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Abstract The interspecific transmission of pathogens can occur frequently in the environment. Among wild bees, the main spillover cases are caused by pathogens associated with Apis mellifera, whose colonies can act as reservoirs. Due to the limited availability of data in Italy, it is challenging to accurately assess the impact and implications of this phenomenon on the wild bee populations. In this study, a total of 3372 bees were sampled from 11 Italian regions within the BeeNet project, evaluating the prevalence and the abundance of the major honey bee pathogens (DWV, BQCV, ABPV, CBPV, KBV, Nosema ceranae, Ascosphaera apis, Crithidia mellificae, Lotmaria passim, Crithidia bombi). The 68.4% of samples were positive for at least one pathogen. DWV, BQCV, N. ceranae and CBPV showed the highest prevalence and abundance values, confirming them as the most prevalent pathogens spread in the environment. For these pathogens, Andrena, Bombus, Eucera and Seladonia showed the highest mean prevalence and abundance values. Generally, time trends showed a prevalence and abundance decrease from April to July. In order to predict the risk of infection among wild bees, statistical models were developed. A low influence of apiary density on pathogen occurrence was observed, while meteorological conditions and agricultural management showed a greater impact on pathogen persistence in the environment. Social and biological traits of wild bees also contributed to defining a higher risk of infection for bivoltine, communal, mining and oligolectic bees. Out of all the samples tested, 40.5% were co-infected with two or more pathogens. In some cases, individuals were simultaneously infected with up to five different pathogens. It is essential to increase knowledge about the transmission of pathogens among wild bees to understand dynamics, impact and effects on pollinator populations. Implementing concrete plans for the conservation of wild bee species is important to ensure the health of wild and human-managed bees within a One-Health perspective.

Abstract In recent years molecular techniques have been used on museum material as integrative support for classic taxonomy. This cumulative systematics approach is especially for rare or extinct specimens, and genetic analysis may be useful to discern information that is not possible to glean from live materials or morphology. To date, the extraction of DNA required at least a partial destruction of the specimens, which is not possible for all individuals, especially the types. In this study, we described a novel method to extract mitochondrial DNA (mtDNA) from pinned museum bee individuals to avoid any external morphological damage. This method was able to amplify the mtDNA Cytochrome C oxidase subunit I (COI) gene in bee samples collected up to 27 years ago. We tested the efficacy of this method on 72 preserved be specimens belonging to nine species among four families, it could be used on many museums’ rare and/or extinct bee species because it does not provide external morphological damages. The method could be helpful for providing ecological, taxonomic, and phylogenetic information about specimens preserved in museum collections.

Diseases contribute to the decline of pollinator populations, which may be aggravated by the interspecific transmission of honey bee pests and pathogens. Flowers increase the risk of transmission, as they expose the pollinators to infections during the foraging activity. In this study, both the prevalence and abundance of 21 honey bee pathogens (11 viruses, 4 bacteria, 3 fungi, and 3 trypanosomatids) were assessed in the flower-visiting entomofauna sampled from March to September 2021 in seven sites in the two North-Italian regions, Emilia-Romagna and Piedmont. A total of 1,028 specimens were collected, identified, and analysed. Of the twenty-one pathogens that were searched for, only thirteen were detected. Altogether, the prevalence of the positive individuals reached 63.9%, with Nosema ceranae, deformed wing virus (DWV), and chronic bee paralysis virus (CBPV) as the most prevalent pathogens. In general, the pathogen abundance averaged 5.15 * 106 copies, with CBPV, N. ceranae, and black queen cell virus (BQCV) as the most abundant pathogens, with 8.63, 1.58, and 0.48 * 107 copies, respectively. All the detected viruses were found to be replicative. The sequence analysis indicated that the same genetic variant was circulating in a specific site or region, suggesting that interspecific transmission events among honey bees and wild pollinators are possible. Frequently, N. ceranae and DWV were found to co-infect the same individual. The circulation of honey bee pathogens in wild pollinators was never investigated before in Italy. Our study resulted in the unprecedented detection of 72 wild pollinator species as potential hosts of honey bee pathogens. Those results encourage the implementation of monitoring actions aiming to improve our understanding of the environmental implications of such interspecific transmission events, which is pivotal to embracing a One Health approach to pollinators’ welfare.

