First National Prevalence in Italian Horse Population and Phylogenesis Highlight a Fourth Sub-Type Candidate of Equine Hepacivirus.

Equine hepacivirus (EqHV, Flaviviridae, hepacivirus) is a small, enveloped RNA virus generally causing sub-clinical hepatitis with occasional fatalities. EqHV is reported in equids worldwide, but for Italy data are limited. To address this, a survey study was set up to estimate prevalence at a national level and among different production categories (equestrian; competition; work and meat; reproduction) and national macro-regions (North, Central, South, and Islands). Data obtained testing 1801 horse serum samples by Real-Time RT PCR were compared within the categories and regions. The NS3 fragment of the PCR-positive samples was sequenced by Sanger protocol for phylogenetic and mutational analysis. The tertiary structure of the NS3 protein was also assessed. The estimated national prevalence was 4.27% [1.97-6.59, 95% CI] and no statistical differences were detected among production categories and macro-regions. The phylogenesis confirmed the distribution in Italy of the three known EqHV subtypes, also suggesting a possible fourth sub-type that, however, requires further confirmation. Mutational profiles that could also affect the NS3 binding affinity to the viral RNA were detected. The present paper demonstrates that EqHV should be included in diagnostic protocols when investigating causes of hepatitis, and in quality control protocols for blood derived products due to its parental transmission.

Histiocytic Sarcoma in a Captive Hybrid Orangutan (Pongo sp.): Morphological and Immunohistochemical Features.

Histiocytic sarcoma (HS), an infrequent highly aggressive hematopoietic tumor, has been observed in diverse animal species, with isolated occurrences in non-human primates. This study describes the first case of disseminated HS in a 45-year-old female hybrid captive orangutan. The clinical profile mirrored symptoms seen in human HS cases, encompassing anorexia and ascites. Detailed histopathological examination demonstrated characteristic features of this tumor and immunohistochemistry, using markers such as Iba-1 and HLA-DR, confirmed the diagnosis. Significantly, the absence of CD163 and CD204 expression challenges their diagnostic utility in non-human primates. This investigation enhances our understanding of HS diagnosis in non-human primates, underscoring the necessity for standardized markers and diagnostic protocols.

Integrated Pest Management Strategies to Control Varroa Mites and Their Effect on Viral Loads in Honey Bee Colonies.

Honey bee viruses in combination with varroa mite are very damaging for honey bee colonies worldwide. There are no effective methods to control the viral load in honey bee colonies except regular and effective control of mites. Integrated Pest Management strategies are required to effectively control mites with veterinary medicines based on organic compounds. We evaluated the effect of two brood interruption techniques, queen caging (QC) and trapping comb (TC), followed by an oxalic acid treatment, on the mite fall, colony strength, and viral load of Deformed Wing Virus (DWV) and Acute Bee Paralysis Virus (ABPV). In this paper, we report the data obtained in two experimental sites, in Slovenia and Italy, in terms of the varroacide efficacy, colony strength, and viral load. The number of adult bees after the adoption of the two techniques showed similar decreasing trends in both locations. The viral load of Acute Bee Paralysis Virus did not show any significant reduction after 25 days, reported as the number of Real-Time PCR cycles needed to detect the virus. The viral load of DWV also did not show a significant reduction after 25 days. The acaricidal efficacy of the applied protocols was high in both experimental groups and in both apiaries. Both the queen caging and trapping comb techniques, followed by an oxalic acid treatment, can be considered effective varroa treatment strategies, but further studies should be carried out to evaluate the long-term effects on viral loads to plan the Integrated Pest Management strategy with the right timing before wintering.

IPM Strategy to Control EFB in Apis mellifera: Oxytetracycline Treatment Combined with Partial Shook Swarm and Queen Caging.

