Proizvodnja, praksa i stavovi pčelara u Hrvatskoj

We conducted a survey on the beekeeping production, practices and attitudes of 117 beekeepers in Croatia, via a questionnaire. The beekeepers were divided into three groups: full-time (professionals), part-time (side-liners) and hobby (hobbyists) beekeepers. The questions covered the purpose and size of their beekeeping production, the production interests of the beekeepers, treatment of the economically most important honeybee diseases, and the possibilities of the growth of their operation. Professional beekeepers were the youngest (39 years of age on average), with the largest average number of beehives per beekeeper (135 beehives), the largest proportion of LR hives and migratory apiaries, and the largest yearly per hive production of 17.04 kg of honey. The results show that the most marketable beekeeping product was honey, and that professional beekeepers also value propolis as equally interesting for production. They all inherited their beekeeping operations from their predecessors. Varroosis is perceived as the largest cause of honeybee colony death, followed by bad beekeeping practices. About 56% of the beekeepers are prepared to switch to organic/ecological beekeeping. Almost all the beekeepers (96.46%) support the introduction of the early diagnostics of American foulbrood. We found that professional beekeepers prefer the conservative economic model of growth based on their own equity, while hobbyists prefer a more expansive but riskier model of growth based on financial leverage (debt). Professional beekeepers are not inclined to create a mutual beekeepers’ fund to cover losses. On the other hand, they strongly support the idea of creating an alliance for the purpose of joint market penetration.Upitnikom smo proveli istraživanje o pčelarskoj proizvodnji, praksi i stavovima 117 pčelara u Hrvatskoj. Pčelari su bili podijeljeni u tri skupine: profesionalci, pčelari iz dopunske djelatnosti i hobisti. Pitanja su obuhvatila svrhu i veličinu pčelarske proizvodnje, proizvodne interese pčelara, liječenje ekonomski najvažnijih bolesti pčela i mogućnosti za rast djelatnosti. Profesionalni pčelari bili su najmlađi (u prosjeku 39 godina), imali su najveći prosječan broj košnica po pčelaru (135 košnica), imali su najveći udio LR košnica i pokretnih pčelinjaka te su imali i najveću proizvodnju meda po košnici od 17,04 kg. Rezultati pokazuju da je najprodavaniji pčelarski proizvod med, dok profesionalni pčelari i propolis smatraju jednako zanimljivim za proizvodnju. Za razliku od drugih skupina, svi su pčelarstvo naslijedili od svojih predaka. Varooza se smatra najvećim uzrokom uginuća zajednica, a slijedi je loša pčelarska praksa. Oko 56 % pčelara spremno je prijeći na ekološko (organsko) pčelarstvo. Gotovo svi pčelari (96,46 %) podržavaju uvođenje rane dijagnostike američke gnjiloće pčelinjeg legla. Utvrdili smo da profesionalni pčelari preferiraju konzervativni ekonomski model rasta temeljen na vlastitom kapitalu, dok hobisti preferiraju ekspanzivniji, ali i rizičniji model rasta koji se temelji na financijskoj poluzi (dugu). Profesionalni pčelari nisu skloni stvaranju zajedničkoga pčelarskog fonda za pokrivanje gubitaka. S druge strane, snažno podupiru ideju udruživanja sa svrhom zajedničkog prodora na tržište

EFFECT OF DISINFECTANTS ON BACTERIA PAENIBACILLUS LARVAE UNDER LABORATORY CONDITIONS

merička gnjiloća medonosne pčele (AGMP) je zarazna bolest nepoklopljenog i poklopljenog pčelinjeg legla koja pčelarstvu nanosi višestruke štete. Uzročnik bolesti je bakterija Paenibacillus larvae koja u nepovoljnim životnim uvjetima tvori dugo živuće i otporne spore. Patogeneza, klinička slika i stupanj virulentnosti kod AGMP ovise o genotipu bakterije P. larvae. Dosad je utvrđeno pet genotipova P. larvae (ERIC I, ERIC II, ERIC III, ERIC IV, ERIC V) koji se međusobno razlikuju u morfologiji, biokemijskim čimbenicima, virulenciji te čimbenicima koji utječu na virulenciju. Poznavanje raširenosti i dinamike pojavnosti pojedinih genotipova P. larvae na određenom području pruža uvid u patofiziološke procese na razini ličinke / pčelinje zajednice te utječe na procjenu rizika od AGMP jer postoji značajna korelacija između genotipa i pojavnosti vidljivih kliničkih znakova. Tvrdokoran tijek bolesti, otpornost uzročnika, poteškoće u kliničkoj dijagnostici i suzbijanju učinili su AGMP jednom od najtežih u svijetu. Bolesne pčelinje zajednice bez provedbe posebnih mjera ne mogu ozdraviti, a primjena antibiotika u liječenju bolesti nije dozvoljena zbog moguće pojave rezidua u pčelinjim proizvodima, rezistencije uzročnika te spoznaje da antibiotici djeluju samo na vegetativne oblike bakterije P. larvae, a ne uništavaju spore što doprinosi horizontalnom širenju bolesti u pčelinjacima. Ponekad je bolesnu pčelinju zajednicu najbolje sanirati spaljivanjem zajedno s košnicom i onečišćenim priborom. Nakon provedenih sanacijskih mjera nužno je primjeniti učinkovitu završnu dezinfekciju opreme, pribora i pčelinjaka čiji uspjeh ovisi o izboru dezinficijensa, preporučenoj koncentraciji radnih otopina, načinu i dužini trajanja aplikacije, vrsti mikroorganizama koji se moraju ukloniti te površini/materijalu koji se dezinficira. Cilj ovog istraživanja bila je provedba genotipizacije bakterije P. larvae na pedeset terenskih izolata izdvojenih iz karakteristično promijenjenih uginulih pčelinjih ličinki skupljenih u razdoblju od jedanaest godina (2010. – 2020.), te tako utvrditi raširenost i učestalost određenih genotipova u Republici Hrvatskoj (RH). Također, cilj je bio i utvrđivanje učinka deset komercijalno dostupnih i uobičajeno korištenih dezinficijensa u pčelarstvu, na terenske i certificirane sojeve bakterije P. larvae, u laboratorijskim uvjetima te uspoređivanje dobivenih rezultata među pojedinim genotipovima bakterije P. larvae. Genotip ERIC I bakterije P. larvae utvrđen je u pčelinjim zajednicama na području RH u visokoj prevalenciji od 90,3 %, a genotip ERIC II u niskoj prevalenciji od 7,3 % od ukupno uspješno analiziranih terenskihXI izolata. Za jedan izolat postavljena je sumnja na genotip ERIC IV s prevalencijom od 2,4 % te je potrebna daljnja verifikacija takvog nalaza. Istraživani su učinci Genoxa, Genolla s pjenom, Ecocid S, Sekusept aktiv, Incidin OxyFoam S, Bee Protect H forte, Bee Protect F, Despadac, Despadac Secure i EM® PROBIOTIK ZA PČELE u testu stvaranja zone inhibicije, suspenzijskom testu učinka na vijabilne bakterije P. larvae, testu dezinficijskog djelovanja na površinama i suspenzijskom testu učinka na spore bakterije P. larvae. Učinak dezinficijensa Genoxa na bakteriju