Cytokine mRNA expression in the bronchoalveolar lavage cells from horses affected by different equine asthma subtypes.

Equine asthma (EA) is a respiratory syndrome associated with the increase of different leukocyte populations in the bronchoalveolar lavage fluid (BALF). Its pathogenetic mechanisms remain unclear. This study aimed to evaluate the associations between the mRNA expression of different cytokines in the BALF, different EA subtypes and lung function. Fifteen horses underwent physical examination, airway endoscopy, BALF cytology and lung function testing (8/15). One horse did not have evidence of EA and was used as healthy reference, while the others were classified as affected by neutrophilic or mixed granulocytic EA. Cells isolated from the residual BALF were used for IL-1ß, IL-2, IFN-γ, IL-4, IL-17A genes expression by quantitative RT-PCR., Cytokine expression was compared between groups, and their correlations with BALF leukocyte and lung function were evaluated. IL-1ß expression was positively correlated with BALF neutrophils count (p=0.038, r=0.56) and with increased expiratory resistance (p=0.047, r=0.76). IFN-γ was correlated with BALF mast cells (p=0.029, r=0.58). IL-4 was higher in horses with mixed granulocytic EA than neutrophilic (p=0.008), positively correlated with BALF mast cells (p=0.028, r=0.59) and inversely with whole-breath (p=0.046, r=-0.76) and expiratory reactance (p=0.003, r=-0.93). Finally, IL-17A was inversely correlated with expiratory reactance (p=0.009, r=-0.92). These results support that multiple immune responses are involved in EA pathogenesis; innate, Th2, and Th17 responses. Innate immunity appeared associated with neutrophilic inflammation, and Th2 response with increased mast cells. The role of Th1 response in EA remains questionable.

How Do Alternative Protein Resources Affect the Intestine Morphology and Microbiota of Atlantic Salmon?

The availability and cost of fishmeal constitute a bottleneck in Atlantic salmon production expansion. Fishmeal is produced from wild fish species and constitutes the major feed ingredient in carnivorous species such as the Atlantic salmon. These natural stocks are at risk of depletion and it is therefore of major importance to find alternative protein sources that meet the nutritional requirements of the Atlantic salmon, without compromising the animals' health. Terrestrial animal by-products have been used in aquaculture feed, but their use is limited by the lack of several essential amino acids and consumer acceptance. In the case of plant ingredients, it is necessary to take into account both their concentration and the extraction methodologies, since, if not dosed correctly, they can cause macro- and microscopic alterations of the structure of the gastrointestinal tract and can also negatively modulate the microbiota composition. These alterations may compromise the digestive functions, growth of the animal, and, ultimately, its well-being. An updated revision of alternative protein sources is provided, with the respective impact on the intestine health in terms of both morphology and microbiota composition. Such information may constitute the premise for the choice and development of Atlantic salmon feeds that guarantee fish health and growth performance without having a significant impact on the surrounding environment, both in terms of depletion of the fish's natural stocks and in terms of pressure on the terrestrial agriculture. The sustainability of aquaculture should be a priority when choosing next-generation ingredients.

Red mark syndrome: Is the aquaculture water microbiome a keystone for understanding the disease aetiology?

Aquaculture significantly contributes to the growing demand for food worldwide. However, diseases associated with intensive aquaculture conditions, especially the skin related syndromes, may have significant implications on fish health and industry. In farmed rainbow trout, red mark syndrome (RMS), which consists of multiple skin lesions, currently lacks recognized aetiological agents, and increased efforts are needed to elucidate the onset of these conditions. Most of the past studies were focused on analyzing skin lesions, but no study focused on water, a medium constantly interacting with fish. Indeed, water tanks are environmental niches colonized by microbial communities, which may be implicated in the onset of the disease. Here, we present the results of water and sediment microbiome analyses performed in an RMS-affected aquaculture facility, bringing new knowledge about the environmental microbiomes harbored under these conditions. On the whole, no significant differences in the bacterial community structure were reported in RMS-affected tanks compared to the RMS-free ones. However, we highlighted significant differences in microbiome composition when analyzing different samples source (i.e., water and sediments). Looking at the finer scale, we measured significant changes in the relative abundances of specific taxa in RMS-affected tanks, especially when analyzing water samples. Our results provide worthwhile insight into a mostly uncharacterized ecological scenario, aiding future studies on the aquaculture built environment for disease prevention and monitoring.

