The effects of various antimicrobials and supplements on the expression of immune-related genes, oxidative stress and survival of honey bee Apis mellifera infected with microsporidium Nosema ceranae

Nosema ceranae je микроспоридијa којa паразитира код Европске медоносне пчеле, Apis mellifera, и доминантна је врста рода Nosema у југоисточној Европи. У зависности од степена инфекције и периода године, овај ендопаразит може имати далекосежне последице по пчеле, њихово здравље и продуктивност. Последњих година посебно је истраживан утицај N. ceranae на имунитет пчела а закључци појединих истраживања издвајају ефекат супресије гена значајних за имунитет пчела, односно доказују имуносупресивне последице инфекције врстом N. ceranae. Терапија ноземозе подразумева употребу антибиотика фумагилина, који има доказану ефикасност у сузбијању инфекције али његови споредни ефекти могу представљати проблем за саме пчеле и квалитет пчелињих производа, те је стога је његова примена у многим земљама ограничена. Имајући ово на уму константно се трага за природним и синтетским компонентама које би биле замена за фумагилин. Тестиране су многе супстанце, а међу онима које су показале најбољи ефекат издвајају се екстракти биљака и алги и синтетске витаминско-минералне мешавине. У овом раду је у кавезном експерименту тестирано пет препарата: (1) антибиотик фумагилин, (2) биљни екстракт тимол; и три адитива у исхрани пчела: (3) комерцијални препарат Beewell AminoPlus који представља витаминско-аминокиселинску мешавину, (4) комерцијални препарат Medenko forte који садржи екстракте храстове коре, пелена и жалфије и (5) полисахаридни екстракт гљиве Agaricus blazei. Пчеле су у свим третираним групама трећег дана живота инфициране спорама N. ceranae, а различите групе су третиране препаратима од првог, трећег и шестог дана. Код третираних пчела је праћен ефекат на преживљавање пчела, степен Nosema инфекције, нивое експресије гена значајних за имунитет пчела (абецин, дефензин, хименоптецин, апидецин и вителогенин) и ниво оксидативног стреса праћен кроз активност антиоксидативних ензима (каталазе, супероксид дизмутазе и глутатион С-трансферазе) и концентрације малондиалдехида...Apis mellifera, and the dominant Nosema species in Southeast Europe. Depending on the infection level it could have significant consequences on bees, their health and productivity. The impact of N. ceranae on honey bees’ immunity has been particularly investigated in recent years. Some conclusions of this research underline N. ceranae-induced suppression of immune-related genes, proving its immunosuppressive impact. Treatment for nosemosis includes the use of the antibiotic fumagillin. Fumagillin has proven effect in Nosema control, but its side effects may pose a problem for the bees and the quality of their products, which is why its application in many countries have been limited. Hence, researchers are constantly looking for natural and synthetic components which could be suitable substitutes for fumagillin. Many products have been tested and plant/algae extracts and synthetic vitamin-mineral complexes showed the best effects. This laboratory (cage) experiment included testing of five products: (1) antibiotic fumagillin (2) a plant extract - thymol, and three dietary supplements: (3) a commercial amino acid and vitamin complex named Beewell AminoPlus, (4) a commercial mixture of oak bark, sage and absinth extracts named Medenko forte, and (5) a polysaccharide extract of Agaricus blazei mushroom. Bees in all treatment groups were infected with N. ceranae spores on day 3, and treated with the listed products from day 1, 3 or 6 after emergence. Bee survival, Nosema loads, levels of immune-related genes (abaecin, apidaecin, hymenoptaecin, defensin and vitellogenin) expression and the level of oxidative stress, assessed through the activity of antioxidative enzymes (catalase, glutathione S-transferase and superoxide dismutase) and the concentration of malondialdehyde, were measured in all experimental groups. The obtained values were compared with those of control (noninfected and infected) groups..