P.larvae nije pokazao poželjan sporocidni profil zbog predugog vremena koje je potrebno da bi se ostvario sporocidni učinak te je isti limitiran dok proizvod Genoll s pjenom nije uopće pokazao sporocidno djelovanje. Proizvodi iz linije Despadac u suspenzijskom testu i u testu na površinama pokazali su baktericidno djelovanje, ali sporocidni učinak nije zadovoljavajući zbog slabijeg učinka u kontaktnom vremenu od 30 minuta. Suspenzijskim testom nije utvrđen zadovoljavajući sporocidni učinak Bee protect proizvoda, a proizvod EM® PROBIOTIK ZA PČELE nije pokazao u testu agar gel difuzije značajan baktericidni učinak. Sekusept aktiv u 2% koncentraciji i Incidin OxyFoam S u suspenzijskom testu sporocidnog djelovanja pokazali su zadovoljavajući sporocidni učinak na sva četiri genotipa bakterije P. larvae (ERIC I do ERIC IV).INTRODUCTION American foulbrood (AFB) is a contagious disease of sealed and unsealed honeybee brood that causes multiple damage to beekeeping. The causative agent of the disease is the bacterium Paenibacillus larvae, which in unfavorable life conditions forms long-lived and resistant spores. The infectious forms of P. larvae are spores, and susceptible to infection are honeybee larvae at the age when they are taking food. It takes only ten spores to infect one honeybee larvae younger than one day, but with the time passing by, susceptibility decreases and more than ten million spores are needed to infect a larva between four and five days old. Moreover, the number of spores required to cause infection in the later stages of honeybee larvae development is so high that a naturally infection is not possible. One dead larva can contain up to 2.5 billion newly created infectious spores. The pathogenesis, clinical signs, and degree of virulence in AFB depend on the P. larvae genotype. Recently, five genotypes of P. larvae (ERIC I, ERIC II, ERIC III, ERIC IV, ERIC V) have been identified, which differ in morphology, biochemical parameters, virulence, and factors influencing virulence. The virulence of P. larvae is conditioned by the possibility of infecting the honeybee larva and the time required until the death of the infected larva. The formation of a large amount of longlived and resistant spores, together with the possibility of multiplication and development of vegetative forms of the bacterium allows a high probability of P. larvae infection. The analysis of pathogenicity or virulence showed significant differences between genotypes ERIC I to ERIC V. The ERIC I genotype of P. larvae takes 12 days to cause death of all infected larvae (LT100 = 12 days), while the genotypes ERIC II to ERIC IV take only seven days. Therefore, according to the rate of larval death, these P. larvae genotypes are classified into three groups: ERIC I - slow leads to death of infected honeybee larvae, ERIC II - moderately fast leads to death of infected larvae, and ERIC III to ERIC V - are genotypes of pathogens that quickly lead to the death of the infected larva. The LT100 results for genotype ERIC II indicated that all infected larvae would die before the brood cells could be sealed. In this way, the adult honeybee workers have enough time to perform their hygienic skills, removing the dead larvae from brood cells. At the same time, the process of creating spores at the level of the honey bee colony would be disrupted, contributing to slow down the spread and development of the disease. However, the ERIC I genotype is less virulent at the level of a single larva because infected larvae die after sealing the cells. Consequently, the removal of dead larvae is reduced in such cases, and the possibility of producing and spreading the causative spores is significantly increased. Ultimately, genotype ERIC I is highly virulent for the honeybee colony leading to its rapid decline when compared to ERIC II which shows lower virulence at the honeybee colony level and slower decline of honeybee colony thereby showing a negative correlation of P. larvae virulence at the larval level, and consequently at the honeybee colony level. The knowledge of the distribution and dynamics of occurrence of individual P. larvae genotypes in a given area provides insight into pathophysiological processes at the level of the larva or honeybee colony and influences the risk assessment of AFB once there is a significant correlation between genotype and clinical signs. Vegetative rods of P. larvae have long, peritrichous arranged cilia that allow active movement in the form of a swarm motility. It was found that the P. larvae ERIC II genotype can move superficially in the form of a swarm and form a free-floating biofilm, while the P. larvae ERIC I genotype can form a biofilm but cannot move in the form of a swarm. These facts, that P. larvae is able to produce biofilm and actively move in the form of a swarm, requires new approaches in diagnostic and disenfection measures. The persistent course of the disease, resistance of pathogens, difficulties in clinical diagnosis and control, have made AFB one of the most difficult honeybee disease in the world. Infected honeybee colonies can hardly recover without implementation of special measures. The use of antibiotics in the treatment of diseases is prohibited due to the appearance of residues in honeybee products, resistance of the pathogen and the knowledge that antibiotics act only on vegetative forms of P. larvae, thereby providing horizontal spread of disease in apiaries. Most of the times, the best way to sanitate an infected honeybee colony it’s by burning it together with the hive and hive tools. During and after the implementation of eradication measures, it is necessary to carry out final disinfection, which success depends on the choice of effective disinfectant, recommended concentration of solutions, method and duration of application, type of microorganisms to be removed, surfaces and material to be disinfected. From the epizootiological point of view, disinfection can be preventive or focal. Preventive disinfection includes procedures and measures when infectious disease is not present in the apiary or its immediate surroundings. It is regularly carried out within the guidelines of good beekeeping practice and is an integral part of normal hygiene in the apiary. Keeping beehives, equipment, hive tools, food and water for bees clean is the basis of preventive disinfection. Focal disinfection is carried out when an infectious disease is present in the apiary and aims to remove microorganisms from the foci of infection, thus preventing its further spread. Depending on the method of execution, it can