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INNOVATIVE THERAPEUTIC APPROACHES FOR THE CONTROL AND PREVENTION OF HONEYBEE NOSEMOSIS: IMPLICATIONS IN THE BEE'S LIFE AND THE QUALITY OF THEIR PRODUCTS.

1. INTRODUCTION

The importance of bees and their involvement in natural ecosystems, in agricultural crops and human health are known from ancient times worldwide. Among the factors involved in bee colony loss, the parasitosis and their spread in the world are one of the most serious problems affecting modern beekeeping nowadays. Honeybee nosemosis, produced by Nosema ceranae and Nosema apis, is a serious disease which develops diarrhea, reducing honey production, increased mortality in winter and underdeveloped colony passed through illness (Brenna, E., Fell, R., 2011, Cox-Foster, D. et al., 2007, Fries, I., 2007, Higes, I. et al., 2010).

The disease has an enzootic character, with acute clinical evolution at the end of the cold season, being influenced by intrinsic and extrinsic factors interacting with each other (Balint, A., 2011).

The disease develops worldwide, the infestation between colonies is easy, so diagnosis of infection levels and preventive and curative treatments, applied rationally and on time, are absolutely necessary (Mederle, Narcisa et al., 2014).

For treatment, more than 200 compounds were tested, but only fumagillin and several organic compounds of mercury gave acceptable results. The organic compounds of mercury have reduced efficacy against parasites and are more toxic to bees than fumagillin. Fumagillin is an antibiotic isolated from cultures of Aspergillus fumigatus, which inhibits parasite development but shows some disadvantages: Nosema spores (element infestation of bees) does not affect; residues in bee products (Botias, C. et al., 2013, Giacobino, A. et al., 2016, Mendoza, Y. et al., 2017).

According to EU requirements, in our country it is mandatory to give up allopathic therapies that leave residues in these products and thus to find natural remedies that are effective, but which do not affect the hive (Balint, A., 2011, Mederle, Narcisa et al., 2014, Mederle, Narcisa et al., 2015, Mederle, Narcisa et al., 2017).

In this context, we have evaluated the therapeutic efficacy of an original natural product and to determine the role of the product components in the bee's life and their products.

2. MATERIALS AND METHODS

The medical steps were: identification of the apiary and establishment the studied lots; nosemosis diagnosis using the qualitative method; therapeutic product administration after a predetermined protocol; statistical processing of results.

The study was performed in Romania, in July and August, to an apiary from Timis County consisting of 10 colonies of bees (600 bee samples). Bee samples were examined in the Parasitology Laboratory--Faculty of Veterinary Medicine Timisoara by qualitative method.

Bee colonies were divided into 2 groups: group I treated with plant dietary supplement and group 2 - control group that received only sugar syrup 1: 1. From each family were collected samples bee before and after treatment and were kept in alcohol 60[degrees]. In order to establish the level of infection with Nosema spp., before and after treatment, were examined individually bee intestines through macroscopic and microscopic examinations. For the microscopic qualitative examination of the preparation between the slides, the 40 X magnification was used.

The qualitative examination method using dead or live bees followed to establish the levels of bees infestation: +++ numerous spores that occupy the entire microscopic field and in each microscopic field or massive infection (fig 1); ++ one or more spores in each microscopic field or moderate infection; + one or more spores, but not in each microscopic field or poor infection; - absence of spores or lack of infection.

Group I was treated with plant dietary supplement. The plant dietary supplement of the present invention contains sugar syrup and herbal infusion: nettle, chamomile, yarrow, thyme, rattles and mint. This invention is based on the use of conventional active principles from different herbs and digestive tract content acidification of bees with vinegar.

The plant dietary supplement is administered in therapeutically nutritious at 200 ml/ colony in the first treatment day and 100 ml/colony in the second and third therapeutic activity. For spraying inside the hive, apple vinegar, fresh grape wine and garlic tincture are added to the dietary supplement. The plant dietary supplement is a Patent application A000113 / 14.02.2014, OSIM registered (fig. 2).

