Ixodes ricinus and Ixodes hexagonus but virtually absent from Dermacentor reticulatus. In rodents, “Candidatus N. mikurensis” was significantly more prevalent than A. phagocytophilum, whereas in cats, only A. phagocytophilum was found.