First autochthonous Dirofilaria immitis (Leidy, 1856) infection in a dog in Hungary

AbstractA 4 year-old, male Hungarian Vizsla dog which had never been abroad was referred with poor general condition, decrease in body weight, haematemesis and jaundice to the Central Clinic of Veterinary Science University, Budapest. After symptomatic treatment abdominal ultrasonography and diagnostic laparatomy were carried out. The dog was humanely euthanized two days later following owner's consent because of sudden worsening of clinical conditions. Two adult heartworms (Dirofilaria immitis) were found in the right ventricle partially coiling around the tricuspid valve. PCR on blood was positive for both D. immitis and Dirofilaria repens while only D. repens microfilariae were found by modified Knott's test and the serological test was negative for D. immitis antigens. This is the first, confirmed report of autochthonous canine heartworm infection in Hungary

Veterinari i pčelarstvo: Koje su uloge, očekivanja i perspektive u budućnosti? – pregledni rad.

Apiculture is an important economy sector, facing a real health crisis. Honeybees, as well as other insect pollinator populations have been in decline over recent decades, and diseases are one of the principal challenges. To face this situation, public and private veterinary services are needed to manage surveillance, control, eradication and prevention measures of honeybee diseases within their territories, in close collaboration with beekeepers and farmers. It is necessary urgently to clarify the current problems and threats to apiculture in the public health sector. Due to possible interactions between bee health and production, the negative effects of environmental xenobiotics and climate change, this field must be considered as an interdisciplinary research issue. Moreover, honeybee veterinary medicine is increasingly engaged in the One Health approach, due to the possibility that the poor health of honeybee colonies affects human life. This review identified the key veterinarian roles, gaps in their higher education curriculum, as well as the necessity for research linkage between expectations and professional perspectives.Pčelarstvo je važan sektor gospodarstva koji se suočava sa zdravstvenom krizom. Zajednice medonosne pčele, kao i populacije drugih kukaca oprašivača se smanjuju posljednjih desetljeća. Bolesti se smatraju jednim od glavnih uzroka gubitaka u pčelarstvu. Kako bi se suočili s tim problemom potrebno je povoditi veterinarski nadzor, kontroliranje, suzbijanje i preveniranje bolesti pčela uključivanjem javnih i privatnih veterinarskih organizacija na području njihova djelovanja i u bliskoj suradnji s pčelarima i poljoprivrednicima. Nužno je potrebno osvijestiti aktualnu problematiku i ugroženost pčelarstva u javnozdravstvenom sektoru. Zbog mogućih negativnih učinaka ksenobiotika iz okoliša i klimatskih promjena na zdravlje i proizvodnju pčelinjih zajednica ovo područje zahtjeva provedbu intradisciplinarnih istraživanja. Štoviše, veterinarska medicina u području zdravlja zajednica medonosne pčele zauzima iznimno važan pristup Jednog zdravlja upravo zbog mogućnosti da narušeno zdravlje pčelinjih zajednica može negativno utjecati na zdravlje ljudi. U ovom radu istaknute su glavne uloge veterinara u pčelarstvu, nedostatci u kurikulima studijskih programa veterinarske medicine visokoobrazovnih ustanova i nužnosti provedbe istraživanja koja povezuju očekivanja i stručne perspektive

Expression monitoring of relevant sensitivity genes in honey bee antennae and their relationship with Hygienic Behavior

Honeybees are very important microlivestock, not only for the economic value of their productions but also for the crucial role they fulfill as pollinators. Recently, colony losses have been recorded throughout Europe and the reasons underpinning such phenomenon can be addressed to agro-chemicals and pathogens. Among the latter, the parasitic mite Varroa destructor is considered the principal mortality cause. Hygienic behavior (HB) in honeybee (Apis mellifera) involves the detection and removal of brood affected by bacterial, fungal diseases, and parasitization. This behavior is part of a series of strategies evolved by social insects known as social immunity, which confers disease resistance thanks to the persistent elimination of pathogens and parasites from the hive, limiting their multiplication and the infection of other bees. It has previously reported that physiological changes in peripheral sense tissues of insects influence the behavioral state of individuals, and it has been suggested that changes in gene expression at antennal level can contribute to shifts in the behavioral states of honeybees (Vergoz et al., 2009). Moreover, it has previously reported that antennae hold a key role in the process of recognition of abnormalities in the brood by honeybees (Mondet et al., 2015). The aim of the work was to investigate the expression level of selected genes (Obp3, Obp4, Obp16, Obp18, Act5C, Mblk-1) through RealTime-PCR in honeybee antennae. These targets are reported as potential biomarkers of HB in previous transcriptomic and proteomic studies (Mondet et al., 2015; Guarna et al., 2015). Hygienic behavior has been measured in the field through optimized freeze-killed-brood method on 10 colonies, from which workers of known age (15 days old) have been collected for molecular analysis (Arathi et al., 2000). Preliminary results show that Obp3 is the least expressed among the tested genes, but its expression pattern is linked to the HB value; in particular, highly hygienic colonies express Obp3 significantly at higher rate with respect to the lower HB group of colonies

