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Infect Immun. Jun 2007; 75(6): 2959–2964.
Published online Apr 9, 2007. doi:  10.1128/IAI.00284-07
PMCID: PMC1932854

Identification of Midgut and Salivary Glands as Specific and Distinct Barriers to Efficient Tick-Borne Transmission of Anaplasma marginale[down-pointing small open triangle]

Abstract

Understanding the determinants of efficient tick-borne microbial transmission is needed to better predict the emergence of highly transmissible pathogen strains and disease outbreaks. Although the basic developmental cycle of Anaplasma and Ehrlichia spp. within the tick has been delineated, there are marked differences in the ability of specific strains to be efficiently tick transmitted. Using the highly transmissible St. Maries strain of Anaplasma marginale in Dermacentor andersoni as a positive control and two unrelated nontransmissible strains, we identified distinct barriers to efficient transmission within the tick. The Mississippi strain was unable to establish infection at the level of the midgut epithelium despite successful ingestion of infected blood following acquisition feeding on a bacteremic animal host. This inability to colonize the midgut epithelium prevented subsequent development within the salivary glands and transmission. In contrast, A. marginale subsp. centrale colonized the midgut and then the salivary glands, replicating to a titer indistinguishable from that of the highly transmissible St. Maries strain and at least 100 times greater than that previously associated with successful transmission. Nonetheless, A. marginale subsp. centrale was not transmitted, even when a large number of infected ticks was used for transmission feeding. These results establish that there are at least two specific barriers to efficient tick-borne transmission, the midgut and salivary glands, and highlight the complexity of the pathogen-tick interaction.

Arthropod vectors transmit a wide diversity of microbial pathogens, including viruses, bacteria, and protozoa, to humans and animals (2, 12, 20, 25, 28). Although simple mechanical transmission may occur, efficient transmission usually requires pathogen replication and development within the vector (1, 10, 16). In most pathogen-vector interactions, this involves the invasion of one or more organs of the vector, survival in face of the arthropod immune response, intra- or extracellular replication, and the development of infectivity prior to transmission effected by the vector biting or feeding on a susceptible human or animal host (2, 9, 27). Understanding how the pathogen mediates each of these steps and how this varies among pathogen strains is fundamentally important for better predicting vector-borne disease patterns and for developing new strategies to block transmission.

Pathogens in the genera Anaplasma and Ehrlichia are transmitted by ixodid ticks that initially feed on an infected animal host and then, following interhost transmission, feed on a susceptible human or animal (8, 9, 14, 17). This transmission reflects a complex development within the tick. Following the initial acquisition feeding and ingestion of the blood meal into the midgut lumen, the bacterium enters the midgut epithelial cells and undergoes a first round of replication within a membrane-bound vacuole (11, 22). This is followed by migration to and invasion of the salivary glands (9, 10). A second round of replication in the salivary gland acinar cells, apparently dependent on resumption of tick feeding on a mammalian host, is followed by transmission via the saliva (9, 13). Although this basic development cycle is broadly conserved among the Anaplasma and Ehrlichia spp., it is also clear that not all pathogen strains are equally or efficiently tick transmitted. Our interest is to identify the specific developmental steps within the tick vector that affect pathogen transmissibility, with the long-term goal of mapping specific pathogen determinants of high versus low transmissibility.

Within these two genera, only a single pathogen strain with a defective transmission phenotype has been examined in detail to date. The Florida strain of Anaplasma marginale is nontransmissible by Dermacentor andersoni, Dermacentor variabilis, Rhipicephalus (Boophilus) microplus, and Rhipicephalus (Boophilus) annulatus, all competent vectors for other A. marginale strains and the natural vectors within North America (6, 23). The defect in the Florida strain occurs at the level of the midgut following initial acquisition feeding. In vitro studies suggest a failure to bind to the midgut epithelial cells, while in vivo studies are more consistent with entry into the midgut epithelium followed by clearance without detectable first-round replication (6, 23). Whether this defect represents the sole determinant of transmissibility or transmission efficiency is unknown. In the present study, we use additional pathogen strains to demonstrate that transmission efficacy is determined at the levels of both the tick midgut and salivary glands.

MATERIALS AND METHODS

Pathogen and vector strains.

