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

Article in Vector Borne and Zoonotic Diseases · January 2017 DOI: 10.1089/vbz.2016.1960

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Cornelia Silaghi Ana Sofia Santos Friedrich Loeffler Institute National Institute of Health Dr. Ricardo Jorge

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The user has requested enhancement of the downloaded file. VECTOR-BORNE AND ZOONOTIC DISEASES Volume 17, Number 1, 2017 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2016.1960

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

Cornelia Silaghi,1,* Ana Sofia Santos,2,* Jacinto Gomes,3 Iva Christova,4 Ioana Adriana Matei,5 Gernot Walder,6 Ana Domingos,7 Lesley Bell-Sakyi,8 Hein Sprong,9 Friederike D. von Loewenich,10 Jose´ A. Oteo,11 Jose´ de la Fuente,12,13 and J. Stephen Dumler14,15

Abstract

The genus Anaplasma (: ) comprises obligate intracellular Gram-negative that are mainly transmitted by , and currently includes six : , Anaplasma centrale, Anaplasma marginale, Anaplasma phagocytophilum, , and Anaplasma ovis. These have long been known as etiological agents of veterinary diseases that affect domestic and wild animals worldwide. A zoonotic role has been recognized for A. phagocytophilum, but other species can also be pathogenic for humans. Anaplasma are usually challenging to diagnose, clinically presenting with nonspecific symptoms that vary greatly depending on the agent involved, the affected host, and other factors such as immune status and coinfections. The substantial economic impact associated with livestock infection and the growing number of human cases along with the risk of transfusion-transmitted infections, determines the need for accurate laboratory tests. Because hosts are usually seronegative in the initial phase of infection and serological cross-reactions with several Anaplasma species are observed after seroconversion, direct tests are the best approach for both case definition and epidemiological studies. Blood samples are routinely used for Anaplasma spp. screening, but in persistently infected animals with intermittent or low-level bacteremia, other tissues might be useful. These guidelines have been developed as a direct outcome of the COST action TD1303 EURNEGVEC (‘‘European Network of Neglected Vectors and Vector-Borne Diseases’’). They review the direct laboratory tests (microscopy, nucleic acid-based detection and in vitro isolation) currently used for Anaplasma detection in ticks and and their application.

Keywords: Anaplasma spp., direct diagnosis, in vitro isolation, microscopy, PCR, ticks, hosts

1National Center for Vector Entomology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland. 2Center for Vector and Infectious Diseases Research, National Institute of Health Doutor Ricardo Jorge, A´ guas de Moura, Portugal. 3Animal Health and Production Unit, National Institute for Agrarian and Veterinary Research, Oeiras, Portugal. 4Department of Microbiology, National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria. 5Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Cluj-Napoca, Romania. 6Department of Hygiene, Medical Microbiology and Social Medicine, Innsbruck Medical University, Innsbruck, Austria. 7Global Health and Tropical Medicine, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal. 8The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey, United . 9Center for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands. 10Department of Medical Microbiology and Hygiene, University of Mainz, Medical Center, Mainz, Germany. 11Infectious Diseases Department, Center of Rickettsioses and -Borne Diseases, Hospital San Pedro- CIBIR, Logron˜o, Spain. 12SaBio. Instituto de Investigacio´n de Recursos Cinege´ticos, IREC-CSIC-UCLM-JCCM, Ciudad Real, Spain. 13Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma. 14Departments of Pathology and Microbiology and Immunology, University of Maryland, School of Medicine, Baltimore, Maryland. 15Department of Pathology, Uniformed Services University for the Health Sciences ‘‘America’s Medical School,’’ Bethesda, Maryland. *These authors contributed equally to this work.

