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Appl Environ Microbiol. Jan 2011; 77(1): 330–334.
Published online Nov 5, 2010. doi:  10.1128/AEM.02096-10
PMCID: PMC3019711

Stability and Tick Transmission Phenotype of gfp-Transformed Anaplasma marginale through a Complete In Vivo Infection Cycle [down-pointing small open triangle]

Abstract

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.

Obligate intracellular, tick-transmitted bacterial pathogens, which include Anaplasma spp., Ehrlichia spp., and Rickettsia spp., cause significant morbidity and mortality in humans and animals. Pathogen transmission relies on successful transition between the vertebrate and invertebrate hosts, which includes the exit from and entrance into markedly different host cell types (25). In the case of Anaplasma marginale, bacteria reside in metabolically quiescent mature erythrocytes within the mammalian host, and then, following acquisition of a blood meal by the tick vector, must transition to colonize tick midgut epithelial cells, which are phagocytic and digestive (8, 15, 16, 25). To complete the cycle, the bacteria transit from the midgut epithelium to the salivary glands and are shed into the saliva during feeding on a new mammalian host (13, 14, 27, 28). Given time, the new host is able to control but not clear the infection. Thus, persistently infected cattle serve as the reservoir for continued tick transmission (4, 10, 25). The molecular mechanisms by which these transitions occur are largely unknown for this group of pathogens, and the genetic tools, including targeted gene knockouts and the creation of mutant libraries, which are required for identification of these mechanisms, are unavailable or in the early stages of development. Several vector-borne Rickettsia spp. and Anaplasma spp. have been transformed using transposon mutagenesis and shown to be stable in vitro (1, 2, 6, 7, 17, 22, 23). However, the stability, infectivity, tick transmissibility, and ability to establish persistent infection within the natural host species, critical components of a valid model system for many of these pathogens, have not been tested. Using transformed A. marginale (6), we tested its biological relevance as a model organism by tracking this organism through a complete infection cycle, including determination of tick infection rates and levels, tick transmissibility, and establishment of persistent infection.

Initial animal infections for tick acquisition feed.

The transformant used in these experiments was described by Felsheim et al. (6). Briefly, this transformant was derived through a single homologous recombination event at nucleotide position 1026363 of the St. Maries genome. The transformant carries a 4.5-kb insert which includes the TurboGFP and spectinomycin/streptomycin resistance genes to assist in selection (6). To establish the initial A. marginale infection with this transformant, two splenectomized calves were inoculated intravenously with either 1011 green fluorescent protein (GFP)-transformed A. marginale (animal 36282) or a control strain of A. marginale (animal 36361). Both inoculums were derived from the St. Maries strain of A. marginale and maintained in ISE6 cells cultured at 34°C (5, 6, 19, 20). The dose was determined using quantitative real-time PCR (qPCR) for msp5, a single-copy gene (5, 8, 18).

Establishment of infection in both animals was determined by using light microscopic examination of Giemsa-stained blood smears and two separate PCR assays (Fig. (Fig.1).1). In the first assay, based on published methods, oligonucleotides specific for msp5 were used to detect both the control (wild-type [wt]) strain of A. marginale and the gfp-transformed A. marginale strain (referred to here as the gfp strain) (26). The second assay, again based on published methods, used oligonucleotide primers to test for the presence of the A. marginale gfp strain by specific amplification of a 705-bp fragment flanking the 5′ end of the insert (AmTss PCR A FOR and Turbo up primers) (Table (Table1)1) (6). As expected, only msp5 was detected in animal 36361 receiving the A. marginale wt, while both amplicons were detected in animal 36282 inoculated with the A. marginale gfp strain. Additionally, both animals developed an anti-Msp5 antibody response, as determined using a commercially available competitive enzyme-linked immunosorbent assay (cELISA) kit (12, 29, 30). Green fluorescence, using fluorescence microscopy, was detected within erythrocytes of only the animal (animal 36282) receiving the A. marginale gfp strain (data not shown).

FIG. 1.
Confirmation of infection. PCR to confirm infection with the A. marginale gfp strain (a) and the A. marginale wt (b) in needle-inoculated animals (AF), tick midguts (MG), and salivary glands (SG) and in animals infected by tick transmission feeding (TF). ...
TABLE 1.
Oligonucleotides used in PCR to confirm infection with and differentiate between the A. marginale gfp and wt strains

Tick colonization and transmission.

