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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 In Italy, information on dictyocaulosis in wildlife is limited and often outdated.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.

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.

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.

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.

Simple Summary Roe deer, whose populations are increasing and expanding throughout Europe, are suitable hosts for the maintenance of a variety of infectious tick-vectored microorganisms, that can infect both animals and humans. In this study the presence of tick-transmitted pathogens was investigated in roe deer recovered by a wildlife rescue center based in Italy. This kind of samples represents a convenient material for investigations under several aspects for both animals and researchers. Notably, no live trapping or killing are required to obtain samples, as they are collected in the context of the rescue activities and aimed to frame the health status of the animal. The investigated blood samples showed high positive rates to typical roe deer-related microorganisms (such as Babesia capreoli), and to the zoonotic agent Babesia venatorum. Roe deer were also positive to Anaplasma phagocytophilum strains mainly considered apathogenic or limited to wild ungulates. The obtained results underline the importance of a constant investigation on circulating tick-borne pathogens in roe deer, and generally speaking, in wild animal species, due to their potential role as a key factor in the endemic cycle of important infectious agents for domestic and wild animals, as well as humans. Abstract 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.

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.

Red mark syndrome (RMS) is a disease of farmed rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), one of the most widespread freshwater farmed species in Europe. The disease emerges at water temperatures below 16 °C and consists of one or more bright red skin lesions on the fish body. Mortality due to RMS is reportedly rare, but the disease leads to downgrading of the product and subsequent economic losses. Despite the disease impact, the causative agent for RMS is still formally undetermined although increasing evidence points to a bacterium ascribed to the Midichloriaceae family (order Rickettsiales), hereafter referred to as RMS-Midichloria like organism (RMS-MLO). Intriguingly, recently deposited sequences revealed the presence of RMS-MLO-like bacteria associated with Ichthyophthirius multifiliis, a common protozoan skin parasite of freshwater fish frequently harboring bacterial endosymbionts. Therefore, we hypothesized that I. multifiliis could be a vehicle for RMS-MLO. This was tested by infecting RMS-diseased rainbow trout with I. multifiliis theronts, and subsequently investigating the presence of RMS-MLO in tomonts detached from the fish. Real time PCR analyses showed clearly that I. multifiliis previously exposed to RMS-affected fish become positive to RMS-MLO suggesting that this bacterium can be at least transiently acquired and carried by the protozoan. Moreover, statistical analyses suggested a possible level of vertical transmission in I. multifiliis from one trophic stage to the next one. Further studies will be necessary to prove whether I. multifiliis has a role in the horizontal transfer of RMS-MLO bacteria from diseased RMS fish to healthy ones.

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.

International audience; Midichloria mitochondrii is a tick-borne intracellular bacterium of the order Rickettsiales, found with high prevalence in the sheep tick (Ixodes ricinus). Midichloria mitochondrii is capable of vertical transmission in the tick, but recently evidence of potential horizontal transmission to the tick hosts through the blood meal has been reported. We investigated the presence of the bacterium in the blood of roe deer (Capreolus capreolus) collected from an area known to be highly infested with I. ricinus ticks. We collected blood and sera samples for 3 yr in Gardouch (Haute Garonne, France) and subjected them to molecular screening through PCR and to serological investigation using enzyme-linked immunosorbent assay and western blot. Bacterial DNA was detected in the blood of four of seven animals, but only at one or two points in time, whereas all sera were positive for M. mitochondrii antigens at all times. Our results indicated that the presence of the bacterium in the blood is transient, but the antibody response appeared to be long-lasting, possibly due to constant exposure to tick bites, and thus to repeated injection of bacteria. The role of M. mitochondrii in the mammalian host, and its interaction with other tick-borne bacteria, remains unknown.