We tested an integrated pest management (IPM) strategy to control European foulbrood (EFB) in honey bees. Colonies affected by EFB were assigned to two homogenous groups: an oxytetracycline-treated group (1.5 g OTC/hive) that underwent partial shook swarm (PSS) in combination with queen caging (QC) and an untreated group where only two beekeeping techniques, PSS and QC, were applied. The consumption of sucrose solution, the strength of the colonies, side effects of the mentioned techniques, clinical as well as subclinical relapses of EFB, and the amount of OTC residues in the honey were assessed over a 7-month-long monitoring period. Regarding the consumption of the sucrose solution, there was no significant difference between the OTC-treated and untreated groups. The strength of the untreated colonies was consistently but not significantly higher than those treated with OTC. PSS combined with QC resulted in various side effects in both groups: queen loss (52%), absconding (8%), and drone-laying queen (4%). Untreated colonies (16.7%) showed clinical EFB relapses 4 months after the application of PSS along with QC, while 15.4% of the OTC-treated colonies were confirmed EFB-positive by PCR. OTC residues were detected in the honey yielded in the cases of both groups. Two months after the PSS, the amount of OTC residues in the untreated group was 0.6 ± 0.2 µg/kg, while that in the OTC-treated group amounted to 5.8 ± 11.6 µg/kg; both results are below the maximum residue limit (MRL) of 100 ppb considered in the EU for cascade use.

Seroconversion of a Swine Herd in a Free-Range Rural Multi-Species Farm against HPAI H5N1 2.3.4.4b Clade Virus.

Starting from October 2021, several outbreaks of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 were reported in wild and domestic birds in Italy. Following the detection of an HPAIV in a free-ranging poultry farm in Ostia, province of Rome, despite the lack of clinical signs, additional virological and serological analyses were conducted on samples collected from free-ranging pigs, reared in the same holding, due to their direct contact with the infected poultry. While the swine nasal swabs were all RT-PCR negative for the influenza type A matrix (M) gene, the majority (%) of the tested pigs resulted serologically positive for the hemagglutination inhibition test and microneutralization assay, using an H5N1 strain considered to be homologous to the virus detected in the farm. These results provide further evidence of the worrisome replicative fitness that HPAI H5Nx viruses of the 2.3.4.4b clade have in mammalian species. Moreover, our report calls for additional active surveillance, to promptly intercept occasional spillover transmissions to domestic mammals in close contact with HPAI affected birds. Strengthened biosecurity measures and efficient separation should be prioritized in mixed-species farms in areas at risk of HPAI introduction.

Articular Cartilage Regeneration by Hyaline Chondrocytes: A Case Study in Equine Model and Outcomes.

Cartilage injury defects in animals and humans result in the development of osteoarthritis and the progression of joint deterioration. Cell isolation from equine hyaline cartilage and evaluation of their ability to repair equine joint cartilage injuries establish a new experimental protocol for an alternative approach to osteochondral lesions treatment. Chondrocytes (CCs), isolated from the autologous cartilage of the trachea, grown in the laboratory, and subsequently arthroscopically implanted into the lesion site, were used to regenerate a chondral lesion of the carpal joint of a horse. Biopsies of the treated cartilage taken after 8 and 13 months of implantation for histological and immunohistochemical evaluation of the tissue demonstrate that the tissue was still immature 8 months after implantation, while at 13 months it was organized almost similarly to the original hyaline cartilage. Finally, a tissue perfectly comparable to native articular cartilage was detected 24 months after implantation. Histological investigations demonstrate the progressive maturation of the hyaline cartilage at the site of the lesion. The hyaline type of tracheal cartilage, used as a source of CCs, allows for the repair of joint cartilage injuries through the neosynthesis of hyaline cartilage that presents characteristics identical to the articular cartilage of the original tissue.

Complete Genome of African Swine Fever Virus Genotype II in Central Italy.

We report here the whole-genome sequence of the African swine fever virus (ASFV) genotype II, strain 20355/RM/2022_Italy, identified in a wild boar in the city of Rome (Lazio region, Italy) in April 2022.

Evidence of transplacental transmission of equine piroplasms Theileria equi and Babesia caballi in an Italian breed mare.

Equine piroplasmosis (EP) is a vector borne disease caused by apicomplexans protists Babesia caballi (Nuttal et Strickland, 1910) and Theileria equi (Laveran, 1901). Carrier mares may transmit the infection transplacental resulting in neonatal piroplasmosis or abortions. This event has been described for T. equi by several authors over the world, but no evidence for B. caballi has been reported in Europe. In this study, vertical transmission for both parasites in an Italian breed mare has been confirmed using molecular and microscopic tools. Transplacental transmission is an underestimated problem mainly in endemic areas as it not only contributes to the spread and maintenance of the infection, but also produces significant economic losses.

Detection of viruses from feces of wild endangered Macaca maura: a potential threat to moor macaque survival and for zoonotic infection.