Uticaj različitih antimikrobnih preparata i aditiva na ekspresiju gena značajnih za imunitet, oksidativni stres i preživljavanje pčela Apis mellifera inficiranih mikrosporidijom Nosema ceranae

Nosema ceranae is a microsporidian endoparasite of the European honey bee, Apis mellifera, and the dominant Nosema species in Southeast Europe. Depending on the infection level it could have significant consequences on bees, their health and productivity. The impact of N. ceranae on honey bees’ immunity has been particularly investigated in recent years. Some conclusions of this research underline N. ceranae-induced suppression of immune-related genes, proving its immunosuppressive impact. Treatment for nosemosis includes the use of the antibiotic fumagillin. Fumagillin has proven effect in Nosema control, but its side effects may pose a problem for the bees and the quality of their products, which is why its application in many countries have been limited. Hence, researchers are constantly looking for natural and synthetic components which could be suitable substitutes for fumagillin. Many products have been tested and plant/algae extracts and synthetic vitamin-mineral complexes showed the best effects. This laboratory (cage) experiment included testing of five products: (1) antibiotic fumagillin (2) a plant extract - thymol, and three dietary supplements: (3) a commercial amino acid and vitamin complex named Beewell AminoPlus, (4) a commercial mixture of oak bark, sage and absinth extracts named Medenko forte, and (5) a polysaccharide extract of Agaricus blazei mushroom. Bees in all treatment groups were infected with N. ceranae spores on day 3, and treated with the listed products from day 1, 3 or 6 after emergence. Bee survival, Nosema loads, levels of immune-related genes (abaecin, apidaecin, hymenoptaecin, defensin and vitellogenin) expression and the level of oxidative stress, assessed through the activity of antioxidative enzymes (catalase, glutathione S-transferase and superoxide dismutase) and the concentration of malondialdehyde, were measured in all experimental groups. The obtained values were compared with those of control (noninfected and infected) groups...Nosema ceranae je mikrosporidija koja parazitira kod Evropske medonosne pčele, Apis mellifera, i dominantna je vrsta roda Nosema u jugoistočnoj Evropi. U zavisnosti od stepena infekcije i perioda godine, ovaj endoparazit može imati dalekosežne posledice po pčele, njihovo zdravlje i produktivnost. Poslednjih godina posebno je istraživan uticaj N. ceranae na imunitet pčela a zaključci pojedinih istraživanja izdvajaju efekat supresije gena značajnih za imunitet pčela, odnosno dokazuju imunosupresivne posledice infekcije vrstom N. ceranae. Terapija nozemoze podrazumeva upotrebu antibiotika fumagilina, koji ima dokazanu efikasnost u suzbijanju infekcije ali njegovi sporedni efekti mogu predstavljati problem za same pčele i kvalitet pčelinjih proizvoda, te je stoga je njegova primena u mnogim zemljama ograničena. Imajući ovo na umu konstantno se traga za prirodnim i sintetskim komponentama koje bi bile zamena za fumagilin. Testirane su mnoge supstance, a među onima koje su pokazale najbolji efekat izdvajaju se ekstrakti biljaka i algi i sintetske vitaminsko-mineralne mešavine. U ovom radu je u kaveznom eksperimentu testirano pet preparata: (1) antibiotik fumagilin, (2) biljni ekstrakt timol; i tri aditiva u ishrani pčela: (3) komercijalni preparat Beewell AminoPlus koji predstavlja vitaminsko-aminokiselinsku mešavinu, (4) komercijalni preparat Medenko forte koji sadrži ekstrakte hrastove kore, pelena i žalfije i (5) polisaharidni ekstrakt gljive Agaricus blazei. Pčele su u svim tretiranim grupama trećeg dana života inficirane sporama N. ceranae, a različite grupe su tretirane preparatima od prvog, trećeg i šestog dana. Kod tretiranih pčela je praćen efekat na preživljavanje pčela, stepen Nosema infekcije, nivoe ekspresije gena značajnih za imunitet pčela (abecin, defenzin, himenoptecin, apidecin i vitelogenin) i nivo oksidativnog stresa praćen kroz aktivnost antioksidativnih enzima (katalaze, superoksid dizmutaze i glutation S-transferaze) i koncentracije malondialdehida..

