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Environmental Microbiology Reports (2012)
doi:10.1111/j.1758-2229.2012.00386.x
Temporal study of Nosema spp. in a cold climate Eva Forsgren* and Ingemar Fries Department of Ecology, Swedish University of Agricultural Sciences, PO Box 7044, SE-750 07 Uppsala, Sweden. Summary In a nationwide Swedish survey, 967 honey bee colonies from 521 beekeepers were sampled in the spring of 2007 and the samples assayed for Nosema spp. infections. Of the 319 positive samples, only 32 samples contained a proportion of N. ceranae DNA in mixed infections with both Nosema spp. above the cut-off point chosen for comparisons of 1%. Only one pure N. ceranae infection was found, with the rest 284 infected samples assayed being pure N. apis infections. In 2009 and 2011, beekeepers or bee inspectors providing N. ceranae mixed positive bee samples in 2007 were again asked to submit samples (2009, n = 96; 2011, n = 83). No trend of an increased proportion of N. ceranae-infected samples could be found. The proportion of N. ceranae DNA in samples with mixed infection did not increase between 2007 and 2011. It is concluded that N. apis is still the dominating Microsporidia infection in honey bees in Sweden and that there is no tendency for one species replacing the other. Introduction The microsporidian intestinal parasite Nosema ceranae was first described from the Asian honey bee Apis cerana in 1996 (Fries et al., 1996). Although it was known at this time that this parasite was also infective for the European honey bee, Apis mellifera, in laboratory studies (Fries, 1997), natural infections were believed to be restricted to the Asian honey bee. However, two independent observations in 2005 documented the occurrence of natural infections of N. ceranae in European honey bees, one in Taiwan from an apiary containing both A. cerana and A. mellifera (Huang et al., 2007) and one in Spain where sequencing of the 16S ribosomal RNA gene revealed that 10 out of 11 samples sequenced were identical to the initial deposition in 1996 in GenBank for this species (Higes et al., 2006). Klee and colleagues (2007) soon Received 21 March, 2012; accepted 9 August, 2012. *For correspondence. E-mail
[email protected]; Tel. (+46) 18 672083; Fax (+46) 18 672890.
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demonstrated that N. ceranae could be found in A. mellifera in South America, North America, Asia, and in many European countries. Later its presence had been confirmed from Australia (Giersch et al., 2009) and New Zealand (G. Munro, personal information, 2011). From available data, it appears likely that N. ceranae is a comparatively recent parasite of the European honey bee. Where this parasite first became a parasite in the field of A. mellifera will probably never be revealed. The oldest confirmed documentation to date of N. ceranae in A. mellifera comes from South America and pre-dates 1990 (Invernizzi et al., 2009). Several investigations using historical samples suggest a replacement process, where N. ceranae successively replace N. ceranae (Klee et al., 2007; Martin-Hernandez et al., 2007; Paxton et al., 2007) and in some areas this replacement appears to have occurred (Higes et al., 2009; Invernizzi et al., 2009; Stevanovic et al., 2011). However, in other areas no replacement seem to occur (Gisder et al., 2010) and it has been suggested that climate may influence disease epidemiology and alter transmission opportunities for N. ceranae compared with N. apis (Fries, 2010). Indeed, the viability of N. ceranae spores is significantly reduced following one week in a deep freezer, which is not the case for N. apis (Fries, 2010) and even storage at refrigerator temperature decreases viability of N. ceranae compared with N. apis (Gisder et al., 2010). This difference in temperature sensitivity between parasite species probably decreases transmission opportunities for N. ceranae in cold climate at least on wax exposed to freezing. The objective of this study was to survey and monitor the prevalence of N. ceranae infections in the Swedish honey bee population. Our study investigates the change in relative proportions over time of N. apis and N. ceranae infections in honey bee colonies in Sweden. The collected data are an extension of the BEE DOC project, studying temporal occurrence of Nosema spp. in different European climates. Results and discussion In the spring of 2007 (April and May), Swedish beekeepers and Swedish bee inspectors were asked to send in samples of adult bees to the honey bee pathology laboratory at the Swedish University of Agricultural Sciences, Uppsala, Sweden. The instructions were to collect bees from the top of the wintered hives before emerging bees would dominate the samples. The bee samples were