be continuous and final. Continuous disinfection involves systematic and repetitive procedures from the moment of the outbreak of infection in the apiary. It can be combined with veterinary administrative measures to cure diseases, such as burning bee colonies. The final disinfection has been considered as a one-step procedure after the implemented measures of disease remediation. Disinfection is often carried out by mechanical, physical and chemical procedures. Mechanical processes, such as cleaning, scraping, and washing, remove impurities and organic matter in which microorganisms are incorporated, thus facilitating the disinfection process. It has been observed that many disinfectants are ineffective in the presence of impurities and organic matter. Cleaning agents - detergents, soaps and abrasive powders - reduce the number of microorganisms, removing them from surfaces and objects. Physical disinfection procedures involve the use of moist or dry heat and radiation, where moist heat acts faster in a shorter period of time and is more efficient compared to dry heat. Burning hives, honeybee colonies and other accessories equipment and tools is an effective way of sanitation of AFB. Although the scorching process completely destroys the P. larvae spores on the surface of wooden hives, a significant number of infectious spores still remain active in internal wood structures. The wood fibers behave like organic matter which, already in a concentration of 2 %, significantly reduces the effect of surface disinfection. Boiling in water under normal pressure for thirty minutes with the addition of 1 to 2 % sodium carbonate or boiling in water under pressure for twenty minutes successfully destroys P. larvae spores in the internal wood structures. Chemical disinfection processes include the use of various disinfectants whose choice depends on the spectrum of the microorganism to be destroyed, the presence of organic matter on the surface, environmental conditions, and the toxicity of the disinfectant to humans, animals, and the environment. The aldehydes, halogen compounds and oxidants show an effect on bacterial endospores while inorganic acids, alkaline salts and phenols have a limited effect. High and rapid sporocidal activity of glutaraldehyde, sodium hypochlorite and caustic soda on P. larvae spores was found. The aim of this study was to implement genotyping of P. larvae on fifty field samples isolated from characteristically altered dead honeybee larvae over a period of eleven years (2010-2020), and thus determine the prevalence and frequency of certain genotypes in the Republic of Croatia. The effect of ten commercially available and commonly used conditions was also determined. The ultimate goal was to perform the comparison of the results obtained on the effect of tested disinfectants with individual genotypes of P. larvae. MATERIAL AND METHODS The P. larvae bacteria used in the study came from two sources. One was a validated strain (German Collection of Microorganism and Cell Culture - DSMZ; Braunschweig, Germany) of which four genotypes were used (DSM 7030 (ERIC I), DSM 25430a (ERIC II), LMG 16252 (ERIC III) and LMG 16247 (ERIC IV)). For an additional verification of the obtained results, genotyped strains collected in the Republic of Croatia for several years were used. The P. larvae strains were cultured on Columbia sheep blood agar (BD), and for liquid culture, P. larvae strains were grown in brain heart infusion medium (BHIM). For the genotyping study, the primers ERIC1R and ERIC2 were used, and the method of repetitive extragenic paliandromic sequence-PCR (REP-PCR) was performed to determine which genotype our samples belonged to. The isolation of genomic DNA was performed with a commercial QIAamp Mini DNA blood and tissue kit according to the manufacturer's instructions, with special preparation of bacteria for isolation. The commercially disinfectants were selected based on the recommendations of producers and beekeepers, as well as their availability on the market. The following disinfectants were used: Bee Protect products (Bee Protect H forte and Bee Protect F), Genox, Genoll with foam, Despadac, Despadac Secure, Ecocid S, Sekusep aktiv, Incidin Oxyfoam S and EM® probiotic for bees. Selected products were tested by 1) determining the zone of inhibition in agar diffusion test, 2) suspension test for viable bacteria, 3) surface disinfectant test, and 4) sporocidal effect in suspension test for all four genotypes of P. larvae (ERIC I to ERIC IV). RESULTS The research and sampling on apiaries in the Republic of Croatia during the period from 2010 to 2020, of the total successfully analyzed P. larvae samples (n = 41), most belonged to the ERIC I genotype (90.3 %), while only three samples belonged to genotype ERIC II (7.3 %). Nine samples were not suitable for interpretation. The finding of one sample suspected to be of ERIC IV genotype (2.4%) would need further verification, especially from the point of view that ERIC III and ERIC IV genotypes have not been isolated from field samples for decades and are present only in archived collections of bacterial cultures. Since there is no anamnestic data, it can be assumed that the beekeeper used old equipment, wax, honey and / or food additives of unknown origin. The extremely high proportion of isolates belonging to the ERIC I genotype can be explained by the lack of systematic monitoring on AFB such as regular clinical examinations before moving bees on honey pasture, or early diagnosis thereby examining honey, adult bees or hive debris from the botom board for P. larvae spores. Moreover, the subject samples were taken from honeybee colonies where beekeepers and veterinarians had already raised the suspicion of AFB based on typical clinical signs. This correlates with the comprehension that the ERIC I genotype is less virulent at the level of a single larva, so infected honeybee larvae die after sealing cells and clinical signs of disease can be clearly seen because workers have not removed the dead larvae. Therefore, it is a logical recommendation to develop a new model for monitoring honeybee colonies on AFB in the Republic of Croatia, which would include active and passive monitoring and early diagnosis of AFB as an act of determining honeybee colonies germ carriers or reservoirs of disease, as well as prevention of disease in apiaries. The suspension test showed a significant effect of undiluted Genox after 15 minutes of application depending on the duration of exposure, while in the test of disinfectant the effect on surfaces was seen after 30 