Three strains of A. marginale were used: the St. Maries and Mississippi strains are A. marginale senso stricto strains, and the Israel vaccine strain is currently classified as A. marginale subsp. centrale (7). The St. Maries strain was used as a reference strain, as it has previously been shown to be efficiently acquired and transmitted by adult male Dermacentor and Rhipicephalus (Boophilus) ticks (9). The Mississippi strain was obtained from an acute clinical case, and it has not been shown to be transmitted by D. andersoni (G. H. Palmer, unpublished data). A. marginale subsp. centrale was derived from the Theiler vaccine strain and has been used continuously as a vaccine in Israel since 1953 (7, 19). The transmission phenotype of A. marginale subsp. centrale by D. andersoni is unknown; however, it lacks the major surface protein 1a (MSP1a) N-terminal repeat region (24) proposed to be required for entry into the tick midgut and thus was predicted to be nontransmissible (5, 6). The colony of D. andersoni was originally collected from Owyhee County, ID, and has been maintained at the USDA-ARS Animal Disease Research Unit (23). The colony efficiently transmits multiple strains of A. marginale, including the St. Maries strain, and is free of other pathogens (8, 9, 26).

Tick acquisition feeding on A. marginale-infected animals.

Age-matched (5 to 6 months old) Holstein calves were utilized for infection with the three strains and subsequent tick acquisition feeding. The calves were confirmed to be free of A. marginale, as determined by an MSP5 serologic competitive enzyme-linked immunosorbent assay (cELISA) (VMRD, Pullman, WA) and nested PCR targeting msp5 (22). These assays will detect all A. marginale strains, including A. marginale subsp. centrale. Calves were inoculated with one of the three A. marginale strains by intravenous inoculation of 109 organisms. The development of acute A. marginale bacteremia was assessed by microscopic examination of Giemsa-stained blood smears. During acute bacteremia, defined as levels of >108 organisms per ml of blood, adult male D. andersoni ticks were allowed to acquisition feed on each calf for 7 days. Immediately after removal, a cohort of ticks was dissected and the total midgut, including the luminal blood meal, was collected and DNA extracted, as previously described (13, 28), to confirm the ingestion of A. marginale by using quantitative PCR (see below).

Development and replication of A. marginale in the tick following transmission feeding.

Following the acquisition feeding, the ticks were incubated at 26°C in 94% relative humidity for an additional 7 days prior to transmission feeding (9, 23). This interval ensures that the blood meal is completely digested, which occurs within the first 72 h, and thus, subsequent detection of bacteria within the midgut can be used to confirm entry into the epithelium and quantification can be used to test whether replication has occurred (9). After this incubation period, D. andersoni ticks were transmission fed on naïve age-matched (5 to 6 months old) Holstein calves, confirmed free of A. marginale as determined by MSP5 cELISA and nested msp5 PCR (22). As a positive control using the known transmissible St. Maries strain, 10 ticks infected with the St. Maries strain were fed on one calf and 35 on a second calf. In contrast, 100 D. andersoni ticks acquisition fed on the Mississippi strain-infected calf were transmission fed on each of two naïve calves. As with the Mississippi strain, 100 D. andersoni ticks acquisition fed on the A. marginale subsp. centrale-infected calf were transmission fed on each of two naïve calves. Following 7 days of transmission feeding, the ticks were removed and individual salivary glands and the midgut were dissected separately. DNA was extracted and used both for quantitative PCR and for confirmation of strain identity from positive samples.

Detection and quantification of A. marginale.

Bacteria were detected using a specific msp5 PCR as previously described (9). The msp5 sequences are identical in the St. Maries and Mississippi strains and 87% identical in A. marginale subsp. centrale (15). Importantly, the primer sequences (forward, 5′-TACACGTGCCCTACCGAGTTA-3′; reverse, 5′-TCCTCGCCTTGGCCCTCAGA-3′) are conserved in all examined A. marginale strains and A. marginale subsp. centrale and amplify a fragment of 343 bp (22). The identity of the msp5 amplicons was confirmed by sequencing.