12 ANAPLASMA SPP. DIRECT DETECTION 13

Introduction Peripheral blood smears directly prepared after fingerstick or superficial vein puncture or from venous blood collected embers of the genus Anaplasma (Rickettsiales: into anticoagulant, obtained during the early acute phase of MAnaplasmataceae) are non-motile, obligate intracellu- symptoms and before initiation of effective antimicrobial lar Gram-negative bacteria residing in membrane-bound therapy, are best for visualization of bacteria in both animals cytoplasmic vacuoles of the host cell where they form ag- and humans. This time frame for sample collection is crucial glomerates called morulae (Latin morus = mulberry). Fol- for all direct tests, including molecular detection and in vitro lowing the reorganization of the order Rickettsiales in 2001, cultivation, as it covers the stage of infection when sufficient the genus Anaplasma contains at least six species: Anaplasma numbers of bacteria are present in the circulating blood. For bovis, Anaplasma centrale, Anaplasma marginale, Anaplas- leukocytotropic species (A. bovis and A. phagocytophilum), ma phagocytophilum, Anaplasma platys, and Anaplasma ovis buffy coat smears are preferred to regular whole blood pre- (Dumler et al. 2001, 2005). In future, recently identified parations, as due to leukopenia very few infected leukocytes agents such as Anaplasma odocoilei and Anaplasma capra may be present. Representing a leukocyte- and platelet- could be included (Tate el al. 2013, Li et al. 2015). Anaplasma enriched fraction, buffy coat is also considered useful for spp. are etiologic agents of veterinary diseases affecting detection of A. platys morulae within platelets (Eddlestone domestic ruminants, equines, dogs, and cats worldwide. et al. 2007). In any case, bacterial detection is best achieved if A. phagocytophilum is also regarded as an emerging human smears are prepared immediately after blood collection. Al- with growing importance in the Northern Hemi- ternatively, anticoagulated samples can be refrigerated and sphere (Jin et al. 2012, Bakken and Dumler 2015). Reports processed preferably within 24–48 h. At post-mortem exam- of human infections caused by other species such as A. platys, ination, tissue impressions or smears (spleen, liver, kidney, A. ovis, and the putative A. capra, suggest a broader medical heart, lung and, in particular, blood vessels) can be performed relevance of this taxon (Chochlakis et al. 2010, Maggi et al. in an attempt to visualize erythrocytotropic Anaplasma spp. 2013, Arraga-Alvarado et al. 2014, Breitschwerdt et al. 2014, (Kocan et al. 2010, OIE 2015). After being dried and fixed in Li et al. 2015). methanol, smears are stable for at least 2 months. Differential Anaplasma exhibit a biological cycle involving infection staining is achieved with Eosin Azure (Romanovsky)-type of both invertebrate and vertebrate hosts. Ticks are regarded dyes such as Giemsa and Diff-Quik; Anaplasma morulae as primary vectors, with different cell types targeted by these typically appear in the host cells as dark blue to purple for- agents in a replication cycle, including invasion of salivary mations by light microscopic examination at 400· or 1000· glands and transmission in saliva released during blood magnification. Open access reference images can be found in feeding. Alternative transmission routes include mechanical the literature of A. bovis (Liu et al. 2012), A. centrale (Bell-

transfer by other hematophagous or fomites such Sakyi et al. 2015), A. marginale (Kocan et al. 2003), A. pha- as contaminated veterinary instruments, and transfusion- gocytophilum (Annen et al. 2012, Henniger et al. 2013), A. transmitted infections (Leiby and Gill 2004, Aubry and Geale platys (Dyachenko et al. 2012), and A. ovis (Yasini et al. 2012). 2011, Renneker et al. 2013, Shields et al. 2015). Anaplasma Light microscopy is the most inexpensive and quickest spp. display unique cell tropisms in vertebrate hosts and de- laboratory test, but also the least sensitive, and is highly pending on the species, different cells of the hematopoietic dependent on examiner experience, relative quantity of lineage are specifically infected (erythrocytes, monocytes/ target cells, bacteremia levels, the degree of neutropenia, macrophages, granulocytes, or platelets). The most important monocytopenia, thrombocytopenia, or anemia and infection biological, ecological, and epidemiological features of Ana- status. It is commonly used for the erythrocytotropic Ana- plasma spp. are shown in Table 1. plasma spp. with good results in recently acquired infec- is a challenging disease in terms of diag- tions (with the examination of up to 100 microscopic fields, nosis because clinical presentation may vary greatly de- *100,000 cells), except in cases of severe anemia (Potgieter pending on the agent involved, the affected host, and other and Stoltsz 1994). However, it has limited value in persis- factors such as immune status and coinfections (Kocan et al. tently infected animals that usually present low-level bac- 2010, Gaunt et al. 2010, Aubry and Geale 2011, Renneker teremia (Eriks et al. 1989, Palmer et al. 1998, Kocan et al. et al. 2013, Bakken and Dumler 2015). The substantial eco- 2010). For A. platys, light microscopy presents low sensi- nomic impact associated with livestock infection, the zoonotic tivity due to the cyclic character of thrombocytopenia and potential, and the risk of transfusion-transmitted infection, the low percentage of infected cells (between 0.5% and 5%); determines the need for accurate direct laboratory tests. Al- therefore, it is recommended to examine between 2,000 and though histopathological investigations can provide sugges- 20,000 platelets (Kontos et al. 1991, Chang et al. 1996, tive diagnoses, and immunohistochemical stains can provide Brown et al. 2006, Eddlestone et al. 2007). For the granulo- more definitive information, these guidelines review the most cytotropic species, morulae can also be sparsely distributed frequent and currently used methods for the direct detection of and difficult to detect, particularly in human samples from Anaplasma in ticks, humans, and other animals and their ap- which at least 800–1,000 granulocytes should be examined plication in laboratory diagnosis and surveillance. (Aguero-Rosenfeld 2002), although at least one study dem- onstrates identification in all human cases after examination of only 200 granulocytes (Rand et al. 2014). For ruminants, Microscopy the examination of 400 granulocytes are generally regarded In vertebrates, microscopical observation of blood smears as sufficient to detect infected leucocytes in recent disease, has traditionally been used for diagnosing clinical anaplas- but blood smears from persistently infected animals may give mosis and to a lesser extent for surveillance purposes, for negative results (Stuen et al. 2002, 2006). Thus, a negative example, wildlife screening and identification of reservoirs. result does not rule out infection and microscopy should

