Guidelines for the Direct Detection of Anaplasma spp. in Diagnosis and Epidemiological Studies

Introduction

Members of the genus Anaplasma (Rickettsiales: Anaplasmataceae) are non-motile, obligate intracellular Gram-negative bacteria residing in membrane-bound cytoplasmic vacuoles of the host cell where they form agglomerates called morulae (Latin mōrus = mulberry). Following the reorganization of the order Rickettsiales in 2001, the genus Anaplasma contains at least six species: Anaplasma bovis, Anaplasma centrale, Anaplasma marginale, Anaplasma phagocytophilum, Anaplasma platys, and Anaplasma ovis (Dumler et al. 2001, 2005). In future, recently identified agents such as Anaplasma odocoilei and Anaplasma capra could be included (Tate el al. 2013, Li et al. 2015). Anaplasma spp. are etiologic agents of veterinary diseases affecting domestic ruminants, equines, dogs, and cats worldwide. A. phagocytophilum is also regarded as an emerging human pathogen with growing importance in the Northern Hemisphere (Jin et al. 2012, Bakken and Dumler 2015). Reports of human infections caused by other species such as A. platys, A. ovis, and the putative A. capra, suggest a broader medical relevance of this taxon (Chochlakis et al. 2010, Maggi et al. 2013, Arraga-Alvarado et al. 2014, Breitschwerdt et al. 2014, Li et al. 2015).

Anaplasma exhibit a biological cycle involving infection of both invertebrate and vertebrate hosts. Ticks are regarded as primary vectors, with different cell types targeted by these agents in a replication cycle, including invasion of salivary glands and transmission in saliva released during blood feeding. Alternative transmission routes include mechanical transfer by other hematophagous arthropods or fomites such as contaminated veterinary instruments, and transfusion-transmitted infections (Leiby and Gill 2004, Aubry and Geale 2011, Renneker et al. 2013, Shields et al. 2015). Anaplasma spp. display unique cell tropisms in vertebrate hosts and depending on the species, different cells of the hematopoietic lineage are specifically infected (erythrocytes, monocytes/macrophages, granulocytes, or platelets). The most important biological, ecological, and epidemiological features of Anaplasma spp. are shown in Table 1.

Anaplasmosis is a challenging disease in terms of diagnosis because clinical presentation may vary greatly depending on the agent involved, the affected host, and other factors such as immune status and coinfections (Kocan et al. 2010, Gaunt et al. 2010, Aubry and Geale 2011, Renneker et al. 2013, Bakken and Dumler 2015). The substantial economic impact associated with livestock infection, the zoonotic potential, and the risk of transfusion-transmitted infection, determines the need for accurate direct laboratory tests. Although histopathological investigations can provide suggestive diagnoses, and immunohistochemical stains can provide more definitive information, these guidelines review the most frequent and currently used methods for the direct detection of Anaplasma in ticks, humans, and other animals and their application in laboratory diagnosis and surveillance.

Microscopy

In vertebrates, microscopical observation of blood smears has traditionally been used for diagnosing clinical anaplasmosis and to a lesser extent for surveillance purposes, for example, wildlife screening and identification of reservoirs.

Peripheral blood smears directly prepared after fingerstick or superficial vein puncture or from venous blood collected into anticoagulant, obtained during the early acute phase of symptoms and before initiation of effective antimicrobial therapy, are best for visualization of bacteria in both animals and humans. This time frame for sample collection is crucial for all direct tests, including molecular detection and in vitro cultivation, as it covers the stage of infection when sufficient numbers of bacteria are present in the circulating blood. For leukocytotropic species (A. bovis and A. phagocytophilum), buffy coat smears are preferred to regular whole blood preparations, as due to leukopenia very few infected leukocytes may be present. Representing a leukocyte- and platelet-enriched fraction, buffy coat is also considered useful for detection of A. platys morulae within platelets (Eddlestone et al. 2007). In any case, bacterial detection is best achieved if smears are prepared immediately after blood collection. Alternatively, anticoagulated samples can be refrigerated and processed preferably within 24–48 h. At post-mortem examination, tissue impressions or smears (spleen, liver, kidney, heart, lung and, in particular, blood vessels) can be performed in an attempt to visualize erythrocytotropic Anaplasma spp. (Kocan et al. 2010, OIE 2015). After being dried and fixed in methanol, smears are stable for at least 2 months. Differential staining is achieved with Eosin Azure (Romanovsky)-type dyes such as Giemsa and Diff-Quik; Anaplasma morulae typically appear in the host cells as dark blue to purple formations by light microscopic examination at 400× or 1000× magnification. Open access reference images can be found in the literature of A. bovis (Liu et al. 2012), A. centrale (Bell-Sakyi et al. 2015), A. marginale (Kocan et al. 2003), A. phagocytophilum (Annen et al. 2012, Henniger et al. 2013), A. platys (Dyachenko et al. 2012), and A. ovis (Yasini et al. 2012).