For quantification of the St. Maries and Mississippi strains, the previously described TaqMan real-time PCR assay was used (9, 23). The primer sequences (forward, 5′-CTTCCGAAGTTGTAAGTGAGGGCA-3′; reverse, 5′-CTTATCGGCATGGTCGCCTAGTTT-3′) were selected to amplify a fragment of 202 bp and a TaqMan probe (5′-GCCTCCGCGTCTTTCAACAATTTGGT-3′) designed to bind within the amplicon. Full-length A. marginale msp5 cloned into pCR-4 TOPO vector (Invitrogen Corporation, Carlsbad, CA) was utilized to construct the standard curve, as previously described (23). Due to the polymorphism in the msp5 gene of A. marginale subsp. centrale (15), the real-time assay was modified. The primer sequences (forward, 5′-CACCTCCGAGGTTGTGAGTGA-3′; reverse, 5′-GGTAGGGCACGTGTACTTGCA-3′) were selected for the amplification of a fragment from nucleotides 114 to 264, and a fluorogenic probe (5′-TTACCGTCAGCAGCAGCGATTTTGG-3′) was designed to anneal between nucleotides 182 and 207 of A. marginale subsp. centrale msp5. The real-time PCR was carried out under the following conditions: 95°C for 10 min, 55 cycles of 95°C for 20 s, 59°C for 10 s, and 72°C for 10 s, final extension at 72°C for 30 s, and holding at 10°C. The real-time PCRs were conducted using a PCR mixture containing 10 mM Tris (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 200 μM of each dATP, dCTP, dGTP, and dTTP, 0.2 μM of each primer, 0.2 μM fluorogenic probe, and 1.25 U of AmpliTaq Gold (PE Applied Biosystems, Foster City, CA). All reactions were performed using the iCycler iQ real-time PCR detection system (Bio-Rad, Hercules, CA). Full-length A. marginale subsp. centrale msp5 was cloned into the pCR-4 TOPO vector and used to construct a standard curve for the real-time assay. As an internal standard for the extraction of DNA, amplification, and probe binding, known numbers of organisms of each strain were used as a source for DNA extraction and amplification and the results from the real-time assay compared.

Confirmation of strain identity.

The identity of A. marginale subsp. centrale in the blood and tick tissues could be confirmed by sequencing of the amplified msp5 due to the polymorphism compared to that of the A. marginale St. Maries and Mississippi strains. To distinguish between the St. Maries and Mississippi strains, the repeat region of msp1α was amplified and sequenced. This region differs in the number and sequence of repeats among strains. Briefly, the primer sequences (forward, 5′-GTGCTTATGGCAGACATTTCC-3′; reverse, 5′-CTCAACACTCGCAACATTGG-3′) were designed to amplify the conserved regions flanking msp1a repeats of A. marginale strains (18). The strain identity was confirmed by amplicon sequencing.

Transmission of A. marginale to naïve animals.

The development of infection in the calves following transmission feeding was monitored by microscopic examination of Giemsa-stained blood smears, msp5 PCR, and MSP5 cELISA. Weekly examination continued until 100 days post-tick feeding, representing >3 standard deviations from the mean time to A. marginale detection in the calves transmission fed with ticks infected with the positive control St. Maries strain.

RESULTS

Anaplasma level in the peripheral blood and tick midgut during acquisition feeding.

Ticks were acquisition fed on infected calves during acute bacteremia, with levels of >108 organisms per ml of blood (Table (Table1).1). All fed ticks ingested A. marginale as demonstrated by PCR amplification of msp5 from the total midgut dissected from a cohort of ticks immediately after removal. The total midgut includes both the epithelium and lumen and thus cannot discriminate between A. marginale bacteria that have already entered and replicated in epithelial cells and those remaining in the luminal blood meal. Quantification of the total midgut A. marginale bacteria revealed the highest levels in ticks acquisition fed on the St. Maries strain-infected calf and the lowest levels in ticks fed on the A. marginale Mississippi-infected calf (Table (Table1).1). There was no positive association between the total midgut levels immediately after removal and the A. marginale bacteremia levels in the calves during acquisition feeding (Table (Table11).

TABLE 1.
A. marginale levels in the peripheral blood and within the D. andersoni total midgut during acquisition feeding

Development and replication of A. marginale in the tick following transmission feeding.

The ability of each strain to colonize the midgut epithelium was measured by both determining the infection rate and quantifying the A. marginale load in positive midguts. Consistent with the high transmissibility of the St. Maries strain, 100% of the ticks exposed to this strain were msp5 positive and contained a mean of 106.8 organisms per midgut (Table (Table2).2). A. marginale subsp. centrale also colonized the midgut epithelium although with a lower efficiency (Table (Table2).2). In contrast, only 3 of 150 ticks that ingested the Mississippi strain had positive midguts and the levels in these three were below the level of detection (<5 × 101 organisms) by real-time PCR.

TABLE 2.
A. marginale infection rates and levels within D. andersoni following transmission feeding

Both the St. Maries strain and A. marginale subsp. centrale effectively colonized the salivary glands during transmission feeding (Table (Table2).2). The levels exceeding 107 organisms per salivary gland pair reflect replication following invasion, as previously shown for efficiently transmitted strains (9, 13). Consistent with the lack of colonization in the midgut epithelium, a prerequisite to invasion of the salivary glands, the Mississippi strain was not detected in the salivary glands of any of the 150 fed ticks examined.

Confirmation of strain identity.