minutes and was not dependent on further prolongation of contact time. Genoll with foam didn't show sporocidal effect while Genox disinfectant at a concentration of 10% reduced the number of spores by 1 logarithm, reaching a reducting of three logarithms in 30 and 60 minutes when 100% concentration was used. The observed effect of Genox disinfectant on P. larvae didn't show a desirable sporocidal profile due to the too long time required to achieve a sporocidal effect. In the suspension test and in the surface test, Incidin OxyFoam S showed antimicrobial activity on vegetative forms of P. larvae after only one minute, and sporocidal action at the level of reduction of 6 logarithms after 30 minutes of contact time. The tested effect of the disinfectant Incidin OxyFoam S on P. larvae showed a satisfactory sporocidal effect on all four genotypes of P. larvae (ERIC I to ERIC IV). The products from the Despadac line showed bactericidal activity in the suspension test and in the surface test, but the sporocidal effect was not satisfactory due to the poorer effect in the contact time of 30 minutes. The bactericidal effect of Ecocid S and Sekusept aktiv on P. larvae was determined by agar gel diffusion test and suspension test. The ATP bioluminescence test in the surface test of Ecocid S and Sekusept aktiv showed a bactericidal effect whereas Sekusept aktiv showed a desirable sporocidal effect in the suspension test at a concentration of 2% on all four genotypes of P. larvae (ERIC I to ERIC IV). The Bee protect products showed in the agar diffusion test a bactericidal effect on P. larvae. In the suspension test for viable bacteria, the bactericidal effect for Bee protect H forte was determined, while in the test of action on surfaces, Bee protect products didn't show any bactericidal effect on P. larvae. The suspension test didn'tXVII show any sporocidal effect of Bee protect products. The EM probiotic product for bees didn't show significant bactericidal effect in the agar gel diffusion test. DISCUSSION In our research on apiaries in the Republic of Croatia in the period from 2010 to 2020, most field isolates belonged to the ERIC I genotype (90.3%), while only three isolates belonged to the ERIC II genotype (7.3%). In one isolate of P. larvae, the ERIC IV genotype (2.4%) was suspected and further verification of such a finding is required. The extremely high proportion of P. larvae isolates belonging to the ERIC I genotype can be explained by the lack of systematic monitoring for the presence of AFB in apiaries. Sodium hypochlorite (NaOCl) is a frequently used disinfectant and its effectiveness depends on the concentration of available chlorine and the pH of the solution. Genox and Genoll show an inhibitory effect on vegetative forms of P. larvae, but the effect of Genox after 60 minutes is slightly attenuated, which may be related to the fact that all biocides that have chlorine as an active component have time-limited effects due to its consumption during exposure to environmental factors. Hydrogen peroxide leads to the oxidation of lipids and proteins of the outer layer of the bacterial cell. The sporocidal action of Incidin OxyFoam S was determined at the level of reduction by six logarithms after 30 minutes of contact. Previous studies performed with hydrogen peroxide did not include a combination with other substances, or even auxiliary substances, which results in differences from our study. Quaternary ammonium salts have been in use for many years in disinfection. Products from the Despadac line (Despadac and Despadac Secure) showed bactericidal activity during exposure for 30 minutes, after which there was a reduction in the number of germinating spores of P. larvae, but only by one or two logarithms. Such a result in disinfection conditions is not satisfactory, but the use of Despadac products in sanitation can be considered. Peracetic acid is considered a potent biocide, even at low concentrations in the presence of organic residues, and is degraded to non-toxic substances. The action of peracetic acid as a disinfectant is not dependent on ambient temperature. Studies have shown that the effectiveness of peracetic acid varies depending on whether the microorganisms are in suspension or on the surface, which was not the case in our study during the study Sekusept Aktiv and Ecocida S. The Bee protect products caused a certain effect that increased with the time of exposure of bacteria to disinfectant. However, this effect did not meet the set standards and Bee protect products can't be recommended for use in the final disinfection of equipment, utensils and apiaries after remediation of clinically visible AFB. The effective microorganisms are used in agriculture,XVIII forestry, livestock, aquaculture, beekeeping, environmental protection and medicine. The inhibitory effect of the bee food supplement EM® PROBIOTIC FOR BEES on reducing the number of P. larvae bacteria was minimal and therefore this product can't be considered a classic disinfectant or biocide in the true sense of the word. However, the actio

EFFECT OF DISINFECTANTS ON BACTERIA PAENIBACILLUS LARVAE UNDER LABORATORY CONDITIONS

merička gnjiloća medonosne pčele (AGMP) je zarazna bolest nepoklopljenog i poklopljenog pčelinjeg legla koja pčelarstvu nanosi višestruke štete. Uzročnik bolesti je bakterija Paenibacillus larvae koja u nepovoljnim životnim uvjetima tvori dugo živuće i otporne spore. Patogeneza, klinička slika i stupanj virulentnosti kod AGMP ovise o genotipu bakterije P. larvae. Dosad je utvrđeno pet genotipova P. larvae (ERIC I, ERIC II, ERIC III, ERIC IV, ERIC V) koji se međusobno razlikuju u morfologiji, biokemijskim čimbenicima, virulenciji te čimbenicima koji utječu na virulenciju. Poznavanje raširenosti i dinamike pojavnosti pojedinih genotipova P. larvae na određenom području pruža uvid u patofiziološke procese na razini ličinke / pčelinje zajednice te utječe na procjenu rizika od AGMP jer postoji značajna korelacija između genotipa i pojavnosti vidljivih kliničkih znakova. Tvrdokoran tijek bolesti, otpornost uzročnika, poteškoće u kliničkoj dijagnostici i suzbijanju učinili su AGMP jednom od najtežih u svijetu. Bolesne pčelinje zajednice bez provedbe posebnih mjera ne mogu ozdraviti, a primjena antibiotika u liječenju bolesti nije dozvoljena zbog moguće pojave rezidua u pčelinjim proizvodima, rezistencije uzročnika te spoznaje da antibiotici djeluju samo na vegetativne oblike bakterije P. larvae, a ne uništavaju spore što doprinosi horizontalnom širenju bolesti u pčelinjacima. Ponekad je bolesnu pčelinju zajednicu najbolje sanirati