Group II was the control group that received only sugar syrup 1: 1.

Infestation levels were correlated with symptoms more or less obvious (fig. 3), and the data obtained before and after treatment were statistically analyzed using the Microsoft Office Excel.

3. RESULTS AND DISCUSSIONS

Bee samples collected were positive with variations in the level of infestation by Nosema from + (poor infestation) to +++ (massive infestation). Table 1 presents the results of the honeybee samples laboratory test belonging to the two groups before and after treatment.

Following administration of the Plant dietary supplement there is a remarkable increase in negative samples and free of Nosema colony (tables 2, 3).

Tables 3 and 4 that after the administration of Plant dietary supplement (PDS), the number of healthy bees increased from an average of 22.8 to 39.4 after treatment, the difference being statistically significant (P = 0.006). On average, the number of bees with mild infection decreased significantly from 30.6 to 20.6 (P = 0.01). The number of bees with ++ infection decreased from 6 to 1.2, but not statistically significant. After treatment, bees with strong infection were not identified (Table 4).

In Tables 5, 6 and 7, the levels of Nosema infection in the control group before and after consumption of syrup, where no significant differences were found during the experiment, are reported. The number of bees with infection level ++ increased from 5.8 to 10.2. There were no differences in heavily infected bees.

The research studies conducted in Argentina and Uruguay about nosemosis therapy with Fumagillin clarified that fumagillin treatment should be at least reviewed and that further research should be conducted to acquire a more complete perspective of Nosemosis disease (Giacobino, A. et al., 2016; Mendoza, Y. et al., 2017).

Considering that Fumagillin is the only veterinary medicament recommended by the World Organization for Animal Health (OIE) to suppress infections by Nosema, but the use of this antibiotic is prohibited in the European Union, Botias, C. et al., (2013) and Nanetti, A. et al. (2015) propose few therapeutic alternatives to control the disease: Nosestat[R], Phenyl salicylate, Vitafeed Gold and oxalic acid. The authors emphasized the low efficacy of those products, but , no remanence hive products. More over, in the absence of commercial products approved in several countries to control nosemosis, oxalic acid syrup appears promising in the development of alternative management strategies (Nanetti, A. et al., 2015).

In the same context, the study performed by Bravo, J. et al. (2017) showing that Cryptocarya alba oil may be a candidate for the treatment or prevention of nosemosis.

Other study highlights that the supplementation of honeybees' diet with improperly selected probiotics or both probiotics and prebiotics does not prevent nosemosis development, can de-regulate insect immune systems, and may significantly increase bee mortality (Ptaszynska, A. et al., 2016). In Romania, Balint, A. et al. (2011), Mederle, Narcisa et al. (2014), Mederle, Narcisa et al. (2015), Mederle, Narcisa et al. (2017) highligted that the plant extract products are the best therapy alternative to control nosemosis disease versus Fumagillin.

4. CONCLUSIONS

The plant dietary supplement used for prevention and control of nosemosis in bees contains only natural ingredients with anti-inflammatory, calming, astringent, antiseptic and antibiotic properties.

The dietary supplement is recommended as a good oportunity for beekeepers and veterinary doctors who can prevent or treat a very important desease of honeybees.

The risk of side-effects and residues in hive products is eliminated, is known as the residues can be dangerous to human's health.

REFERENCES

Balint, A., (2011), Parazitozele albinelor in vestul Romaniei. Strategii de control, teza de doctorat, USAMVBT, Facultatea de Medicina Veterinara, Timisoara.

Botias, C., Martin-Hernandez, R., Meana, A., Higes, M., (2013), Screening alternative therapies to control Nosemosis type C in honey bee (Apis mellifera iberiensis) colonies, Res Vet Sci., 95(3), 1041-5.

