Biological compatibility between two temperate lineages of brown dog ticks, Rhipicephalus sanguineus (sensu lato)

Background: The brown dog tick Rhipicephalus sanguineus (sensu stricto) is reputed to be the most widespread tick of domestic dogs worldwide and has also been implicated in the transmission of many pathogens to dogs and humans. For more than two centuries, Rh. sanguineus (s.s.) was regarded as a single taxon, even considering its poor original description and the inexistence of a type specimen. However, genetic and crossbreeding experiments have indicated the existence of at least two distinct taxa within this name: the so-called "temperate" and "tropical" lineages of Rh. sanguineus (sensu lato). Recent genetic studies have also demonstrated the existence of additional lineages of Rh. sanguineus (s.l.) in Europe and Asia. Herein, we assessed the biological compatibility between two lineages of Rh. sanguineus (s.l.) found in southern Europe, namely Rhipicephalus sp. I (from Italy) and Rhipicephalus sp. II (from Portugal). Methods: Ticks morphologically identified as Rh. sanguineus (s.l.) were collected in southern Portugal and southern Italy. Tick colonies were established and crossbreeding experiments conducted. Morphological, biological and genetic analyses were conducted. Results: Crossbreeding experiments confirmed that ticks from the two studied lineages were able to mate and generate fertile hybrids. Hybrid adult ticks always presented the same genotype of the mother, confirming maternal inheritance of mtDNA. However, larvae and nymphs originated from Rhipicephalus sp. I females presented mtDNA genotype of either Rhipicephalus sp. I or Rhipicephalus sp. II, suggesting the occurrence of paternal inheritance or mitochondrial heteroplasmy. While biologically compatible, these lineages are distinct genetically and phenotypically. Conclusions: The temperate lineages of Rh. sanguineus (s.l.) studied herein are biologically compatible and genetic data obtained from both pure and hybrid lines indicate the occurrence of paternal inheritance or mitochondrial heteroplasmy. This study opens new research avenues and raises question regarding the usefulness of genetic data and crossbreeding experiments as criteria for the definition of cryptic species in ticks

Flotac and Mini-Flotac for uro-microscopic diagnosis of Capillaria plica (syn. Pearsonema plica) in dogs

Background: Capillaria plica (syn. Pearsonema plica) is a nematode that resides in the urinary bladder and rarely in ureters or in the kidney pelvis of various carnivores, especially foxes and dogs. Urine sedimentation technique is actually the only diagnostic tool that permits the identification of C. plica eggs, but its sensitivity is low and when an infection is suspected (or when it is necessary to confirm treatment efficacy) more than one examination of urine sediment should be performed. The present paper reports a clinical case of natural C. plica infection in a dog from southern Italy. In addition, two new techniques, FLOTAC and Mini-FLOTAC, were used for the diagnosis of C. plica in dog urine and compared with the technique of sedimentation. Results: Using FLOTAC with fresh urine and sodium chloride as flotation solution, were obtained the best results for the diagnosis of C. plica in dog urine in term of eggs counted (mean eggs per 10 ml of urine = 70.3 FLOTAC vs 40.3 Mini FLOTAC vs 32.8 sedimentation) and coefficient of variation (CV%) (6.2 FLOTAC vs 13.4 Mini-FLOTAC vs 32.9 sedimentation). Conclusions: The FLOTAC was the more sensitive method, but also the Mini-FLOTAC could be a valid alternative diagnostic method because gave better results than the classical sedimentation and can be used in place of the FLOTAC in laboratories where the centrifugation step cannot be performed. © 2014 Maurelli et al.; licensee BioMed Central Ltd

Human osteoarthritic cartilage shows reduced in vivo expression of IL-4, a chondroprotective cytokine that differentially modulates IL-1β-stimulated production of chemokines and matrix-degrading enzymes in vitro

open10noThis work was supported by grants from Rizzoli Orthopaedic Institute (Ricerca Corrente); University of Bologna (RFO); MIUR (FIRB-RBAP10KCNS); “Cinque per mille” Funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.BACKGROUND: In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β. METHODOLOGY/PRINCIPAL FINDINGS: The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13. CONCLUSIONS/SIGNIFICANCE: Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.openAssirelli E.; Pulsatelli L.; Dolzani P.; Platano D.; Olivotto Eleonora .; Filardo G.; Trisolino G.; Facchini A.; Borzì R.M.; Meliconi R.Assirelli E.; Pulsatelli L.; Dolzani P.; Platano D.; Olivotto Eleonora .; Filardo G.; Trisolino G.; Facchini A.; Borzì R.M.; Meliconi R

