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Typing of Melissococcus plutonius isolated from European and Japanese honeybees suggests spread of sequence types across borders and between different Apis species
Veterinary Microbiology 171 (2014) 221–226
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Typing of Melissococcus plutonius isolated from European and Japanese honeybees suggests spread of sequence types across borders and between different Apis species Daisuke Takamatsu a,b,*, Keiko Morinishi c, Rie Arai b,d, Aya Sakamoto e, Masatoshi Okura a, Makoto Osaki a a
Bacterial and Parasitic Diseases Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-0856, Japan The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Gifu 501-1193, Japan c Kagawa Prefecture Eastern Regional Livestock Hygiene Service Center, Miki, Kagawa 761-0701, Japan d Saitama Prefectural Chuo Livestock Hygiene Service Center, Saitama, Saitama 331-0821, Japan e Matsumoto Livestock Hygiene Service Center, Matsumoto, Nagano 390-0851, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 November 2013 Received in revised form 20 March 2014 Accepted 24 March 2014
Melissococcus plutonius is an important pathogen of honeybee larvae and causes European foulbrood (EFB) not only in European honeybees (Apis mellifera) but also in other native honeybees. We recently confirmed the first EFB case in Japanese native honeybees (Apis cerana japonica) and isolated M. plutonius from this case. In this study, to obtain a better understanding of the ecology of M. plutonius and the epidemiology of EFB, we analyzed M. plutonius isolates that originated from European and Japanese honeybees in Japan using an existing multilocus sequence typing scheme. These analyzed Japanese isolates were resolved into six sequence types (STs), three of which were novel STs. Among these six STs, ST3 and ST12 were the two most common and found in isolates from both European and Japanese honeybees (or their environment). Moreover, these two STs were identified not only in Japan but also in other countries, suggesting the spread of some STs across borders and different honeybee species. ß 2014 Elsevier B.V. All rights reserved.
Keywords: European foulbrood Japanese honeybee European honeybee Melissococcus plutonius MLST
1. Introduction European foulbrood (EFB) is an important bacterial disease of honeybee larvae. The causative agent, Melissococcus plutonius, had been thought to be remarkably homogeneous (Forsgren, 2010). However, Arai et al. (2012) recently reported the presence of atypical M. plutonius, which is phenotypically and genetically distinct from typical M. plutonius strains, in Japan. Unlike typical M.
* Corresponding author at: Bacterial and Parasitic Diseases Research Division, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. Tel.: +81 29 838 7754; fax: +81 29 838 7754. E-mail address: [email protected] (D. Takamatsu). http://dx.doi.org/10.1016/j.vetmic.2014.03.036 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
plutonius, atypical M. plutonius did not require highpotassium conditions for growth, was positive for bglucosidase activity, and produced acid from L-arabinose, D-cellobiose, and salicin. In addition, the typical and atypical strains were separately grouped into two genetically distinct clusters by pulsed-field gel electrophoresis analysis (Arai et al., 2012). Interestingly, although artificially cultured atypical M. plutonius strains killed most of the experimentally infected larvae within five days, cultured typical M. plutonius strains hardly affected larvae at least in the testing period under the conditions tested (Arai et al., 2012), suggesting that these two types might have different mechanisms to regulate their virulence and have different impacts on apiculture. Recently, Haynes et al. (2013) developed a modified multilocus sequencing
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subcultured on KSBHI agar and identified as typical or atypical M. plutonius according to the morphological and cultural characteristics of the isolates (Arai et al., 2012) and by regular (Govan et al., 1998) and duplex (Arai et al., 2014) M. plutonius-specific PCR assays.
typing (MLST) scheme for M. plutonius and analyzed international isolates. Budge et al. (2014) then added MLST data of UK isolates and showed the presence of three clonal complexes (CCs) in the M. plutonius population. Intriguingly, one of the CCs (CC12 or atypical group in Haynes et al., 2013) included not only a Japanese atypical strain (DAT561) but also isolates from the UK, the USA, Brazil and the Netherlands, suggesting that so-called atypical strains are distributed globally. M. plutonius has not only been isolated from the European honeybee (Apis mellifera), but also from diseased larvae of Apis cerana (Diwan et al., 1971; Bailey, 1974; Zhou et al., 2000; Rana et al., 2012) and Apis laboriosa (Allen et al., 1990). However, because isolates from native honeybee species have not been characterized well, further heterogeneity might be present in the population of M. plutonius. In Japan, besides DAT561, many typical and atypical M. plutonius have been isolated from diseased European honeybee larvae. In addition, we recognized the first EFB case in Japanese native honeybees (Japanese honeybee; Apis cerana japonica) in 2013 and isolated M. plutonius isolates from this case. In this study, to obtain a better understanding of the population structure and epidemiology of this important honeybee pathogen, we analyzed Japanese M. plutonius isolates by MLST and investigated the relationships of M. plutonius isolates from different honeybee species and different countries.
MLST was performed by sequencing four genes as described previously (Haynes et al., 2013). Sequencing was carried out with a BigDye Terminator v3.1 cycle sequencing kit using 3130x Genetic Analyzer (Applied Biosystems). The allelic numbers and STs of the isolates were determined by comparing their sequences with those in the M. plutonius MLST database (http://pubmlst.org/mplutonius/) developed recently by Budge et al. (2014). Novel alleles and STs were assigned through submission of the data to the database. The novel allele sequences of argE and gbpB have also been deposited in the DDBJ/EMBL/GenBank database. Potential patterns of evolutionary descent between STs were calculated using the goeBURST algorithm (Francisco et al., 2009) in the PHYLOViZ program (Francisco et al., 2012), a modification of the earlier eBURST algorithm (Feil et al., 2004). When constructing the goeBURST tree, MLST data of 86 isolates from the current study were added to those of 61 isolates from Haynes et al. (2013) and 205 isolates from Budge et al. (2014).