The identity of the A. marginale strains colonized in the salivary glands was confirmed using amplification and sequencing of msp5 and msp1α. For comparison, msp5 amplicons were generated from the blood of each of the calves used for original tick acquisition feeding (Fig. (Fig.1A).1A). Similarly, the msp1α repeat region was amplified from the blood of the calves infected with the St. Maries and Mississippi strains but not with A. marginale subsp. centrale, which completely lacks this repeat region (24). The msp1α amplicons represented the expected size for the three repeats and five repeats of the St. Maries and Mississippi strains, respectively (Fig. (Fig.1A).1A). msp5 was amplified from the salivary glands colonized by both the St. Maries strain and A. marginale subsp. centrale, and msp1α was amplified from the salivary glands colonized only by the St. Maries strain. Neither msp5 nor msp1α was amplified from the Mississippi strain, which failed to colonize the salivary glands (Fig. (Fig.1A).1A). The sequences of the msp5 and msp1α amplicons matched those previously reported for each strain, thus confirming strain identity. The msp5 sequences of the St. Maries (blood and salivary glands) and Mississippi (blood) strains were identical to each other and to the genome sequence of the St. Maries msp5 strain (GenBank accession no. NC_004842). These two strains were discriminated and the identity of the St. Maries strain within the salivary glands confirmed by sequencing of the msp1α amplicon; the amplicon sequence from the salivary glands was identical to that in the blood of the St. Maries-infected calf and to that previously reported for this strain (GenBank accession no. NC_004842). This msp1α sequence differs from that of the Mississippi strain in both the number and sequence of repeats (GenBank accession no. AY010243) (Fig. (Fig.1B).1B). The identity of A. marginale subsp. centrale in the salivary glands was confirmed by the msp5 sequence; the sequences from the blood and salivary glands were identical to each other and to that previously reported for the Israel vaccine strain (GenBank accession no. AY054384).

FIG. 1.
(A) Confirmation of A. marginale strain identity in the acquisition feed blood and tick salivary glands by PCR amplification of msp5 and msp1α. AF blood, blood collected at time of acquisition feeding; D. andersoni SG, salivary glands following ...

Transmission of A. marginale to naïve animals.

D. andersoni colonized with the St. Maries strain successfully transmitted infection to naïve calves following transmission feeding of either 10 or 35 ticks (Table (Table3).3). Infection followed a typical course of acute bacteremia, detectable by microscopic examination of Giemsa-stained blood smears and accompanied by seroconversion. Both animals progressed to persistent infection within 60 days, characterized by bacteremia of ≤107 A. marginale bacteria per ml of blood, detectable by PCR but not microscopic examination of blood smears (Table (Table3).3). In contrast, none of the calves, each transmission fed with 100 ticks exposed to either the Mississippi strain or A. marginale subsp. centrale, were infected as documented throughout the 100-day observation period following attempted transmission.

TABLE 3.
Tick transmission of A. marginale to naïve calves

DISCUSSION

We have identified the tick salivary glands as a second and distinct barrier, in addition to the midgut, for efficient Anaplasma marginale transmission. The entry and replication of A. marginale subsp. centrale in the midgut and then salivary glands recapitulated the basic developmental cycle of the pathogen within the tick (8, 9); however, this was not sufficient for transmission. As a positive control, the St. Maries strain of A. marginale was transmitted using either 10 or 35 D. andersoni ticks, consistent with a previously published study in which three infected ticks were sufficient for transmission (21) and with data (G. A. Scoles, unpublished data) showing that a single infected tick transmitted the St. Maries strain. In contrast, feeding of 100 D. andersoni ticks, of which >70% had positive salivary glands, did not transmit A. marginale subsp. centrale. Notably, this did not reflect a failure to replicate within the salivary glands during transmission feeding. Replication in the salivary glands to levels of ≥104 A. marginale bacteria has previously been associated with successful transmission (9, 13). In the present study, A. marginale subsp. centrale replicated to a mean level of 107.4 bacteria per salivary gland pair and each individual positive tick had >106 A. marginale bacteria per salivary gland pair, a minimum of 100 times the number of organisms previously linked with successful transmission (9). While there was no significant difference in the numbers of organisms within the salivary glands at transmission feeding between the St. Maries strain and A. marginale subsp. centrale, the transmission of only the St. Maries strain reveals that a previously unsuspected mechanism within the salivary glands is required for efficient transmission. This may involve the site of replication (i.e., at the cellular level within the correct acinar cell type or intracellular compartmentalization), vector pathogen signaling for the development of infectivity, efficient secretion in the saliva, or early survival in the mammalian host following transmission tick feeding. The identification of A. marginale subsp. centrale as being replication competent but defective for efficient transmission by D. andersoni provides a needed tool to identify these required mechanisms.