Table 1. Biological, Ecological, and Epidemiological Features of Anaplasma Species Ixodid , confirmed or Affected vertebrates Target cell in Species potential vectorsa (rare cases) vertebrates Disease Geographic distribution Anaplasma bovis Haemaphysalis spp. Domestic ruminants Monocytes Bovine ‘‘’’ Worldwide, excluding (H. longicornis, H. punctata, (dogs)/several wild Australia H. concinna, H. megaspinosa, ruminant species H. langrangei, H. leporispalustris); Less frequent in: Dermacentor spp. (D. occidentalis, D. andersoni), Rhipicephalus spp. (R. sanguineus; R. turanicus; R. evertsi) Anaplasma centrale Rhipicephalus simus Erythrocytes Anaplasmosis South Africa In South America, Australia, Africa, the Middle East, and South-East Asia as vaccine strain Anaplasma Up to 20 species involvedb Cattle/wild ruminants Erythrocytes Bovine anaplasmosis Worldwide in tropical and marginale Examples are: Dermacentor spp. subtropical regions of the (D. andersoni, D. variabilis), New World, Europe, Rhipicephalus spp. (R. [B.] Africa, Asia, and Australia

14 annulatus, R. [B.] microplus, R. sanguineus, R. simus) Anaplasma ovis Dermacentor spp. (D. marginatus, Domestic small Erythrocytes Ovine anaplasmosis Mainly tropical and D. andersoni, D. occidentalis ruminants/wild subtropical regions, also D. niveus), Haemaphysalis ruminants (humans) Mediterranean spp. (H. concinna, H. punctata), Hyalomma spp., Ixodes ricinus, Rhipicephalus spp. (R. bursa, R. sanguineus) Anaplasma platys Rhipicephalus spp. (R. sanguineus, Dogs (cats, humans, Platelets and platelet Infectious cyclic Worldwide in tropical and sub- R. turanicus, R. evertsi) ruminants)/wild precursors thrombocytopenia tropical areas few reports canids (other wild from Africa, not reported animals) from northern part of the world (Canada, Northern Europe, and Russia) Anaplasma Up to 20 species involvedc Domestic ruminants, Neutrophils (rarely Tick-borne fever, equine, Europe, North America, Asia phagocytophilum Examples are: Ixodes spp. horses, dogs, cats, eosinophils, canine, feline, and rare reports from Africa (I. ricinus, I. persulcatus, humans/several wild basophils, and human granulocytic and South America I. pacificus, I. scapularis, animals monocytes) anaplasmosis I. ovatus), Dermacentor spp. (D. variabilis, D. occidentalis, D. silvarum), Haemaphysalis concinna