Understanding the health status of bees is crucial in assessing the epidemiology of pathogens that cause diseases in honey bee (Apis mellifera) colonies and wild bees [...]

The recent decades witnessed the collapse of honey bee colonies at a global level. The major drivers of this collapse include both individual and synergic pathogen actions, threatening the colonies’ survival. The need to define the epidemiological pattern of the pathogens that are involved has led to the establishment of monitoring programs in many countries, Italy included. In this framework, the health status of managed honey bees in the Emilia–Romagna region (northern Italy) was assessed, throughout the year 2021, on workers from 31 apiaries to investigate the presence of major known and emerging honey bee pathogens. The prevalence and abundance of DWV, KBV, ABPV, CBPV, Nosema ceranae, and trypanosomatids (Lotmaria passim, Crithidia mellificae, Crithidia bombi) were assessed by molecular methods. The most prevalent pathogen was DWV, followed by CBPV and N. ceranae. Trypanosomatids were not found in any of the samples. Pathogens had different peaks in abundance over the months, showing seasonal trends that were related to the dynamics of both bee colonies and Varroa destructor infestation. For some of the pathogens, a weak but significant correlation was observed between abundance and geographical longitude. The information obtained in this study increases our understanding of the epidemiological situation of bee colonies in Emilia–Romagna and helps us to implement better disease prevention and improved territorial management of honey bee health.

Leptospirosis is a re-emerging and globally spread zoonosis caused by pathogenic genomospecies of Leptospira. Wild boar (Sus scrofa) are an important Leptospira host and are increasing in population all over Europe. The aim of this investigation was to evaluate Leptospira spp. infection in the reproductive systems of wild boar hunted in two Italian regions: Tuscany and Sardinia. From 231 animals, reproductive system tissue samples (testicles, epididymides, uteri) as well as placentas and fetuses were collected. Bacteriological examination and Real-Time PCR were performed to detect pathogenic Leptospira (lipL32 gene). Leptospires were isolated from the testicles and epididymides of one adult and two subadult wild boar. Four isolates from the two subadult males were identified as Leptospira interrogans serogroup Australis by MLST, whereas Leptospira kirschneri serogroup Grippotyphosa was identified from the adult testicles and epididymis. Using Real-Time PCR, 70 samples were positive: 22 testicles (23.16%) and 22 epididymides (23.16%), 10 uteri (7.35%), 3 placentas (6.66%), and 13 fetuses (28.88%). Amplification of the rrs2 gene identified L. interrogans and L. kirschneri species. The results from this investigation confirmed that wild boar represent a potential source of pathogenic Leptospira spp. Isolation of Leptospira serogroups Australis and Grippotyphosa from the male reproductive system and the positive Real-Time PCR results from both male and female samples could suggest venereal transmission, as already demonstrated in pigs. Furthermore, placentas and fetuses were positive for the lipL32 target, and this finding may be related to a possible vertical transmission of pathogenic Leptospira.

The microsporidian Nosema ceranae is a severe threat to the western honey bee Apis mellifera, as it is responsible for nosemosis type C, which leads the colonies to dwindle and collapse. Infection quantification is essential to clinical and research aims. Assessment is made often with molecular assays based on rRNA genes, which are present in the N. ceranae genome as multiple and polymorphic copies. This study aims to compare two different methods of Real-Time PCR (qPCR), respectively relying on the 16S rRNA and Hsp70 genes, the first of which is described as a multiple and polymorphic gene. Young worker bees, hatched in the laboratory and artificially inoculated with N. ceranae spores, were incubated at 33 °C and subject to different treatment regimens. Samples were taken post-infection and analyzed with both qPCR methods. Compared to Hsp70, the 16S rRNA method systematically detected higher abundance. Straightforward conversion between the two methods is made impossible by erratic 16s rRNA/Hsp70 ratios. The 16s rRNA polymorphism showed an increase around the inoculated dose, where a higher prevalence of ungerminated spores was expected due to the treatment effects. The possible genetic background of that irregular distribution is discussed in detail. The polymorphic nature of 16S rRNA showed to be a limit in the infection quantification. More reliably, the N. ceranae abundance can be assessed in honey bee samples with methods based on the single-copy gene Hsp70.