Light microscopy is the most inexpensive and quickest laboratory test, but also the least sensitive, and is highly dependent on examiner experience, relative quantity of target cells, bacteremia levels, the degree of neutropenia, monocytopenia, thrombocytopenia, or anemia and infection status. It is commonly used for the erythrocytotropic Anaplasma spp. with good results in recently acquired infections (with the examination of up to 100 microscopic fields, ∼100,000 cells), except in cases of severe anemia (Potgieter and Stoltsz 1994). However, it has limited value in persistently infected animals that usually present low-level bacteremia (Eriks et al. 1989, Palmer et al. 1998, Kocan et al. 2010). For A. platys, light microscopy presents low sensitivity due to the cyclic character of thrombocytopenia and the low percentage of infected cells (between 0.5% and 5%); therefore, it is recommended to examine between 2,000 and 20,000 platelets (Kontos et al. 1991, Chang et al. 1996, Brown et al. 2006, Eddlestone et al. 2007). For the granulocytotropic species, morulae can also be sparsely distributed and difficult to detect, particularly in human samples from which at least 800–1,000 granulocytes should be examined (Aguero-Rosenfeld 2002), although at least one study demonstrates identification in all human cases after examination of only 200 granulocytes (Rand et al. 2014). For ruminants, the examination of 400 granulocytes are generally regarded as sufficient to detect infected leucocytes in recent disease, but blood smears from persistently infected animals may give negative results (Stuen et al. 2002, 2006). Thus, a negative result does not rule out infection and microscopy should always be combined with other laboratory diagnostic tests, complemented by the screening of other sample types if persistent infections are suspected, as discussed below. Moreover, false-positive interpretations can occur due to Döhle and Howell–Jolly bodies, other inclusions, contaminant particles or platelets, and nuclear fragments superimposed on leukocytes. In addition, agent identification can be misinterpreted in hosts known to be affected by different Anaplasma spp. or related agents with identical cell tropism. For example, wild ruminants can be infected by the erythrocytotropic species A. marginale/A. centrale and A. ovis worldwide, by the granulocytotropic A. phagocytophilum and Ehrlichia ewingii in North America, and by the monocytotropic A. bovis, Ehrlichia chaffeensis, and Ehrlichia canis in North America and in the Far East or Ehrlichia ruminantium in Africa that can also target granulocytic leukocytes as well as endothelial cells (Dumler et al. 2005). The identification of the causative agent in uncommon infections or atypical locations or hosts can also be limited if relying solely on light microscopy.

Other light and electron microscopic techniques (transmission electron microscopy, scan electron microscopy, confocal microscopy) have been used to study host samples for specific research purposes. The same is true for the microscopic detection of Anaplasma in vectors, which has been most useful for life cycle investigation rather than for diagnosis and epidemiological studies.

Molecular Detection

Both experimental and field studies have pointed out the utility of molecular laboratory diagnosis for sensitive and specific identification of Anaplasma infections (Eriks et al. 1989, Palmer et al. 1998, Eddlestone et al. 2007, Haigh et al. 2008, Hing et al. 2014). The growing number of high-performance molecular protocols with increased potential for automation and multiplex detection has resulted in these becoming indispensable laboratory tools.