Oxidative Stress, Endoparasite Prevalence and Social Immunity in Bee Colonies Kept Traditionally vs. Those Kept for Commercial Purposes

Commercially and traditionally managed bees were compared for oxidative stress (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and malondialdehyde (MDA)), the prevalence of parasites (Lotmaria passim, Crithidia mellificae and Nosema ceranae/apis) and social immunity (glucose oxidase gene expression). The research was conducted on Pester plateau (Serbia—the Balkan Peninsula), on seemingly healthy colonies. Significant differences in CAT, GST and SOD activities (p p L. passim and N. ceranae was significantly (p p L. passim was detected in honey bee brood. In commercial colonies, the prevalence of L. passim was significantly (p p < 0.01) higher, which probably results from their increased need to strengthen their social immunity. Commercially kept colonies were under higher oxidative stress, had higher parasite burdens and higher GOX gene transcript levels. It may be assumed that anthropogenic influence contributed to these differences, but further investigations are necessary to confirm that

Exposure of honey bee larvae to thiamethoxam and its interaction with Nosema ceranae infection in adult honey bees

During their lifetime honey bees (Apis mellifera) rarely experience optimal conditions. Sometimes, a simultaneous action of multiple stressors, natural and chemical, results in even greater effect than of any stressor alone. Therefore, integrative investigations of different factors affecting honey bees have to be carried out. In this study, adult honey bees exposed to thiamethoxam in larval and/or adult stage and infected with Nosema ceranae were examined. Newly emerged bees from colonies, non-treated or treated with thiamethoxam, were organized in six groups and kept in cages. Thiamethoxam treated bees were further exposed to either thiamethoxam or Nosema (groups TT and TN), or simultaneously to both (group TTN). Newly emerged bees from non-treated colonies were exposed to Nosema (group CN). From both, treated and non-treated colonies two groups were organized and further fed only with sugar solution (groups C and TC). Here, we present the expression profile of 19 genes in adult worker honey bees comprising those involved in immune, detoxification, development and apoptosis response. Results showed that gene expression patterns changed with time and depended on the treatment. In group TC at the time of emergence the majority of tested genes were downregulated, among which nine were significantly altered. The same gene pattern was observed on day six, where the only significantly upregulated gene was defensin-1. On day nine most of analyzed genes in all experimental groups showed upregulation compared to control group, where upregulation of antimicrobial peptide genes abaecin, defensin-1 and defensin-2 was significant in groups TT and TTN. On day 15 we observed a similar pattern of expression in groups TC and TT exposed to thiamethoxam only, where most of the detoxification genes were downregulated. Additionally RNA loads of Nosema and honey bee viruses were recorded. We detected a synergistic interaction of thiamethoxam and Nosema, reflected in lowest honey bee survival

Efficacy of plant-derived formulation argus ras in varroa destructor control

Varroa destructor is the most important honey bee parasite. There are various methods used in the control of this mite, but none of them meets all requested criteria, to be safe, effective and easy to apply. The objective of this study was to evaluate the varroacidal efficacy of newly created plant-derived formulation Argus Ras (mixture of extracts of Sophora flavescens, Ginkgo biloba, Gleditsia chinensis and Teucrium chamaedrys) in a field trial. The investigation was conducted on 240 Apis mellifera colonies equalized in respect of brood amount, adult bee population and food reserves. Efficiency was evaluated by applying Argus Ras consecutively with two other acaricides, amitraz and oxalic acid. Average acaricidal efficacy of Argus Ras was 80.89%, being higher of other previously tested essential oils. Besides, it showed a potential in knocking down the mites resistant to other acaricides. It should not be neglected that Argus Ras requires a smaller number of treatments and financial investments than other formulations used for the control of Varroa mites