Spirocerca lupi in the stomach of two Andean foxes (Lycalopex culpaeus) from Chile

The genus Spirocerca includes nematodes that parasitize the stomach and the oesophagus of carnivores, chiefly canids. Herein, we provide new data about the morphological, histopathological, and molecular characterization of Spirocerca sp. in Andean foxes (Lycalopex culpaeus) in Chile. Intact immature worms, identified as Spirocerca sp., were recovered in the lumen of the stomach from two foxes. Histologically, worms morphologically consistent with spirurid nematodes were present within the wall of the stomach and surrounded by nodular areas of inflammation with central necrotic debris. Molecular analysis of the cox1 gene yielded 19 sequences and 5 nucleotide sequence types with 99.95 to 99.98% similarity, being shared between both foxes. Nucleotide similarity ranged from 93.1 (with genotype 2 of S. lupi and S. vulpis) to 95.8% (with genotype 1 of S. lupi), a higher similarity than noted from sequences of S. lupi from an Andean fox from Peru (91.0 to 93.3%). However, the Poisson Tree Processes for species delineation did not support the existence of a new species Spirocerca. Phylogenetic and nucleotide analyses suggest that these specimens belong to a new variant or genotype of S. lupi or to a cryptic species. Whether the presence of the worms in the stomach has to do with genotypic differences in parasites or host or some combination is uncertain. Spirocerca lupi has never been found in Chilean dogs and must be investigated

Native strains of Beauveria bassiana for the control of Rhipicephalus sanguineus sensu lato

Background Rhipicephalus sanguineus sensu lato ticks are widespread worldwide due to their adaptability to survive under different environmental conditions. They may act as vectors of a wide range of pathogens to humans and animals and their control is based on the use of chemical products on dogs and in the environment. Alternative control strategies, such as the use of entomopathogenic fungi as bio-control agents have also been investigated. The ability of native strains of Beauveria bassiana sensu lato in causing mortality in different tick species (e.g., Amblyomma cajennense and Rhipicephalus microplus) has been demonstrated. However, limited studies have assessed the use of B. bassiana for the control of R. sanguineus s.l. and none of them have employed native strains of this fungus. Here we investigated the pathogenicity of a native strain of B. bassiana (CD1123) against all developmental stages of R. sanguineus s.l.. Methods Batches of eggs, larvae, nymphs and adult ticks were immersed in a suspension of 107 conidia/ml of B. bassiana s.l., isolated from a R. sanguineus s.l. engorged female. All treatment and control groups were observed for 20 days, and the biological parameters (i.e., mortality, hatching, moulting percentage, pre-oviposition period, oviposition period and rate, eggs production efficiency, reproductive efficiency and fitness indexes) were assessed. Results The effect of the B. bassiana strain tested herein on eggs, larvae, nymphs and adults showed a significantly higher mortality than those of the control groups (p < 0.05) at 5 days post-infection. No infected eggs hatched and no infected larvae moulted. Only 15% of infected nymphs moulted into adults. All biological parameters of treated groups differed significantly (p < 0.001) from those of control groups. Conclusions This study demonstrates that a suspension containing 107 conidia/ml of a native B. bassiana strain is highly virulent towards all life-cycle developmental stages of R. sanguineus s.l. and may be of potential interest as a biological control agent against these ticks

First evidence of pyrethroid resistance in Italian populations of West Nile virus vector Culex pipiens