aNot all are experimentally proven; this list also includes tick species in which Anaplasma spp. have been detected by PCR only. bReviewed in detail by Kocan et al. (2004). cReviewed in detail by Stuen et al. (2013). ANAPLASMA SPP. DIRECT DETECTION 15 always be combined with other laboratory diagnostic tests, species that are difficult to obtain in a questing state, as for complemented by the screening of other sample types if per- example the one-host ticks Rhipicephalus (Boophilus) mi- sistent infections are suspected, as discussed below. Moreover, croplus and R. (B.) annulatus, it should always be kept in false-positive interpretations can occur due to Do¨hle and mind that tick positivity could either result from the remnant Howell–Jolly bodies, other inclusions, contaminant particles of infected host blood meal or from an established infection or platelets, and nuclear fragments superimposed on leuko- in the tick tissues (Estrada-Pen˜a et al. 2013). Since transo- cytes. In addition, agent identification can be misinterpreted in varial transmission of Anaplasma spp. is not known to occur hosts known to be affected by different Anaplasma spp. or in naturally infected ticks, questing larvae are not useful in related agents with identical cell tropism. For example, wild prevalence studies. However, it is important to note that at- ruminants can be infected by the erythrocytotropic species tached larvae with PCR-positive results might be of potential A. marginale/A. centrale and A. ovis worldwide, by the gra- value for the identification of infected hosts. nulocytotropic A. phagocytophilum and ewingii in Ticks should be identified to species level by examining North America, and by the monocytotropic A. bovis, Ehrlichia morphological characters before being processed for mo- chaffeensis,andEhrlichia canis in North America and in the lecular analysis. Otherwise, any conclusion drawn regarding Far East or Ehrlichia ruminantium in Africa that can also target vector–pathogen associations could be subject to substan- granulocytic leukocytes as well as endothelial cells (Dumler tial errors (Estrada-Pen˜a et al. 2013). Identification can also et al. 2005). The identification of the causative agent in un- be confirmed by molecular methods, by sequencing 12S or common infections or atypical locations or hosts can also be 16S rRNA gene fragments (Mangold et al. 1998, Beati and limited if relying solely on light microscopy. Keirans 2001). However, tick-derived sequences in the Other light and electron microscopic techniques (trans- GenBank database are still far from being comprehensive, mission electron microscopy, scan electron microscopy, preventing accurate classification based solely on molecular confocal microscopy) have been used to study host samples toolsinmanycases. for specific research purposes. The same is true for the mi- Ticks can be used freshly for DNA extraction, with only croscopic detection of Anaplasma in vectors, which has been short-term storage at 4C. For partially fed ticks removed most useful for life cycle investigation rather than for diag- from hosts, no more than 1–2 days is recommended, but for nosis and epidemiological studies. unfed ticks this might be as long as a month; alternatively, unfed ticks can be kept for longer at 12–16C, 85% relative humidity. For longer time periods, samples can be immersed Molecular Detection in RNAlater or ‡ 70% ethanol and maintained at 4Cor Both experimental and field studies have pointed out the frozen immediately at -20Cor-80C. Decontamination of

utility of molecular laboratory diagnosis for sensitive and the tick surface should be performed before DNA extraction by specific identification of Anaplasma infections (Eriks et al. a sequence of 5-min immersions in sterile distilled water/PBS, 1989, Palmer et al. 1998, Eddlestone et al. 2007, Haigh et al. 70–80% ethanol, and again water/PBS, ending by air drying 2008, Hing et al. 2014). The growing number of high- (or drying on sterile filter paper). Ticks should be handled with performance molecular protocols with increased potential for sterile forceps in between each step and after decontamination. automation and multiplex detection has resulted in these Molecular testing of blood, tissue samples, and ticks (or becoming indispensable laboratory tools. other arthropods) is usually performed on total DNA. Ex- Anticoagulated whole blood and buffy coat are the best traction can be manual or automatic, with ready-to-use com- samples for molecular screening of Anaplasma-infected human mercial kits or prepared solutions, but in any case it is highly and nonhuman vertebrates. Ethylenediaminetetraacetic acid recommended to include DNA purification to remove PCR or citrate are preferred to heparin as anticoagulant, since the inhibitors, particularly in the case of blood and tick samples latter is considered to interfere with PCR (Hebels et al. 2014, (Schwartz et al. 1997). For fully engorged female ticks, DNA Sa´nchez-Fito and Oltra 2015). Spleen samples are equally should be extracted from only one half of the specimen after good and highly recommended for persistently infected ani- longitudinal bisection, to avoid excess sample and to prevent mals, especially in wildlife studies or as an additional sample PCR inhibition due to the high erythrocyte concentration to rule out intermittent or low-level bacteremia in blood- during extraction. Prior disruption or homogenization of ticks negative individuals (Eddlestone et al. 2007). Other samples and tissue samples is very important for efficient DNA ex- reported in the literature with variable results for Anaplasma traction and can be performed using automatic homogenizers screening include serum/plasma, liver, lung, lymph nodes, (e.g., Precyllis or Mixer Mill benchtop units) or manually, bone marrow, and skin biopsies (Massung et al. 1998, Ed- using sterile tools, such as scalpel blades or pestles (one per dlestone et al. 2007, Gaunt et al. 2010, Blanˇarova´ et al. 2014, sample). For these samples, if an enzymatic digestion step is Szekeres et al. 2015). To increase the sensitivity of Anaplasma included it should last at least 1 h at 56C or can be extended to detection in clinical cases, it is important to pay attention to an overnight incubation, facilitating organization of work. the previously mentioned time frame for sample collection (as Several commercial kits have been used by the authors in mentioned in the Microscopy section). Again, samples should diagnosis and surveillance studies with good results (Table 2 be processed as soon as possible after collection, at least to a footnote). Less expensive protocols using the TRI Reagent stage at which they can be maintained below -20C until re- protocol (Sigma-Aldrich) and alkaline hydrolysis with 1.25% quired (e.g., buffy-coat separation, preparation of aliquots with ammonium solution (Schouls et al. 1999) have also been used volume/weight suitable for nucleic acid extraction) to avoid in tick surveillance studies. The former is a time-consuming repeated freeze and thaw cycles. protocol, although it yields good-quality DNA and also en- For studies of prevalence of Anaplasma spp. in vectors, it ables protein and RNA isolation. The alkaline hydrolysis is advisable to use questing (unfed) ticks. In the case of method is easy to perform and uses intact ticks, but results in a