Honey bee health is a very important topic that has recently raised the interest of researchers [...]

The microsporidian fungus Nosema ceranae represents one of the primary bee infection threats worldwide and the antibiotic fumagillin is the only registered product for nosemosis disease control, while few alternatives are, at present, available. Natural bioactive compounds deriving from the glucosinolate–myrosinase system (GSL–MYR) in Brassicaceae plants, mainly isothiocyanates (ITCs), are known for their antimicrobial activity against numerous pathogens and for their health-protective effects in humans. This work explored the use of Brassica nigra and Eruca sativa defatted seed meal (DSM) GSL-containing diets against natural Nosema infection in Apis mellifera colonies. DSM patties from each plant species were obtained by adding DSMs to sugar candy at the concentration of 4% (w/w). The feeding was administered in May to mildly N. ceranae-infected honey bee colonies for four weeks at the dose of 250 g/week. In the treated groups, no significant effects on colony development and bee mortality were observed compared to the negative controls. The N. ceranae abundance showed a slight but significant decrease. Furthermore, the GSL metabolism in bees was investigated, and MYR hydrolytic activity was qualitatively searched in isolated bee midgut and hindgut. Interestingly, MYR activity was detected both in the bees fed DSMs and in the control group where the bees did not receive DSMs. In parallel, ITCs were found in gut tissues from the bees treated with DSMs, corroborating the presence of a MYR-like enzyme capable of hydrolyzing ingested GSLs. On the other hand, GSLs and other GSL hydrolysis products other than ITCs, such as nitriles, were found in honey produced by the treated bees, potentially increasing the health value of the final product for human consumption. The results are indicative of a specific effect on the N. ceranae infection in managed honey bee colonies depending on the GSL activation within the target organ.

Honey bees, and pollinators in general, play a major role in the health of ecosystems. There is a consensus about the steady decrease in pollinator populations, which raises global ecological concern. Several drivers are implicated in this threat. Among them, honey bee pathogens are transmitted to other arthropods populations, including wild and managed pollinators. The western honey bee, Apis mellifera, is quasi-globally spread. This successful species acted as and, in some cases, became a maintenance host for pathogens. This systematic review collects and summarizes spillover cases having in common Apis mellifera as the mainteinance host and some of its pathogens. The reports are grouped by final host species and condition, year, and geographic area of detection and the co-occurrence in the same host. A total of eighty-one articles in the time frame 1960–2021 were included. The reported spillover cases cover a wide range of hymenopteran host species, generally living in close contact with or sharing the same environmental resources as the honey bees. They also involve non-hymenopteran arthropods, like spiders and roaches, which are either likely or unlikely to live in close proximity to honey bees. Specific studies should consider host-dependent pathogen modifications and effects on involved host species. Both the plasticity of bee pathogens and the ecological consequences of spillover suggest a holistic approach to bee health and the implementation of a One Health approach.

Honey bee health is a crucial issue that has recently received increased interest from researchers, stakeholders, and citizens [...]

The deformed wing virus (DWV) is one of the most common honey bee pathogens. The virus may also be detected in other insect species, including Bombus terrestris adults from wild and managed colonies. In this study, individuals of all stages, castes, and sexes were sampled from three commercial colonies exhibiting the presence of deformed workers and analysed for the presence of DWV. Adults (deformed individuals, gynes, workers, males) had their head exscinded from the rest of the body and the two parts were analysed separately by RT-PCR. Juvenile stages (pupae, larvae, and eggs) were analysed undissected. All individuals tested positive for replicative DWV, but deformed adults showed a higher number of copies compared to asymptomatic individuals. Moreover, they showed viral infection in their heads. Sequence analysis indicated that the obtained DWV amplicons belonged to a strain isolated in the United Kingdom. Further studies are needed to characterize the specific DWV target organs in the bumblebees. The result of this study indicates the evidence of DWV infection in B. terrestris specimens that could cause wing deformities, suggesting a relationship between the deformities and the virus localization in the head. Further studies are needed to define if a specific organ could be a target in symptomatic bumblebees.