The tick midgut epithelium also represents a barrier to A. marginale transmission but, unlike the salivary glands, functions at the time of tick acquisition feeding (6, 23). This was originally described using the Florida strain of A. marginale (6, 23), and in the present study, the transmission phenotype of the Mississippi strain displayed a similar early defect. Although the Mississippi strain-infected calf had the highest level of bacteremia during acquisition feeding and all fed ticks ingested A. marginale (Table (Table1),1), the levels within the total midgut (lumen and epithelium) were significantly lower than in ticks that ingested either the St. Maries strain or A. marginale subsp. centrale. This suggested that while the last two strains entered the midgut epithelium early and initiated replication, the Mississippi strain total midgut levels reflected only the luminal A. marginale ingested with the blood meal. Consistent with this early defect, the Mississippi strain did not progress to invade the salivary glands and was not transmitted.

Whether the very few (3/150) Mississippi strain-positive midgut epithelium samples (Table (Table2),2), all of which had fewer than 50 bacteria per midgut, indicate that entry into the midgut epithelium did occur but was inefficient, or that entry occurred but there was no subsequent survival or replication, is unknown and highlights the current gap in knowledge regarding these early events. Similar to what has been observed with the Mississippi strain, the failure of the Florida strain to be transmitted has been proposed to reflect either an inability to bind the midgut epithelial cells for subsequent internalization or a failure to survive and replicate within the midgut epithelial cells (6, 23). These possibilities are not necessarily mutually exclusive, as blood digestion in ixodid ticks occurs intracellularly within the midgut epithelium, rather than in the lumen, and thus passive entrance of cell-associated A. marginale into the epithelium may occur in addition to any receptor-ligand interactions of cell-free bacteria.

The outer membrane protein MSP1a has been proposed to mediate the binding of cell-free A. marginale to the midgut epithelial cells and to allow for the prediction of the transmission phenotype of A. marginale strains based on the number and sequence of MSP1a repeats (5, 6). Our results are not entirely supportive of this proposed mechanism. First, A. marginale subsp. centrale completely lacks the MSP1a repeat region (24) yet invades and replicates within the midgut epithelium prior to colonization of the salivary glands. This demonstrates that MSP1a N-terminal repeats are not uniformly required for entry and colonization in Dermacentor sp. ticks. Whether this phenotype will be representative of other A. marginale strains is unknown. Multiple genome-level comparisons have revealed an unexpected high level of polymorphism among A. marginale strains (3), a wide spectrum that may well include A. marginale subsp. centrale. However, to date all A. marginale senso stricto strains encode the MSP1a N-terminal repeats, and thus, A. marginale subsp. centrale could represent an outlier that uses an alternative, non-MSP1a mechanism to invade the midgut epithelium as a cell-free bacterium. Second, the Mississippi strain used in the present study has an MSP1a repeat type identical to that identified as being transmission competent (5), yet this strain failed to colonize the D. andersoni midgut and was consequently not transmitted. This indicates that even within A. marginale senso stricto strains, the sequence of MSP1a N-terminal repeats is not consistently predictive of midgut invasion in Dermacentor spp. The previously reported predictive association between the MSP1a repeat sequence and transmission phenotype was based on transmission data for D. andersoni as well as a closely related tick, D. variabilis (4, 5, 6); thus, it is possible that an association remains valid for the latter species. While studies with the St. Maries, South Idaho, Puerto Rico, and Florida strains of A. marginale and both Dermacentor and Rhipicephalus species suggest that the transmission phenotypes, both positive and negative, are conserved, there may be differences among tick species in the efficiency or mechanism of the interaction at the midgut level (9, 20, 23, 26).

In summary, there are both early (midgut) and late (salivary glands) barriers to efficient tick-borne transmission of A. marginale. The identification of specific strains that fail to traverse these barriers provides needed tools to dissect the mechanisms involved in transmission not only to define the defect of these specific strains but to better understand the epidemiology of highly transmissible strains responsible for disease outbreaks.

Acknowledgments

We thank Ralph Horn, Nancy Kumpula-McWhirter, and James Allison for their excellent technical assistance. Also, we thank Terry F. McElwain for critical review of the manuscript.

This work was supported by BARD US-3315-02C, NIH R01 AI44005, USDA ARS-CRIS 5348-32000-027-00D, and the Wellcome Trust GR075800M. Massaro W. Ueti was supported by NIH T32 AI007025.

Notes

Editor: R. P. Morrison

Footnotes

[down-pointing small open triangle]Published ahead of print on 9 April 2007.

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