Obp4 and Act5C gene expression is related to Hygienic behavior in honeybee families

Honeybees (Apis mellifera) evolved social immunity mechanisms based on coordinated behavioral mechanisms for reducing the spread of pathogens and parasites. In particular, Hygienic Behavior (HB) is an actively participation of family defense by uncapping and removal of dead, diseased or parasitized brood (Cremer et al., 2007). Currently, HB evaluation is performed only by in-field empirical assays, and suitable molecular markers that could allow the discrimination between families with High and Non-High HB score are not yet available. The research activity was characterized by a transversal approach: from in-field phenotypic characterization, to expression profiling of selected coding genes in honeybee brains. The expression analysis was performed on pools of 15 days-old honeybees collected in spring 2017 from 10 different colonies (5 with high HB and 5 with low HB scores). The brains were dissected and total RNA was purified. After quantitation and quality evaluation, the extracted RNAs were reverse transcribed to cDNAs and Real Time PCR was performed on different target genes (Act5C, Mblk-1, Obp3, Obp4, Obp16 and Obp18) previously showed by transcriptomic and proteomic studies to be involved in HB (Cristino et al., 2014; Takeuchi et al., 2001; Chandrasekaran et al., 2011). The comparative analysis of RT-PCR threshold cycles (Ct) showed that Mblk-1 was the least expressed gene among the analyzed targets. Most importantly, statistical analysis of expression data validated (p<0.05) the delta-Ct between Obp4 and Act5C as a predictive index of high vs low HB score. These results confirmed the findings obtained through a preliminary experiment performed in spring 2016 (Dell\u2019Orco et al., 2016)

Candidate molecular markers of hygienic behavior in honeybees (Apis mellifera): an expression study

Honeybees (Apis mellifera) evolved social immunity mechanisms based on coordinated behavioral mechanisms for reducing the disease risk in the hive. Among these, Hygienic Behavior (HB) leads to the uncapping and removal of dead, diseased or parasitized brood (Cremer, 2007). The characterization of expression markers of HB could greatly assist in-field selection still incomplete. This work was characterized by a transversal approach: from in-field phenotypic characterization, to expression profiling of selected honeybee brain non-coding (microRNAs) and coding (RNAm) genes to assess differential expression of selected gene targets between selected honeybee families with high and low HB, respectively. After a preliminary work performed in 2015, during spring 2016 honeybee nurses were sampled from 28 hives simultaneously with the evaluation of HB score through freeze-killed brood assay performed with the cooperation of professional honeybee breeders (E. Bonfanti, L. Sesso). The nurses from 16 families (8 with the highest and 8 with the lowest HB scores, respectively) underwent expression analysis of the following target genes potentially linked to HB and/or learning processes: ame-miR-219, ame-miR-276, let-7a (Behura, 2010) ame-miR-932 (Cristino, 2014), and Act5C, Mblk-1 (Takeuchi, 2001) and Obp4 (Chandrasekaran, 2011). The experimental workflow was as follows: 1) dissection and pooling of brains, 2) total RNA extraction, quantification and retrotranscription, 3) RT-quantitative Real Time PCR. The differential expression analysis of the candidate markers, performed after selection of the normalizing gene through geNorm and NormFinder algorithms, highlighted a correlation of OBP4 and ACT5C expression level with HB score