Domestic dogs (Canis lupus familiaris) used for wild boar (Sus scrofa) hunting may represent incidental hosts for several zoonotic pathogens. This investigation aimed to evaluate the presence of anti-Leptospira antibodies and the occurrence, antimicrobial resistance, and virulence of Salmonella spp., Yersinia enterocolitica, and Listeria monocytogenes in sera and rectal swabs collected from 42 domestic hunting dogs in the Tuscany region (Italy). Regarding Leptospira, 31 out of 42 serum samples (73.8%) were positive and serogroup Pomona was the most detected (71.4%) at titers between 1:100 and 1:400. Four Salmonella isolates (9.52%) were obtained, all belonging to serotype Infantis; two of them showed antimicrobial resistance to streptomycin, while pipB and sopE presence was assessed in all but one isolate. Concerning Yersinia enterocolitica, seven isolates (16.7%) were obtained, six belonging to biotype 1 and one to biotype 4. Resistance to amoxicillin–clavulanic acid, cephalothin, and ampicillin was detected. Biotype 4 presented three of the virulence genes searched (ystA, ystB, inv), while isolates of biotype 1 showed only one gene. No Listeria monocytogenes was isolated from dog rectal swabs. The results suggest that hunting dogs are exposed to different bacterial zoonotic agents, potentially linked to their work activity, and highlight the possible health risks for humans.

Nosema ceranae is a widespread parasite responsible for nosemosis Type C in Apis mellifera honey bees, reducing colony survival. The antibiotic fumagillin is the only commercial treatment available, but concerns are emerging about its persistence, safety, and pathogen resistance. The use of natural substances from Brassicaceae defatted seed meals (DSMs) with known antimicrobial and antioxidant properties was explored. Artificially infected bees were fed for 8 days with candies enriched with two concentrations, 2% and 4%, of two DSMs from Brassica nigra and Eruca sativa, containing a known amount of different glucosinolates (GSLs). The food palatability, GSL intake, bee survival, and treatment effects on N. ceranae spore counts were evaluated. Food consumption was higher for the two 2% DSM patties, for both B. nigra and E. sativa, but the GSL intake did not increase by increasing DSM to 4%, due to the resulting lower palatability. The 2% B. nigra patty decreased the bee mortality, while the higher concentration had a toxic effect. The N. ceranae control was significant for all formulates with respect to the untreated control (312,192.6 +/− 14,443.4 s.e.), and was higher for 4% B. nigra (120,366.3 +/− 13,307.1 s.e.). GSL hydrolysis products, the isothiocyanates, were detected and quantified in bee gut tissues. Brassicaceae DSMs showed promising results for their nutraceutical and protective effects on bees artificially infected with N. ceranae spores at the laboratory level. Trials in the field should confirm these results.

Leptospirosis is a re-emerging and worldwide diffused zoonosis. Recently, the high importance of their epidemiology was explained by the intermediate Leptospira strains. Among these strains, Leptospira fainei was the first intermediate strain detected in domestic and wild swine. Wild boars (Sus scrofa) are well known as a reservoir, as well as all swine, for pathogenic Leptospira, but very little information is available concerning intermediate Leptospira infection. The investigation aim was to evaluate if intermediate Leptospira can infect the reproductive systems of wild boars hunted in the Tuscany region (Italy), as previously demonstrated for pathogenic ones. The reproductive system tissue (testicles, epididymides, uteri), and placentas and fetuses, were collected from 200 regularly hunted animals. Bacteriological examination and real-time PCR were performed to detect intermediate Leptospira DNA. Unfortunately, no isolates were obtained. Using real-time PCR, in six (3%) male organs (both testicles and epididymis), intermediate Leptospira DNA was found. The amplification of the 16S rRNA gene identified that all DNA obtained belong to Leptospira fainei. The results of this investigation highlighted for the first time the localization of Leptospira fainei in the male wild boar reproductive system, opening up a new avenue to further investigate.