Tendon injuries are severe burdens in clinics. The poor tendon healing is related to an ineffective response of resident cells and inadequate vascularization. Thanks to the high proliferation and multi-lineage differentiation capability, bone marrow-derived mesenchymal stem cells (BMSCs) are a promising cell source to support the tendon repair. To date, the association of various growth factors to induce the in vitro tenogenic differentiation of multipotent progenitor cells is poorly investigated. This study aimed to investigate the tenogenic differentiation of rabbit BMSCs by testing the combination of bone morphogenetic proteins (BMP-12 and 14) with transforming growth factor beta (TGF-β) and vascular endothelial growth factor (VEGF) both in 2D and 3D cultures within fibrin-based constructs. After 7 and 14 days, the tenogenic differentiation was assessed by analyzing cell metabolism and collagen content, the gene expression of tenogenic markers and the histological cell distribution and collagen deposition within 3D constructs. Our results demonstrated that the association of BMP-14 with TGF-β3 and VEGF enhanced the BMSC tenogenic differentiation both in 2D and 3D cultures. This study supports the use of fibrin as hydrogel-based matrix to generate spheroids loaded with tenogenic differentiated BMSCs that could be used to treat tendon lesions in the future.

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.

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 Hsp90β - 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.

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.

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.

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.

International audience; Ticks are important vectors of a variety of pathogens affecting humans and other animals, but they also harbor numerous microorganisms whose role is still limitedly investigated. Ixodes ricinus harbors the endosymbiont Midichloria mitochondrii, which is localized in ovaries and in salivary glands. The bacterium is vertically transmitted and is present in 100% of wild adult females, while prevalence values drop after some generations under laboratory conditions. Molecular and serological evidences showed that M. mitochondrii molecules are transmitted to the vertebrate hosts by I. ricinus during the blood meal. Our work was focused on monitoring M. mitochondrii antigens and DNA in a vertebrate model after infestation with I. ricinus for a time-span of four months. Two groups of rabbits were infested with I. ricinus females, respectively from the wild (naturally infected with the symbiont) and laboratory strain (lab; considered devoid of M. mitochondrii after quantitative PCR investigations) and screened using molecular and serological assays at nine time points. M. mitochondrii presence was detected in rabbits infested with wild I. ricinus ticks, but surprisingly also in those infested with lab ticks, albeit at later time points. This result prompted a more sensitive molecular screening of lab ticks, which were found to harbor very low symbiont loads. Our results indicate that transmission of the bacterium occurs even at low bacterial loads, and that antibody response against M. mitochondrii antigens begins within one week post-infestation with wild I. ricinus. Circulating DNA was detected in the blood of rabbits belonging to both groups up to the end of the experiment, suggesting a replication of the symbiont inside the vertebrate host.

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.

Rheinheimera sp. strain EpRS3, isolated from the rhizosphere of Echinacea purpurea, is already known for its ability to produce antibacterial compounds. By use of culture experiments, we verified and demonstrated its harmful effect against the ciliated protist Euplotes aediculatus (strain EASCc1), which by FISH experiments resulted to harbor in its cytoplasm the obligate bacterial endosymbiont Polynucleobacter necessarius (Betaproteobacteria) and the secondary endosymbiont "Candidatus Nebulobacter yamunensis" (Gammaproteobacteria). In culture experiments, the number of ciliates treated both with liquid broth bacteria-free (Supernatant treatment) and bacteria plus medium (Tq treatment), decreases with respect to control cells, with complete disappearance of ciliates within 6 h after Tq treatment. Results suggest that Rheinheimera sp. EpRS3 produces and releases in liquid culture one or more bioactive molecules affecting E. aediculatus survival. TEM analysis of control (not treated) ciliates allowed to morphologically characterize both kind of E. aediculatus endosymbionts. In treated ciliates, collected soon after the arising of cell suffering leading to death, TEM observations revealed some ultrastructural damages, indicating that P. necessarius endosymbionts went into degradation and vacuolization after both Supernatant and Tq treatments. Additionally, TEM investigation showed that when the ciliate culture was inoculated with Tq treatment, both a notable decrease of P. necessarius number and an increase of damaged and degraded mitochondria occur. FISH experiments performed on treated ciliates confirmed TEM results and, by means of the specific probe herein designed, disclosed the presence of Rheinheimera sp. EpRS3 both inside phagosomes and free in cytoplasm in ciliates after Tq treatment. This finding suggests a putative ability of Rheinheimera sp. EpRS3 to reintroduce itself in the environment avoiding ciliate digestion.