spaljivanjem zajedno s košnicom i onečišćenim priborom. Nakon provedenih sanacijskih mjera nužno je primjeniti učinkovitu završnu dezinfekciju opreme, pribora i pčelinjaka čiji uspjeh ovisi o izboru dezinficijensa, preporučenoj koncentraciji radnih otopina, načinu i dužini trajanja aplikacije, vrsti mikroorganizama koji se moraju ukloniti te površini/materijalu koji se dezinficira. Cilj ovog istraživanja bila je provedba genotipizacije bakterije P. larvae na pedeset terenskih izolata izdvojenih iz karakteristično promijenjenih uginulih pčelinjih ličinki skupljenih u razdoblju od jedanaest godina (2010. – 2020.), te tako utvrditi raširenost i učestalost određenih genotipova u Republici Hrvatskoj (RH). Također, cilj je bio i utvrđivanje učinka deset komercijalno dostupnih i uobičajeno korištenih dezinficijensa u pčelarstvu, na terenske i certificirane sojeve bakterije P. larvae, u laboratorijskim uvjetima te uspoređivanje dobivenih rezultata među pojedinim genotipovima bakterije P. larvae. Genotip ERIC I bakterije P. larvae utvrđen je u pčelinjim zajednicama na području RH u visokoj prevalenciji od 90,3 %, a genotip ERIC II u niskoj prevalenciji od 7,3 % od ukupno uspješno analiziranih terenskihXI izolata. Za jedan izolat postavljena je sumnja na genotip ERIC IV s prevalencijom od 2,4 % te je potrebna daljnja verifikacija takvog nalaza. Istraživani su učinci Genoxa, Genolla s pjenom, Ecocid S, Sekusept aktiv, Incidin OxyFoam S, Bee Protect H forte, Bee Protect F, Despadac, Despadac Secure i EM® PROBIOTIK ZA PČELE u testu stvaranja zone inhibicije, suspenzijskom testu učinka na vijabilne bakterije P. larvae, testu dezinficijskog djelovanja na površinama i suspenzijskom testu učinka na spore bakterije P. larvae. Učinak dezinficijensa Genoxa na bakteriju P.larvae nije pokazao poželjan sporocidni profil zbog predugog vremena koje je potrebno da bi se ostvario sporocidni učinak te je isti limitiran dok proizvod Genoll s pjenom nije uopće pokazao sporocidno djelovanje. Proizvodi iz linije Despadac u suspenzijskom testu i u testu na površinama pokazali su baktericidno djelovanje, ali sporocidni učinak nije zadovoljavajući zbog slabijeg učinka u kontaktnom vremenu od 30 minuta. Suspenzijskim testom nije utvrđen zadovoljavajući sporocidni učinak Bee protect proizvoda, a proizvod EM® PROBIOTIK ZA PČELE nije pokazao u testu agar gel difuzije značajan baktericidni učinak. Sekusept aktiv u 2% koncentraciji i Incidin OxyFoam S u suspenzijskom testu sporocidnog djelovanja pokazali su zadovoljavajući sporocidni učinak na sva četiri genotipa bakterije P. larvae (ERIC I do ERIC IV).INTRODUCTION American foulbrood (AFB) is a contagious disease of sealed and unsealed honeybee brood that causes multiple damage to beekeeping. The causative agent of the disease is the bacterium Paenibacillus larvae, which in unfavorable life conditions forms long-lived and resistant spores. The infectious forms of P. larvae are spores, and susceptible to infection are honeybee larvae at the age when they are taking food. It takes only ten spores to infect one honeybee larvae younger than one day, but with the time passing by, susceptibility decreases and more than ten million spores are needed to infect a larva between four and five days old. Moreover, the number of spores required to cause infection in the later stages of honeybee larvae development is so high that a naturally infection is not possible. One dead larva can contain up to 2.5 billion newly created infectious spores. The pathogenesis, clinical signs, and degree of virulence in AFB depend on the P. larvae genotype. Recently, five genotypes of P. larvae (ERIC I, ERIC II, ERIC III, ERIC IV, ERIC V) have been identified, which differ in morphology, biochemical parameters, virulence, and factors influencing virulence. The virulence of P. larvae is conditioned by the possibility of infecting the honeybee larva and the time required until the death of the infected larva. The formation of a large amount of longlived and resistant spores, together with the possibility of multiplication and development of vegetative forms of the bacterium allows a high probability of P. larvae infection. The analysis of pathogenicity or virulence showed significant differences between genotypes ERIC I to ERIC V. The ERIC I genotype of P. larvae takes 12 days to cause death of all infected larvae (LT100 = 12 days), while the genotypes ERIC II to ERIC IV take only seven days. Therefore, according to the rate of larval death, these P. larvae genotypes are classified into three groups: ERIC I - slow leads to death of infected honeybee larvae, ERIC II - moderately fast leads to death of infected larvae, and ERIC III to ERIC V - are genotypes of pathogens that quickly lead to the death of the infected larva. The LT100 results for genotype ERIC II indicated that all infected larvae would die before the brood cells could be sealed. In this way, the adult honeybee workers have enough time to perform their hygienic skills, removing the dead larvae from brood cells. At the same time, the process of creating spores at the level of the honey bee colony would be disrupted, contributing to slow down the spread and development of the disease. However, the ERIC I genotype is less virulent at the level of a single larva because infected larvae die after sealing the cells. Consequently, the removal of dead larvae is reduced in such cases, and the possibility of producing and spreading the causative spores is significantly increased. Ultimately, genotype ERIC I is highly virulent for the honeybee colony leading to its rapid decline when compared to ERIC II which shows lower virulence at the honeybee colony level and slower decline of honeybee colony thereby showing a negative correlation of P. larvae virulence at the larval level, and consequently at the honeybee colony level. The knowledge of the distribution and dynamics of occurrence of individual P. larvae genotypes in a given area provides insight into pathophysiological processes at the level of the larva or honeybee colony and influences the risk assessment of AFB once there is a significant correlation between genotype and clinical signs. Vegetative rods of P. larvae have long, peritrichous arranged cilia that allow active movement in the form of a swarm motility. It was found that the P. larvae ERIC II genotype can move superficially in the form of a swarm and form a free-floating biofilm, while the P. larvae ERIC I genotype can form a biofilm but cannot move in the form of a swarm. These facts, that P. larvae is able to produce biofilm and actively move in the form of a