Molecular characterization of Dictyocaulus nematodes in wild red deer Cervus elaphus in two areas of the Italian Alps.

Nematodes of the genus Dictyocaulus are the causative agents of parasitic bronchitis and pneumonia in several domestic and wild ungulates. Various species have been described in wild cervids, as the case of Dictyocaulus cervi in red deer, recently described as a separate species from Dictyocaulus eckerti. In Italy, information on dictyocaulosis in wildlife is limited and often outdated. In this work, 250 red deer were examined for the presence of Dictyocaulus spp. in two areas of the Italian Alps (n = 104 from Valle d'Aosta, n = 146 from Stelvio National Park), and the retrieved lungworms were molecularly characterized. Lungworms were identified in 23 and 32 animals from Valle d'Aosta and Stelvio National Park, respectively. The nematodes, morphologically identified as D. cervi, were characterized molecularly (18S rDNA, ITS2, and coxI). Consistently, almost all specimens were found to be phylogenetically related to D. cervi. Three individuals, detected from both study sites and assigned to an undescribed Dictyocaulus sp., clustered with Dictyocaulus specimens isolated from red deer and fallow deer in previous studies. Within each of D. cervi and the undescribed Dictyocaulus sp., the newly isolated nematodes phylogenetically clustered based on their geographical origin. This study revealed the presence of D. cervi in Italian red deer, and an undetermined Dictyocaulus sp. that should be more deeply investigated. The results suggest that further analyses should be focused on population genetics of cervids and their lungworms to assess how they evolved, or co-evolved, throughout time and space and to assess the potential of transmission towards farmed animals.

A Novel High Discriminatory Protocol for the Detection of Borrelia afzelii, Borrelia burgdorferi Sensu Stricto and Borrelia garinii in Ticks.

Bacteria of the Borrelia burgdorferi sensu lato complex are the causative agents of Lyme borreliosis (LB). Even if the conventional diagnosis of LB does not rely on the species itself, an accurate species identification within the complex will provide a deepened epidemiological scenario, a better diagnosis leading to a more targeted therapeutic approach, as well as promote the general public's awareness. A comparative genomics approach based on the 210 Borrelia spp. genomes available in 2019 were used to set up three species-specific PCR protocols, able to detect and provide species typing of Borrelia afzelii, Borrelia burgdorferi sensu stricto (s.s.) and Borrelia garinii, the three most common and important human pathogenic Lyme Borrelia species in Europe. The species-specificity of these protocols was confirmed on previously identified B. afzelii, B. burgdorferi s.s. and B. garinii specimens detected in Ixodes ricinus samples. In addition, the protocols were validated on 120 DNA samples from ticks collected in Sweden, showing 88% accuracy, 100% precision, 72% sensitivity and 100% specificity. The proposed approach represents an innovative tool in epidemiological studies focused on B. burgdorferi s.l. occurrence in ticks, and future studies could suggest its helpfulness in routine diagnostic tests for health care.

Rearing Environment during the Endogenous Feeding Stage of Acipenser baerii.

The aim of this study was to evaluate behaviour, growth, lipid composition, muscle development, and stress status of Siberian sturgeon larvae reared with two types of substrate: Bioballs1 (BB1) and Bioballs2 (BB2), when compared to no substrate (CTR). Sampling points were: hatching (T0), schooling (T1), and yolk-sac full absorption (T2). BB1 larvae were less active and showed no schooling behaviour. At T1 and at T2, BB1 larvae showed a significantly higher weight and total length than larvae reared in either CTR or BB2 (p < 0.05). The lipid content of larvae decreased over time, with little relevant differences between groups. At T2, total muscle area, slow muscle area and fast muscle area were significantly higher in larvae reared in BB1 (p < 0.05). No significant differences in muscle proliferation were found between groups. Real Time PCR was used for evaluating the relative expression of a pool of genes: myod, myog, mrf4, igf2, hsp70, hsp90a, hsp90b, and glut2. The expression of these genes did not seem to be much affected by the type of rearing substrate, except for myog and hsp70 at T1, which was greater in BB2 larvae. Our data suggest that the presence of a substrate during this developmental period seems to have positive effects but further studies would be necessary during the exogenous feeding stage.

