Nosema ceranae, Silva, S. B. Slemenda & N. J. Pieniazek, 1996

Teixeira, Érica Weinstein, Guimarães-Cestaro, Lubiane, Alves, Maria Luisa Teles Marques Florêncio, Message, Dejair, Martins, Marta Fonseca, Luz, Cynthia Fernandes Pinto da & Serrão, José Eduardo, 2018, Spores of Paenibacillus larvae, Ascosphaera apis, Nosema ceranae and Nosema apis in bee products supervised by the Brazilian Federal Inspection Service, Revista Brasileira de Entomologia 62 (3), pp. 188-194 : 191-192

Nosema ceranae and N. apis

Our results showed that N. ceranae was the most prevalent pathogen, detected in 80% of royal jelly, 94.1% of honey and 85.7% of pollen samples, while N. apis was detected in 10% of royal jelly, 35% of honey and 28.6% of pollen samples ( Table 1).

N. ceranae was identified in Apis cerana ( Fries et al., 1996) and has rapid dissemination in A. mellifera , occurring in five continents ( Higes et al., 2006; Klee et al., 2007; Paxton et al., 2007; Fries, 2010). This dispersion may be due to transit of bees, either naturally transported legally or illegally, without accurate sanitary screening, in addition to trade in bee products contaminated with the microsporidium or by use contaminated wax. The importation of queens without health control also is a potential risk for pathogen dispersion.

In a long-term laboratory cage study, Williams et al. (2014) demonstrated that parasitism by Nosema , in particular by the invasive N. ceranae compared to the historic N. apis , increased honey bee worker mortality. They also observed higher spore intensity in honey bees parasitized by N. ceranae compared to N. apis , and a numerical response in spore production during co-infection; this is likely important to inter-host horizontal parasite transmission that relies on ingestion of spores. Recently, McGowan et al. (2016) showed that although the median infective dose of N. ceranae was 149 spores per bee, the minimum dose capable of causing a detectable infection was very low (1.28 spores), suggesting that differences in reproduction and intra-host competition may explain apparent heterogeneous exclusion of the historic parasite N. apis (Williams et al., 2014) . Therefore, even only a few spores in bee products could spread the pathogen and be one of the relevant forms of dispersion of the disease.

Teixeira et al. (2013), stated that simultaneous contamination with two species of Nosema in same colony is rare, since from 637 samples collected between 2009 and 2012, in 47 municipalities in 10 different Brazilian states, 0.63% were contaminated simultaneously with N. apis and N. ceranae , the former found in 0.31% of the samples.

Although this pathogen is present in several parts of the world ( Klee et al., 2007; Paxton et al., 2007; Fries, 2010), there are no official techniques for their detection in bee’s products indicated by the World Organization for Animal Health in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (OIE, 2016). Another widely used as reference is the Beebook, which is a collection of standard protocols for research on bee’s pathogens, where detection of Nosema spp. ( Fries et al., 2013) and of A. apis ( Jensen et al., 2013) in bee products are lacking.

Besides the PCR used here, Giersch et al.(2009) indicate centrifugation at 3000 × g for 45 min before PCR for detect Nosema spp. in honey. Although the technique here presented higher centrifugation time and speed, it is noteworthy that it is performed in order to simultaneously detect the three pathogens analyzed.

Spores of N. ceranae are found not only in midgut cells, but also in hypopharyngeal glands ( Chen and Huang, 2010) and in samples of royal jelly from China ( Cox-Foster et al., 2007). Our findings show that honey may be a dispersing vehicle of N. ceranae . Giersch et al. (2009) found this microsporidium in honey samples from Australia and some other countries and Hamiduzzaman et al. (2010) report detection of Nosema spp. in honey with molecular analyzes. Botias et al. (2012) stated a positive correlation between increase in N. ceranae infection and its occurrence in honey.

Differential immune activation may be involved if the higher dose triggered a stronger larval immune response that resulted in fewer adult spores but imposed a cost, reducing lifespan ( Eiri et al., 2015).

Paenibacillus larvae

In relation to occurrence of P. larvae in honey, the first molecular protocol that addresses the sensitivity of the technique, was provided by Bakonyi et al. (2003), where the sensitivity of PCR was evaluated by dilutions of DNA isolated directly from the bacterial colonies (thus requiring the use of culture media) and not directly from bee’s products. Although some spores detected by the molecular technique may be dead, it is a more practical, quick and lower cost analysis, indicating the presence of the pathogen and ensuring rapid decision-making by the Official Veterinary Agencies.

In artificially contaminated honey, Bassi et al. (2010) compared microbiological and molecular methods for the diagnosis of P. larvae . Both methods were sensitive to values greater than 8 CFU/mL of contaminated honey. The authors claim that PCR provides a fast rate to obtain result (<24 h) when compared to the microbiological method (eight days). In Brazil there are similar problem, considering that the official microbiological technique ( Brasil, 2003) adopted by the Ministry of Agriculture, to detect the pathogen in honey can take up to nine days. Our proposal contemplates the adaptation of multiplex PCR technique to identify different pathogens and their spores that affect bees with the use of conventional PCR technique.