2. Materials and methods
3. Results and discussion
2.1. Bacterial isolates and DNA extraction
3.1. Sequence types of M. plutonius isolates from European honeybees in Japan
A total of 86 M. plutonius isolates (29 typical and 57 atypical isolates) were analyzed by MLST in this study (Table S1). All isolates except for DAT569 isolated in Paraguay were isolated in various areas of Japan. Fortynine isolates (48 isolates from Japan and one from Paraguay) from diseased individual and bulk European honeybee (A. mellifera) larvae were described previously (Arai et al., 2012, 2014), and the other isolates were isolated from individual and bulk larval samples of European honeybees and individual larval and pupal samples and the environments of Japanese honeybees (A. c. japonica), as described below. Genomic DNA of bacterial isolates cultured on KSBHI agar [brain heart infusion (BHI; Becton Dickinson, Sparks, MD, USA)-based medium supplemented with 0.15 M KH2PO4, 1% soluble starch, and 1.5% agar] (Arai et al., 2012) under anaerobic conditions was extracted using InstaGene Matrix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2014.03.036.
2.3. MLST and data analysis
A total of 58 M. plutonius isolates (26 typical and 32 atypical isolates) isolated from diseased individual or bulk European honeybee larvae in Japan (Table S1) were classified into four STs by the MLST analysis. ST3 and ST12 were the two most common in the population. Twenty-three isolates with typical cultural characteristics were assigned into ST3, whereas only three typical isolates were found to be ST26, a novel single-locus variant of ST13. On the other hand, 31 isolates with atypical cultural characteristics were classified into ST12, and the remaining one atypical isolate had a novel allele sequence of argE (argE-6; DDBJ/EMBL/GenBank accession number, AB853996) and was assigned into ST25, a novel singlelocus variant of ST12 (Tables 1 and 2, and Table S1). Of note, although atypical strain DAT561 was reported to be ST10 on the basis of the genome sequence data by Haynes et al. (2013), the strain was assigned into ST12 in this study. Moreover, as far as we tested, no ST10 isolate was found in Japan (see below and Table 1). 3.2. First EFB case in Japanese honeybees and sequence types of isolated M. plutonius
2.2. Isolation and identification of M. plutonius Homogenized individual or bulk larvae, honey, and swabs of beehives were streaked on KSBHI agar and incubated at 37 8C under anaerobic conditions for 3 or 4 days. Bacterial colonies considered to be M. plutonius were
In the spring of 2013, a beekeeper noticed that nurse bees neglected larvae in his Japanese honeybee colonies. In the first inspection in May, a decrease in the number of larvae and an increase of larvae that could not pupate were observed in all seventeen colonies inspected. Approximately
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Table 1 Japanese M. plutonius isolates analyzed in this study. Source
Typical/atypicala
MLST resultsb
No. of strains/isolates
Individual and bulk diseased larval samples of European honeybees (A. mellifera)c
Typical
ST3 ST26d ST12e ST25d
23 3 31 1
ST3 ST4 ST12 ST27d
1 1 21 4
Atypical
Individual larval and pupal samples and the environments (honey and beehives) of Japanese honeybee (A. c. japonica) coloniesc with or without clinical signs of EFB
Typical Atypical
a M. plutonius isolates were classified into two types (typical and atypical) by cultural characteristics reported by Arai et al. (2012) and the duplex PCR developed by Arai et al. (2014). b MLST analysis was performed by sequencing four genes as described by Haynes et al. (2013). c All the honeybee colonies were managed by beekeepers. d Novel STs found in this study. The ST numbers were assigned through submission of the data to the M. plutonius MLST database (http://pubmlst.org/ mplutonius/) developed by Budge et al. (2014). e Although strain DAT561 isolated in Japan was reported as ST10 by Haynes et al. (2013), it was assigned into ST12 in this study.
four to five-day-old unsealed larvae were mainly affected, and some of them were displaced from their normal coiled position in the bottom and twisted around the walls or stretched out lengthways. Empty cells scattered randomly among the affected larvae were also observed. Dead larvae showed a light yellow or brown color and dissolved into a semi-liquid mass (Fig. 1). Some larval remains were dried and easily removed from the cells. Two of the seventeen
colonies were further examined in a regional veterinary diagnostic laboratory, and M. plutonius was detected from individual larvae of the two colonies, whereas other bee pathogens including Paenibacillus larvae (the causative agent of American foulbrood), sacbrood virus (the causative agent of sacbrood disease), and Acarapis woodi (tracheal mite; the causative agent of acarine disease) were not detected from the larval samples tested. On the basis of
Table 2 Updated MLST typing scheme and distribution of M. plutonius strains/isolates analyzed so far. STa
Profileb
No. of strains/isolatesc
Origin (No. of strains/isolates)c
England and Wales (2), Italy (1), Scotland (1), Thailand (2) England and Wales (13), Scotland (1) England and Wales (67), France (1), Italy (2), Japan (24), Paraguay (1), Republic of Ireland (1), The Netherlands (1), USA (5) Australia (1), France (4), Japan (1) England and Wales (60) England and Wales (5) England and Wales (9) England and Wales (4) England and Wales (2) England and Wales (1), USA (1) England and Wales (8) England and Wales (1), Japan (52), USA (1) Denmark (1), England and Wales (19), Poland (3) Poland (1) India (1) Brazil (1) Tanzania (1) Scotland (1) The Netherlands (1) England and Wales (2) England and Wales (6) England and Wales (4) England and Wales (29) England and Wales (2) Japan (1) Japan (3) Japan (4)
argE
galK
gbpB
purR
1 2 3
1 2 2
1 3 3
1 2 2
1 2 4
6 14 102
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1 2 2 2 1 1 4 3 4 1 1 5 4 1 1 4 1 4 2 2 2 6 1 4
1 3 2 3 1 1 4 3 4 1 5 1 6 3 1 4 1 4 7 3 3 4 1 4
2 4 2 5 7 6 3 4 3 1 8 1 9 1 2 10 8 11 2 12 13 3 5 14
4 4 2 4 3 4 4 4 5 4 4 4 4 4 1 5 4 5 2 4 4 5 4 5
6 60 5 9 4 2 2d 8 54d 23 1 1 1 1 1 1 2 6 4 29 2 1 3 4
a
Sequence type. argE, putative acetylornithine deacetylase gene; galK, putative galactokinase gene; gbpB, putative secreted antigen gene; purR, putative purine operon repressor gene. c Eighty-five Japanese and one Paraguayan isolates were analyzed in this study, and the other data was from previous studies (Haynes et al., 2013; Budge et al., 2014). d Although strain DAT561 isolated in Japan was reported as ST10 by Haynes et al. (2013), it was assigned into ST12 in this study. b
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Fig. 1. A representative beehive and a dead larva of Japanese honeybee (A. c. japonica) affected by M. plutonius infection.