BACKGROUND: To date, there is a scarcity of information and literature on Macaca maura health status relative to viral diseases. The objectives of the present study were to investigate on the potential spread of enteric and non-enteric viruses shed in the environment through a wild macaque feces and to understand the possible interrelation in the spread of zoonotic viruses in a poorly studied geographical area, the Sulawesi Island. This study will also contribute providing useful information on potential threats to the health of this endangered species. METHODS: The sampling was conducted between 2014 and 2016 in the Bantimurung Bulusaraung National Park, in the south of the Sulawesi Island and non-invasive sampling methods were used to collect fresh stools of the M. maura, one of the seven macaque species endemic to the island of Sulawesi, Indonesia. The population under study consisted in two wild, neighboring social macaque groups with partially overlapping home ranges; twenty-four samples were collected and examined using negative staining electron microscopy and a panel of PCR protocols for the detection of ten RNA and two DNA viruses. RESULTS: Viral particles resembling parvovirus (5 samples), picornavirus (13 samples) and calicivirus (13 samples) were detected by electron microscopy whereas the PCR panel was negative for the 12 viruses investigated, except for one sample positive for a mosquito flavivirus. The results did not correlate with animal sex; furthermore, because all of the animals were clinically healthy, it was not possible to correlate feces consistency with viral presence. CONCLUSIONS: As information on viral infections in wild moor macaques remains limited, further studies are yet required to identify the fecal-oral and blood transmitted potentially zoonotic viruses, which may infect the moor macaque and other macaque species endemic to the South Sulawesi Island.

Comparison of direct and indirect methods to maximise the detection of Babesia caballi and Theileria equi infections in Central Southern Italy.

Equine piroplasmosis is a disease of equids, caused by tick-borne apicomplexan protozoan pathogens Babesia caballi and Theileria equi, which, according to the World Organisation for Animal Health (OIE), can be diagnosed by enzyme-linked immunosorbent assay (ELISA), immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR). The present study was conducted to evaluate and compare the assays available for the diagnosis of equine piroplasmosis. Data employed were obtained from 1300 blood samples collected between 2012-2014 from asymptomatic and symptomatic equines (horses and donkeys) of central-southern regions of Italy and analyzed by ELISA, IFAT, PCR (one commercial and one from literature) and blood smear microscopic examination. Statistical differences of the proportions of positivity for each parasite and group (asymptomatic and symptomatic) among the methods were verified by the z test to identify the most sensitive. The concordance between each pair of methods - for each parasite and within the groups - and trends in detection of suspect samples of four hypothetical diagnostic algorithms using serological and biomolecular assays were evaluated to identify the most suitable laboratory diagnostic workflow. The results of this study highlighted a lower capacity to detect suspect samples of commercial ELISA for B. caballi in all groups when compared to biomolecular methods and IFAT; and of the commercial PCRs in asymptomatic animals, identifying a PCR from literature and IFAT as the best choice for a combined diagnosis. For T. equi, IFAT detected more suspect samples than ELISA, even if the latter showed good performance and some samples were positive only by the ELISA and PCR, indicating that their simultaneous employment is still advantageous. Host-parasite interaction, amino-acid/genetic diversity and differences in detection limits among the assays could be among the reasons in explaining the present results. In view of further studies, ELISA should be used in combination with PCR, that should regularly be included in the laboratory diagnosis to maximise the detection of early infections and support the evaluation of pharmacological treatment.

Comparison of PCR-based methods for the detection of Babesia caballi and Theileria equi in field samples collected in Central Italy.

Equine piroplasmosis (EP) is a disease of equids caused by Theileria equi and Babesia caballi, members of the order Piroplasmida, transmitted by several species of ticks. As the disease is endemic in many countries, a clinical examination or a serological test are required prior to movement of horses to prove freedom from infection and to avoid the introduction of EP with its sanitary and economic impact, especially in areas where it is absent. Currently, numerous diagnostic PCR protocols are available, some of which are recommended by the World Organisation for Animal Health (OIE). In order to adopt this diagnostic method, the Italian National Reference Centre for Equine Diseases (NRC-ED) conducted a preliminary comparison between an end-point PCR, nested PCR, real-time PCR, and commercial real-time PCR, for the detection of T. equi and B. caballi, respectively. One hundred and three field samples, collected during spring-summer 2013 in Latium and Tuscany regions, were employed for the study, and results discordant between detection assays were confirmed by sequencing. The reference assay was defined as that showing the highest sensitivity, and the relative sensitivity (rSe) and specificity (rSp) of the other methods were estimated referring to this assay. Agreement between methods was estimated by calculating the concordance between each pair of methods. Although no statistical differences were detected among PCR-based methods, the non-commercial real-time PCR assays seemed to be the most suitable for detection of T. equi and B. caballi, respectively. An important advantage of direct PCR detection of the pathogen, in comparison to indirect detection using serological methods, is that it allows specific treatment against the causative pathogen species responsible of the infection as well as for the definition of the infectious status of an animal for international movement.