In Brazil, the American foulbrood is considered an exotic disease, and one outbreak was officially detected in the state of Paraná at 2006 (MAPA, 2006) following its eradication. In this outbreak, samples were collected for analysis in 14 apiaries with 69 colonies contaminated following the burning of all 246 hives, without the identification of the origin of primary focus (E. Kruger, personal communication, SFA-PR/MAPA).

For detection of P. larvae bacteria in honey, the OIE (2016) indicates microbiological analysis with honey sample diluted in an equal volume of PBS and centrifuged at 6000 × g for 40 min. However, in the Brazilian official technique ( Brasil, 2003) the centrifugation is 3000 × g for 30 min. In the protocol proposed in this research samples were centrifuged at 12,500 × g for 40 min in order to detect not only P. larvae , but also Nosema spp. and A.apis , because use of samples with mild centrifugation have spores in the supernatant, and the aliquot analyzed was the pellet.

In pollen samples, we obtained a successful filtration of 10 g pollen with the aid of a vacuum pump. In the technique recommended by the OIE (2016), however, it is suggested to dilute 1 g of pollen in 10 mL of sterile distilled water and simple filtration with Whatman-1 filter paper. However the use of a vacuum pump allowed rapid filtration of greater pollen samples (10 g), resulting in high accuracy, because it increases the possibility of detection due to greater amount of sample analyzed as well as of smaller number of spores retained on filter paper, due to the use of vacuum pump. It is noteworthy that the filter paper used was Whatman-1 with 11 µm pores, in contrast with Whatman-2 indicated by Brazilian official technique with 8 µm pores, retaining more spores associated with pollen ( Higes et al., 2008b; Fries et al., 2013; Pettis et al., 2013).

For molecular detection of the bacterium in royal jelly, the OIE (2016) indicates the same methodology used for larvae, adult and wax, but samples of this product can be submitted to PCR after centrifugation of 6000 × g for 30 min using 1–5 µL of the supernatant as DNA template in a 50 µL PCR reaction. Our spore recovery tests, artificially inoculated in sterile royal jelly, showed that the speed of 12,500 × g for 40 min was better, with greater recovery of the pathogen in few amount (5 g) of royal jelly.

The contamination of 80% of the royal jelly samples with P. larvae and 50% with A. apis suggest that royal jelly has not antibiotic effect against spores of these two pathogens. The peptide royalisin found in the royal jelly has been reported as a potent antibiotic against gram-positive bacteria ( Fujiwara et al., 1990), P. larvae and fungus ( Bíliková et al., 2001), avoiding growth of bacteria and fungi ( Fujiwara et al., 1990; Bíliková et al., 2001; Fontana et al., 2004). However, the bee’s products here analyzed have spores and not by the vegetative forms.

It is expected that bee’s products such as honey and pollen from diseased hives from countries with occurrence of American foulbrood have P. larvae spores. Honey extracted from colonies infected with 25 million spores per gram of bee pollen has about 4.5 million spores per gram ( Gochnauer, 1981). However, the presence of P. larvae spores in honey formally marketed with Federal registration in the Brazil, where the disease is not recognized is not expected. Our molecular analyzes are not quantitative, but show the presence of the pathogen in the products, which suggests both the possibility of the disease in Brazilian apiary suppliers of such products (we must investigate if clinical signs are present or if spores are present, even in the absence of clinical signs), as well as that there may be commercialization of contaminated products imported. In the case of royal jelly, pollen analyses indicate the second hypothesis is more suitable because pollen grains in this bee’s product were from many exotic plants rarely cultivated or not cultivated in Brazil (unpublished data).

In the samples of honey and bee pollen contaminated, pollen analyses showed the presence only of native plants (unpublished data). Thus the contamination may be due mixtures of several samples in the warehouses. This fact may have impaired the evaluation of the original pollen spectrum, with predominance of the pollen types of native plants mixed. As the production of royal jelly in Brazil became economically unviable after the importation of Chinese royal jelly in 1990 decade, the analyzed samples are not mixed with Brazilian royal jelly .

The dispersion of spores by imported bee’s product without sanitary evaluation is the principal concern associated with routes of contamination: (i) food supplementation with infected products during periods of food resources decrease; (ii) use of contaminated royal jelly (purchased at low prices in trade from China) for queen production, (iii) handling of imported contaminated product and posterior use of tools in healthy colonies, (iv) bees’ access to debris of contaminated products discarded in the vicinity of colonies (e.g. pollen dryers equipment, where pollen from different places are dehydrated), and (v) use of contaminated wax. Current management practices such as the transfer of combs between colonies, as well as the use of the same tools and trophallaxis among bees could spread contamination too.

Future studies should also quantify pathogen levels, besides identify specific haplotypes to estimate potential risk of spread.