Transporter gene expression and Wolbachia quantification in adults of Dirofilaria immitis treated in vitro with ivermectin or moxidectin alone or in combination with doxycycline for 12 h.

Due to their marked larvicidal activity, macrocyclic lactones (MLs) are used for the prevention of heartworm disease ( Dirofilaria immitis) in dogs. They have also been shown to eliminate adult parasites after long-term administration, with a so-called "slow-kill" effect. In addition, recent studies have established that a combination of doxycycline, which eliminates the endosymbiont Wolbachia, and MLs has superior adulticide effects when compared to MLs alone. It has been hypothesized that the apparent synergism between doxycycline/MLs may be due to interaction with drug efflux transport proteins. The aim of the present study was to evaluate gene expression of several transport proteins in D. immitis adults treated in vitro either with doxycycline alone, ivermectin alone, moxidectin alone, or a combination of ivermectin or moxidectin with doxycycline for 12 h. Quantitative PCR analysis showed a sex-dependent response to treatments. In female worms, Dim-pgp-10, Dim-haf-1 and Dim-haf-5 were upregulated compared to controls with doxycycline alone and when combined with ivermectin. Moxidectin did not induce any changes in gene expression. In males, moxidectin administered alone induced a slight increase in Dim-pgp-10, Dim-pgp-11and Di-avr-14, while ivermectin in combination with doxycycline produced significant upregulation of the ML receptor Di-avr-14. These results suggest possible synergism between the two drug classes and different susceptibility of males vs. females to adulticide effects.

A dual endosymbiosis supports nutritional adaptation to hematophagy in the invasive tick Hyalomma marginatum.

Many animals are dependent on microbial partners that provide essential nutrients lacking from their diet. Ticks, whose diet consists exclusively on vertebrate blood, rely on maternally inherited bacterial symbionts to supply B vitamins. While previously studied tick species consistently harbor a single lineage of those nutritional symbionts, we evidence here that the invasive tick Hyalomma marginatum harbors a unique dual-partner nutritional system between an ancestral symbiont, Francisella, and a more recently acquired symbiont, Midichloria. Using metagenomics, we show that Francisella exhibits extensive genome erosion that endangers the nutritional symbiotic interactions. Its genome includes folate and riboflavin biosynthesis pathways but deprived functional biotin biosynthesis on account of massive pseudogenization. Co-symbiosis compensates this deficiency since the Midichloria genome encompasses an intact biotin operon, which was primarily acquired via lateral gene transfer from unrelated intracellular bacteria commonly infecting arthropods. Thus, in H. marginatum, a mosaic of co-evolved symbionts incorporating gene combinations of distant phylogenetic origins emerged to prevent the collapse of an ancestral nutritional symbiosis. Such dual endosymbiosis was never reported in other blood feeders but was recently documented in agricultural pests feeding on plant sap, suggesting that it may be a key mechanism for advanced adaptation of arthropods to specialized diets.

Molecular and Immunohistochemical Expression of LTA4H and FXR1 in Canine Oral Melanoma.

Oral melanoma is a common canine tumor whose prognosis is considered ominous, but poorly predicted by histology alone. In the present study the gene and protein expression of Leukotriene A4 hydrolase (LTA4H) and Fragile-X-mental retardation-related protein1 (FXR1), both reported as related to metastatic potential in different tumors, were investigated in canine oral melanoma. The main aim of the study was to confirm and quantify the presence of LTA4H and FXR1 genes and protein in oral melanomas. A secondary aim was to investigate their association with histologic prognostic criteria (mitotic count, Ki-67 index). Formalin-fixed-paraffin-embedded canine oral melanomas (36) were collected and histopathological evaluation carried out. Immunolabelling for LTA4H and FXR1 and Ki-67 were performed. RT-PCR evaluated LTA4H and FXR1 gene expressions. Histologically, most tumors were epithelioid cell melanomas (19/36) and were amelanotic, mildly or moderately pigmented (5, 12 and 13/36 respectively), only 6 were highly pigmented. Mitotic count ranged 1-106, Ki-67 index ranged 4.5-52.3. Thirty-two (32/32) melanomas immunolabelled for LTA4H and 33/34 for FXR1. RT-PCR values ranged 0.76-5.11 ΔCt for LTA4H and 0.22-6.24 ΔCt for FXR1. Molecular and immunohistochemical expression of both LTA4H and FXR1 did not statically correlate with mitotic count or Ki-67 index. The present study demonstrates LTA4H and FXR1 gene and protein in canine oral melanoma, however their expression is apparently unrelated to histopathologic prognostic criteria. Although LTA4H and FXR1 seem unrelated to tumor behavior, their extensive expression in the present cohort of cases suggest that they may play a role in canine oral melanoma oncogenesis.