Table 2. PCR Assays for Detection of Anaplasma spp. DNA in Tick and Host Materials Target gene Method Primers/Probe 5¢-3¢ Reference Anaplasma spp.a rrs, 345 bp Conventional EHR16SR: GGTACCYACAGAAGAAGTCC Parola et al. (2000) EHR16SD: TAGCACTCATCGTTTACAGC rrs, 421 bp Conventional 16SANA-F: CAGAGTTTGATCCTGGCTCAGAACG Stuen et al. (2003) 16SANA-R: GAG TTT GCC GGG ACT TCTTCT GTA A. centrale/A. marginale/ msp4, 842 bp Conventional MSP43: CCGGATCCTTAGCTGAACAGGAATCTTGC de la Fuente et al. (2002) A. ovis MSP45: GGGAGCTCCTATGAATTACAGAGAATTGTTTAC A. centrale msp3 989 bp Conventional Acent_F: CCAGAAGGTGAAGGAGAAGAT Shkap et al. (2009) Acent_R: AAGCATTTACAGGAAAGGAA GC groEL 77 bp Real-time AC-For: CTATACACGCTTGCATCTC Decaro et al. (2008) AC-Rev: CGCTTTATGATGTTGATGC AC-Probe: Texas Red ATCATCATTCTTCCCCTTTACCTCGT BHQ2 A. marginale msp1ß 95 pb Real-time AM-forward: TTGGCAAGGCAGCAGCTT Carelli et al. (2007) AM-reverse: TTCCGCGAGCATGTGCAT AM-probe: 6FAM TCGGTCTAACATCTCCAGGCTTTCAT BHQ1 A. phagocytophilum/ groEL, 442 bp Nested 1st amplification Sumner et al. (1997) A. platys HS1: TGGGCT GGTAMTGAAAT HS6: CCICCIGGIACIAYACCT TC 2nd amplification

16 HS43: ATWGCWAARGAAGCATAGTC HS45: ACTTCACGYYTCATAGAC Rrs 98 bp Real-time EPlat-19f: CGGATTTTTGTCGTAGCTTGCTAT Teglas et al. (2005) EPlat-117r: CCATTTCTAGTGGCTATCCCATACTACT EPlat-55p-S1: FAM-TGGCAGACGGGTGAGTAATGCATAGGA-BHQ1 A. phagocytophilum msp4, 849 bp Conventional MSP4AP3: TTAATTGAAAGCAAATCTTGCTCCTATG de la Fuente et al. (2005) MAP4AP5: ATGAATTACAGAGAATTGCTTGTAGG rrs, 497 bp Nested 1st amplification Massung et al. (1998) ge3a: CACATGCAAGTCGAACGGATTATTC ge10r: TTCCGTTAAGAAGGATCTAATCTCC 2nd amplification ge9f: AACGGATTATTCTTTATAGCTTGCT ge2: GGCAGTATTAAAAGCAGCTCCAGG msp2; 77 bp Real-time ApMSP2f: ATGGAAGGTAGTGTTGGTTATGGTATT Courtney et al. (2004) ApMSP2r: TTGGTCTTGAAGCGCTCGTA ApMSP2p: FAM TGGTGCCAGGGTTGAGCTTGAGATTG TAMRA or BHQ A. platys rrs, 359 bp Conventi onal EPLAT5: TTTGTCGTAGCTTGCTATGAT Murphy et al. (1998) EPLAT3: CTTCTGTGGGTACCGTC

Examples of commercial kits for total DNA extraction that have been widely used by the authors with good results in diagnosis and surveillance studies include: QIAamp and DNeasy Kits (Qiagen, Hilden, Germany; manual/automated protocol for both blood and tissue), High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland; manual protocol for blood), MagCore Genomic DNA kits (RBCBioscience, New Taipai City, Taiwan; automated protocols for blood or tissue) and Maxwell 16 LEV Blood DNA Kit (Promega GmbH, Madison, Wisconsin; automated protocol). aThis protocol also amplifies other Anaplasmataceae species. msp, major surface protein. ANAPLASMA SPP. DIRECT DETECTION 17