A Canine Distemper Virus Retrospective Study Conducted from 2011 to 2019 in Central Italy (Latium and Tuscany Regions).

Canine distemper virus (CDV) is a highly lethal contagious viral pathogen mainly found in domestic and wild canids and mustelids. Although, in Italy, circulating strains of Europe 1, Europe wildlife and Arctic type are reported, data relating to Latium and Tuscany regions are limited. In view of this, through passive surveillance, we investigated the presence of CDV and which strains were circulating in these Regions. From March 2017 to October 2019, a group of 122 subjects were tested for CDV using a PCR protocol described in the literature, with 12 detected positive; analyses were carried out on a set of target samples (brain and lung, conjunctival, nasal and rectal swabs, urine or swab from bladder and intracardiac clot) that was defined for the detection of CDV in both live and dead animals. The rectal swab, easily collected also from live animals, represented the most suitable sample for CDV diagnosis, with 9 positive of the 11 (81.82%) tested. In addition, brain and lung of 15 subjects out of 181 susceptible animals collected between 2011 and 2018, during post mortem investigations in routine diagnostic activity, were CDV positive. Molecular analyses of all positive samples, using a 287 bp fragment located within the conserved N terminus of the morbillivirus nucleoprotein gene, detected the circulation of strain CDV599/2016 (KX545421.1) belonging to the "Europe wildlife" lineage, and of strain CDV12254/2015 (KX024709.1), belonging to the Arctic-lineage, thus confirming the co-circulation of the two lineages, as already noted in previous studies.

Investigation of Ixodid ticks as vectors of Babesia caballi and Theileria equi (Protozoa: Apicomplexa) in central Italy.

Babesia caballi and Theileria equi are widely recognized as causative agents of equine pirolasmosis (EP), an acute, sub-acute, and chronic disease of equines, with relevant economic impact on horse trade worldwide. Although several studies on EP prevalence from central Italy have been published, data on ticks responsible for its transmission are still lacking. In this study, we identified a potential competent vector, investigating main features of its ecology together with EP infection rates. A two-year sampling of questing ticks was carried out for the first time in Italy in an area known for high EP prevalence in horse sera, detecting the association between Rhipicephalus bursa and causative agents of EP. Most of the positive pools harbored a single infection (91.1%); mixed infections were also detected (8.9%). The infection rate for T. equi slightly decreased among years; B. caballi showed a lower, but increasing, infection rate. Tick phenology, climate variables, and peaks of EP prevalence indicated late May and second half of June as periods with the highest risk of new infections, especially during warm and dry days.

Detection of Felis catus papillomavirus type-2 DNA and viral gene expression suggest active infection in feline oral squamous cell carcinoma.

Papillomavirus (PV) infection is associated with development of epithelial cancer in different species, including domestic cat (Felis catus). Felis catus PV type-2 (FcaPV-2) is considered the causative agent of a proportion of feline cutaneous squamous cell carcinoma (SCC), through the transforming properties of its E6 and E7 oncogenes. However, the possible role of FcaPVs in the aetiology of feline oral SCC (FOSCC) is still unclear. The aim of this study was to assess the presence and gene expression of FcaPV-2 in FOSCC samples. We detected FcaPV-2 DNA in 10/32 (31%) of the analysed FOSCC by the use of PCR methods. Importantly, viral mRNA was detected by RT-PCR in 7/10 (70%) of DNA positive samples. In particular, FcaPV-2 L1, E2 and E6E7 genes were found to be expressed in 5/10 (50%), 3/10 (33%) and 5/10 (50%) samples, respectively. Viral DNA was also detected in non neoplastic oral ulcerative lesions (ULs) (4/11, 36%); qPCR suggested a difference in viral load between ULs and FOSCCs, particularly in those expressing E6E7, although it was not statistically significant. These data suggest, but do not definively prove, a possible role of FcaPV-2 in the development of a proportion of FOSCC. Moreover, L1 and E2 gene expression results indicate that FcaPV-2 infection associated with these tumours may possibly be productive.