13openInternationalItalian coauthor/editorCulex pipiens (Linnaeus), one of the most abundant mosquito species in Europe, plays a crucial role in the endemic transmission of West Nile virus and caused the large outbreak with >1600 human cases in 2018. Although evidence of resistance to pyrethroids has been reported for Cx. pipiens populations from Spain and Greece, resistance monitoring has been largely neglected in Italy. Herein, we investigate susceptibility of Italian Cx. pipiens populations to the pyrethroids permethrin and deltamethrin. Results from WHO-tube-bioassays revealed mortalities ranging from 14–54%, indicating high levels of resistance, in four out of 10 populations exposed to permethrin (0.75%) and of 63% in one of three populations exposed to deltamethrin (0.05%). Reduced susceptibility (mortality<98%) was detected in almost all other populations. A clear association is shown between the resistant phenotype and the presence of kdr-alleles in position 1014 of the VSSC, strongly suggesting its role in reducing susceptibility. The study provides the first evidence of pyrethroid-resistance in Italian Cx. pipiens populations and reports levels of resistance paralleled in the European region only in Turkey. This highlights the urgent need to implement insecticide-resistance management plans to restore the efficacy of the nowadays only chemical weapon available to control arbovirus transmission in Europe.openPichler, Verena; Giammarioli, Carola; Bellini, Romeo; Veronesi, Rodolfo; Arnoldi, Daniele; Rizzoli, Annapaola; Lia, Riccardo Paolo; Otranto, Domenico; Ballardini, Marco; Cobre, Pietro; Serini, Paola; Della Torre, Alessandra; Caputo, BeniaminoPichler, V.; Giammarioli, C.; Bellini, R.; Veronesi, R.; Arnoldi, D.; Rizzoli, A.; Lia, R.P.; Otranto, D.; Ballardini, M.; Cobre, P.; Serini, P.; Della Torre, A.; Caputo, B

First evidence of resistance to pyrethroid insecticides in Italian Aedes albopictus populations after 26 years since invasion

Aedes albopictus has spread during the last decades all over the world. This has increased significantly the risk of exotic arbovirus transmission (e.g. Chikungunya, Dengue, and Zika) also in temperate areas, as testified by the Chikungunya 2007- and 2017-outbreaks in north-east and central Italy. Insecticides represent a main tool for limiting the circulation of these mosquito-borne viruses. The aim of the present study is to start filling the current gap of knowledge on pyrethroid insecticide resistance of European Ae. albopictus populations focusing on populations from Italy, Albania and Greece

Phylogenetic analysis of Haemaphysalis erinacei Pavesi, 1884 (Acari: Ixodidae) from China, Turkey, Italy and Romania

Background: Haemaphysalis erinacei is one of the few ixodid tick species for which valid names of subspecies exist. Despite their disputed taxonomic status in the literature, these subspecies have not yet been compared with molecular methods. The aim of the present study was to investigate the phylogenetic relationships of H. erinacei subspecies, in the context of the first finding of this tick species in Romania. Results: After morphological identification, DNA was extracted from five adults of H. e. taurica (from Romania and Turkey), four adults of H. e. erinacei (from Italy) and 17 adults of H. e. turanica (from China). From these samples fragments of the cytochrome c oxidase subunit 1 (cox1) and 16S rRNA genes were amplified via PCR and sequenced. Results showed that cox1 and 16S rRNA gene sequence divergences between H. e. taurica from Romania and H. e. erinacei from Italy were below 2%. However, the sequence divergences between H. e. taurica from Romania and H. e. turanica from China were high (up to 7.3% difference for the 16S rRNA gene), exceeding the reported level of sequence divergence between closely related tick species. At the same time, two adults of H. e. taurica from Turkey had higher 16S rRNA gene similarity to H. e. turanica from China (up to 97.5%) than to H. e. taurica from Romania (96.3%), but phylogenetically clustered more closely to H. e. taurica than to H. e. turanica. Conclusions: This is the first finding of H. erinacei in Romania, and the first (although preliminary) phylogenetic comparison of H. erinacei subspecies. Phylogenetic analyses did not support that the three H. erinacei subspecies evaluated here are of equal taxonomic rank, because the genetic divergence between H. e. turanica from China and H. e. taurica from Romania exceeded the usual level of sequence divergence between closely related tick species, suggesting that they might represent different species. Therefore, the taxonomic status of the subspecies of H. erinacei needs to be revised based on a larger number of specimens collected throughout its geographical range

Vertical transmission of Anaplasma platys and Leishmania infantum in dogs during the first half of gestation.