swarm, requires new approaches in diagnostic and disenfection measures. The persistent course of the disease, resistance of pathogens, difficulties in clinical diagnosis and control, have made AFB one of the most difficult honeybee disease in the world. Infected honeybee colonies can hardly recover without implementation of special measures. The use of antibiotics in the treatment of diseases is prohibited due to the appearance of residues in honeybee products, resistance of the pathogen and the knowledge that antibiotics act only on vegetative forms of P. larvae, thereby providing horizontal spread of disease in apiaries. Most of the times, the best way to sanitate an infected honeybee colony it’s by burning it together with the hive and hive tools. During and after the implementation of eradication measures, it is necessary to carry out final disinfection, which success depends on the choice of effective disinfectant, recommended concentration of solutions, method and duration of application, type of microorganisms to be removed, surfaces and material to be disinfected. From the epizootiological point of view, disinfection can be preventive or focal. Preventive disinfection includes procedures and measures when infectious disease is not present in the apiary or its immediate surroundings. It is regularly carried out within the guidelines of good beekeeping practice and is an integral part of normal hygiene in the apiary. Keeping beehives, equipment, hive tools, food and water for bees clean is the basis of preventive disinfection. Focal disinfection is carried out when an infectious disease is present in the apiary and aims to remove microorganisms from the foci of infection, thus preventing its further spread. Depending on the method of execution, it can be continuous and final. Continuous disinfection involves systematic and repetitive procedures from the moment of the outbreak of infection in the apiary. It can be combined with veterinary administrative measures to cure diseases, such as burning bee colonies. The final disinfection has been considered as a one-step procedure after the implemented measures of disease remediation. Disinfection is often carried out by mechanical, physical and chemical procedures. Mechanical processes, such as cleaning, scraping, and washing, remove impurities and organic matter in which microorganisms are incorporated, thus facilitating the disinfection process. It has been observed that many disinfectants are ineffective in the presence of impurities and organic matter. Cleaning agents - detergents, soaps and abrasive powders - reduce the number of microorganisms, removing them from surfaces and objects. Physical disinfection procedures involve the use of moist or dry heat and radiation, where moist heat acts faster in a shorter period of time and is more efficient compared to dry heat. Burning hives, honeybee colonies and other accessories equipment and tools is an effective way of sanitation of AFB. Although the scorching process completely destroys the P. larvae spores on the surface of wooden hives, a significant number of infectious spores still remain active in internal wood structures. The wood fibers behave like organic matter which, already in a concentration of 2 %, significantly reduces the effect of surface disinfection. Boiling in water under normal pressure for thirty minutes with the addition of 1 to 2 % sodium carbonate or boiling in water under pressure for twenty minutes successfully destroys P. larvae spores in the internal wood structures. Chemical disinfection processes include the use of various disinfectants whose choice depends on the spectrum of the microorganism to be destroyed, the presence of organic matter on the surface, environmental conditions, and the toxicity of the disinfectant to humans, animals, and the environment. The aldehydes, halogen compounds and oxidants show an effect on bacterial endospores while inorganic acids, alkaline salts and phenols have a limited effect. High and rapid sporocidal activity of glutaraldehyde, sodium hypochlorite and caustic soda on P. larvae spores was found. The aim of this study was to implement genotyping of P. larvae on fifty field samples isolated from characteristically altered dead honeybee larvae over a period of eleven years (2010-2020), and thus determine the prevalence and frequency of certain genotypes in the Republic of Croatia. The effect of ten commercially available and commonly used conditions was also determined. The ultimate goal was to perform the comparison of the results obtained on the effect of tested disinfectants with individual genotypes of P. larvae. MATERIAL AND METHODS The P. larvae bacteria used in the study came from two sources. One was a validated strain (German Collection of Microorganism and Cell Culture - DSMZ; Braunschweig, Germany) of which four genotypes were used (DSM 7030 (ERIC I), DSM 25430a (ERIC II), LMG 16252 (ERIC III) and LMG 16247 (ERIC IV)). For an additional verification of the obtained results, genotyped strains collected in the Republic of Croatia for several years were used. The P. larvae strains were cultured on Columbia sheep blood agar (BD), and for liquid culture, P. larvae strains were grown in brain heart infusion medium (BHIM). For the genotyping study, the primers ERIC1R and ERIC2 were used, and the method of repetitive extragenic paliandromic sequence-PCR (REP-PCR) was performed to determine which genotype our samples belonged to. The isolation of genomic DNA was performed with a commercial QIAamp Mini DNA blood and tissue kit according to the manufacturer's instructions, with special preparation of bacteria for isolation. The commercially disinfectants were selected based on the recommendations of producers and beekeepers, as well as their availability on the market. The following disinfectants were used: Bee Protect products (Bee Protect H forte and Bee Protect F), Genox, Genoll with foam, Despadac, Despadac Secure, Ecocid S, Sekusep aktiv, Incidin Oxyfoam S and EM® probiotic for bees. Selected products were tested by 1) determining the zone of inhibition in agar diffusion test, 2) suspension test for viable bacteria, 3) surface disinfectant test, and 4) sporocidal effect in suspension test for all four genotypes of P. larvae (ERIC I to ERIC IV). RESULTS The research and sampling on apiaries in the Republic of Croatia during the period from 2010 to 2020, of the total successfully analyzed P. larvae samples (n = 41), most belonged to the ERIC I genotype (90.3 %), while only three samples belonged to genotype ERIC II (7.3 %). Nine samples were not