Management of Mycobacterium avium subsp. paratuberculosis in dairy farms: Selection and evaluation of different DNA extraction methods from bovine and buffaloes milk and colostrum for the establishment of a safe colostrum farm bank.

The aim of this study was to develop and validate different innovative DNA extraction methods to detect Mycobacterium avium subsp. paratuberculosis (MAP) DNA from bovine and buffalo colostrum. Paratuberculosis is a chronic inflammatory infection of domestic and wild animals, especially ruminants, caused by MAP. The primary route of disease transmission is feces, but MAP can also be excreted in milk and colostrum. In 2015, the Italian Ministry of Health has issued a voluntary control plan of MAP in order to allow risk-based certification of bovine and buffaloes farms. In addition to the annual diagnostic screening and to the clinical surveillance of animals the plan includes the adoption of biosecurity and management measures to progressively mitigate the incidence of MAP. To achieve this goal it is crucial to ensure the accuracy of the methods used to detect the presence of MAP in bovine and buffaloes milk and colostrum, in order to: (1) support a "safe colostrum farm-bank" set-up and thus prevent the main within-farm MAP transmission route and (2) to allow the MAP-free certification of milk products for export purposes. To achieve these goals, seven different DNA extraction protocols were identified from bibliography, out of which three methods were finally selected after the adoption of an evaluation procedure aimed at assessing the efficiency of extraction of DNA, the purity of DNA and the adaptability of the DNA amplification: NucleoSpin® Food Kit (Macherey-Nagel), NucleoSpin® Food Kit (Macherey-Nagel) combined with the magnetic beads, and QIAamp Cador Pathogen Mini kit (QIAGEN). In particular, the NucleoSpin® Food Kit (Macherey-Nagel) and the QIAamp Cador Pathogen Mini kit (QIAGEN) were tested on bovine and buffalo colostrum, showing a LOD between 4 × 104 (2.6 × 106  cfu/ml) and 4.08 (26.7 cfu/ml) IS900 target copies and a LOD between 5.3 × 105 (4.1 × 106  cfu/ml) and 53 (4.1 × 103  cfu/ml) IS900 target copies, respectively.

Novel dolphin morbillivirus (DMV) outbreak among Mediterranean striped dolphins Stenella coeruleoalba in Italian waters.

An unusual mortality event (UME) of striped dolphins Stenella coeruleoalba occurred in the period July to December 2016 along the Italian Ionian coastline. We conducted a complete postmortem examination on 28 specimens and detected dolphin morbillivirus (DMV), by means of biomolecular analyses, in the target tissues of 17 animals. Unlike previous outbreaks occurring in the Mediterranean Sea in 2011 and 2013, we observed typical pathological changes suggestive of morbilliviral infection in an acute/subacute phase and immunohistochemical reactivity. The same findings were observed in 13 other specimens beached along the Italian coastline during 2016 with no temporal and geographical relationship with the ongoing epidemic outbreak. Molecular characterization and phylogenetic analysis showed that DMV sequences detected in Italy in 2016 clustered with those identified in Portugal and Galicia (Spain), representing a novel DMV strain of Atlantic origin which entered the Mediterranean Sea and affected a naïve striped dolphin population. DMV sequences detected in the previous Mediterranean outbreaks exhibited a marked genetic relatedness and diverged from those detected in cetaceans stranded along the Galician and Portuguese coasts since 2007.

Monitoring honey bee health in five natural protected areas in Italy.

The health status of the honey bee populations has attracted a great amount of interest in recent years. We investigated honey bee health in five natural protected areas in Italy from October 2009 to December 2010. Areas were selected to represent a wide range of bio­geographical zones including alpine, continental, and Mediterranean. Within each of these natural protected areas, one apiary of 20 colonies near potential pollution sources (e.g., agricultural areas, industrial areas, or urban settlements) and another apiary of 20 colonies far from possible sources of pollutants have been placed. To monitor honey bee health, colony mortality was related to: honey bee pathologies, environment (Naturality Index, plant protection products and heavy metal exposure), and apiary management. Anthropogenic pollutants and pathogens did not have significant effects on colony mortality while environment and the poor colony management skills of the beekeepers did.