clinical symptoms of classic EFB observed in the affected colonies and larvae and isolation of M. plutonius from the larvae, the abnormal colonies were diagnosed with EFB. The beekeeper placed beehives for Japanese honeybees at several different locations in an area within an approximately 2.5-kilometer radius. Because similar symptoms were also observed in some of the other colonies at different locations, first follow-up examination was performed in early June for all 119 colonies in the area by visual inspection, M. plutonius-specific PCR, and isolation of the causative strains. Colonies from which M. plutonius was detected were destroyed. In late June, second follow-up examination was performed for 13 colonies that showed clinical signs of EFB such as those described above, and colonies from which M. plutonius was detected were destroyed. Consequently, EFB in the Japanese honeybee colonies had ended by early August. Although an apiary managing nine European honeybee colonies was located at a distance of two kilometers from the apiary inspected in May, no clinical signs were observed in these colonies, and M. plutonius isolation was negative. During the examinations, in total, 27 M. plutonius isolates were isolated from 13 individual larvae, one pupa, one honey sample, and 12 beehives of the colonies with or without clinical signs of EFB in five apiaries (apiaries A–E) (Table 1 and Table S1). Interestingly, two and twenty-five isolates were identified as typical and atypical M. plutonius, respectively, on the basis of the cultural characteristics (Arai et al., 2012) and the results of the duplex PCR assay (Arai et al., 2014). In addition, the two typical isolates were further divided into ST3 and ST4 by MLST, and 21 and 4 atypical isolates were assigned into ST12 and ST27 (a novel single-locus variant of ST12, which has a novel allele sequence of gbpB [gbpB-14; DDBJ/EMBL/GenBank accession number, AB853997]), respectively (Tables 1 and 2, and Table S1); that is, at least four genetically different M. plutonius strains were prevalent in the Japanese honeybee colonies raised in a limited area by the same beekeeper. Although only a single ST was detected from each colony by culture methods in this case, more than one ST was
found to be present in a single affected apiary (e.g., DAT869 of ST4, DAT870 of ST27, and DAT872 of ST12 in apiary A; DAT879 of ST12 and DAT885 of ST27 in apiary C) (Table S1). Because the beekeeper has moved some beehives and captured swarms between different apiaries, this might contribute to the spread of different strains among different apiaries. Arai et al. (2014) recently reported that both typical and atypical M. plutonius were detected from approximately 50% of larval samples collected from European honeybee colonies with clinical signs of EFB in Japan. In the study, each sample contained several larvae, but all the larvae in a single sample were retrieved from a single colony. Therefore, regardless of honeybee species, the participation of several different strains in a single outbreak may be typical for EFB at least in Japan. In this case, atypical M. plutonius was isolated more frequently than typical M. plutonius. Because atypical M. plutonius grows more rapidly and forms larger colonies than typical M. plutonius (Arai et al., 2012), if both types are present in a single sample, typical M. plutonius tends to be missed due to its small colony size or the growth of atypical strains over the small colonies of typical strains (Arai et al., 2014). Therefore, typical strains might be more prevalent in the Japanese honeybee colonies than detected. The two most common STs (ST3 and ST12) in M. plutonius isolates from European honeybees in Japan were also found in the isolates from Japanese honeybees or their environment. In addition, although ST4 found in an isolate from a larva in a diseased Japanese honeybee colony has not been isolated from European honeybees in Japan, this ST has been found in isolates from European honeybees (A. mellifera) in Australia and France (Haynes et al., 2013; http://pubmlst.org/mplutonius/). Because some isolates that showed different SmaI-digested profiles by pulsedfield gel electrophoresis in our previous study (Arai et al., 2012) were classified into the same ST in this study (e.g., DAT580 and DAT585 of ST26; DAT561, DAT571, and DAT607 of ST12) (Figure 1 in Arai et al., 2012 and Table S1), even if different isolates are found to be the same ST, they may have slightly different genetic backgrounds. However, our present results suggest that some M. plutonius strains may have the ability to cause EFB in different honeybee species. Of note, the analyzed M. plutonius isolates from Japanese honeybees and those from European honeybees were isolated in different prefectures of Japan. Therefore, it remains unclear at present whether the transmission of M. plutonius between the two honeybee species has occurred in Japan. 3.3. Population structure of Japanese and international M. plutonius isolates In addition to Japanese M. plutonius isolates, a typical M. plutonius isolate (DAT569) from a diseased European honeybee larva in Paraguay was analyzed by MLST and found to be ST3 (Table S1). Consequently, a total of 352 M. plutonius strains/isolates from 16 countries and regions analyzed in this and previous (Haynes et al., 2013; Budge et al., 2014) studies were resolved into 27 STs (Table 2 and Table S1). Although three novel STs were found in this study, the overall topology of the goeBURST tree was
D. Takamatsu et al. / Veterinary Microbiology 171 (2014) 221–226
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Fig. 2. Updated goeBURST tree of STs found in M. plutonius isolates from different countries and honeybee species. MLST data of 86 isolates from the current study were added to those reported previously (61 isolates from Haynes et al., 2013 and 205 isolates from Budge et al., 2014) (Table S1). Each circle represents a different ST, with lines linking closest relatives. Black lines indicate a single allelic change between STs. Light gray lines indicate differences at two loci. Circles ringed with a green outline indicate putative founder genotypes. Colors within circles show the proportion of isolates of a particular type that were found in the countries/honeybee species indicated.