Brucellosis is a zoonosis caused by different Brucella species. Wild boar (Sus scrofa) could be infected by some species and represents an important reservoir, especially for B. suis biovar 2. This study aimed to investigate the prevalence of Brucella spp. by serological and molecular assays in wild boar hunted in Tuscany (Italy) during two hunting seasons. From 287 animals, sera, lymph nodes, livers, spleens, and reproductive system organs were collected. Within sera, 16 (5.74%) were positive to both rose bengal test (RBT) and complement fixation test (CFT), with titres ranging from 1:4 to 1:16 (corresponding to 20 and 80 ICFTU/mL, respectively). Brucella spp. DNA was detected in four lymph nodes (1.40%), five epididymides (1.74%), and one fetus pool (2.22%). All positive PCR samples belonged to Brucella suis biovar 2. The results of this investigation confirmed that wild boar represents a host for B.suis biovar. 2 and plays an important role in the epidemiology of brucellosis in central Italy. Additionally, epididymis localization confirms the possible venereal transmission.

Knowledge regarding the honey bee pathogens borne by invasive bee pests remains scarce. This investigation aimed to assess the presence in Aethina tumida (small hive beetle, SHB) adults of honey bee pathogens belonging to the following groups: (i) bacteria (Paenibacillus larvae and Melissococcus plutonius), (ii) trypanosomatids (Lotmaria passim and Crithidia mellificae), and (iii) viruses (black queen cell virus, Kashmir bee virus, deformed wing virus, slow paralysis virus, sacbrood virus, Israeli acute paralysis virus, acute bee paralysis virus, chronic bee paralysis virus). Specimens were collected from free-flying colonies in Gainesville (Florida, USA) in summer 2017. The results of the molecular analysis show the presence of L. passim, C. mellificae, and replicative forms of deformed wing virus (DWV) and Kashmir bee virus (KBV). Replicative forms of KBV have not previously been reported. These results support the hypothesis of pathogen spillover between managed honey bees and the SHB, and these dynamics require further investigation.

Leptospirosis is a re-emerging worldwide zoonotic disease. Even though the primary serological test for diagnosis and surveying is the microscopic agglutination test (MAT), isolation remains the gold-standard test to detect Leptospira infections. The leptospirosis transmission is linked to maintenance and accidental hosts. In the epidemiology of Leptospira some serovar are strictly related to specific maintenance hosts; however, in recent years, the bacterium was isolated from an even wider spectrum of species. The aim of this review is to report the isolation of Leptospira strains in animals which could be recognized as “unconventional” hosts, analyzing studies from 1960 to 2020 that highlighted the Leptospira isolation. This scientific literature aimed to provide evidence of infection in several animal species including of the Carnivora, Didelphimorphia, Rodentia, Cetacea, Cingulata, Afrosoricida, Chiroptera and Primate orders, as well as in Reptilia and Amphibia classes. In conclusion, the spreading of Leptospira is attention-worthy because the infection could occur in all the animal species ranging in a specific area. Further screening and isolations are needed to collect all necessary data to gain a complete understanding of leptospirosis epidemiology and its modifications.

Wild boar and domestic swine share several pathogens, including viruses responsible for reproductive failures, representing an important sanitary and economic risk for the swine industry. Among them, suid herpesvirus 1 (SuHV-1), porcine circovirus 2 (PCV2) and porcine parvovirus 1 (PPV1) are widely diffused in the wild boar population. Unfortunately, little is known about their pathogenetic mechanisms and impact on the reproductive parameters of wild animals. This study aims to investigate the presence of viruses responsible for reproductive failure in pregnant wild boar sows and their foetuses. The investigation was conducted on 46 pregnant wild boar and their foetuses by molecular analysis; a phylogenetic study was performed on the positive samples. All of the investigated pathogens were identified in sows, while only herpesvirus and circovirus were detected in the tissues of their foetuses. Phylogenetic analysis revealed that the viral sequences obtained from the positive wild boars were closely related to those previously identified in domestic swine belonging to the same study areas. The results suggest that SuHV-1 and PCV2 can infect wild boar foetuses, with a possible impact on wild boar reproductive performance. Moreover, our data highlight the importance of continuous monitoring of swine pathogens circulating in wild environments, so as to carry out adequate sanitary actions.