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frozen and stored at - 20°C until analysis. A total of 967 sampled colonies from 521 beekeepers were analysed by light microscopy for the presence of Microsporidia infections. Positive samples were stored as spore suspensions at - 20°C for later species determination using molecular tools as discussed below. Of the 967 samples from individual colonies analysed in 2007, 319 were positive for Microsporidia using light microscopy. Briefly, 60 bees from each sample were pooled, double-distilled water was added at a rate of 0.5 ml per bee, the bees were ground to a homogenate and checked under a light microscope at 400 ¥ magnification for the presence of Nosema spores. This sample size provides a 95% confidence of detecting a 5% infection level in the colony (Linder, 1947). Figure 1 illustrates the distribution and number of samples across Sweden, the number of samples with mixed infections of N. apis and N. ceranae and the number of samples positive for Microsporidia infections from the sampling in 2007. The distribution of beekeepers with samples on all three sampling occasions is also indicated. There are no samples from the four most northern counties, but the sampled counties represent approximately 95% of the honey bee colonies in Sweden (Anonymous, 2011). The data presented in Fig. 1 suggest that the proportion of samples with mixed infections may be higher in the most southern county (M = Skåne) compared with the pooled data for the rest of the country (P < 0.01, chi-square). Whether this is because of climatic differences, management differences or other differences is not known. The identification and quantification of the microscopically positive Nosema spp. was carried out using a quantitative real-time PCR method (as described in Forsgren and Fries, 2010). Briefly, 1 ml of the homogenates was subjected to DNA extraction and processed using the previously published SYBR-green assay. The amplified products were confirmed using melting curve analysis, and each set of PCR assays comprised serial dilutions of recombinant DNA templates of the N. apis and N. ceranae PCR fragments as external standards, covering a Log10 = 6 dynamic range. Of the 319 samples assayed using quantitative PCR, the analysis of two samples failed for technical reasons, 43 samples contained traces of N. ceranae DNA whereas only 32 samples contained a proportion of N. ceranae DNA in mixed samples above the cut-off point chosen for comparisons of both parasites of 1%. This cut-off point was chosen as a safeguard against false positive results. One case of pure N. ceranae infection was found, with the remaining 284 infected samples being pure N. apis infections. To monitor a possible change in the prevalence of N. ceranae over time, beekeepers with apiaries where N. ceranae was detected in 2007 were contacted and
asked to send in further samples of adult bees during April and May both in 2009 and 2011. Again, samples were assayed for Microsporidia infections using light microscopy, and positive samples stored for later species determination. In several cases, beekeepers sent in different number of samples in different years and some bee inspectors sent in samples from different beekeepers in his or her district in different years. Figure 2 presents the results from all mixed infection (samples containing a proportion of N. ceranae DNA in mixed infections) in samples received 2007, 2009 and 2011. In some cases (n = 10) it was possible to monitor samples from the same beekeepers over the whole period (2007, 2009 and 2011). Table 1 presents data on N. ceranae prevalence in mixed infections from the same beekeepers having N. ceranae positive samples in 2007. Different number of samples from different beekeepers may skew the interpretations of the results. Nevertheless, the complete absence of N. ceranae DNA in samples from 2011 is obvious. From Fig. 1 it can be seen that the beekeepers followed for the entire sampling period were distributed throughout several counties, including the most southern county with the highest prevalence in general of mixed infections. We have no explanation for the complete absence of mixed infections in these apiaries at the last sampling occasion in spite of previous findings of mixed infections; however, since N. ceranae is more sensitive to low temperatures (Fries, 2010; Gisder et al., 2010), one can speculate that cold winters may have had an impact. At several places in Sweden, 2010 is ranked as one of the 10 coldest in the last 110 years. For the country as a whole the year was the coldest since 1987 (http://www.smhi.se). The presented data cannot be interpreted as if N. ceranae is replacing N. apis in Sweden. It is striking that where we did receive samples from the same beekeepers for the whole sampling period, not a single N. ceranae mixed infection was found in 2011. This is in contrast to the data presented by Klee and colleagues (2007) where the hypothesis was forwarded that N. ceranae is replacing N. apis. Obviously, the parasite is present in the Swedish honey bee population, but the prevalence is low and there is no indication of an increase over time in the sampled material, as reported from southern Europe (Higes et al., 2009) or other parts of the world (Klee et al., 2007; Chen et al., 2008; Invernizzi et al., 2009; Stevanovic et al., 2011). In this survey, we found only one single case of pure N. ceranae infection, and the highest proportion of N. ceranae DNA reported in mixed infections was 0.21 in 2007, 0.65 in 2009 and 0.33 in 2011, in all cases with different beekeepers involved. Fries (2010) forwarded the hypothesis that climate differences could be important for determining N. ceranae
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
Temporal study of Nosema spp.