Retrospective analysis of the bluetongue outbreak in Serbia

Bluetongue, a vector-born disease caused by the Bluetongue virus (BTV) and transmitted by Culicoides biting midges, is considered to be one of the most important diseases of domestic ruminants. The first outbreak of bluetongue in Serbia was reported in 2001, when BTV serotype 9 was identified in sampled materials. In 2014, outbreak of BTV-4 in Serbia caused considerable economic losses affecting sheep, cattle and goats. During this outbreak, BTV-4 was recorded in 644 outbreaks within 49 municipalities, part of 17 administrative regions. From the total number of sheep kept in areas affected by bluetongue (n=1 748 110), 2 083 cases (0.2%) were proven to be BTV-4 infected. Total of 206 infected cattle and 24 infected goats were reported during this investigation period, which represents 0.06% and 0.03% of the total number of cattle and goats kept in affected areas, respectively. The highest incidence of infected sheep, cattle and goats was recorded on the territory covered by veterinary institute of Nis. Recorded lethality in cattle, sheep and goats was 18.45% (n=38), 48.10% (n=1002) and 54.17% (n=13), respectively. The peak of the outbreak was in September and October when 94.43% of the confirmed positive cases, regardless of the species, was recorded. Monitoring of bluetongue disease in Serbia relies on active surveillance programmes aimed at: (i) identification and tracing of susceptible and potentially infected animals and (ii) detection, distribution and prevalence of insect vectors. Vaccination of sheep is planned to be implemented as a control measure against bluetongue in Serbia

Improved DNA-based identification of cervidae species in forensic investigations

The main reasons for wildlife forensic research are animal poaching, illegal trade, and falsified game meat products. Small trace amounts, old and degraded materials present the most common samples in revealing criminal activities in this field. This is the reason why it is crucial to use adequate and reliable methods and samples to identify animal species killed outside the hunting season or species protected by law. In this study, different endpoint PCR and real-time PCR protocols were compared in the identification of three Cervidae species (Capreolus capreolus, Cervus elaphus, Dama dama) from old and damaged material found in an enclosed area where the animals were kept. From a total of 129 samples, end point PCR provided results for 119 samples, while real-time PCR was successful in all cases. Also, we created and tested a protocol for simultaneous analyses of different types of samples, which is of great importance as when the amplification is carried out simultaneously it is more cost efficient and speeds up the process

Clinical babesiosis and molecular identification of babesia canis and babesia gibsoni infections in dogs from Serbia

Canine babesiosis is a frequent and clinically significant tick-borne disease. Sixty symptomatic dogs with clinical findings compatible with babesiosis were included in this study conducted in Serbia. After clinical examination, blood samples were taken for microscopic examination, complete blood count (CBC), Canine SNAP 4Dx Test, DNA analyses and sequencing. The main clinical signs included apathy, anorexia, fever, brown/red discoloration of urine, pale mucous membranes, icterus, splenomegaly, and vomiting. The main clinicopathological findings in Babesia infections were a slight to severe thrombocytopenia and a mild to very severe normocytic normochromic anaemia. Microscopic evaluation revealed 58 positive samples with the presence of large and small intraerythrocytic piroplasms in 57 and 1 sample(s), respectively. No co-infections were found using SNAP test. Two Babesia species, B. canis (58/60) and B. gibsoni (2/60), were differentiated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Species identification was further confirmed by sequencing PCR products of B. gibsoni samples and six randomly selected B. canis samples. All dogs were treated with imidocarb dipropionate (6.6 mg/kg of body weight), given intramuscularly twice at an interval of 14 days. This report presents the first molecular evidence of the occurrence of B. gibsoni and B. canis, confirmed by DNA sequencing, in sick dogs from Serbia

Efikasnost kumafosa protiv varoe i njegov uticaj na oksidativni stres i preživljavanje medonosne pčele