Wild boar is an animal the population of which constantly increases in Europe. This animal plays an important role as a reservoir for several pathogens, including three of the most important zoonoses: salmonellosis, yersiniosis and listeriosis. The aim of this investigation was to evaluate the occurrence of antimicrobial-resistant and virulence factor genes of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes isolated from wild boar in Tuscany (Central Italy). During two consequent hunting seasons (2018/2019 and 2019/2020), rectal swabs, spleens and livers were collected from 287 hunted wild boar to isolate strains. Each isolate was tested to investigate its antimicrobial resistance and to detect virulence factor genes by PCR. Eighteen Salmonella strains (6.27%) were isolated. Of these, 66.7% were resistant to streptomycin, 13.4% to cephalothin, 6.67% to imipenem and one isolate (6.67%) was resistant simultaneously to five antimicrobials. Moreover, the most detected genes were sopE (73.4%), pipB (66.7%), sodCI (53.3%), spvR and spvC (46.7%). In total, 54 (17.8%) Yersinia enterocolitica were isolated; of them, 26 (48.1%), 9 (16.7%), 17 (31.5%), 1 (1.85%) and 1 (1.85%) belonged to biotypes 1, 2, 3, 4 and 5, respectively. All strains (100%) demonstrated resistance to cephalothin and 70.4% to amoxicillin-clavulanic acid, 55.6% to ampicillin, and 37.0% to cefoxitin. Additionally, the most detected genes were ystA (25.9%), inv (24.1%), ail (22.2%), ystB (18.5%) and virF (14.8%). Finally, only one Listeriamonocytogenes isolate (0.35%) was obtained, belonging to serogroup IVb, serovar 4b, and it was found to be resistant to cefoxitin, cefotaxime and nalidixic acid. The results highlighted the role of wild boar as a carrier for pathogenic and antimicrobial-resistant Salmonella spp., Yersinia enterocolitica and Listeria monocytogens, representing a possible reservoir for domestic animals and human pathogens.

The employment of natural substances such as beehive products with a preventive and therapeutic purpose has been a widespread custom since ancient times. In this investigation, the antibacterial activity of 41 honey samples from different Ukraine regions has been evaluated. For each honey, melissopalynological and physico-chemical analysis were performed in order to determine botanical origin, pH, glucose and fructose contents and free acidity. So, antibacterial activity against Staphylococcusaureus CCM 4223, Listeria monocytogenes ATCC 7644, Salmonella enterica serovar Typhimurium CCM 3807 and Escherichia coli ATCC 25922 was assessed through the determination of MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values by the microdilutions method. The results show that the most susceptible bacterial strain was L. monocytogenes. Its growth was inhibited at a honey concentration ranging from 0.094 to 0.188 g/mL. The most resistant bacterial strain was S. aureus. As concerns MBC values, L. monocytogenes was the most susceptible bacteria, while S. aureus was the most resistant. Helianthus spp. honeys was the most effective against all tested bacterial strains, followed by Robinia spp. and multifloral honeys. Promising results for MIC tests have been found for Brassica spp.

Environmental resistance is an important factor for understanding the epidemiology of leptospirosis. Recently, new Leptospira hosts were identified, including also marine mammals. Moreover, halotolerant Leptospira strain, isolated from the environment and animals, highlighted the capability of this microorganism to persist in the seawater. The aim of this research was to investigate the bacteriostatic and bactericidal effect of salt on Leptospira strains belonging to 16 different serovars. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were verified through the microdilutions method starting from a 20% sodium chloride concentration. MIC values obtained were between 0.3125% and 10% of salt, while MBC values between 0.625% and >20%. Icterohaemorrhagiae (MIC: 0.3125%; MBC: 0.625%) resulted the most inhibited serovar, while the most resistant was Tarassovi (MIC: 10%; MBC: >20%). Interestingly, trends were reported for Pomona (MIC: 1.25%; MBC: >20%) and Bratislava (MIC: 0.625%; MBC: 20%), highlighting low MIC values but high MBC values. This is the first investigation aimed at the in vitro effect of salt on the growth of Leptospira spp. reference strains.