Sera from Dirofilaria immitis-experimentally infected dogs treated with a combination of ivermectin/doxycycline were analysed for doxycycline levels by HPLC and anti-Wolbachia Surface Protein (rWSP) antibodies by ELISA and compared with sera from dogs treated with doxycycline alone. Results show that doxycycline levels were not statistically different between the two groups. Circulating anti-WSP antibody titres were markedly lower in both treatment groups when compared to control D. immitis infected dogs, indicating that doxycycline is able to reduce Wolbachia and prevent the immune response against the bacteria. The combination treatment protocol has been shown to be highly adulticidal and further studies are needed to better understand the interaction between doxycycline and ivermectin in D. immitis infected dogs.

“Candidatus Fokinia solitaria” is an obligate intracellular endosymbiont of a unicellular eukaryote, a ciliate of the genus Paramecium. Here, we present the genome sequence of this bacterium and subsequent analysis. Phylogenomic analysis confirmed the previously reported positioning of the symbiont within the “Candidatus Midichloriaceae” family (order Rickettsiales), as well as its high sequence divergence from other members of the family, indicative of fast sequence evolution. Consistently with this high evolutionary rate, a comparative genomic analysis revealed that the genome of this symbiont is the smallest of the Rickettsiales to date. The reduced genome does not present flagellar genes, nor the pathway for the biosynthesis of lipopolysaccharides (present in all the other so far sequenced members of the family “Candidatus Midichloriaceae”) or genes for the Krebs cycle (present, although not always complete, in Rickettsiales). These results indicate an evolutionary trend toward a stronger dependence on the host, in comparison with other members of the family. Two alternative scenarios are compatible with our results; “Candidatus Fokinia solitaria” could be either a recently evolved, vertically transmitted mutualist, or a parasite with a high host-specificity.

Background Ticks are obligate haematophagous ectoparasites of vertebrates and frequently parasitize avian species that can carry them across continents during their long-distance migrations. Ticks may have detrimental effects on the health state of their avian hosts, which can be either directly caused by blood-draining or mediated by microbial pathogens transmitted during the blood meal. Indeed, ticks host complex microbial communities, including bacterial pathogens and symbionts. Midichloria bacteria (Rickettsiales) are widespread tick endosymbionts that can be transmitted to vertebrate hosts during the tick bite, inducing an antibody response. Their actual role as infectious/pathogenic agents is, however, unclear. Methods We screened for Midichloria DNA African ticks and blood samples collected from trans-Saharan migratory songbirds at their arrival in Europe during spring migration. Results Tick infestation rate was 5.7%, with most ticks belonging to the Hyalomma marginatum species complex. Over 90% of Hyalomma ticks harboured DNA of Midichloria bacteria belonging to the monophylum associated with ticks. Midichloria DNA was detected in 43% of blood samples of avian hosts. Tick-infested adult birds were significantly more likely to test positive to the presence of Midichloria DNA than non-infested adults and second-year individuals, suggesting a long-term persistence of these bacteria within avian hosts. Tick parasitism was associated with a significantly delayed timing of spring migration of avian hosts but had no significant effects on body condition, whereas blood Midichloria DNA presence negatively affected fat deposits of tick-infested avian hosts. Conclusions Our results show that ticks effectively transfer Midichloria bacteria to avian hosts, supporting the hypothesis that they are infectious to vertebrates. Bird infection likely enhances the horizontal spread of these bacteria across haematophagous ectoparasite populations. Moreover, we showed that Midichloria and tick parasitism have detrimental non-independent effects on avian host health during migration, highlighting the complexity of interactions involving ticks, their vertebrate hosts, and tick-borne bacteria. Electronic supplementary material The online version of this article (10.1186/s13071-018-2669-z) contains supplementary material, which is available to authorized users.

The diagnosis of hepatic cystic echinococcosis is based on ultrasonography and confirmed by serology. However, no biological marker of cyst viability is currently available implying years-long patient follow-up, which is not always feasible in endemic areas. We characterized the performance of an immunoblotting test based on human hydatid cyst fluid with particular regard to its ability to distinguish between cyst stages. Sera from patients with cysts in different stages showed distinctive band pattern recognition. Most importantly, the test discriminated in 80% of cases CE3a from CE3b transitional cysts, known to have different viability profiles. Interestingly, we observed a rapid change in band pattern recognition of sera from one patient at time points when his cyst passed from active to transitional to inactive stages. Further identification of different antigens expressed by different cyst stages will support the development of diagnostic tools that could early define cyst viability, to guide clinical decision making, and shorten patient follow-up.