Anticoagulated whole blood and buffy coat are the best samples for molecular screening of Anaplasma-infected human and nonhuman vertebrates. Ethylenediaminetetraacetic acid or citrate are preferred to heparin as anticoagulant, since the latter is considered to interfere with PCR (Hebels et al. 2014, Sánchez-Fito and Oltra 2015). Spleen samples are equally good and highly recommended for persistently infected animals, especially in wildlife studies or as an additional sample to rule out intermittent or low-level bacteremia in blood-negative individuals (Eddlestone et al. 2007). Other samples reported in the literature with variable results for Anaplasma screening include serum/plasma, liver, lung, lymph nodes, bone marrow, and skin biopsies (Massung et al. 1998, Eddlestone et al. 2007, Gaunt et al. 2010, Blaňarová et al. 2014, Szekeres et al. 2015). To increase the sensitivity of Anaplasma detection in clinical cases, it is important to pay attention to the previously mentioned time frame for sample collection (as mentioned in the Microscopy section). Again, samples should be processed as soon as possible after collection, at least to a stage at which they can be maintained below −20°C until required (e.g., buffy-coat separation, preparation of aliquots with volume/weight suitable for nucleic acid extraction) to avoid repeated freeze and thaw cycles.

For studies of prevalence of Anaplasma spp. in vectors, it is advisable to use questing (unfed) ticks. In the case of species that are difficult to obtain in a questing state, as for example the one-host ticks Rhipicephalus (Boophilus) microplus and R. (B.) annulatus, it should always be kept in mind that tick positivity could either result from the remnant of infected host blood meal or from an established infection in the tick tissues (Estrada-Peña et al. 2013). Since transovarial transmission of Anaplasma spp. is not known to occur in naturally infected ticks, questing larvae are not useful in prevalence studies. However, it is important to note that attached larvae with PCR-positive results might be of potential value for the identification of infected hosts.

Ticks should be identified to species level by examining morphological characters before being processed for molecular analysis. Otherwise, any conclusion drawn regarding vector–pathogen associations could be subject to substantial errors (Estrada-Peña et al. 2013). Identification can also be confirmed by molecular methods, by sequencing 12S or 16S rRNA gene fragments (Mangold et al. 1998, Beati and Keirans 2001). However, tick-derived sequences in the GenBank database are still far from being comprehensive, preventing accurate classification based solely on molecular tools in many cases.

Ticks can be used freshly for DNA extraction, with only short-term storage at 4°C. For partially fed ticks removed from hosts, no more than 1–2 days is recommended, but for unfed ticks this might be as long as a month; alternatively, unfed ticks can be kept for longer at 12–16°C, 85% relative humidity. For longer time periods, samples can be immersed in RNAlater or ≥70% ethanol and maintained at 4°C or frozen immediately at −20°C or −80°C. Decontamination of the tick surface should be performed before DNA extraction by a sequence of 5-min immersions in sterile distilled water/PBS, 70–80% ethanol, and again water/PBS, ending by air drying (or drying on sterile filter paper). Ticks should be handled with sterile forceps in between each step and after decontamination.

Molecular testing of blood, tissue samples, and ticks (or other arthropods) is usually performed on total DNA. Extraction can be manual or automatic, with ready-to-use commercial kits or prepared solutions, but in any case it is highly recommended to include DNA purification to remove PCR inhibitors, particularly in the case of blood and tick samples (Schwartz et al. 1997). For fully engorged female ticks, DNA should be extracted from only one half of the specimen after longitudinal bisection, to avoid excess sample and to prevent PCR inhibition due to the high erythrocyte concentration during extraction. Prior disruption or homogenization of ticks and tissue samples is very important for efficient DNA extraction and can be performed using automatic homogenizers (e.g., Precyllis or Mixer Mill benchtop units) or manually, using sterile tools, such as scalpel blades or pestles (one per sample). For these samples, if an enzymatic digestion step is included it should last at least 1 h at 56°C or can be extended to an overnight incubation, facilitating organization of work. Several commercial kits have been used by the authors in diagnosis and surveillance studies with good results (Table 2 footnote). Less expensive protocols using the TRI Reagent protocol (Sigma-Aldrich) and alkaline hydrolysis with 1.25% ammonium solution (Schouls et al. 1999) have also been used in tick surveillance studies. The former is a time-consuming protocol, although it yields good-quality DNA and also enables protein and RNA isolation. The alkaline hydrolysis method is easy to perform and uses intact ticks, but results in a crude DNA extraction, and elimination of PCR inhibitors and long-term DNA storage stability are not guaranteed.