suitable for interpretation. The finding of one sample suspected to be of ERIC IV genotype (2.4%) would need further verification, especially from the point of view that ERIC III and ERIC IV genotypes have not been isolated from field samples for decades and are present only in archived collections of bacterial cultures. Since there is no anamnestic data, it can be assumed that the beekeeper used old equipment, wax, honey and / or food additives of unknown origin. The extremely high proportion of isolates belonging to the ERIC I genotype can be explained by the lack of systematic monitoring on AFB such as regular clinical examinations before moving bees on honey pasture, or early diagnosis thereby examining honey, adult bees or hive debris from the botom board for P. larvae spores. Moreover, the subject samples were taken from honeybee colonies where beekeepers and veterinarians had already raised the suspicion of AFB based on typical clinical signs. This correlates with the comprehension that the ERIC I genotype is less virulent at the level of a single larva, so infected honeybee larvae die after sealing cells and clinical signs of disease can be clearly seen because workers have not removed the dead larvae. Therefore, it is a logical recommendation to develop a new model for monitoring honeybee colonies on AFB in the Republic of Croatia, which would include active and passive monitoring and early diagnosis of AFB as an act of determining honeybee colonies germ carriers or reservoirs of disease, as well as prevention of disease in apiaries. The suspension test showed a significant effect of undiluted Genox after 15 minutes of application depending on the duration of exposure, while in the test of disinfectant the effect on surfaces was seen after 30 minutes and was not dependent on further prolongation of contact time. Genoll with foam didn't show sporocidal effect while Genox disinfectant at a concentration of 10% reduced the number of spores by 1 logarithm, reaching a reducting of three logarithms in 30 and 60 minutes when 100% concentration was used. The observed effect of Genox disinfectant on P. larvae didn't show a desirable sporocidal profile due to the too long time required to achieve a sporocidal effect. In the suspension test and in the surface test, Incidin OxyFoam S showed antimicrobial activity on vegetative forms of P. larvae after only one minute, and sporocidal action at the level of reduction of 6 logarithms after 30 minutes of contact time. The tested effect of the disinfectant Incidin OxyFoam S on P. larvae showed a satisfactory sporocidal effect on all four genotypes of P. larvae (ERIC I to ERIC IV). The products from the Despadac line showed bactericidal activity in the suspension test and in the surface test, but the sporocidal effect was not satisfactory due to the poorer effect in the contact time of 30 minutes. The bactericidal effect of Ecocid S and Sekusept aktiv on P. larvae was determined by agar gel diffusion test and suspension test. The ATP bioluminescence test in the surface test of Ecocid S and Sekusept aktiv showed a bactericidal effect whereas Sekusept aktiv showed a desirable sporocidal effect in the suspension test at a concentration of 2% on all four genotypes of P. larvae (ERIC I to ERIC IV). The Bee protect products showed in the agar diffusion test a bactericidal effect on P. larvae. In the suspension test for viable bacteria, the bactericidal effect for Bee protect H forte was determined, while in the test of action on surfaces, Bee protect products didn't show any bactericidal effect on P. larvae. The suspension test didn'tXVII show any sporocidal effect of Bee protect products. The EM probiotic product for bees didn't show significant bactericidal effect in the agar gel diffusion test. DISCUSSION In our research on apiaries in the Republic of Croatia in the period from 2010 to 2020, most field isolates belonged to the ERIC I genotype (90.3%), while only three isolates belonged to the ERIC II genotype (7.3%). In one isolate of P. larvae, the ERIC IV genotype (2.4%) was suspected and further verification of such a finding is required. The extremely high proportion of P. larvae isolates belonging to the ERIC I genotype can be explained by the lack of systematic monitoring for the presence of AFB in apiaries. Sodium hypochlorite (NaOCl) is a frequently used disinfectant and its effectiveness depends on the concentration of available chlorine and the pH of the solution. Genox and Genoll show an inhibitory effect on vegetative forms of P. larvae, but the effect of Genox after 60 minutes is slightly attenuated, which may be related to the fact that all biocides that have chlorine as an active component have time-limited effects due to its consumption during exposure to environmental factors. Hydrogen peroxide leads to the oxidation of lipids and proteins of the outer layer of the bacterial cell. The sporocidal action of Incidin OxyFoam S was determined at the level of reduction by six logarithms after 30 minutes of contact. Previous studies performed with hydrogen peroxide did not include a combination with other substances, or even auxiliary substances, which results in differences from our study. Quaternary ammonium salts have been in use for many years in disinfection. Products from the Despadac line (Despadac and Despadac Secure) showed bactericidal activity during exposure for 30 minutes, after which there was a reduction in the number of germinating spores of P. larvae, but only by one or two logarithms. Such a result in disinfection conditions is not satisfactory, but the use of Despadac products in sanitation can be considered. Peracetic acid is considered a potent biocide, even at low concentrations in the presence of organic residues, and is degraded to non-toxic substances. The action of peracetic acid as a disinfectant is not dependent on ambient temperature. Studies have shown that the effectiveness of peracetic acid varies depending on whether the microorganisms are in suspension or on the surface, which was not the case in our study during the study Sekusept Aktiv and Ecocida S. The Bee protect products caused a certain effect that increased with the time of exposure of bacteria to disinfectant. However, this effect did not meet the set standards and Bee protect products can't be recommended for use in the final disinfection of equipment, utensils and apiaries after remediation of clinically visible AFB. The effective microorganisms are used in agriculture,XVIII forestry, livestock, aquaculture, beekeeping, environmental protection and medicine. The inhibitory effect of the bee food supplement EM® PROBIOTIC FOR BEES on reducing the number of P. larvae bacteria was minimal and therefore this product can't be considered a classic disinfectant or biocide in the true sense of the word. However, the actio