Ticks can harbor complex and highly variable microbial communities. Among these microorganisms, there are important pathogens of humans and animals that can be transmitted through the blood meal. Less is known about the other members of the microbial community of Ixodida, those that do not cause overt diseases. Among these, Midichloria mitochondrii, symbiont of the tick Ixodes ricinus, is the first described member of the family Midichloriaceae, order Rickettsiales. This bacterium is present in 100% females and is vertically transmitted (Sassera, 2008). The possibility of horizontal transmission is suggested by serological and molecular analyses showing positivity of mammalian blood and sera to M. mitochondrii (Mariconti, 2012; Bazzocchi, 2013). However, its role is still unknown. Recent reports are expanding the view of this family, now including bacteria of great biological and medical interest, indicating a widespread distribution with an increasing range of hosts, with ticks being strongly represented (Epis, 2008). Here we present a molecular screening of 17 tick species (for a total of 92 individuals), detecting and quantifying bacteria closely related to M. mitochondrii in seven of them, including the first report of a midichloriacea in a soft tick species, Ornithodoros maritimus. Based on sequence identity and phylogenetic analysis we propose that these bacteria could constitute the genus Midichloria. The performed screening highlights different prevalence levels in different tick species including one, Ixodes aulacodi, where the bacteria is present in all examined individuals, like in I. ricinus. This result prompts us to hypothesize different roles of Midichloria bacteria in different tick species., International Journal of Health, Animal Science and Food Safety, Vol 3, No 1s (2016)

The tick Ixodes ricinus is vector of many pathogens important for human and animal health (Parola and Raoult, 2001; Socolovschi, 2009). Midichloria mitochondrii (order Rickettsiales; family Midichloriaceae) is an endosymbiont present in the salivary glands of 100% of Ixodes ricinus females. Two lines of evidence suggest a transmission of M. mitochondrii in mammalians during the tick bite: 1) detection of circulating DNA in blood samples of different animal species; 2) seropositivity toward a M. mitochondrii protein (FliD) in humans and dogs exposed to tick bite (Mariconti 2012; Bazzocchi 2013). Here we present serological and molecular results demonstrating the circulation of M. mitochondrii also in Capreolus capreolus (the host of choice for adult and nymph stages of I. ricinus), confirming that this host is a good subject to study the spread of tick-borne pathogens. Based on these results, FliD protein and other M. mitochondrii markers could thus be extremely useful to determine the risk of infection by I. ricinus pathogens in given areas, and for investigating the epidemiological association of a variety of pathological alterations with this tick. Here we show results of the screening of 218 human sera (50 from non endemic areas used as negative controls and 168 from subjects exposed to tick bite) collected in different areas of Germany. Results showed that 48 out of 168 sera were positive to M. mitochondrii. These results have posed the basis for the development of a serological test for investigating the exposure of humans and animals to this tick species., International Journal of Health, Animal Science and Food Safety, Vol 3, No 1s (2016)

Midichloria mitochondrii is an intracellular bacterium found in the hard tick Ixodes ricinus. In this arthropod, M. mitochondrii is observed in the oocytes and in other cells of the ovary, where the symbiont is present in the cell cytoplasm and inside the mitochondria. No studies have so far investigated whether M. mitochondrii is present in the salivary glands of the tick and whether it is transmitted to vertebrates during the tick blood meal. To address the above issues, we developed a recombinant antigen of M. mitochondrii (to screen human sera) and antibodies against this antigen (for the staining of the symbiont). Using these reagents we show that (i) M. mitochondrii is present in the salivary glands of I. ricinus and that (ii) seropositivity against M. mitochondrii is highly prevalent in humans parasitized by I. ricinus (58%), while it is very low in healthy individuals (1·2%). These results provide evidence that M. mitochondrii is released with the tick saliva and raise the possibility that M. mitochondrii is infectious to vertebrates. Besides this, our study indicates that M. mitochondrii should be regarded as a package of antigens inoculated into the human host during the tick bite. This implies that the immunology of the response toward the saliva of I. ricinus is to be reconsidered on the basis of potential effects of M. mitochondrii and poses the basis for the development of novel markers for investigating the exposure of humans and animals to this tick species.