Bravo, J., Carbonell, V., Sepulveda, B., Delporte, C., Valdovinos, C., Martin-Hernandez, R., Higes, M., (2017), Antifungal activity of the essential oil obtained from Cryptocarya alba against infection in honey bees by Nosema ceranae, J Invertebr Pathol., 149, 141-147.

Brenna, E., Fell, R., (2011), Nosema ceranae in drone honey bees (Apis mellifera), J. Invert. Pathol., 107, 3, 234-236.

Cox-Foster, D., Conlan, S., Holmes, E., Palacios, G., Evans, J., Moran, N., (2007), A metagenomic survey of microbes in honey bee colony collapse disorder, Science, 318, 283-287.

Fries, I., (2007), Nosema ceranae has infected Apis mellifera Europe in at least 1998 and may be more virulent than Nosema apis, Apidologie, 38, 558-565.

Giacobino, A., Rivero, R., Molineri, A., Cagnolo, N., Merke, J., Orellano, E., Salto, C., Signorini, M., (2016), Fumagillin control of Nosema ceranae (Microsporidia:Nosematidae) infection in honey bee (Hymenoptera:Apidae) colonies in Argentina, Vet Ital., 30; 52(2),145-51.

Higes, M., Hernandez, R., M., Meana, A., (2010), Nosema ceranae in Europe: an emergent type C nosemosis, Apidologie, 41, 3, 375-392.

Mederle, Narcisa, Morariu, S., Darabus, Gh., Bogdan, A., Craciun, C., (2014), Supliment alimentar din plante utilizat in prevenirea si combaterea nosemozei la albine, Cerere de brevet Nr. A/00113 din 14.02.2014.

Mederle, Narcisa, Balint, A., Morariu, S., Hora, F.S., Mederle, O., Marincu, I., Darabus, Gh., (2015), Research on the prevalence of honey bees nosemosis in Arad County, J. of Biotechnology, 208, S5-S 120.

Mederle, Narcisa, Kaya, A., Balint, A., Morariu, S., Oprescu, I., Ilie, M., Imre, Mirela, Ciobanu, Gh., Darabus, Gh., (2017), Epidemiological investigations on honey bees nosemosis in Timis County, Lucr. Stiint. Med. Vet. Timisoara, L (2), 142-147.

Mendoza, Y., Diaz-Cetti, S., Ramallo, G., Santos, E., Porrini, M., Invernizzi, C., (2017), Nosema ceranae Winter Control: Study of the Effectiveness of Different Fumagillin Treatments and Consequences on the Strength of Honey Bee (Hymenoptera: Apidae) Colonies, J Econ Entomol.,1;110(1), 1-5.

Nanetti, A., Rodriguez-Garcia, C., Meana, A., Martin-Hernandez, R., Higes, M., (2015), Effect of oxalic acid on Nosema ceranae infection, Res Vet Sci., 102, 167-72.

Ptaszynska, A., Borsuk, G., Zdybicka-Barabas, A., Cytrynska, M., Malek, W., (2016), Are commercial probiotics and prebiotics effective in the treatment and prevention of honeybee nosemosis C? Parasitol Res., 115(1), 397-406.

Narcisa MEDERLE Banat's University of Agricultural Sciences and Veterinary Medicine "King Michael I of Romania", Timisoara, Romania Email: narcisa.mederle@yahoo.com

Alpaslan KAYA

"Mustafa Kemal" University, Antakya-Hatay, Turkey Email:dak1976@msn.com

Sorin MORARIU

Banat's University of Agricultural Sciences and Veterinary Medicine "King Michael I of Romania", Timisoara, Romania Email: sorin.morariu@fmvt.ro

Catalin CRACIUN

Banat's University of Agricultural Sciences and Veterinary Medicine "King Michael I of Romania", Timisoara, Romania Email: catalincraciun@yahoo.com

Adrian BALINT

Banat's University of Agricultural Sciences and Veterinary Medicine "King Michael I of Romania", Timisoara, Romania Email: balintadrian26@yahoo.com