Molecular Survey of Babesia spp. and Anaplasma phagocytophilum in Roe Deer from a Wildlife Rescue Center in Italy.

Babesia ssp. and Anaplasma spp. are tick-borne microorganisms representing a possible health risk for domestic and wild animals, as well as humans. Roe deer serve as a suitable reservoir host for some species ascribed to Babesia spp. and Anaplasma phagocytophilum taxa, also due to its important role in the maintenance of large populations of Ixodes ricinus, the main tick vector of these pathogens in Europe. Roe deer populations have been recently expanding throughout Europe, namely in Italy. However, the collection of samples from free-ranging wild animals for diagnostic investigations often includes several practical issues. This problem can be overcome using samples provided by wildlife rescue centers making them available for investigations following routine analyses. The presence of Babesia spp. and Anaplasma spp. in blood samples of 43 roe deer rescued by a wildlife rescue center in Emilia-Romagna region (Italy) was molecularly investigated. PCR screening revealed the presence of at least one pathogen in 86.05% of the animals, while co-infection occurred in 18.92% of the tested individuals. Zoonotic Babesia venatorum was found in 6.98% of the samples, while Babesia capreoli and Anaplasma phagocytophilum were detected in 74.42% and in 20.93%, respectively. No hematological signs compatible with clinical anaplasmosis or piroplasmosis, as well as absence of intracellular circulating microorganisms in blood smears, were observed, suggesting asymptomatic infection in the tested animals. These results confirm the usefulness of wild rescued animals as convenient source of biological samples for tick-borne pathogens investigation and the role of roe deer as a key factor in the endemic cycle of Babesia species and A. phagocytophilum.

Protocol optimization for simultaneous DNA and RNA co-extraction from single hard tick specimens.

Hard ticks are important vectors of DNA- and RNA-based infectious microorganisms, but they also host complex microbial communities in which pathogens and symbionts can interact among each other and with the arthropod host itself. Molecular investigations on ticks and their hosted microorganisms are important for human and animal health. These analyses often imply the use of both DNA and RNA, with prompt preservation of nucleic acids after collection, and safe handling in case of low-level containment. Several commercial kits are available for the co-extraction of DNA and RNA; however, cost can be a limiting factor for the choice of this method, which also require additional reagents for nucleic acids preservation before extraction. Additionally, extraction buffers provided in commercial kits do not guarantee the safe handling in case of hazardous biological material. With the aim of epidemiological screenings for tick-borne pathogens and gene expression analyses focused on the relationship among ticks and their microbial communities, an optimized protocol for DNA and RNA co-extraction from single adult hard tick specimens (Ixodidae) has been developed using TRIzolⓇ LS Reagent.A method for•DNA/RNA co-extraction from adult hard tick specimens;•Safe sample handling;•Obtaining DNA and RNA simultaneously for diagnostic procedures and RNA for gene expression/transcriptomic analyses.

Investigation of Tick-Borne Pathogens in Ixodes ricinus in a Peri-Urban Park in Lombardy (Italy) Reveals the Presence of Emerging Pathogens.