We present a protocol for the preparation of DNA suitable for genomic studies, starting from uncultured intracellular bacteria. The study was conducted on Midichloria mitochondrii, the intramitochondrial symbiont of the tick Ixodes ricinus. Single oocytes, which carry a high load of symbionts, were microdissected from the ovary of a semi-engorged tick specimen. After short incubation of oocytes in hypotonic medium, the cytoplasm that extruded from the cells was collected and used as template for multiple displacement amplification (MDA). Eight MDA preparations were examined with real-time PCR, targeted on seven loci of M. mitochondrii (to assess the uniformity of the genome amplification) and on four loci of I. ricinus (to select DNA preparations showing minimal presence of host DNA). The two MDA products that presented minimal biases in terms of symbiont genes and minimal host-DNA presence were pooled and cloned in a plasmid library. Fifty clones were then sequenced. Only 6% of the sequences were of arthropod origin, while the vast majority could be inferred to have originated from M. mitochondrii. In summary, the developed protocol allowed us to generate micrograms of symbiont DNA, with minimal biases in genome representation and minimal presence of host DNA.

There is still a pressing need for effective adulticide treatment for human and animal filarial infections. Like many filarial nematodes, Dirofilaria immitis, the causative agent of canine heartworm disease, harbours the bacterial endosymbiont Wolbachia, which has been shown to be essential for worm development, fecundity and survival. Here the authors report the effect of different treatment regimens in dogs experimentally infected with adult D. immitis on microfilariemia, antigenemia, worm recovery and Wolbachia content. Treatment with ivermectin (IVM; 6 microg/kg per os weekly) combined with doxycycline (DOXY; 10 mg/kg/day orally from Weeks 0-6, 10-12, 16-18, 22-26 and 28-34) resulted in a significantly faster decrease of circulating microfilariae and higher adulticidal activity compared with either IVM or DOXY alone. Quantitative PCR analysis of ftsZ (Wolbachia DNA) and 18S rDNA (nematode DNA) absolute copy numbers showed significant decreases in Wolbachia content compared with controls in worms recovered from DOXY-treated dogs that were not, however, associated with worm death. Worms from IVM/DOXY-treated dogs, on the other hand, had Wolbachia/nematode DNA ratios similar to those of control worms, suggesting a loss of both Wolbachia and nematode DNA as indicated by absolute copy number values. Histology and transmission electron microscopy of worms recovered from the IVM/DOXY combination group showed complete loss of uterine content in females and immunohistochemistry for Wolbachia was negative. Results indicate that the combination of these two drugs causes adult worm death. This could have important implications for control of human and animal filarial infections.

Candidatus Midichloria mitochondrii, symbiont of the sheep tick Ixodes ricinus , was the first described member of the family Candidatus Midichloriaceae , order Rickettsiales. Recent reports are expanding our view of this family, now including numerous bacteria of great biological and medical interest, indicating a widespread distribution with an increasing range of hosts, with ticks being strongly represented. Here we present a molecular screening of 17 tick species, detecting and quantifying bacteria of the family Midichloriaceae in seven of them, including the first report of a representative of this family in a soft tick species (Argasidae), Ornithodoros maritimus . Based on sequence identity and phylogenetic analysis we propose that all these bacterial symbionts of ticks could be members of the genus Midichloria . The performed screening highlights different prevalence levels and variable bacterial loads in different tick species including one, Ixodes aulacodi , where the bacterium is present in all examined individuals, like in I. ricinus . This result prompts us to hypothesize different roles of Midichloria bacteria in different tick species.