almost the same as that constructed in previous studies (Haynes et al., 2013; Budge et al., 2014) (Fig. 2), and all Japanese atypical isolates (ST12, ST25 and ST27) belonged to a group corresponding to the atypical group (Haynes et al., 2013) or CC12 (Budge et al., 2014) in previous studies with isolates from the UK, the USA, Brazil, and the Netherlands. Of 20 STs that include multiple isolates, seven were found to contain isolates from more than one country or region (Table 2 and Fig. 2). Among the seven STs, three (ST3, ST4, and ST12) were identified in Japan. In particular, ST3 was the most globally distributed type and found in eight countries. As described above, ST4 found in a Japanese honeybee larva in Japan was identified in France and Australia (Haynes et al., 2013). In addition, ST12 found in both Japanese and European honeybee colonies in Japan was found in the USA and the UK (Table 2, Table S1, and Fig. 2) (Haynes et al., 2013; Budge et al., 2014). Moreover, although ST26 has been identified only in Japan, this ST was a single-locus variant of ST13, and ST13 was found in Denmark, Poland, and the UK (Haynes et al., 2013; Budge et al., 2014).
4. Conclusion In this study, we analyzed M. plutonius isolates isolated from European and Japanese honeybees in Japan by MLST and showed the spread of some STs across borders and honeybee species. European honeybees are now commonly used in apiculture globally and, in areas where different honeybee species may interact, cross-species pathogen transmission could occur. Varroa destructor, which was transmitted from the Eastern honeybee A. cerana to the European honeybee A. mellifera, is a well-known example (Oldroyd, 1999; Rosenkranz et al., 2010). After the host shift, the mite has spread worldwide and is almost
cosmopolitan today (Oldroyd, 1999; Ellis and Munn, 2005; Rosenkranz et al., 2010). Recently, Kojima et al. (2011) suggested possible transmission of Israeli acute paralysis virus between European and Japanese honeybees in Japan on the basis of the sequence data of viruses detected from Japanese honeybee colonies and neighboring European honeybee colonies. However, as described above, it is not clear at present whether the cross-species transmission of M. plutonius has indeed occurred in Japan. Even if it has, the direction of the transmission between European and Japanese honeybees is unclear. In addition, epidemiological relationships among M. plutonius isolates of the same ST found in different countries are unknown. For a more comprehensive understanding of the ecology and epidemiology of M. plutonius including possible crossspecies transmission and international spread of specific M. plutonius strains, more extensive studies of different honeybee species from geographically diverse regions and detailed characterization of isolated M. plutonius strains will be necessary. Acknowledgements A part of this study was supported by a Grant-in-Aid for Scientific Research (B) (25292200) from the Japan Society for the Promotion of Science. This paper made use of the M. plutonius MLST database (http://pubmlst.org/mplutonius/) (Budge et al., 2014) curated by Giles E. Budge. References Allen, M.F., Ball, B.V., Underwood, B.A., 1990. An isolate of Melissococcus pluton from Apis laboriosa. J. Invertebr. Pathol. 55, 439–440. Arai, R., Miyoshi-Akiyama, T., Okumura, K., Morinaga, Y., Wu, M., Sugimura, Y., Yoshiyama, M., Okura, M., Kirikae, T., Takamatsu, D., 2014. Development of duplex PCR assay for detection and differentiation of typical and atypical Melissococcus plutonius strains. J. Vet. Med. Sci. 76, 491–498.