Ticks are important vectors of a great range of pathogens of medical and veterinary importance. Lately, the spread of known tick-borne pathogens has been expanding, and novel ones have been identified as (re)emerging health threats. Updating the current knowledge on tick-borne pathogens in areas where humans and animals can be easily exposed to ticks represents a starting point for epidemiological studies and public awareness. A PCR screening for tick-borne pathogens was carried out in Ixodes ricinus ticks collected in a peri-urban recreational park in Ticino Valley, Italy. The presence of Rickettsia spp., Borrelia burgdorferi senso latu complex, Anaplasma spp. and Babesia spp. was evaluated in a total of 415 I. ricinus specimens. Rickettsia spp. (R monacensis and R. helvetica) were detected in 22.96% of the samples, while B. burgdorferi s.l. complex (B. afzelii and B. lusitaniae) were present in 10.94%. Neoehrlichia mikurensis (1.99%) and Babesia venatorum (0.73%) were reported in the area of study for the first time. This study confirmed the presence of endemic tick-borne pathogens and highlighted the presence of emerging pathogens that should be monitored especially in relation to fragile patients, the difficult diagnosis of tick-borne associated diseases and possible interactions with other tick-borne pathogens.

Development of a PCR for Borrelia burgdorferi sensu lato, targeted on the groEL gene.

Borrelia burgdorferi sensu lato (s.l.) is the etiological agent of Lyme disease, transmitted by ticks of the genus Ixodes Latreille. Diagnosis of Lyme disease in humans is often difficult and a detailed knowledge of the circulation of B. burgdorferi s.l. in tick hosts is therefore fundamental to support clinical procedures. Here we developed a molecular approach for the detection of B. burgdorferi s.l. in North Italian Ixodes ricinus (Linnaeus). The method is based on the amplification of a fragment of the groEL gene, which encodes a heat-shock protein highly conserved among B. burgdorferi s.l. species. The tool was applied in both qualitative and Real-time PCR approaches testing ticks collected in a North Italian area. The obtained results suggest that this new molecular tool could represent a sensitive and specific method for epidemiological studies aimed at defining the distribution of B. burgdorferi s.l. in I. ricinus and, consequently, the exposure risk for humans.

How Different Stocking Densities Affect Growth and Stress Status of Acipenser baerii Early Stage Larvae.

In the present study, a multidisciplinary approach was used in order to evaluate growth, muscle development, and stress status in Siberian sturgeon Acipenser baerii larvae at schooling (T1) and complete yolk sac absorption (T2), reared at three stocking densities (low, medium, and high). Larvae growth, morphological muscle development, and whole-body cortisol levels were assessed. The expression of genes involved in the growth process (igf1 and igf2), in the myogenesis (myog), and in the regulation of cellular stress (glut1, glut2, glut4, and hsp70) was analyzed using a quantitative PCR. Larvae reared at lower densities showed a higher Specific Growth Rate and showed a physiological muscle development. Cortisol levels were low and did not differ significantly, both in different time sampling and across densities, suggesting that either the considered densities are not stressors in this species in the early stages of development or the hypothalamus-pituitary-adrenal (HPA) axis is not yet fully mature. Gene expression of glut1, igf1, and igf2 showed an up-regulation in both developmental stages at all the rearing densities considered, while myog significantly up-regulated at T1 at the highest density. Considering all of the results, it would seem that lower densities should be used in these stages of development, as these showed a higher growth rate, even if it is not economically feasible in commercial hatcheries. Therefore, choosing an intermediate stocking density could be a good compromise between larval performance and economical feasibility.

How different rearing temperatures affect growth and stress status of Siberian sturgeon Acipenser baerii larvae.

Environmental temperature is one of the critical factors affecting fish development. The aim of this study was to examine the impact of three different rearing temperatures (16, 19 and 22°C) throughout the endogenous feeding phase of the Siberian sturgeon Acipenser baerii. This was performed by assessing (a) larval survival and growth; (b) immunofluorescence localization and expression of genes involved in muscle development and growth - myog and Igf1; and (c) stress status through the expression of thermal stress genes - Hsp70, Hsp90α and Hsp90ß - and whole body cortisol. Overall survival rate and larval weight did not differ significantly across temperatures. Larvae subjected to 22°C showed faster absorption of the yolk-sac than larvae subjected to 19 or 16°C. Both at schooling and at the end of the trial, larvae reared at 16°C showed significantly lower levels of cortisol than those reared at 19 or 22°C. IGF-1 immunopositivity was particularly evident in red muscle at schooling stage in all temperatures. The expression of all Hsps as well as the myog and Igf1 genes was statistically higher in larvae reared at 16°C but limited to the schooling stage. Cortisol levels were higher in larvae at 22°C, probably because of the higher metabolism demand rather than a stress response. The observed apparent incongruity between Hsps gene expression and cortisol levels could be due to the lack of a mature system. Further studies are necessary, especially regarding the exogenous feeding phase, in order to better understand if this species is actually sensitive to thermal stress.