Table 3. Commonly Used and Potentially Useful Cell Lines for Anaplasma spp. Isolation and Cultivation

Cell line Medium and supplementation Culture conditions DH82 (ATCC CRL-10389) Type: canine MEM medium Cells grow as adherent monolayer macrophage-like a Origin: dog with 10% or 5% heat-inactivated FBS Incubation at 37C with 5% CO2 malignant histiocytosis atmosphere Wellman et al. (1988) 2 mM L-glutamine, Can also be grown at 32C 1 mM sodium pyruvate Change of 1/3 medium volume every 2– 3 days 0.1–1 mM nonessential amino acids Subculture once a week is advised for stock cultures HL60 (ATCC CCL-240) Type: human RPMI-1640 medium Cells grow in suspension promyelocyte a Origin: human with 5% or 2% FBS Incubation at 37C with 5% CO2 promyelocytic atmosphere leukemia Collins et al. 2 mM L-glutamine Change medium every 2–3 days, (1977) adjusting cell concentration to 4 · 105 cell/mL or to 2 · 105 cell/mLa IDE8 (ATCC CRL-11973) Type: tick cell line L-15B medium Munderloh and Kurtti (1989) Cells grow in loosely adhered layers Origin: Ixodes scapularis 5% FBS Incubation at 32C (at 34Ca) in sealed embryos Munderloh container, under normal atmospheric et al. (1994) conditions 10% tryptose phosphate broth Change of 3/4 medium volume once or 0.1% bovine lipoprotein concentrate twice a week. 100 IU/mL penicillinb 100 lg/mL streptomycin Additional supplementationa: 0.1% NaHCO3 10 mM HEPES Adjust to pH 7.5 ISE6 (ATCC CRL-11974) Type: tick cell line L-15B300 medium Munderloh et al. (1999) Cells grow in loosely adhered layers Origin: Ixodes scapularis 5% FBS Incubation at 32C (at 34Ca) in sealed embryos Kurtti et al. 10% tryptose phosphate broth container, under normal atmospheric (1996) conditions 0.1% bovine lipoprotein concentrate Change of 3/4 medium volume once or 2 mM L-glutamine twice a week. 100 IU/mL penicillinb 100 lg/mL streptomycin Additional supplementationa: 0.1% NaHCO3 10 mM HEPES Adjust to pH 7.5 IRE/CTVM20c Type: tick cell line 1:1 mixture of L-15 (Leibovitz) medium Cells grow predominantly in suspension and L-15B medium Bell-Sakyi (2004) Origin: Ixodes ricinus 15% FBS Incubation at 28–32C, in sealed embryos Bell-Sakyi container, under normal atmospheric et al. (2007) conditions 10% tryptose phosphate broth Change 3/4 of medium volume once a 0.05% bovine lipoprotein concentrate week. 2 mM L-glutamine 100 IU/mL penicillinb 100 lg/mL streptomycin IRE/CTVM19c Type: tick cell line L-15 (Leibovitz) medium Cells grow predominantly in suspension 20% FBS

(continued) 18 SILAGHI ET AL.

Table 3. (Continued) Cell line Medium and supplementation Culture conditions Origin: Ixodes ricinus 10% tryptose phosphate broth Incubation at 28–32C, in sealed embryos Bell-Sakyi et al. container, under normal atmospheric (2007) conditions 2 mM L-glutamine Change 3/4 of medium volume once a 100 IU/mL penicillinb week. 100 lg/mL streptomycin RAE25c Type: tick cell line L-15B medium Munderloh and Kurtti (1989) Cells grow in loosely adherent sheets and clumps Origin: Rhipicephalus 5% FBS Incubation at 32C, in sealed container, appendiculatus under normal atmospheric conditions embryos Kurtti and 10% tryptose phosphate broth Change 3/4 of medium volume once a Munderloh (1982) 0.1% bovine lipoprotein concentrate week. 2 mM L-glutamine 100 IU/mL penicillinb 100 lg/mL streptomycin Additional supplementationa: 0.1% NaHCO3 10 mM HEPES Adjust to pH 7.5

aFor isolation attempts and propagation of infected cells. bUninfected cultures can be maintained with antibiotics (penicillin and streptomycin) if required, whereas infected cultures can be supplemented with an antimycotic (amphotericin B) if required for the first few weeks to minimize fungal contamination, but antibiotics should be avoided if the target Anaplasma sp. is known or suspected to be sensitive to penicillin or streptomycin. cAvailable from the Tick Cell Biobank http://tickcells.pirbright.ac.uk FBS, fetal bovine serum. crude DNA extraction, and elimination of PCR inhibitors and Sensitivity of molecular detection depends on several