Evaluation of the extraction process is an important requirement. Negative controls (i.e., sterile water) should be included in each group of samples during extraction, to monitor the occurrence of contamination by DNA carryover. Quality and quantity of nucleic acids can be ascertained with a spectrophotometer and degradation of DNA by gel electrophoresis. PCRs targeting host (e.g., β-actin, albumin, human β-globin) or vector (e.g., Ixodes cox gene, 12S or 16S rRNA genes) housekeeping genes can also be performed to validate extraction and to confirm the absence of PCR inhibitors (Ausubel et al. 1998, Mangold et al. 1998, Beati and Keirans 2001, Schwaiger and Casinotti 2003). Alternatively, validation can be achieved by spiking the samples with nonrelated bacterial suspensions before extraction, and subsequently targeting the corresponding DNA by specific PCR. Bacillus thuringiensis commercial suspensions are widely used for this purpose (De Bruin et al. 2011).

Despite being closely related species, few molecular approaches have been designed to target the entire Anaplasma genus. One of the reasons is that some Anaplasma spp. are ecologically divergent and not found in the same hosts or vectors. Moreover, broader-range PCR assays are usually less sensitive and prone to selectively amplifying the predominant Anaplasma spp. or genetically similar agents (e.g., other members of the Anaplasmataceae or other alpha-proteobacteria) that might be present in the samples in higher concentrations. However, genus-specific primers could be used when, for example, nothing is known about the Anaplasma species present in a given area. Furthermore, broad-range PCRs are indispensable when attempting to identify clades within the Anaplasma genus.

Sensitivity of molecular detection depends on several factors such as: (1) sample nature and quality, that is, plasma and serum usually present much lower bacterial loads than blood due to the intracellular nature of Anaplasma; (2) the genomic copy number of target genes, for example, the major surface protein (msp) families include multiple copy genes; and (3) the amplicon length (e.g., short sequences are generally preferred to long ones for screening). Among the most targeted genes of Anaplasma spp. are those for 16S rRNA (rrs), heat shock protein (groEL), citrate synthase (gltA), and major surface proteins (msp1, msp2, msp4, msp5). Protocols targeting some of these genes are suggested in Table 2. For molecular screening, the sensitive multicopy msp approaches are preferred over single copy genes, whereas for sequence comparison and database crossmatch, conservative or moderately conservative rrs and groEL strategies are regarded as the best choice. Nested PCRs are now generally replaced by less time-consuming and more sensitive real-time assays. However, further confirmation of positive results by sequencing is still highly advised. In most cases, this requires an additional conventional PCR, as real-time PCR targets that are most suitable for screening are usually very short (<150 bp), yielding sequence data of limited phylogenetical value.

For the confirmation of a potential new strain/variant/species it is strongly encouraged to rely on a multilocus approach and, if possible, in vitro isolation. Generally, DNA sequences from fully characterized Anaplasma variants are a reliable source for typing (Huhn et al. 2014, Guillemi et al. 2015).

In Vitro Isolation

Isolation and in vitro propagation of Anaplasma species provides a valuable tool for the study of their biology. It is, however, less used in direct routine diagnosis and surveillance as the previously mentioned approaches. Still, it is of great value for proving the etiology in atypical/new clinical occurrences or fatal cases. It is also an indispensable procedure to claim discovery of a new bacterial species, enabling proper taxonomic classification and the attribution of a formal scientific name (Cabezas-Cruz et al. 2012, Zweygarth et al. 2013). Due to ethical issues regarding animal experimentation, there is a strong incentive for isolation and propagation of Anaplasma in continuously cultured cell lines.

In vitro culture of Anaplasma spp. in mammalian cells relies on the availability of cellular systems that are similar to or could mimic the intracellular environment found in natural host cells. This was first achieved for the granulocytotropic A. phagocytophilum using the human promyelocytic leukemia cell line HL-60 (ATCC CCL-240) (Goodman et al. 1996). Since then, HL-60 cells have been routinely used for A. phagocytophilum culture, although other mammalian cell lines are also reported to sustain its growth. These include the human monocytic THP-1 (ATCC TIB-202) and microvascular endothelial HMEC-1 and MVEC cell lines, the bovine corneal BCE C/D1-b (ATCC CRL-2048) cell line and the Rhesus monkey retina choroid RF/6A (ATCC CRL-1780) endothelial cell line (Munderloh et al. 2004, Garcia-Garcia et al. 2009). Continuous growth of the intraerythrocytic A. marginale was also achieved in both BCE C/D1-b and RF/6A cells, but only after establishment in tick cell lines (Munderloh et al. 2004). Additionally, HL-60 and THP-1 cells have enabled the isolation of the newly reported zoonotic A. capra (Li et al. 2015).