BVDV permissiveness and lack of expression of co-stimulatory molecules on PBMCs from calves pre-infected with BVDV.

Bovine viral diarrhea virus (BVDV) has been detected in peripheral blood mononuclear cells (PBMCs) of immunocompetent animals, not being clear whether the development of a specific humoral immune response can prevent BVDV infection. The aim of this study was to evaluate the ability of non-cytopathic BVDV to replicate and produce infectious virus in PBMCs from calves pre-infected with BVDV and to elucidate the immunomodulatory effect of BVDV on these cells in an in vitro model. Quantification of virus was by quantitative PCR, while its replicative capacity and shedding into the extracellular environment was evaluated by viral titration. Apoptosis was assessed by flow cytometry analysis of annexin V and propidium iodide, and by expression of caspase-3/7. Flow cytometry was used to analyze the expression of CD14/CD11b/CD80, CD4/CD8/CD25, MHC-I/MHC-II and B-B2 markers. Our results showed that PBMCs from cattle naturally infected with BVDV were more susceptible to in vitro BVDV infection and showed a more severe apoptosis response than those from naïve animals. Non-cytopathic BVDV in vitro infection also resulted in a lack of effect in the expression of antigen presentation surface markers. All these findings could be related to the immunosuppressive capacity of BVDV and the susceptibility of cattle to this infection.

Optimized protocol for DNA/RNA co-extraction from adults of Dirofilaria immitis.

Dirofilaria immitis, the etiologic agent of canine heartworm disease, like several other filarial nematodes, harbors the bacterial endosymbiont Wolbachia. To investigate metabolic and functional pathways of D. immitis and Wolbachia individually, along with their interactions, the use of both transcriptomic and genome analysis has becoming increasingly popular. Although several commercial kits are available for the single extraction of either DNA or RNA, no specific protocol has been described for simultaneous extraction of DNA and RNA from such a large organism like an adult D. immitis, where female worms generally reach ∼25 cm in length. More importantly, adult worms of D. immitis can only be obtained either through necropsy of experimentally infected dogs or by minimally-invasive surgical heartworm removal of naturally infected dogs. This makes each individual worm sample extremely important. Thus, in the context of a project aimed at the evaluation of both gene expression analysis and Wolbachia population assessment following different treatments, an optimized protocol for co-extraction of DNA and RNA from a single sample of adult D. immitis has been developed. •An optimized method for DNA/RNA co-extraction from large size nematodes using TRIzol® reagent.•Allows maximum exploitation of unique samples as adults of D. immitis.

Seropositivity to Midichloria mitochondrii (order Rickettsiales) as a marker to determine the exposure of humans to tick bite.

Ixodes ricinus is the most common tick species parasitizing humans in Europe, and the main vector of Borrelia burgdorferi sensu lato, the causative agent of Lyme disease in the continent. This tick species also harbors the endosymbiont Midichloria mitochondrii, and there is strong evidence that this bacterium is inoculated into the vertebrate host during the blood meal. A high proportion of tick bites remains unnoticed due to rarity of immediate symptoms, implying the risk of occult tick-borne infections in turn a potential risk factor for the onset of chronic-degenerative diseases. Since suitable tools to determine the previous exposure to I. ricinus bites are needed, this work investigated whether seropositivity toward a protein of M. mitochondrii (rFliD) could represent a marker for diagnosis of I. ricinus bite. We screened 274 sera collected from patients from several European countries, at different risk of tick bite, using an ELISA protocol. Our results show a clear trend indicating that positivity to rFliD is higher where the tick bite can be regarded as certain/almost certain, and lower where there is an uncertainty on the bite, with the highest positivity in Lyme patients (47.30%) and the lowest (2.00%) in negative controls. According to the obtained results, M. mitochondrii can be regarded as a useful source of antigens, with the potential to be used to assess the exposure to ticks harboring this bacterium. In prospect, additional antigens from M. mitochondrii and tick salivary glands should be investigated and incorporated in a multi-antigen test for tick bite diagnosis.