long-term DNA storage stability are not guaranteed. factors such as: (1) sample nature and quality, that is, plasma Evaluation of the extraction process is an important re- and serum usually present much lower bacterial loads than quirement. Negative controls (i.e., sterile water) should be blood due to the intracellular nature of Anaplasma; (2) the included in each group of samples during extraction, to genomic copy number of target genes, for example, the major monitor the occurrence of contamination by DNA carryover. surface protein (msp) families include multiple copy genes; Quality and quantity of nucleic acids can be ascertained with and (3) the amplicon length (e.g., short sequences are gen- a spectrophotometer and degradation of DNA by gel elec- erally preferred to long ones for screening). Among the most trophoresis. PCRs targeting host (e.g., b-actin, albumin, targeted genes of Anaplasma spp. are those for 16S rRNA human b-globin) or vector (e.g., Ixodes cox gene, 12S or 16S (rrs), heat shock protein (groEL), citrate synthase (gltA), and rRNA genes) housekeeping genes can also be performed to major surface proteins (msp1, msp2, msp4, msp5). Protocols validate extraction and to confirm the absence of PCR in- targeting some of these genes are suggested in Table 2. For hibitors (Ausubel et al. 1998, Mangold et al. 1998, Beati and molecular screening, the sensitive multicopy msp approaches Keirans 2001, Schwaiger and Casinotti 2003). Alternatively, are preferred over single copy genes, whereas for sequence validation can be achieved by spiking the samples with comparison and database crossmatch, conservative or mod- nonrelated bacterial suspensions before extraction, and sub- erately conservative rrs and groEL strategies are regarded as sequently targeting the corresponding DNA by specific PCR. the best choice. Nested PCRs are now generally replaced Bacillus thuringiensis commercial suspensions are widely by less time-consuming and more sensitive real-time assays. used for this purpose (De Bruin et al. 2011). However, further confirmation of positive results by se- Despite being closely related species, few molecular ap- quencing is still highly advised. In most cases, this requires an proaches have been designed to target the entire Anaplasma additional conventional PCR, as real-time PCR targets that are genus. One of the reasons is that some Anaplasma spp. are most suitable for screening are usually very short (<150 bp), ecologically divergent and not found in the same hosts or yielding sequence data of limited phylogenetical value. vectors. Moreover, broader-range PCR assays are usually For the confirmation of a potential new strain/variant/ less sensitive and prone to selectively amplifying the pre- species it is strongly encouraged to rely on a multilocus approach dominant Anaplasma spp. or genetically similar agents (e.g., and, if possible, in vitro isolation. Generally, DNA sequences other members of the Anaplasmataceae or other alpha- from fully characterized Anaplasma variants are a reliable ) that might be present in the samples in higher source for typing (Huhn et al. 2014, Guillemi et al. 2015). concentrations. However, genus-specific primers could be used when, for example, nothing is known about the Ana- In Vitro Isolation plasma species present in a given area. Furthermore, broad- range PCRs are indispensable when attempting to identify Isolation and in vitro propagation of Anaplasma species clades within the Anaplasma genus. provides a valuable tool for the study of their biology. It is, ANAPLASMA SPP. DIRECT DETECTION 19 however, less used in direct routine diagnosis and surveillance Anaplasma can also be attempted from vertebrate tissue as the previously mentioned approaches. Still, it is of great samples or fresh ticks, after maceration, fragmentation, or value for proving the etiology in atypical/new clinical occur- dissection in culture medium. However, especially for tech- rences or fatal cases. It is also an indispensable procedure to niques which do not discard the tick exoskeleton, contami- claim discovery of a new bacterial species, enabling proper nation with environmental bacteria and fungi can be a taxonomic classification and the attribution of a formal scien- problem for establishment of the culture. In this case, special tific name (Cabezas-Cruz et al. 2012, Zweygarth et al. 2013). attention should be given to external surface decontamination Due to ethical issues regarding animal experimentation, there is of the tick, as previously mentioned for molecular testing, a strong incentive for isolation and propagation of Anaplasma adding an extra 5-min immersion in 0.1% benzalkonium in continuously cultured cell lines. chloride (Sigma) before the ethanol step to ensure decon- In vitro culture of Anaplasma spp. in mammalian cells relies tamination. Samples of 0.1–0.5 mL anticoagulated whole on the availability of cellular systems that are similar to or blood, buffy coat, or tissue samples can be inoculated straight could mimic the intracellular environment found in natural into small cell culture flasks (12.5- or 25-cm2 capacity) or host cells. This was first achieved for the granulocytotropic flat-sided tubes (Nunc) and maintained according to the re- A. phagocytophilum using the human promyelocytic leukemia spective cell line culturing conditions (Table 3). Every 2– cell line HL-60 (ATCC CCL-240) (Goodman et al. 1996). 7 days, fresh medium should be added to cultures and, in the Since then, HL-60 cells have been routinely used for A. pha- case of mammalian cells growing in suspension, cell con- gocytophilum culture, although other mammalian cell lines are centration adjusted. As tick cells tolerate high cell densities also reported to sustain its growth. These include the human and can survive for many months without subculture, there is monocytic THP-1 (ATCC TIB-202) and microvascular en- no need to adjust cell density and inoculated cultures can be dothelial HMEC-1 and MVEC cell lines, the bovine corneal maintained for the long periods (12 weeks or more) required BCE C/D1-b (ATCC CRL-2048) cell line and the Rhesus for adaptation of some Anaplasma spp. to in vitro growth monkey retina choroid RF/6A (ATCC CRL-1780) endothelial (Silaghi et al. 2011, Dyachenko et al. 2013, Bell-Sakyi et al. cell line (Munderloh et al. 2004, Garcia-Garcia et al. 2009). 2015). Cultures should also be periodically evaluated by Continuous growth of the intraerythrocytic A. marginale was microscopy to detect any microbial growth. Infection can be also achieved in both BCE C/D1-b and RF/6A cells, but only assessed by direct observation of cytocentrifuged culture after establishment in tick cell lines (Munderloh et al. 2004). aliquots after staining with Eosin Azure-type dyes as de- Additionally, HL-60 and THP-1 cells have enabled the isola- scribed above. tion of the newly reported zoonotic A. capra (Li et al. 2015). In vitro culturing is a demanding task in terms of time and As an alternative to mammalian-derived cells, tick cell expertise, and only a limited number of research institutions