SUCCESS OF RELEASING AND REINTRODUCING GREY PARTRIDGE (Perdix perdix L.) INTO THE WILDERNESS FROM ARTIFICIAL BREEDING IN HUNTING GROUND OF CENTRAL CROATIA

Mogućnosti ispuštanja i podivljavanja trčke iz umjetnog uzgoja istraživalo se tijekom dvije godine i na području dva otvorena lovišta. Tražio se način, mjesto i vrijeme kojim bi se postigao najveći uspjeh preživljavanja ptica ispuštenih iz umjetnog uzgoja. Tijekom dvije godine ukupno je ispušteno 34 jedinke, od čega je 17 obilježeno telemetrijskom ogrlicom. Trčke iz umjetnog uzgoja ispuštane su u tri različite godišnje periode. Prvo ispuštanje poluiznešenog matičnog jata izvršeno je u lovištu "Ježdovec–Stupnik" (lokalitet 1, 45°45´20,41" N; 15°49´59,36" E) gdje je ukupno ispušteno pet parova trčki. Drugo ispuštanje matičnog jata (8 kljunova) izvršeno je u jesenskom razdoblju na području lovišta "Crnovščak" (lokalitet 2, 45°45´20,41" N; 16°20´30,10" E). Treće ukupno i drugo po redu ispuštanje na području istog lokaliteta izvršeno je u rano proljeće te je tom prilikom ispušteno 16 i obilježeno 8 jedinki. Na ispuštenim lokacijama nije se radila nikakva priprema staništa. Za obilježavanje su korištene telemetrijske ogrlice koje emitiraju signal svake 2 sec. Radijskom antenom svakodnevno su praćene i bilježene lokacije zadržavanja ispuštenih jedinki. Od ukupno 17 obilježenih i ispuštenih jedinki njih 13 je nakon duljeg ili kraćeg razdoblja pronađeno usmrćeno. Utvrđeno je da se duljina preživljavanja ispuštenih jedinki signifikantno razlikuje s obzirom na vrijeme ispuštanja. Najdulje preživljavanje imale su jedinke ispuštene u ljetnom periodu, dok su najkraće preživljavale jedinke ispuštene u rano proljeće. Nakon provedenog istraživanja može se zaključiti da je ispuštanje trčki iz umjetnog uzgoja u svrhu formiranja matičnog fonda upitno i ima svoju opravdanost tek kada su zadovoljeni svi potrebni uvjeti, odnosno uz pogodno stanište kod one brojnosti predatora koja neće ugrožavati opstanak ispuštenih jedinki. Najbolje vrijeme ispuštanja je definirano kao rani ljetni period kada jedinke u staništu pronalaze dovoljno hrane i zaklona, klimatske prilike uz osiguranje vode su također povoljne, međutim ostaje problem preživljavanja tih jedinki nakon uklanjanja poljoprivrednih kultura kada postaju lak plijen za dlakave, a posebice pernate predatore na čiju se brojnost prema važećoj zakonskoj regulativi ne može izravno utjecati.The possibility of releasing and reintroducing artificially breaded partridges in open habitats has been researched through a period of two years, on the territories of two separate sites. During these two years 34 animals have been released from which 17 have been marked with a telemetric collar. Partridges from artificial breeding have been released in three different times of year. First release of a semi-adults has been carried out in hunting ground "Ježdovec–Stupnik" (site 1) where altogether five pairs of partridges have been released. Second release of the mother flock has been done in autumn in the territory of hunting ground "Črnovšćak" (site 2). Third release altogether and second in line has been carried out in early spring in the same territory when 16 animals have been released, and 8 marked. In the release sites preparation of habitat hasn’t been done. Telemetric collars that emit a signal every 2 seconds have been used for marking. Retention location of the released animals has been monitored and recorded on a daily basis with a radio antenna using the triangulation method. From 17 marked and released animals altogether 13 of them have been found dead after a shorter or longer period of time. It has been determined that the length of survival of the released animal is significantly different considering the period of release. The longest survival had the animals released in summer, whereas the shortest survival had animals released in early spring. After the conducted research it can be determined that the release of partridges from artificial breeding with the purposes of creating a mother fund is questionable and it has justification only when all necessary conditions are met or in other words with a suitable habitat with a certain number of predators that won’t endanger the survival of the released animals. Optimal release time has been determined as early summer period when animals in habitat find enough food and shelter, when the climate conditions with enough water are favorable as well, on the other hand the survival problem of these animal remains after removal of agricultural crops when they become easy prey for fury and especially feather predators whose numbers cannot be directly influenced due to binding law regulations

Effects of Disinfectants on Bacterium <i>Paenibacillus larvae</i> in Laboratory Conditions

American foulbrood is an infectious disease of the honeybee brood that causes multiple types of damage to beekeeping. The causative agent of the disease is the bacterium Paenibacillus larvae, which forms resistant infective spores and is viable for decades. After the eradication measures have been implemented, in cases of clinically visible disease, it is necessary to conduct effective final disinfections of equipment and tools. This study aimed to determine the effect of ten commercially available and commonly used disinfectants on certified strains of P. larvae under laboratory conditions, as well as to compare the obtained results among individual genotypes of P. larvae. Selected products were tested by determining the zone of inhibition using an agar diffusion test, a suspension test for viable bacteria, a surface disinfectant test, and a sporicidal effect in the suspension test. Incidin OxyFoam S and Sekusept Aktiv are both effective against all examined genotypes of P. larvae. Despadac and Despadac Secure have a bactericidal effect, but their sporocidal effect is not as satisfactory as that of Genox. Genoll does not exhibit a sporicidal effect, and Ecocide S at 1%, Bee protect H forte, and Bee protect F did not exhibit a satisfactory sporocidal effect. Additionally, EM® PROBIOTIC FOR BEES did not exhibit any bactericidal effect. The effective application of control measures and proper application of final disinfection can reduce the reoccurrence of visible clinical signs of disease, whereas methods of early diagnosis can significantly reduce the incidence of the disease