Apart from the efficiency of coumaphos against Varroa mites, its impact on the oxidative status and survival of the honey bee (Apis mellifera) was assessed. The research was conducted on hives from the same apiary, equalised regarding the number of bees, brood area and food storage. Based on Varroa infestation the hives were allotted to two groups: Non-infested (N) and infested (I). Both groups were either treated (T)-NT and IT, or untreated (U)-NU and IU. The treatment of infested bees was controlled with a follow-up treatment with amitraz. The efficiency of coumaphos was 96-97%. This organophosphate had a negligible effect on bee survival, but it significantly affected their oxidative status: Superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) activities, and the concentrations of malonyl dialdehyde (MDA). Coumaphos significantly (pË 0.0001) decreased SOD activity in non-infested bees, but increased it in those infested. By contrast, both CAT and GST activities, as well as MDA concentrations significantly increased (from pË 0.05 to pË 0.0001) after treatment in all groups, with the exception of IT, where it declined. Coumaphos in non-infested hives caused oxidative stress per se, not unlike varroa in infested colonies. However, in infested colonies it decreased oxidative stress, owing to its efficacy against Varroa mites and contributed to the recovery of bee colonies. In spite of its certain downsides, coumaphos remains an effective anti-varroa substance, but should be used with precaution, not to add to the effects of environmental factors which may cause red-ox misbalance.Procenjivana je efikasnost kumafosa protiv varoe, kao i njegov uticaj na oksidativni status i preživljavanje medonosne pčele (Apis mellifera). Ispitivanje je obavljeno na košnicama iz istog pčelinjaka, ujednačenim po broju pčela, površini legla i količini uskladištene hrane. Na osnovu infestacije varoom, košnice su podeljene u dve grupe: neinfestirane (N) i infestirane (I). Obe grupe su bile ili tretirane (T) – NT i IT, ili netretirane (U) – NU i IU. Tretman infestiranih društava je kontrolisan naknadnom primenom amitraza. Efikasnost kumafosa iznosila je 96-97%. Ovaj organofosfat imao Zikic et al.: Anti-varroa efficiency of coumaphos and its influence on oxidative stress and survival of honey bees 373 je zanemarljiv efekat na preživljavanje pčela, ali je značajno uticao na njihov oksidativni status: aktivnosti superoksid-dismutaze (SOD), katalaze (CAT) i glutation Stransferaze (GST), i koncentracije malonil-dialdehida (MDA). Kumafos je značajno (p˂0,0001) smanjio aktivnost SOD kod neinfestiranih pčela, ali ju je povećao kod infestiranih. Za razliku od toga, aktivnosti CAT i GST, kao i koncentracije MDA značajno su se povećale (od p˂0,05 do p˂0,0001) posle tretmana u svim grupama, sa izuzetkom IT, u kojoj su opale. Kumafos je sam po sebi u neinfestiranim društvima prouzrokovao oksidativni stres kod pčela, slično kao Varroa u infestiranim. Međutim, u infestiranim košnicama on je smanjio oksidativni stress zahvaljujući efikasnosti protiv varoe, čime je doprineo oporavku društava. Uprkos određenim nedostacima, kumafos ostaje efikasna supstanca u borbi protiv varoe, ali ga treba koristiti sa oprezom, da bi se izbeglo dodatno opterećenje pčela prouzrokovano faktorima sredine koji mogu da izazovu red-oks neravnotežu

Experimental evaluation of infection, dissemination, and transmission rates for two West Nile virus strains in European Aedes japonicus under a fluctuating temperature regime

West Nile virus (WNV) is continuously spreading in Eastern and Southern Europe. However, the extent of vector competence of Aedes japonicus (Theobald, 1901) is controversial. In this work, we elucidated the dynamics of virus growth in this invasive mosquito species. Females of Ae. japonicus were reared from eggs collected in the field in Switzerland and fed on bovine blood spiked with two WNV lineage 1 strains (FIN, Italy; NY99, USA). Fully engorged females were incubated for 14 days under a fluctuating temperature regime of 24 ± 7 °C (average 24 °C), 45–90% relative humidity, which is realistic for a Central European mid-summer day. Infection, dissemination, and transmission rates were assessed from individual mosquitoes by analyzing the abdomen, legs and wings, and saliva for the presence of viral RNA. Saliva was also investigated for the presence of infectious virus particles. Overall, 302 females were exposed to WNV strain FIN and 293 to strain NY99. A higher infection rate was observed for NY99 (57.4%) compared to FIN (30.4%) (p = 0.003). There was no statistical evidence that the dissemination rate (viral RNA in legs and wings) was different between females infected with FIN (57.1%) compared to NY99 (35.5%) (p = 0.16). Viral RNA load of FIN compared to NY99 was significantly higher in the hemocoel (p = 0.031) of exposed females but not at other sites (legs and wings, saliva). This is the first study describing the vector competence parameters for two WNV strains in a European population of Ae. japonicus. The high dissemination and transmission rates for WNV under a realistic temperature regime in Ae. japonicus together with recent findings on its opportunistic feeding behavior (mammals and birds) indicate its potential role in WNV transmission in Central Europe where it is highly abundant