When bacteremia in animals 36361 and 36282 reached 107 A. marginale bacteria per ml of blood, as determined by microscopic examination of Giemsa-stained blood smears, 60 adult male Dermacentor andersoni ticks were allowed to feed for 7 days. After acquisition feeding, the ticks were held at 26°C for 4 days to allow for clearance of the blood meal from the mouthparts. The midgut and salivary glands were then removed from 10 ticks. Infection in these tissues was detected using the PCR assays described above to amplify msp5 and gfp. Additionally, qPCR for msp5 was done to determine infection rate (percentage of infected ticks/number of ticks tested) and level in the salivary glands. As expected, only the msp5 amplicon was detected in the midguts and salivary glands of ticks exposed to the A. marginale wt, while both amplicons were detected in the ticks exposed to the A. marginale gfp strain (Fig. (Fig.1).1). The tick infection rates were 100% for both the ticks exposed to the A. marginale wt and the ticks exposed to the A. marginale gfp strain, which, based on published data, is typical for the St. Maries strain in D. andersoni fed on acutely infected animals (Table (Table2)2) (24, 27). The infection level was approximately 2 log10 lower in ticks exposed to the A. marginale gfp strain than in ticks exposed to the A. marginale wt and the published values for A. marginale (Table (Table2)2) (27).

TABLE 2.
Comparison of infection parameters in cattle and ticks infected with St. Maries strains of A. marginale

After the acquisition feed and holding period, fluorescent organisms were identified in the midguts of ticks exposed to the A. marginale gfp strain (Fig. (Fig.2).2). Twenty-four hours after the subsequent transmission feed, colonies of fluorescent bacteria were present in both the midguts and salivary glands in the ticks exposed to the A. marginale gfp strain (Fig. (Fig.2).2). Microscopic detection of colonies in the midgut but not the salivary gland after the acquisition feed is expected, as the midgut is the initial site of colonization and subsequent replication in the salivary glands occurs during transmission feeding (27).

FIG. 2.
Green fluorescing colonies in tick tissues. (A) Autofluorescence in a D. andersoni midgut infected with the A. marginale wt. (B) Green fluorescent colonies in a D. andersoni midgut after acquisition feeding on animal 36282 infected with the A. marginale ...

After the acquisition feed and holding period, the 25 remaining ticks were placed on two spleen-intact calves for 7 days to test for transmission. Animal 36488 was exposed to ticks infected with the A. marginale gfp strain and animal 36432 exposed to ticks infected with the A. marginale wt. Both animals became PCR positive (Fig. (Fig.1)1) and developed an anti-Msp5 specific-antibody response. However, the prepatent period was approximately 2 times longer in the animal infected with the A. marginale gfp strain than in the animal infected with the A. marginale wt (Table (Table22).

Establishment of persistent infection and insert stability.

To test for the stability of the insert and the ability to establish persistent infection in an immune-competent host, 5 months after transmission, 100 ml of whole blood from animal 36488 was inoculated into a naïve, splenectomized animal (animal 31022). Animal 31022 developed a microscopically detectable bacteremia with continued expression of GFP (Fig. (Fig.3)3) and a specific anti-Msp5 antibody response, thus confirming the establishment of persistent infection in animal 36488.

FIG. 3.
Green fluorescing colonies in erythrocytes from animal 30122 subinoculated with blood from persistently infected animal 36488.

Two methods were used to rule out the possibility of loss or rearrangement of the gfp-containing insert in a subset of the A. marginale gfp strain population. First, primers flanking the gfp-containing insert (AmTss PCR A FOR and AmTSS PCR B REV) were used to differentiate between A. marginale bacteria with and without the insert in all animals (Table (Table1)1) (6). The amplicon from the A. marginale wt is expected to be 1,015 bp, while the amplicon from the A. marginale gfp strain is expected to be 5,564 bp. Importantly, amplification is generally biased toward detection of the smaller product, thus increasing the likelihood of detecting A. marginale lacking the insert (9, 11). In all animals infected with the A. marginale gfp strain, only the larger product was detected (Fig. (Fig.4).4). The identity of the amplicons was verified by end sequencing (data not shown).