As an alternative to mammalian-derived cells, tick cell lines are a valuable tool for the cultivation of Anaplasma species. They can be a good option for the isolation of Anaplasma variants found in vectors, but with low or unknown pathogenicity for vertebrates (Massung et al. 2007). Moreover, these cells have already proven their value for bacteria that target mammalian cells that are difficult to continuously propagate in vitro, such as the intraerythrocytic and intrathrombocytic Anaplasma spp. (Munderloh et al. 1994, 2003). The continuous culture of A. marginale was first achieved in an Ixodes scapularis embryo-derived cell line (IDE8) (Munderloh et al. 1994, 1996). Since then, many isolates have been established in the IDE8 cell line, as well as in other tick cell lines (Munderloh et al. 2004, Zivkovic et al. 2010), making possible a more intense study of A. marginale biology, as reviewed by Blouin et al. (2002) and Passos (2012). Very recently, continuous cultures of the vaccine strain of A. centrale were established for the first time in Rhipicephalus appendiculatus RAE25 and Dermacentor variabilis DVE1 cell lines (Bell-Sakyi et al. 2015). A. phagocytophilum cell cultures have also been established in several tick-derived cell lines, including I. scapularis-derived IDE8 and ISE6, Ixodes ricinus-derived IRE/CTVM19 and IRE/CTVM20 (Munderloh et al. 1996, Woldehiwet et al. 2002, Silaghi et al. 2011, Dyachenko et al. 2013, Alberdi et al. 2015), R. appendiculatus-derived RAE25 and I. ricinus-derived IRE11 (Bell-Sakyi, unpublished data). ISE6 cells were also valuable for the isolation of potentially new thrombocytotropic Anaplasma closely related to A. platys (Munderloh et al. 2003, Tate et al. 2013).

As mentioned for the other direct diagnostic techniques, blood collected from animals or humans in the proper time frame is also the best inoculum for in vitro culture (as mentioned in the Microscopy section). Furthermore, growth of Anaplasma can also be attempted from vertebrate tissue samples or fresh ticks, after maceration, fragmentation, or dissection in culture medium. However, especially for techniques which do not discard the tick exoskeleton, contamination with environmental bacteria and fungi can be a problem for establishment of the culture. In this case, special attention should be given to external surface decontamination of the tick, as previously mentioned for molecular testing, adding an extra 5-min immersion in 0.1% benzalkonium chloride (Sigma) before the ethanol step to ensure decontamination. Samples of 0.1–0.5 mL anticoagulated whole blood, buffy coat, or tissue samples can be inoculated straight into small cell culture flasks (12.5- or 25-cm² capacity) or flat-sided tubes (Nunc) and maintained according to the respective cell line culturing conditions (Table 3). Every 2–7 days, fresh medium should be added to cultures and, in the case of mammalian cells growing in suspension, cell concentration adjusted. As tick cells tolerate high cell densities and can survive for many months without subculture, there is no need to adjust cell density and inoculated cultures can be maintained for the long periods (12 weeks or more) required for adaptation of some Anaplasma spp. to in vitro growth (Silaghi et al. 2011, Dyachenko et al. 2013, Bell-Sakyi et al. 2015). Cultures should also be periodically evaluated by microscopy to detect any microbial growth. Infection can be assessed by direct observation of cytocentrifuged culture aliquots after staining with Eosin Azure-type dyes as described above.

In vitro culturing is a demanding task in terms of time and expertise, and only a limited number of research institutions are currently able to perform it. Even so, this might not be a constraint on its use for direct diagnosis at least for some agents such as A. phagocytophilum that can be successfully cultured from infected blood kept for up to 18 days under refrigerated conditions (Kalantarpour et al. 2000). Thus, biological samples can be transported under refrigerated conditions to a referral laboratory, where the appropriate assays can be carried out.

Conclusion

In summary, for the direct detection of Anaplasma spp. in blood and tissue samples, ticks or other vectors, molecular methods are preferred. Specific real-time PCRs offer several advantages over conventional PCR assays for screening purposes, but the confirmation of sequence identity is still often required. Approaches targeting multiple genes can be very useful for phylogeny and taxonomy studies. Other direct methods such as microscopy or in vitro isolation are mostly reserved for research applications, such as experimental studies, transmission trials etc., but can also contribute in specific diagnostic/surveillance investigations and in identifying and characterizing novel Anaplasma spp.