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Arai, R., Tominaga, K., Wu, M., Okura, M., Ito, K., Okamura, N., Onishi, H., Osaki, M., Sugimura, Y., Yoshiyama, M., Takamatsu, D., 2012. Diversity of Melissococcus plutonius from honeybee larvae in Japan and experimental reproduction of European foulbrood with cultured atypical isolates. PLoS ONE 7, e33708. Bailey, L., 1974. An unusual type of Streptococcus pluton from the eastern hive bee. J. Invertebr. Pathol. 23, 246–247. Budge, G.E., Shirley, M.D.F., Jones, B., Quill, E., Tomkies, V., Feil, E.J., Brown, M.A., Haynes, E.G., 2014. Molecular epidemiology and population structure of the honey bee brood pathogen Melissococcus plutonius. ISME J., http://dx.doi.org/10.1038/ismej.2014.20 (advance online publication). Diwan, V.V., Kshirsagar, K.K., Ramana Rao, A.V., Raghunath, D., Bhambure, C.S., Godbole, S.H., 1971. Occurrence of a new bacterial disease of Indian honey-bees Apis indica F. Curr. Sci. 40, 196–197. Ellis, J.D., Munn, P.A., 2005. The worldwide health status of honey bees. Bee World. 86, 88–101. Francisco, A.P., Bugalho, M., Ramirez, M., Carric¸o, J.A., 2009. Global optimal eBURST analysis of multilocus typing data using a graphic matroid approach. BMC Bioinformatics 10, 152. Francisco, A.P., Vaz, C., Monteiro, P.T., Melo-Cristino, J., Ramirez, M., Carric¸o, J.A., 2012. PHYLOViZ: phylogenetic inference and data visualization for sequence based typing methods. BMC Bioinformatics 13, 87. Feil, E.J., Li, B.C., Aanensen, D.M., Hanage, W.P., Spratt, B.G., 2004. eBURST: Inferring patterns of evolutionary descent among clusters of related
bacterial genotypes from multilocus sequence typing data. J. Bacteriol. 186, 1518–1530. Forsgren, E., 2010. European foulbrood in honey bees. J. Invertebr. Pathol. 103, S5–S9. Govan, V.A., Bro¨zel, V., Allsopp, M.H., Davison, S., 1998. A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae. Appl. Environ. Microbiol. 64, 1983–1985. Haynes, E., Helgason, T., Young, J.P.W., Thwaites, R., Budge, G.E., 2013. A typing scheme for the honeybee pathogen Melissococcus plutonius allows detection of disease transmission events and a study of the distribution of variants. Environ. Microbiol. Rep. 5, 525–529. Kojima, Y., Toki, T., Morimoto, T., Yoshiyama, M., Kimura, K., Kadowaki, T., 2011. Infestation of Japanese native honey bee by tracheal mite and virus from non-native European honey bees in Japan. Microb. Ecol. 62, 895–906. Oldroyd, B.P., 1999. Coevolution while you wait: Varroa jacobsoni, a new parasite of western honeybees. Trends Ecol. Evol. 14, 312–315. Rana, B.S., Rao, K.M., Chakravarty, S.K., Katna, S., 2012. Characterization of Melissococcus plutonius causing European foulbrood disease in Apis cerana F. J. Apic. Res. 51, 306–311. Rosenkranz, P., Aumeier, P., Ziegelmann, B., 2010. Biology and control of Varroa destructor. J. Invertebr. Pathol. 103, S96–S119. Zhou, T., Feng, F., Dong, B., 2000. Study on the pathogen of European foulbrood in the Chinese honey bee (Apis cerana cerana F.). Acta Entomol. Sinica 43, 104–108.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Typing of Melissococcus plutonius isolated from European and Japanese honeybees suggests spread of sequence types across borders and between different Apis species Daisuke Takamatsu a,b,*, Keiko Morinishi c, Rie Arai b,d, Aya Sakamoto e, Masatoshi Okura a, Makoto Osaki a a
Bacterial and Parasitic Diseases Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-0856, Japan The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Gifu 501-1193, Japan c Kagawa Prefecture Eastern Regional Livestock Hygiene Service Center, Miki, Kagawa 761-0701, Japan d Saitama Prefectural Chuo Livestock Hygiene Service Center, Saitama, Saitama 331-0821, Japan e Matsumoto Livestock Hygiene Service Center, Matsumoto, Nagano 390-0851, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 November 2013 Received in revised form 20 March 2014 Accepted 24 March 2014
Melissococcus plutonius is an important pathogen of honeybee larvae and causes European foulbrood (EFB) not only in European honeybees (Apis mellifera) but also in other native honeybees. We recently confirmed the first EFB case in Japanese native honeybees (Apis cerana japonica) and isolated M. plutonius from this case. In this study, to obtain a better understanding of the ecology of M. plutonius and the epidemiology of EFB, we analyzed M. plutonius isolates that originated from European and Japanese honeybees in Japan using an existing multilocus sequence typing scheme. These analyzed Japanese isolates were resolved into six sequence types (STs), three of which were novel STs. Among these six STs, ST3 and ST12 were the two most common and found in isolates from both European and Japanese honeybees (or their environment). Moreover, these two STs were identified not only in Japan but also in other countries, suggesting the spread of some STs across borders and different honeybee species. ß 2014 Elsevier B.V. All rights reserved.
Keywords: European foulbrood Japanese honeybee European honeybee Melissococcus plutonius MLST
1. Introduction European foulbrood (EFB) is an important bacterial disease of honeybee larvae. The causative agent, Melissococcus plutonius, had been thought to be remarkably homogeneous (Forsgren, 2010). However, Arai et al. (2012) recently reported the presence of atypical M. plutonius, which is phenotypically and genetically distinct from typical M. plutonius strains, in Japan. Unlike typical M.
* Corresponding author at: Bacterial and Parasitic Diseases Research Division, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. Tel.: +81 29 838 7754; fax: +81 29 838 7754. E-mail address: [email protected] (D. Takamatsu). http://dx.doi.org/10.1016/j.vetmic.2014.03.036 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
plutonius, atypical M. plutonius did not require highpotassium conditions for growth, was positive for bglucosidase activity, and produced acid from L-arabinose, D-cellobiose, and salicin. In addition, the typical and atypical strains were separately grouped into two genetically distinct clusters by pulsed-field gel electrophoresis analysis (Arai et al., 2012). Interestingly, although artificially cultured atypical M. plutonius strains killed most of the experimentally infected larvae within five days, cultured typical M. plutonius strains hardly affected larvae at least in the testing period under the conditions tested (Arai et al., 2012), suggesting that these two types might have different mechanisms to regulate their virulence and have different impacts on apiculture. Recently, Haynes et al. (2013) developed a modified multilocus sequencing
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subcultured on KSBHI agar and identified as typical or atypical M. plutonius according to the morphological and cultural characteristics of the isolates (Arai et al., 2012) and by regular (Govan et al., 1998) and duplex (Arai et al., 2014) M. plutonius-specific PCR assays.
typing (MLST) scheme for M. plutonius and analyzed international isolates. Budge et al. (2014) then added MLST data of UK isolates and showed the presence of three clonal complexes (CCs) in the M. plutonius population. Intriguingly, one of the CCs (CC12 or atypical group in Haynes et al., 2013) included not only a Japanese atypical strain (DAT561) but also isolates from the UK, the USA, Brazil and the Netherlands, suggesting that so-called atypical strains are distributed globally. M. plutonius has not only been isolated from the European honeybee (Apis mellifera), but also from diseased larvae of Apis cerana (Diwan et al., 1971; Bailey, 1974; Zhou et al., 2000; Rana et al., 2012) and Apis laboriosa (Allen et al., 1990). However, because isolates from native honeybee species have not been characterized well, further heterogeneity might be present in the population of M. plutonius. In Japan, besides DAT561, many typical and atypical M. plutonius have been isolated from diseased European honeybee larvae. In addition, we recognized the first EFB case in Japanese native honeybees (Japanese honeybee; Apis cerana japonica) in 2013 and isolated M. plutonius isolates from this case. In this study, to obtain a better understanding of the population structure and epidemiology of this important honeybee pathogen, we analyzed Japanese M. plutonius isolates by MLST and investigated the relationships of M. plutonius isolates from different honeybee species and different countries.