Isidora RADULOV

Banat's University of Agricultural Sciences and Veterinary Medicine "King Michael I of Romania", Timisoara, Romania Email: isidoraradulov@yahoo.com

Gheorghe DARABUS

Banat's University of Agricultural Sciences and Veterinary Medicine "King Michael I of Romania", Timisoara, Romania Email: gheorghe.darabus@fmvt.ro
Table 1. The results of honeybee samples laboratory examination before
and after treatment

Group       Colony  Infestation level  Infestation level
            number  before treatment   after treatment
                    0   +   ++  +++    0   +   ++  +++

Group I     1       36  24   0  0      47  13   0  0
Plant       2       24  30   6  0      36  24   0  0
dietary     3       24  36   0  0      42  18   0  0
supplement  4       12  33  15  0      36  24   6  0
(PDS)       5       18  30   9  3      36  24   0  0
            1       24  36   0  0      21  39   0  0
Control     2       18  30   6  6      12  18  24  6
group       3       30  24   6  0      24  22  16  0
syrop       4       42  18   0  0      42  18   0  0
            5       21  19  17  3      23  22  11  3

Table 2. Descriptive statistics of group I before treatment with PDS

                          Infestation level
Biostatistic parameters   -         +        ++       +++

Medians                       22,8     30,6      6        0,6
Standard error             3,979    1,989    2,845        0,6
Standard deviation         8,899    4,449    6,363    1,341
Samples variance              79,2     19,8     40,5      1,8
Minimum values                12       24        0        0
Maximum values                36       36       15        3
Total                        114      153       30        3
Confidence level (95.0%)  11,050    5,525    7,901    1,665

Table 3. Descriptive statistics of group I after treatment with PDS

                          Infestation level
Biostatistic parameters   -        +        ++       +++

Medians                      39,4     20,6      1,2  0
Standard Error            2,227    2,227        1,2  0
Standard deviation        4,979    4,979    2,683    0
Samples variance             24,8     24,8      7,2  0
Minimum values               36       13        0    0
Maximum values               47       24        6    0
Total                       197      103        6    0
Confidence level (95,0%)  6,183    6,183    3,331    0

Table 4. P value calculated by test of group treated with PDS before and
after treatment

           Before treatment

After           -      +     ++     +++
treatment  -    0,006
           +           0,01
           ++                0,158
           +++                      0,346

Table 5. Descriptive statistics of control group before consumption the
syrup

                          Infestation level
Biostatistic parameters   -       +        ++       +++

Medians                       27     25,4      5,8      1,8
Standard error             4,242      3,4  3,104        1,2
Standard deviation         9,486  7,602    6,942    2,683
Samples variance              90     57.8     48,2      7,2
Minimum                       18     18        0        0
Maximum                       42     36       17        6
Total                        135    127       29        9
Confidence level (95,0%)  11,779  9,439    8,620    3,331

Table 6. Descriptive statistics of control group after consumption the
syrup
                          Infestation level
Biostatistic parameters   -         +         ++        +++

Medians                       24,4      23,8      10,2      1,8
Standard error             4,884     3,903     4,651        1,2
Standard deviance         10,924     8,729    10,401    2,683
Samples variance             119,3      76,2     108,2      7,2
Minimum                       12        18         0        0
Maximum                       42        39        24        6
Total                        122       119        51        9
Confidence level (95.0%)  13,562    10,838    12,915    3,331

Table 7. P value calculated by test of control group before and after
consumption the syrup

           Before treatment

After           -      +      ++     +++
treatment  -    0,698
           +           0,765
           ++                 0,454
           +++                       1
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Author:Mederle, Narcisa; Kaya, Alpaslan; Morariu, Sorin; Craciun, Catalin; Balint, Adrian; Radulov, Isidora
Publication:Geographica Timisiensis
Date:Sep 22, 2017
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