FIG. 4.
PCR demonstrating the stability of transformed A. marginale in acquisition-fed animals (AF), transmission-fed animals (TF), and the animal which was subinoculated (SI) with blood from the persistently infected, transmission-fed animal. Animals 36361 and ...

Second, the total copy number of A. marginale within each animal was determined using qPCR to amplify msp5 and gfp (Table (Table3),3), and these values were compared. Detection of gfp by qPCR employed the oligonucleotides and TaqMan probe listed in Table Table1.1. A standard curve was constructed from serial dilutions of a PCR4 TOPO plasmid containing a 2,249-bp insert that included the full-length TurboGFP gene. The reactions were conducted using a PCR mixture containing 10 mM Tris (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 200 μM each dATP, dCTP, dGTP, and dTTP, 0.2 μM each primer, 0.2 μM fluorogenic probe, and 1.25 U of AmpliTaq Gold. The cycling conditions for the qPCR were as follows: 95°C for 10 min, 95°C for 15 s, 60°C for 30 s, and 72°C for 45 s, followed by a final extension at 72°C for 3 min. If a subset of the transformed A. marginale bacteria no longer carried the insert, the copy number of msp5 would be greater than the copy number of gfp. All animals exposed to the A. marginale gfp strain had higher copy numbers of gfp than of msp5, with differences of less than 1 log10 between the numbers. These differences were not found to be statistically different using a 1-tailed Student's t test with a null hypothesis that the copy number of msp5 is not higher that the copy number of gfp. The reason for higher levels of gfp is unknown, as the qPCR efficiencies were similar (93.4% for gfp and 93.5% for msp5, with a 99% correlation coefficient). However, other parameters which affect amplification, such as primer binding and secondary structures, likely account for this difference. Regardless, these data support the conclusion that the gfp-containing insert is stable within A. marginale through a transmission cycle, including persistent infection. Rearrangements in which the regions flanking the insert and the sequence of the TurboGFP gene remain intact would not be detected by the two tests used to examine stability; however, such rearrangements are unlikely, as A. marginale appears to lack the enzymatic machinery for the DNA excision and repair process (3).

TABLE 3.
Comparison of copy numbers of A. marginale msp5 and gfp in cattle by use of quantitative real-time PCR

The findings presented here demonstrate that the A. marginale gfp strain develops as expected within the tick, with microscopically detectable colonies forming in the midgut after the acquisition feed and within the midgut and salivary glands after the transmission feed. The transformed A. marginale strain has infection rates (100%) in ticks equivalent to those observed for wild-type A. marginale; however, the A. marginale gfp strain replicates to lower levels in the tick salivary gland than wild-type A. marginale (27). Additionally, animal 36488, which was infected with the A. marginale gfp strain via tick feeding, had a longer incubation period and lower peak bacteremia than either the animal infected with the A. marginale wt in this experiment (animal 36432) or animals infected via tick feeding with A. marginale (St. Maries), as determined by collating historical data from nine separate tick feeding experiments (Table (Table2).2). Similarly, the A. marginale gfp strain requires longer subculture intervals than the A. marginale wt grown in ISE6 tick cells (6). Together, these data suggest that the A. marginale gfp strain replicates to lower levels than the A. marginale wt; however, similar to the A. marginale wt, the A. marginale gfp strain is able to establish persistent infection in the immune-competent mammalian host. Additionally, expression of GFP was stable through the infection cycle, which allows for tracking of the organism in both the mammalian host and the tick vector. Transformed A. marginale expressing gfp that establishes infection in the bovine host and is transmitted by the tick vector demonstrates that biologically relevant, stable transformation of Anaplasma spp. is possible. Fluorescent transformants will serve as a tool essential for the dissection of the molecular mechanisms of tick invasion, colonization, and transmission.

Acknowledgments

We acknowledge Ralph Horn, James Allison, and Nancy Kumpula-McWhirter for their outstanding technical assistance.

This work was supported by NIH R01 AI 44005, USDA-ARS CRIS 5348-32000-027-00D, USDA CSREES 35604-15440, and The Wellcome Trust (GR075800M).

Footnotes

[down-pointing small open triangle]Published ahead of print on 5 November 2010.

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