MLST was performed by sequencing four genes as described previously (Haynes et al., 2013). Sequencing was carried out with a BigDye Terminator v3.1 cycle sequencing kit using 3130x Genetic Analyzer (Applied Biosystems). The allelic numbers and STs of the isolates were determined by comparing their sequences with those in the M. plutonius MLST database (http://pubmlst.org/mplutonius/) developed recently by Budge et al. (2014). Novel alleles and STs were assigned through submission of the data to the database. The novel allele sequences of argE and gbpB have also been deposited in the DDBJ/EMBL/GenBank database. Potential patterns of evolutionary descent between STs were calculated using the goeBURST algorithm (Francisco et al., 2009) in the PHYLOViZ program (Francisco et al., 2012), a modification of the earlier eBURST algorithm (Feil et al., 2004). When constructing the goeBURST tree, MLST data of 86 isolates from the current study were added to those of 61 isolates from Haynes et al. (2013) and 205 isolates from Budge et al. (2014).
2. Materials and methods
3. Results and discussion
2.1. Bacterial isolates and DNA extraction
3.1. Sequence types of M. plutonius isolates from European honeybees in Japan
A total of 86 M. plutonius isolates (29 typical and 57 atypical isolates) were analyzed by MLST in this study (Table S1). All isolates except for DAT569 isolated in Paraguay were isolated in various areas of Japan. Fortynine isolates (48 isolates from Japan and one from Paraguay) from diseased individual and bulk European honeybee (A. mellifera) larvae were described previously (Arai et al., 2012, 2014), and the other isolates were isolated from individual and bulk larval samples of European honeybees and individual larval and pupal samples and the environments of Japanese honeybees (A. c. japonica), as described below. Genomic DNA of bacterial isolates cultured on KSBHI agar [brain heart infusion (BHI; Becton Dickinson, Sparks, MD, USA)-based medium supplemented with 0.15 M KH2PO4, 1% soluble starch, and 1.5% agar] (Arai et al., 2012) under anaerobic conditions was extracted using InstaGene Matrix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2014.03.036.
2.3. MLST and data analysis
A total of 58 M. plutonius isolates (26 typical and 32 atypical isolates) isolated from diseased individual or bulk European honeybee larvae in Japan (Table S1) were classified into four STs by the MLST analysis. ST3 and ST12 were the two most common in the population. Twenty-three isolates with typical cultural characteristics were assigned into ST3, whereas only three typical isolates were found to be ST26, a novel single-locus variant of ST13. On the other hand, 31 isolates with atypical cultural characteristics were classified into ST12, and the remaining one atypical isolate had a novel allele sequence of argE (argE-6; DDBJ/EMBL/GenBank accession number, AB853996) and was assigned into ST25, a novel singlelocus variant of ST12 (Tables 1 and 2, and Table S1). Of note, although atypical strain DAT561 was reported to be ST10 on the basis of the genome sequence data by Haynes et al. (2013), the strain was assigned into ST12 in this study. Moreover, as far as we tested, no ST10 isolate was found in Japan (see below and Table 1). 3.2. First EFB case in Japanese honeybees and sequence types of isolated M. plutonius
2.2. Isolation and identification of M. plutonius Homogenized individual or bulk larvae, honey, and swabs of beehives were streaked on KSBHI agar and incubated at 37 8C under anaerobic conditions for 3 or 4 days. Bacterial colonies considered to be M. plutonius were
In the spring of 2013, a beekeeper noticed that nurse bees neglected larvae in his Japanese honeybee colonies. In the first inspection in May, a decrease in the number of larvae and an increase of larvae that could not pupate were observed in all seventeen colonies inspected. Approximately
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Table 1 Japanese M. plutonius isolates analyzed in this study. Source
Typical/atypicala
MLST resultsb
No. of strains/isolates
Individual and bulk diseased larval samples of European honeybees (A. mellifera)c
Typical
ST3 ST26d ST12e ST25d
23 3 31 1
ST3 ST4 ST12 ST27d
1 1 21 4
Atypical
Individual larval and pupal samples and the environments (honey and beehives) of Japanese honeybee (A. c. japonica) coloniesc with or without clinical signs of EFB
Typical Atypical
a M. plutonius isolates were classified into two types (typical and atypical) by cultural characteristics reported by Arai et al. (2012) and the duplex PCR developed by Arai et al. (2014). b MLST analysis was performed by sequencing four genes as described by Haynes et al. (2013). c All the honeybee colonies were managed by beekeepers. d Novel STs found in this study. The ST numbers were assigned through submission of the data to the M. plutonius MLST database (http://pubmlst.org/ mplutonius/) developed by Budge et al. (2014). e Although strain DAT561 isolated in Japan was reported as ST10 by Haynes et al. (2013), it was assigned into ST12 in this study.