BACKGROUND: Leishmania infantum is a canine zoonotic vector-borne protozoan pathogen transmitted by phlebotomine sand flies, whereas Anaplasma platys is a bacterium most likely transmitted by ticks. While vertical transmission of L. infantum from pregnant bitches to their offspring has been documented, thus far no studies have explored the possibility of vertical transmission of A. platys in dogs. This study investigated the occurrence of vertical transmission of L. infantum and A. platys in sheltered dogs during the first half of gestation, in an area of southern Italy characterised by a high incidence of infection by both pathogens. METHODS: The study population included 20 bitches (n = 10 pregnant, at 25-35 days of pregnancy; n = 10 non-pregnant), all subjected to ovariohysterectomy, which were examined for the presence of L. infantum and A. platys via cytological screening of bone marrow and whole blood samples. Infection by L. infantum and A. platys was also tested by immunofluorescence antibody test (IFAT) and quantitative real-time PCR (qPCR) targeting both pathogens. Selected tissue samples (n = 210) collected during surgical procedures from bitches and foetuses (n = 20) were assessed for the presence of L. infantum and A. platys by qPCR targeting a fragment of the kinetoplast minicircle DNA (kDNA) and the 16S rRNA gene, respectively. RESULTS: Leishmania infantum DNA was not amplified from either uteri or ovaries from pregnant bitches or foetal tissue samples, whereas a subset of ovarian (n = 2) and uterine (n = 4) tissue samples from non-pregnant bitches were infected, with parasite loads of up to 3.09 × 10 and 7.51 parasite/PCR reaction, respectively. Conversely, uterine (n = 10) and ovarian (n = 8) tissues from both pregnant and non-pregnant bitches, together with a subset (n = 5) of foetal tissue samples were qPCR positive for A. platys. Leishmania infantum and A. platys nucleic acids were amplified from two uteri from non-pregnant bitches, with parasite loads of up to 2.32 × 10(-3) and 2.05 parasite/per PCR reaction, respectively. CONCLUSIONS: Results from this study suggest that, in contrast to L. infantum, A. platys can be transmitted from pregnant dogs to their offspring during the first half of gestation. This hypothesis remains to be verified, for instance via direct observation of parasites in postpartum foetal tissues.The paper has been sponsored by Bayer Animal Health in the framework of the 11th CVBD World Forum Symposium

Filarial infection caused by Onchocerca boehmi (Supperer, 1953) in a horse from Italy.

Equids can be infected by a range of skin-dwelling filarial nematodes, including four species of the genus Onchocerca. Current literature on equine onchocercosis is fragmentary and often limited to isolated case reports. The present study aimed to describe a clinical case of equine onchocercosis caused by Onchocerca boehmi (Supperer, 1953) (syn. Elaeophora boehmi) in an 8-year-old gelding Belgian show jumper from northern Italy. The horse was presented with a firm and painless mass on the proximal third of the right metacarpal region. Ultrasound examination showed a peritendinous enlargement around the palmaro-lateral area of the tendons, characterized by an elongated hypoechoic and well-defined structure, embedding a coiled hyperechoic line. The metacarpal nodule was resected and histologically examined. Fragments of a parasitic nematode were detected, isolated and examined. The morphological analysis allowed identifying the nematode as O. boehmi. In addition, total genomic DNA was extracted from individual fragments using a commercial kit for the nematode identification and a comparative sequence analysis of the nematode cytochrome oxidase subunit 1 (cox1) sequence with data available in the GenBankTM database revealed the closest identity (i.e. 91 %) with that of Onchocerca lupi. Thus far, O. boehmi has only been reported in Austria and Iran, and information about its life-cycle and vectors is lacking. The systematic position of this species within the genus Onchocerca, not in Elaeophora where it was originally described, is in concordance with the morphological and molecular analysis. In this article, we describe the first autochthonous case of equine onchocercosis in Italy caused by O. boehmi and discuss novel parasitological, clinical, and pathological data on these pathogens of horses.The scanning electron microscope study was supported by the Bulgarian National Endowment Fund “13 Centuries Bulgaria”