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Fig. 1. Map of Sweden with the county regions (sampling sites) indicated. The numbers in each county represent from left to right; number of N. ceranae infections (one pure N. ceranae isolate indicated with an asterisk)/samples positive for Nosema ssp. infection using light microscopy/total number of samples/number of beekeepers. The ‘N = ’ represents the number of beekeepers where samples were collected from the same beekeeper throughout the study (all three sampling occasions; 2007, 2009 and 2011). These figures are also presented in a table to help get an overview.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
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Fig. 2. Graph showing the proportion of Nosema apis and Nosema ceranae DNA respectively in infections where both species were detected.
epidemiology and impact. The hypothesis was based on results demonstrating that the spores of N. ceranae rapidly loose viability when exposed to freezing temperatures (Fenoy et al., 2009; Fries, 2010) and even when stored in sugar solutions in the refrigerator for rather short periods of time (Gisder et al., 2010). Indeed, differential responses between N. apis and N. ceranae to climate factors such as temperature and desiccation have been reported (Fenoy et al., 2009; Martin-Hernandez et al., 2009). Soiled comb is believed to be the primary source of infection for N. apis (Bailey, 1955). Disease transmission through soiled comb is possible since N. apis spores may remain viable in faecal deposits for more than a year (Bailey, 1962), however, the effect of time on the viability of N. ceranae spores in the hive environment is unknown. The typical association with dysenteriae in colonies infected by N. apis (Bailey, 1955; 1967) appears to be absent in N. ceranae infected colonies (Faucon, 2005; Fries et al., 2006; Higes et al., 2008). This observation suggests that the main mode of transmission within colonies may be different for N. ceranae compared with N. apis. Since freezing (Fries, 2010) and low temperatures (Gisder et al., 2010) significantly reduces N. ceranae viability, this may be important for reducing parasite Table 1. Number of samples (N) positive for Nosema spp. infection, proportion of samples with mixed infections of N. apis and N. ceranae and proportion of N. ceranae DNA in mixed infections for three years (2007, 2009 and 2011).
N Number of positive samples Proportion mixed samples Proportion N. ceranae DNA
2007
2009
2011
28 20 0.71 0.24
50 45 0.62 0.12
26 15 0 0
Samples collected from the same beekeepers (n = 10).
transmission similar to N. apis. Combs, even within parts of occupied bee hives, may be exposed to low temperatures posing a negative effect on N. ceranae spore viability. Nevertheless, relatively high incidence of N. ceranae is also reported from some, but not all, regions in Canada (Currie et al., 2010) as well as from Finland (S. Korpela, 2010, pers. comm.). In Germany, a 5-year cohort study revealed that N. ceranae has not replaced N. apis in the northern parts of the country, although the prevalence of N. ceranae has increased (Gisder et al., 2010). The conflicting data on N. ceranae prevalence and distribution in cold climates remain enigmatic. Possibly, bee trade has an impact on the Nosema spp. distribution patterns since trade with live bees from warmer climates has been common in both Canada and Finland, but only recently was allowed in Sweden and still is highly restricted in Norway where the prevalence picture is similar to what is found in Sweden (B. Dahle, 2010, pers. comm.).
Acknowledgements Surveys for Nosema ceranae prevalence had financial support from The Board of Agriculture through the EU-financed National program for production and sale of honey. The sampling in 2011 is included in the EU-funded 7th Framework project BEE DOC, Grant Agreement 244956 investigating Nosema ssp. prevalence in different climates in Europe.
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