four to five-day-old unsealed larvae were mainly affected, and some of them were displaced from their normal coiled position in the bottom and twisted around the walls or stretched out lengthways. Empty cells scattered randomly among the affected larvae were also observed. Dead larvae showed a light yellow or brown color and dissolved into a semi-liquid mass (Fig. 1). Some larval remains were dried and easily removed from the cells. Two of the seventeen
colonies were further examined in a regional veterinary diagnostic laboratory, and M. plutonius was detected from individual larvae of the two colonies, whereas other bee pathogens including Paenibacillus larvae (the causative agent of American foulbrood), sacbrood virus (the causative agent of sacbrood disease), and Acarapis woodi (tracheal mite; the causative agent of acarine disease) were not detected from the larval samples tested. On the basis of
Table 2 Updated MLST typing scheme and distribution of M. plutonius strains/isolates analyzed so far. STa
Profileb
No. of strains/isolatesc
Origin (No. of strains/isolates)c
England and Wales (2), Italy (1), Scotland (1), Thailand (2) England and Wales (13), Scotland (1) England and Wales (67), France (1), Italy (2), Japan (24), Paraguay (1), Republic of Ireland (1), The Netherlands (1), USA (5) Australia (1), France (4), Japan (1) England and Wales (60) England and Wales (5) England and Wales (9) England and Wales (4) England and Wales (2) England and Wales (1), USA (1) England and Wales (8) England and Wales (1), Japan (52), USA (1) Denmark (1), England and Wales (19), Poland (3) Poland (1) India (1) Brazil (1) Tanzania (1) Scotland (1) The Netherlands (1) England and Wales (2) England and Wales (6) England and Wales (4) England and Wales (29) England and Wales (2) Japan (1) Japan (3) Japan (4)
argE
galK
gbpB
purR
1 2 3
1 2 2
1 3 3
1 2 2
1 2 4
6 14 102
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1 2 2 2 1 1 4 3 4 1 1 5 4 1 1 4 1 4 2 2 2 6 1 4
1 3 2 3 1 1 4 3 4 1 5 1 6 3 1 4 1 4 7 3 3 4 1 4
2 4 2 5 7 6 3 4 3 1 8 1 9 1 2 10 8 11 2 12 13 3 5 14
4 4 2 4 3 4 4 4 5 4 4 4 4 4 1 5 4 5 2 4 4 5 4 5
6 60 5 9 4 2 2d 8 54d 23 1 1 1 1 1 1 2 6 4 29 2 1 3 4
a
Sequence type. argE, putative acetylornithine deacetylase gene; galK, putative galactokinase gene; gbpB, putative secreted antigen gene; purR, putative purine operon repressor gene. c Eighty-five Japanese and one Paraguayan isolates were analyzed in this study, and the other data was from previous studies (Haynes et al., 2013; Budge et al., 2014). d Although strain DAT561 isolated in Japan was reported as ST10 by Haynes et al. (2013), it was assigned into ST12 in this study. b
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Fig. 1. A representative beehive and a dead larva of Japanese honeybee (A. c. japonica) affected by M. plutonius infection.
clinical symptoms of classic EFB observed in the affected colonies and larvae and isolation of M. plutonius from the larvae, the abnormal colonies were diagnosed with EFB. The beekeeper placed beehives for Japanese honeybees at several different locations in an area within an approximately 2.5-kilometer radius. Because similar symptoms were also observed in some of the other colonies at different locations, first follow-up examination was performed in early June for all 119 colonies in the area by visual inspection, M. plutonius-specific PCR, and isolation of the causative strains. Colonies from which M. plutonius was detected were destroyed. In late June, second follow-up examination was performed for 13 colonies that showed clinical signs of EFB such as those described above, and colonies from which M. plutonius was detected were destroyed. Consequently, EFB in the Japanese honeybee colonies had ended by early August. Although an apiary managing nine European honeybee colonies was located at a distance of two kilometers from the apiary inspected in May, no clinical signs were observed in these colonies, and M. plutonius isolation was negative. During the examinations, in total, 27 M. plutonius isolates were isolated from 13 individual larvae, one pupa, one honey sample, and 12 beehives of the colonies with or without clinical signs of EFB in five apiaries (apiaries A–E) (Table 1 and Table S1). Interestingly, two and twenty-five isolates were identified as typical and atypical M. plutonius, respectively, on the basis of the cultural characteristics (Arai et al., 2012) and the results of the duplex PCR assay (Arai et al., 2014). In addition, the two typical isolates were further divided into ST3 and ST4 by MLST, and 21 and 4 atypical isolates were assigned into ST12 and ST27 (a novel single-locus variant of ST12, which has a novel allele sequence of gbpB [gbpB-14; DDBJ/EMBL/GenBank accession number, AB853997]), respectively (Tables 1 and 2, and Table S1); that is, at least four genetically different M. plutonius strains were prevalent in the Japanese honeybee colonies raised in a limited area by the same beekeeper. Although only a single ST was detected from each colony by culture methods in this case, more than one ST was
found to be present in a single affected apiary (e.g., DAT869 of ST4, DAT870 of ST27, and DAT872 of ST12 in apiary A; DAT879 of ST12 and DAT885 of ST27 in apiary C) (Table S1). Because the beekeeper has moved some beehives and captured swarms between different apiaries, this might contribute to the spread of different strains among different apiaries. Arai et al. (2014) recently reported that both typical and atypical M. plutonius were detected from approximately 50% of larval samples collected from European honeybee colonies with clinical signs of EFB in Japan. In the study, each sample contained several larvae, but all the larvae in a single sample were retrieved from a single colony. Therefore, regardless of honeybee species, the participation of several different strains in a single outbreak may be typical for EFB at least in Japan. In this case, atypical M. plutonius was isolated more frequently than typical M. plutonius. Because atypical M. plutonius grows more rapidly and forms larger colonies than typical M. plutonius (Arai et al., 2012), if both types are present in a single sample, typical M. plutonius tends to be missed due to its small colony size or the growth of atypical strains over the small colonies of typical strains (Arai et al., 2014). Therefore, typical