Cetacean Poxvirus in Two Striped Dolphins (Stenella coeruleoalba) Stranded on the Tyrrhenian Coast of Italy: Histopathological, Ultrastructural, Biomolecular, and Ecotoxicological Findings.

Tattoo skin disease (TSD) is a poxviral disease typical of cetaceans. Two juvenile and well-preserved male striped dolphins (Stenella coeruleoalba), found stranded along the Tuscany and Latium coasts of Italy in 2015 and 2016, respectively, showed typical skin lesions ascribable to TSD. Histological, ultrastructural and biomolecular investigations confirmed a poxviral aetiology for the aforementioned skin lesions. To our knowledge, this should be the first report of TSD in cetaceans stranded along the Italian coastline. As organochlorines like PCBs and DDTs are known to be highly immunotoxic, the tissue loads of these contaminants were evaluated, in order to increase our knowledge on their potential role as well as on the relationships between the level of exposure to these pollutants and poxviral infection's occurrence.

Genetic diversity of Theileria equi and Babesia caballi infecting horses of Central-Southern Italy and preliminary results of its correlation with clinical and serological status.

Babesia caballi and Theileria equi are tick-borne pathogens causing equine piroplasmosis infecting the Equidae family in which they cause significant sanitary and economic losses. Furthermore, equine piroplasmosis is included in the World Animal Health Organization (OIE) notifiable diseases list with possible movement restrictions for positive horses. Thirty-nine EDTA and whole-blood samples collected during 2013 and 2014 from symptomatic and asymptomatic horses of Central-Southern Italy were included in the present study either because of their strongly positive results in Real Time (RT) PCRs targeting the 18S rRNA gene specific for each piroplasm and/or due to their serological ELISA/18S rRNA RT-PCR discordant results. A nested PCR amplifying the hypervariable V4 region of the 18S rRNA gene of both piroplasms was performed on all samples. T. equi positive samples were also analysed with a PCR targeting the equi merozoite antigen 1-gene (EMA-1). The sequences obtained were thirty for T. equi, 25 of which were for the hypervariable V4 region of the 18S rRNA gene and 13 for the EMA-1 gene, with eight samples positive for both targets, while only six 18S rRNA gene sequences were retrieved for B. caballi. The phylogenetic analysis results are as follows: T. equi sequences of the 18S rRNA gene clustered in three different phylogenetic groups, respectively in the A (15), B (9) and C (1) while those of B. caballi in the A (1), B1 (3) and B2 (2) groups. T. equi sequences for EMA-1 gene clustered in A (11) and in B (2). This analysis confirms that both T. equi and B. caballi sequences present a genetic heterogeneity independently of their geographical location, similar to that reported by other authors. Statistical associations were evaluated between phylogenetic groups of T. equi 18S rRNA gene and each of the following variables, using Fisher's exact test: clinical signs, serological ELISA/18S rRNA RT-PCR discordant results and T. equi EMA-1 negativity. The different groups were found to be statistically related to the presence of signs (less present in group B samples), to ELISA negativity/18S rRNA RT-PCR positivity (more seronegative samples in group B). No statistical analysis was performed for the B. caballi as the number of sequences available was insufficient and for the EMA -1 sequences which almost all grouped in the same cluster. The results here obtained provide additional information about T. equi and B. caballi sequences, which could also be used to verify the performance of serological and molecular diagnostic methods.

RNA-sequence analysis of gene expression from honeybees (Apis mellifera) infected with Nosema ceranae.

Honeybees (Apis mellifera) are constantly subjected to many biotic stressors including parasites. This study examined honeybees infected with Nosema ceranae (N. ceranae). N. ceranae infection increases the bees energy requirements and may contribute to their decreased survival. RNA-seq was used to investigate gene expression at days 5, 10 and 15 Post Infection (P.I) with N. ceranae. The expression levels of genes, isoforms, alternative transcription start sites (TSS) and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation suggesting that bees use a range of tactics to cope with the stress of N. ceranae infection. N. ceranae infection may cause reduced immune function in the bees by: (i)disturbing the host amino acids metabolism (ii) down-regulating expression of antimicrobial peptides (iii) down-regulation of cuticle coatings and (iv) down-regulation of odorant binding proteins.