Differential ABC transporter gene expression in adult Dirofilaria immitis males and females following in vitro treatment with ivermectin, doxycycline or a combination of both.

BACKGROUND: Combination doxycycline/macrocyclic lactone (ML) protocols have been shown to provide a more rapid adulticidal and microfilaricidal effect than either MLs or doxycycline alone, although female worms were reported to have a higher tolerance to treatments compared to male worms. The present study aimed to evaluate how ABC transporters may be involved in the synergic effect of the combination treatment. Adult worms of D. immitis were treated in vitro for 24 hours with doxycycline (DOXY), ivermectin (IVM) and a combination of both, and changes in the modulation of ABC transporter genes were measured. Levels of doxycycline inside different treatment media, post-treatment, were determined through HPLC analysis. RESULTS: Quantitative RT-PCR analysis showed the presence of changes in the modulation of ABC transporter genes evaluated in this study. In particular, in female worms, the combination treatment induced a substantial increase in gene expressions, especially of Dim-pgp-10 and Dim-haf-4; whereas in male worms, the greatest increase in gene expression was observed for Dim-pgp-10 and Dim-pgp-11 when treated with DMSO + IVM and DMSO + DOXY/IVM. HPLC analysis of the DOXY concentrations in the media after in vitro treatments of male worms showed a slight difference between the DMSO + DOXY samples and the combination (DMSO + DOXY + IVM), while no difference was observed among females. CONCLUSIONS: Further studies are required to explain whether the modulation of cellular efflux plays a role, even partially, in the adulticide effect of doxycycline/macrocyclic lactone combinations in heartworm-infected dogs. To the authors' knowledge, this is the first study to evaluate P-gp expression in adult D. immitis.

Tissue tropism and metabolic pathways of Midichloria mitochondrii suggest tissue-specific functions in the symbiosis with Ixodes ricinus.

A wide range of arthropod species harbour bacterial endosymbionts in various tissues, many of them playing important roles in the fitness and biology of their hosts. In several cases, many different symbionts have been reported to coexist simultaneously within the same host and synergistic or antagonistic interactions can occur between them. While the associations with endosymbiotic bacteria have been widely studied in many insect species, in ticks such interactions are less investigated. The females and immatures of Ixodes ricinus (Ixodidae), the most common hard tick in Europe, harbour the intracellular endosymbiont "Candidatus Midichloria mitochondrii" with a prevalence up to 100%, suggesting a mutualistic relationship. Considering that the tissue distribution of a symbiont might be indicative of its functional role in the physiology of the host, we investigated M. mitochondrii specific localization pattern and the corresponding abundance in selected organs of I. ricinus females. We paired these experiments with in silico analysis of the metabolic pathways of M. mitochondrii, inferred from the available genome sequence, and additionally compared the presence of these pathways in seven other symbionts commonly harboured by ticks to try to obtain a comparative understanding of their biological effects on the tick hosts. M. mitochondrii was found to be abundant in ovaries and tracheae of unfed I. ricinus, and in ovaries, Malpighian tubules and salivary glands of semi-engorged females. These results, together with the in silico metabolic reconstruction allow to hypothesize that the bacterium could play multiple tissue-specific roles in the host, both enhancing the host fitness (supplying essential nutrients, enhancing the reproductive fitness, helping in the anti-oxidative defence, in the energy production and in the maintenance of homeostasis and water balance) and/or for ensuring its presence in the host population (nutrients acquisition, vertical and horizontal transmission). The ability of M. mitochondrii to colonize different tissues allows to speculate that distinctive sub-populations may display different specializations in accordance with tissue tropism. Our hypotheses should be corroborated with future nutritional and physiological experiments for a better understanding of the mechanisms underlying this symbiotic interaction.