Nosema ceranae is a worldwide distributed midgut parasite of western honey bees, leading to dwindling colonies and their collapse. As a treatment, only fumagillin is available, causing issues like resistance and hampered bee physiology. This study aimed to evaluate ApiHerb® and Api-Bioxal® as treatments against N. ceranae. The efficacy was tested using two qPCR methods based on the 16S rRNA and Hsp70 genes. In addition, these methods were compared for their aptitude for the quantification of the infection. For this, 19 colonies were selected based on the presence of N. ceranae infections. The colonies were divided into three groups: treated with ApiHerb, Api-Bioxal with previous queen caging and an untreated control. All colonies were sampled pre- and post-treatment. The bees were analyzed individually and in duplicate with both qPCR methods. All bees in the pre-treatment tested positive for N. ceranae. Both treatments reduced the abundance of N. ceranae, but ApiHerb also decreased the prevalence of infected bees. Analysis with the 16S rRNA method resulted in several orders of magnitude more copies than analysis with the Hsp70 method. We conclude that both products are suitable candidates for N. ceranae treatment. From our analysis, the qPCR method based on the Hsp70 gene results as more apt for the exact quantification of N. ceranae as is needed for the development of veterinary medicinal products.

Tigecycline is a relatively new antimicrobial, belonging to glycylcyclines with antimicrobial activity against a large spectrum of bacteria. Very few data are available on its effect on Leptospira spp., which consist in a bacteriostatic mechanism. The aim of this investigation was to evaluate the bacteriostatic and bactericidal effect of tigecycline on reference Leptospira strains belonging to 16 serovars. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined through the microdilutions method, and tetracycline was used as the control. Results showed that tigecycline had higher MIC and MBC values than tetracycline. Obtained MIC values were between 4 and 32 µg/mL, while MBC values between 16 and >128 µg/mL. Patoc (MIC: 4 µg/mL; MBC: 16 µg/mL) resulted in the most susceptible serovar, while the most resistant were Bataviae (MIC: 32 µg/mL; MBC: 64 µg/mL), Bratislava (MIC: 8 µg/mL; MBC 128 µg/mL), and Tarassovi (MIC: 8 µg/mL; MBC: >128 µg/mL). This is the first investigation focused on the effect of tigecycline against Leptospira spp. reference strains. Since tigecycline is used as a treatment for bacteremia and urinary tract disease, and these symptoms could be linked to Leptospira infection, the possibility of using this antibiotic as a treatment for leptospirosis should be evaluated. Further studies are needed to explore the possibility to use tigecycline for in vivo application against Leptospira.

Leptospirosis is a worldwide-distributed, re-emerging zoonosis due to the large variety of wild and domestic animal species that can play the role of natural or accidental host. Currently, specific animal species play an important role as the reservoir for particular Leptospira serovars, although recent investigations have highlighted new host–pathogen interactions involved in Leptospira epidemiology. Furthermore, the constant modification of ecosystems and wildlife habitats and the constantly increasing number of animal species moving towards urban or peri-urban areas are increasing the possibility of direct or indirect contacts between wildlife and domestic animals; furthermore, the constant modification of animal leptospirosis also causes problems for human health. The studies published in this Special Issue have evidenced and confirmed the hidden role of a large variety of animal species, domestic and wild, in the leptospirosis epidemiology. They highlighted the necessity for continuous monitoring and large-scale surveillance studies to better understand this neglected and re-emerging zoonosis.

The effects of veterinary drugs, dietary supplements and non-protein amino acids on the European honey bee (Apis mellifera ligustica Spinola, 1806) ventriculum microbial profile were investigated. Total viable aerobic bacteria, Enterobacteriaceae, staphylococci, Escherichia coli, lactic acid bacteria, Pseudomonas spp., aerobic bacterial endospores and Enterococcus spp. were determined using a culture-based method. Two veterinary drugs (Varromed® and Api-Bioxal®), two commercial dietary supplements (ApiHerb® and ApiGo®) and two non-protein amino acids (GABA and beta-alanine) were administered for one week to honey bee foragers reared in laboratory cages. After one week, E. coli and Staphylococcus spp. were significantly affected by the veterinary drugs (p < 0.001). Furthermore, dietary supplements and non-protein amino acids induced significant changes in Staphylococcus spp., E. coli and Pseudomonas spp. (p < 0.001). In conclusion, the results of this investigation showed that the administration of the veterinary drugs, dietary supplements and non-protein amino acids tested, affected the ventriculum microbiological profile of Apis mellifera ligustica.