“Candidatus Midichloriaceae” (order Rickettsiales) is a family that encompasses obligate intracellular bacteria present in a wide range of hosts, from parasitic arthropods (ticks, fleas, bedbugs) to aquatic animals (e.g. sponges and corals) and protists, including pathogenic amoebae (genus Acanthamoeba). In addition, multiple studies have detected evidence of the presence of “Candidatus Midichloriaceae” in vertebrates, suggesting a capacity to cause infections, with possible pathogenic effects. Two members of the family that are described as symbionts of ticks, i.e. “Candidatus Midichloria mitochondrii” and “Candidatus Lariskella arthropodarum” have also been found in the blood of humans and other mammals, using molecular and serological methods. Consequently, it has been hypothesized that these arthropods could transmit the microorganisms during the blood meal, as is known to happen with pathogenic members of the order Rickettsiales; for example, ticks can be vectors of rickettsiosis or ehrlichiosis. Indeed, clinical cases compatible with these pathologies in humans or animals parasitized by ticks, but characterized by the absence of antibody titers for known pathogenic rickettsiae, could be reinvestigated for the presence of “Candidatus Midichloriaceae”. A third member of the family, a “Candidatus Midichloria”-related organism, has repeatedly been found in association with red mark syndrome in the rainbow trout Oncorhynchus mykiss. Although the etiology of this economically relevant disease is currently unknown, multiple independent studies suggest that this member of “Candidatus Midichloriaceae” is the most probable causative agent.

Human and animal parasitic filarial nematodes, which often are the cause of severe disease, harbor intracellular bacteria of the genus Wolbachia (Rickettsiaceae). It is thought that these bacteria play an important role in the pathogenesis and immune response to filarial infection. In order to determine the possible role of Wolbachia in heartworm disease, dogs naturally infected with Dirofilaria immitis were studied for specific antibody response to Wolbachia surface protein (WSP). Antibody subclasses were analyzed to determine immune response polarization. Dogs that died from heartworm disease were necropsied, and various organs were studied by immunohistochemistry to determine whether Wolbachia-derived molecules were present in tissue from infected dogs. Humoral response to the WSP was present in all infected dogs and appeared to be predominantly of the Th1-type. Several organs, including lung, liver, and kidney, contained positive-staining cells for WSP, confirming that the canine host does come into contact with Wolbachia-derived molecules.

This compilation of articles consists of four papers presented at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP) (held in New Orleans, LA, USA, on 10-14 August 2003) in a symposium session titled "Recent Advances in Heartworm Disease," organized and chaired by John W. McCall and Jorge Guerrero. The first paper (Guerrero) covered the American Heartworm Society's most recent revision of their guidelines for the diagnosis, prevention, and management of heartworm infection in dogs, based on new research and clinical experience, particularly in the areas of heartworm chemoprophylaxis, adulticide therapy, and serologic testing and retesting. The entire updated 2003 "Guidelines" are presented herein. One paper (McCall) reviewed the "soft-kill" adulticidal and "safety-net" (reach-back, retroactive, clinical prophylactic) activity of prolonged dosing of prophylactic doses of macrocyclic lactones, concluding that ivermectin is the most effective in this way, milbemycin oxime is the least effective, and the activity of injectable moxidectin and selamectin lies between that of ivermectin and milbemycin oxime. The two remaining papers provided an overview of the discovery, rediscovery, phylogeny, and biological association between Wolbachia endosymbionts and filarial nematodes (Genchi and co-authors) and compelling evidence that Wolbachia may play a major role in the immunopathogenesis of filarial diseases of man and animals (Kramer and co-authors).

Two healthy buffaloes (Bubalus bubalis) in a herd which had not been vaccinated against infectious bovine rhinotracheitis (IBR), were selected for their seropositivity for anti-bovine herpesvirus type 1 (BoHV-1) glycoprotein E antibodies, and injected intramuscularly daily with dexamethasone for five consecutive days (day 1 to day 5) to reactivate any latent herpesvirus. Blood samples and nasal and vaginal swabs were collected daily from day 5 to day 15 from each buffalo for virological examination. All the vaginal swabs and blood samples were negative, but 13 of the 22 nasal swabs were positive; a cytopathic effect was observed in primary cultures of bovine fetal lung cells, and the viral isolates were identified as a herpesvirus by PCR. The viral strains were characterised by the sequence analysis of the genes coding for glycoproteins D and B, and the gene sequences were then used for phylogenetic analysis. The isolates from both buffaloes appeared identical at the level of the two genes, and were more closely related to bovine herpesvirus type 5 than to BoHV-1.