strains might be more prevalent in the Japanese honeybee colonies than detected. The two most common STs (ST3 and ST12) in M. plutonius isolates from European honeybees in Japan were also found in the isolates from Japanese honeybees or their environment. In addition, although ST4 found in an isolate from a larva in a diseased Japanese honeybee colony has not been isolated from European honeybees in Japan, this ST has been found in isolates from European honeybees (A. mellifera) in Australia and France (Haynes et al., 2013; http://pubmlst.org/mplutonius/). Because some isolates that showed different SmaI-digested profiles by pulsedfield gel electrophoresis in our previous study (Arai et al., 2012) were classified into the same ST in this study (e.g., DAT580 and DAT585 of ST26; DAT561, DAT571, and DAT607 of ST12) (Figure 1 in Arai et al., 2012 and Table S1), even if different isolates are found to be the same ST, they may have slightly different genetic backgrounds. However, our present results suggest that some M. plutonius strains may have the ability to cause EFB in different honeybee species. Of note, the analyzed M. plutonius isolates from Japanese honeybees and those from European honeybees were isolated in different prefectures of Japan. Therefore, it remains unclear at present whether the transmission of M. plutonius between the two honeybee species has occurred in Japan. 3.3. Population structure of Japanese and international M. plutonius isolates In addition to Japanese M. plutonius isolates, a typical M. plutonius isolate (DAT569) from a diseased European honeybee larva in Paraguay was analyzed by MLST and found to be ST3 (Table S1). Consequently, a total of 352 M. plutonius strains/isolates from 16 countries and regions analyzed in this and previous (Haynes et al., 2013; Budge et al., 2014) studies were resolved into 27 STs (Table 2 and Table S1). Although three novel STs were found in this study, the overall topology of the goeBURST tree was
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Fig. 2. Updated goeBURST tree of STs found in M. plutonius isolates from different countries and honeybee species. MLST data of 86 isolates from the current study were added to those reported previously (61 isolates from Haynes et al., 2013 and 205 isolates from Budge et al., 2014) (Table S1). Each circle represents a different ST, with lines linking closest relatives. Black lines indicate a single allelic change between STs. Light gray lines indicate differences at two loci. Circles ringed with a green outline indicate putative founder genotypes. Colors within circles show the proportion of isolates of a particular type that were found in the countries/honeybee species indicated.
almost the same as that constructed in previous studies (Haynes et al., 2013; Budge et al., 2014) (Fig. 2), and all Japanese atypical isolates (ST12, ST25 and ST27) belonged to a group corresponding to the atypical group (Haynes et al., 2013) or CC12 (Budge et al., 2014) in previous studies with isolates from the UK, the USA, Brazil, and the Netherlands. Of 20 STs that include multiple isolates, seven were found to contain isolates from more than one country or region (Table 2 and Fig. 2). Among the seven STs, three (ST3, ST4, and ST12) were identified in Japan. In particular, ST3 was the most globally distributed type and found in eight countries. As described above, ST4 found in a Japanese honeybee larva in Japan was identified in France and Australia (Haynes et al., 2013). In addition, ST12 found in both Japanese and European honeybee colonies in Japan was found in the USA and the UK (Table 2, Table S1, and Fig. 2) (Haynes et al., 2013; Budge et al., 2014). Moreover, although ST26 has been identified only in Japan, this ST was a single-locus variant of ST13, and ST13 was found in Denmark, Poland, and the UK (Haynes et al., 2013; Budge et al., 2014).
4. Conclusion In this study, we analyzed M. plutonius isolates isolated from European and Japanese honeybees in Japan by MLST and showed the spread of some STs across borders and honeybee species. European honeybees are now commonly used in apiculture globally and, in areas where different honeybee species may interact, cross-species pathogen transmission could occur. Varroa destructor, which was transmitted from the Eastern honeybee A. cerana to the European honeybee A. mellifera, is a well-known example (Oldroyd, 1999; Rosenkranz et al., 2010). After the host shift, the mite has spread worldwide and is almost
cosmopolitan today (Oldroyd, 1999; Ellis and Munn, 2005; Rosenkranz et al., 2010). Recently, Kojima et al. (2011) suggested possible transmission of Israeli acute paralysis virus between European and Japanese honeybees in Japan on the basis of the sequence data of viruses detected from Japanese honeybee colonies and neighboring European honeybee colonies. However, as described above, it is not clear at present whether the cross-species transmission of M. plutonius has indeed occurred in Japan. Even if it has, the direction of the transmission between European and Japanese honeybees is unclear. In addition, epidemiological relationships among M. plutonius isolates of the same ST found in different countries are unknown. For a more comprehensive understanding of the ecology and epidemiology of M. plutonius including possible crossspecies transmission and international spread of specific M. plutonius strains, more extensive studies of different honeybee species from geographically diverse regions and detailed characterization of isolated M. plutonius strains will be necessary. Acknowledgements A part of this study was supported by a Grant-in-Aid for Scientific Research (B) (25292200) from the Japan Society for the Promotion of Science. This paper made use of the M. plutonius MLST database (http://pubmlst.org/mplutonius/) (Budge et al., 2014) curated by Giles E. Budge. References Allen, M.F., Ball, B.V., Underwood, B.A., 1990. An isolate of Melissococcus pluton from Apis laboriosa. J. Invertebr. Pathol. 55, 439–440. Arai, R., Miyoshi-Akiyama, T., Okumura, K., Morinaga, Y., Wu, M., Sugimura, Y., Yoshiyama, M., Okura, M., Kirikae, T., Takamatsu, D., 2014. Development of duplex PCR assay for detection and differentiation of typical and atypical Melissococcus plutonius strains. J. Vet. Med. Sci. 76, 491–498.
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