Enhanced diagnostic protocol to identify E.coli VTEC from milk filters

The EHEC (enterohemorrhagic E. coli) are a subgroup of VTEC with strong pathogenicity. The most well-known EHEC serotype is E. coli O157:H7, which has been implicated in many large outbreaks of deadly human diseases. However, EHEC strains of other serotypes have increasingly been implicated in sporadic cases and outbreaks of serious illness in humans, e.g., serotypes O26, O45, O103, O111, O121 and O145. Epidemics studied from 1982 to date have shown that ruminants, and in particular bovine, appear almost always involved in the transmission of these bacteria to humans through direct or indirect fecal contamination of foods. Unpasteurized milk and milk products are considered minor, but important sources of infection. The possible ways to the entrance of VTEC in milk are fecal contamination and mammary excretion during E.coli mastitis. Between the two ways, the first is considered much more frequent in practice, but it cannot be excluded that a small part of EHEC found in milk resulting from mammary gland, as reported. Previous investigation suggested that milk filters used in milking machines could be a useful control point to identify the presence of EHEC in dairy herds. However, conventional methods to identify the presence of EHEC have a poor sensitivity due the high content of fecal bacteria of these filters. In order to set up a monitoring scheme to identify herds at risk, we developed and tested a diagnostic protocol involving VIDAS\uae UP E.coli serogroups (ESPT) which is a method using phage recombinant proteins for the immuno-concentration (IC) of E.coli serogroups O157, O26, O103, O111, O145, O45 and O121 from food, multiplex PCR and high resolution melting analysis (HRMA). Practically, bulk tank milk or washing solution obtained from milk filters after stomacher mixing were analyzed by Vidas ESPT. After incubation, the solution obtained was analyzed by multiplex PCR based on serotype-specific primers coding for O-antigen regions of the seven major VTEC serogroups available in literature. If PCR was positive for any of the seven serogroups, a HRMA-based protocol to detect virulence-predictive SNPs, as discovered by Norman et al., 2012, was applied to confirm the presence of a EHEC strain. The protocol was preliminary validated by inoculation of milk and milk filters with a known concentration of the seven EHEC serotypes (O26, O45, O103, O111, O121, O145, O157). The results confirmed that this protocol was able to identify as low as 101 UFC in both milk and milk filters. The protocol applied to milk and milk filters obtained from 70 dairy herds allow to identify 2 EHEC from milk and 17 from milk filters for one or more of the EHEC serogroups considered. The proposed protocol confirmed to be useful in detecting the presence of EHEC and that milk filters are an important critical control point to identify herd at risk

A new integrated approach to analyze bulk tank milk and raw milk filters for the presence of the E. coli serogroups frequently associated with VTEC status

We optimized a combination of microbiological and molecular methods to quickly identify the presence of the O157 and the six non-O157 serogroups (O26, O45, O103, O111, O121 and O145) most frequently associated with VTEC status, at herd level. The lower detection limit of this methodology is 101 CFU/ml for each of the serogroups tested. We tested 67 bulk tank milk (BTM) and raw milk filters (RMF) derived from dairy herds located in Lombardy and Trentino Alto Adige. We identified 3 positive samples and 20 positive samples out of 67 respectively in the BTM and RMF. Interestingly, several samples showed positivity for more than one serogroups at the same time. We also identified the presence of E. coli O45 and O121 for the first time in raw milk and raw milk filters. Once screened the seven serogroups of interest in our samples, we evaluated the real pathogenicity of our positive, non-O157 samples through two parallel molecular biology methods: virulence gene research by PCR, and HRMA and sequencing. The most frequently isolated serogroups in milk were O157 (2.64%), O103 (2.11%), and O145 (1.06%), while in RMF the frequencies were, respectively 14.92%, 4.48%, and 2.98%. Moreover, this is the first published report in Italy of positive recovery of O45 and O121 serogroups in milk and milk filters. The new diagnostic approach proposed investigate the presence of the O157 and big six non-O157 serogroups at farm level and not to identify VTEC hazard only once the product is processed and/or is ready to be consumed

MicroRNA expression correlated with hygienic behaviour in honeybees

Honeybees (Apis mellifera) play important roles in modern agriculture regarding zootechnical production and crop pollination. Recently, honeybees have received more attention from the public, beekeepers and researchers due to emerging heath issues. Thus, scientific interest for honeybee health and selection resistance to major pathogens is sharply increasing. Honeybees evolved social immunity mechanisms consisting in the cooperation of individuals to control disease level in the hive, and in particular hygienic behavior (HB), as based on the uncapping and removal of dead, diseased or parasitized brood. HB is affected by heritable and environmental factors, and specific neurogenomic states can be inferred based on the coordinated brain expression of transcription factors and their predicted target genes, including Mblk-1 (transcription factor that function in the mushroom body) and Obp4 (sensitive olfactory detection in the antennae of adult bees). Besides, microRNAs are known to influence neurological status linked to age-related social behaviour in honeybees7. In order to investigate the relationship between microRNA expression and HB, the present work performed the expression profile of selected honeybee brain microRNA in individual\u2019s honeybee from field colonies with high HB level compared to low HB level, in comparison with the expression profile of Mblk-1 and Obp4. The genetic information resulting from this project could help to understand the role of microRNAs in HB and to drive honeybee selection schemes for production, health, and behavioral traits favoring pathogen control