Leptospirosis is a re-emerging, worldwide zoonosis, and wild boar (Sus scrofa) are involved in its epidemiology as the reservoir. The aim of this study was to investigate the prevalence of Leptospira with serological, bacteriological, and molecular assays in wild boar hunted in Tuscany (Italy) during two hunting seasons. In total, 287 specimens of sera, kidneys, and liver were collected to perform microscopic agglutination tests (MATs), isolation, and RealTime PCR to detect pathogenic (lipL32 gene), intermediate (16S rRNA gene), and saprophytic (23S rRNA gene) Leptospira. Within sera, 39 (13.59%) were positive to the MAT, and Australis was the most represented serogroup (4.88%), followed by Pomona (4.18%), and Tarassovi (3.14%). Moreover, four Leptospira cultures were positive, and once isolates were identified, one was identified as L. borgpetersenii serovar Tarassovi, and three as L. interrogans serovar Bratislava. Pathogenic Leptospira DNA were detected in 32 wild boar kidneys (11.15%). The characterization through the amplification of the rrs2 gene highlighted their belonging to L. interrogans (23 kidneys), L. borgpetersenii (four), and L. kirschneri (one), while nine kidneys (3.14%) were positive for intermediate Leptospira, all belonging to L. fainei. The results of this study confirmed the importance of wild boar in the epidemiology of leptospirosis among wildlife in Central Italy.

Wild boar are among the most widespread wild mammals in Europe. Although this species can act as a reservoir for different pathogens, data about its role as a carrier of pathogenic and antimicrobial-resistant Escherichia coli are still scarce. The aim of this study was to evaluate the occurrence of antimicrobial-resistant and pathogenic Escherichia coli in wild boar in the Tuscany region of Italy. During the hunting season of 2018–2019, E. coli was isolated from 175 of 200 animals and subjected to antimicrobial resistance tests and PCR for detection of resistance and virulence factor genes. The highest resistance rates were against cephalothin (94.3%), amoxicillin–clavulanic acid (87.4%), ampicillin (68.6%), and tetracycline (44.6%). The most detected resistance genes were blaCMY-2 (54.3%), sul1 (38.9%), sul2 (30.9%), and tetG (24.6%). Concerning genes encoding virulence factors, 55 of 175 isolates (31.4%) were negative for all tested genes. The most detected genes were hlyA (47.4%), astA (29.1%), stx2 (24.6%), eaeA (17.1%), and stx1 (11.4%). E. coli was classified as Shiga toxin-producing E. coli (STEC) (21.7%), enterohemorrhagic E. coli (EHEC) (6.3%), enteroaggregative E. coli (EAEC) (5.1%), and atypical enteropathogenic E. coli (aEPEC) (3.4%). Enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and typical enteropathogenic E. coli (tEPEC) were not detected. Our results show that wild boars could carry pathogenic and antimicrobial-resistant E. coli, representing a possible reservoir of domestic animal and human pathogens.

Pseudorabies, or Aujeszky’s disease, is a notifiable worldwide infection of domestic and feral swine that causes economic losses for the swine industry. In domestic pigs, the virus is responsible for nervous and/or respiratory symptoms; in pregnant sows, it is one of the major causes of stillbirth, mummification, embryonic death, and infertility (SMEDI). It is known that PRV infection in wild boar is associated with low pathogenicity and attenuated or absent symptomatology, but limited information is available about the ability of the virus to infect the foetuses of infected wild boar pregnant sows. Due to scarce information about the reproductive consequences, we investigate the possible intrauterine vertical transmission of the virus in wild boar pregnant sow living in a highly infected area. A number of 54 hunted wild boar were sampled during 2018−2019, and blood, genital and nasal swabs, placenta, and fetuses were collected for serological and molecular investigations. A seroprevalence of 74% (40/54) was detected, while 1/24 pregnant sow and 1/24 pooled foetuses tested positive by PCR (gene gB). This is the first evidence of viral detection in foetuses from seropositive pregnant wild boar. This finding suggests the possible pathogenetic role of PRV on pregnancy in wild boar and the existence of an additional transmission route.