lines are a valuable tool for the cultivation of Anaplasma are currently able to perform it. Even so, this might not be a species. They can be a good option for the isolation of Ana- constraint on its use for direct diagnosis at least for some plasma variants found in vectors, but with low or unknown agents such as A. phagocytophilum that can be successfully pathogenicity for vertebrates (Massung et al. 2007). More- cultured from infected blood kept for up to 18 days under over, these cells have already proven their value for bacteria refrigerated conditions (Kalantarpour et al. 2000). Thus, bio- that target mammalian cells that are difficult to continu- logical samples can be transported under refrigerated condi- ously propagate in vitro, such as the intraerythrocytic and tions to a referral laboratory, where the appropriate assays intrathrombocytic Anaplasma spp. (Munderloh et al. 1994, can be carried out. 2003). The continuous culture of A. marginale was first achieved in an Ixodes scapularis embryo-derived cell line Conclusion (IDE8) (Munderloh et al. 1994, 1996). Since then, many isolates have been established in the IDE8 cell line, as well as In summary, for the direct detection of Anaplasma spp. in in other tick cell lines (Munderloh et al. 2004, Zivkovic et al. blood and tissue samples, ticks or other vectors, molecular 2010), making possible a more intense study of A. marginale methods are preferred. Specific real-time PCRs offer several biology, as reviewed by Blouin et al. (2002) and Passos (2012). advantages over conventional PCR assays for screening pur- Very recently, continuous cultures of the vaccine strain of poses, but the confirmation of sequence identity is still often A. centrale were established for the first time in Rhipicephalus required. Approaches targeting multiple genes can be very appendiculatus RAE25 and Dermacentor variabilis DVE1 useful for phylogeny and studies. Other direct cell lines (Bell-Sakyi et al. 2015). A. phagocytophilum cell cul- methods such as microscopy or in vitro isolation are mostly tures have also been established in several tick-derived cell reserved for research applications, such as experimental lines, including I. scapularis-derived IDE8 and ISE6, Ixodes studies, transmission trials etc., but can also contribute in ricinus-derived IRE/CTVM19 and IRE/CTVM20 (Munderloh specific diagnostic/surveillance investigations and in identi- et al. 1996, Woldehiwet et al. 2002, Silaghi et al. 2011, fying and characterizing novel Anaplasma spp. Dyachenko et al. 2013, Alberdi et al. 2015), R. appendiculatus- derived RAE25 and I. ricinus-derived IRE11 (Bell-Sakyi, un- Acknowledgments published data). ISE6 cells were also valuable for the isolation This work was done under the frame of COST action of potentially new thrombocytotropic Anaplasma closely re- TD1303. The authors thank A. Mathis for some suggestions lated to A. platys (Munderloh et al. 2003, Tate et al. 2013). on the article. As mentioned for the other direct diagnostic techniques, blood collected from animals or humans in the proper time Author Disclosure Statement frame is also the best inoculum for in vitro culture (as men- tioned in the Microscopy section). Furthermore, growth of No competing financial interests exist. 20 SILAGHI ET AL.

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