Bluetongue virus detection in new Culicoides species in Sardinia, Italy.

Bluetongue is an infectious disease transmitted by Culicoides biting midges. Culicoides imicola is considered the main vector in the Mediterranean basin but other species have been implicated in the Bluetongue virus (BTV) transmission. During 2017, BTV serotype 4 re-occurred in Sardinia causing outbreaks in sheep farms. A survey was carried out on affected farms with the aim to detect the virus in field-collected Culicoides Biting midges were morphologically identified, pooled and then assayed with a real time RT-PCR. To evaluate BTV dissemination, some Culicoides were dissected and head, thorax and abdomen were tested singly by PCR. A total of 173,738 Culicoides adults were collected. Viral RNA was detected in 68 out of 77 pools and all species analysed resulted positive. Detection of BTV in parous female body regions (head, thorax and abdomen) confirmed the full dissemination of BTV in all species analysed. During this study, the vector competence of C imicola, C newsteadi s.l. and Obsoletus complex was confirmed. The authors found two new Culicoides species BTV positive, C paolae never associated with BTV transmission and C circumscriptus only recently found BTV positive in Turkey, which could be considered potential vectors.

Entomological surveillance of Zika virus in Sardinia, Italy, 2016.

Zika Virus (ZIKV) is a RNA virus belonging to the genus Flavivirus, family Flaviviridae. This virus is transmitted through bite of Aedes mosquitoes, in particular Ae. aegypti. On February 1st 2016, the World Health Organization (WHO) has declared ZIKV a Public Health Emergency of International Concern. Successively, considering the establishment of Ae. albopictus, WHO has classified Italy as having a moderate likelihood of local transmission of ZIKV, preceded in Europe only by France. For this reason an entomological surveillance plan was been activated in Sardinia in 2016. BG Sentinel Mosquito Traps have been positioned in 29 sites, comprising urban areas and points of entry, as ports and airports. Mosquitoes were collected fortnightly from April to December. A total of 3,089 mosquitoes were collected belonging to 10 species. The most numerous species have been Cx. pipiens s.l. and Ae. albopictus. All mosquitoes sampled have been assayed by real time reverse transcriptase PCR for detection of ZIKV RNA. A total of 584 pool have been analyzed and have been reported no evidence of ZIKV. A permanent entomological surveillance should be implemented principally in the urban areas and points of entry, as ports and airports, because Ae. albopictus, susceptible to ZIKV, is established in Sardinia and also know the recent introduction of invasive mosquitoes species Ae. koericus and Ae. japonicus in Italy.

Testicular Degeneration and Infertility following Arbovirus Infection.

Arboviruses can cause a variety of clinical signs, including febrile illness, arthritis, encephalitis, and hemorrhagic fever. The recent Zika epidemic highlighted the possibility that arboviruses may also negatively affect the male reproductive tract. In this study, we focused on bluetongue virus (BTV), the causative agent of bluetongue and one of the major arboviruses of ruminants. We show that rams that recovered from bluetongue displayed signs of testicular degeneration and azoospermia up to 100 days after the initial infection. Importantly, testicular degeneration was induced in rams experimentally infected with either a high (BTV-1IT2006)- or a low (BTV-1IT2013)-virulence strain of BTV. Rams infected with the low-virulence BTV strain displayed testicular lesions in the absence of other major clinical signs. Testicular lesions in BTV-infected rams were due to viral replication in the endothelial cells of the peritubular areas of the testes, resulting in stimulation of a type I interferon response, reduction of testosterone biosynthesis by Leydig cells and destruction of Sertoli cells and the blood-testis barrier in more severe cases. Hence, BTV induces testicular degeneration and disruption of spermatogenesis by replicating solely in the endothelial cells of the peritubular areas unlike other gonadotropic viruses. This study shows that a naturally occurring arboviral disease can cause testicular degeneration and affect male fertility at least temporarily.IMPORTANCE During the recent Zika epidemic, it has become apparent that arboviruses could potentially cause reproductive health problems in male patients. Little is known regarding the effects that arboviruses have on the male reproductive tract. Here, we studied bluetongue virus (BTV), an arbovirus of ruminants, and its effects on the testes of rams. We show that BTV was able to induce testicular degeneration in naturally and experimentally infected rams. Testicular degeneration was caused by BTV replication in the endothelial cells of the peritubular area surrounding the seminiferous tubules (the functional unit of the testes) and was associated with a localized type I interferon response, destruction of the cells supporting the developing germinal cells (Sertoli cells), and reduction of testosterone synthesis. As a result of BTV infection, rams became azoospermic. This study highlights that problems in the male reproductive tract caused by arboviruses could be more common than previously thought.

Combined larvicidal and adulticidal treatments to control Culicoides biting midges (Diptera: Ceratopogonidae): Results of a pilot study.

Culicoides biting midges can transmit viruses such as bluetongue virus and Schmallenberg virus to ruminants and African horse sickness virus to equines. The control of Culicoides population can be based on physical and/or chemical methods. This study aims to evaluate the efficacy of larvicides and adulticides combined treatments to control the vector Culicoides populations. Field trials have been conducted during 2014 on seven farms in northern Sardinia (Italy). Three insecticide treatments (larvicides and adulticides) have been carried out on three farms, whereas four farms remained unsprayed as untreated controls. For the larval control two commercial formulations, based on diflubenzuron and Bacillus thuringiensis var. israelensis (H-14), have been used. Against adults a deltamethrin based product has been sprayed on the walls and roofs of animal shelters. Adult Culicoides have been collected on each farm twice a week with an Onderstepoort blacklight suction trap. After each treatment a significant reduction in the abundance of total Culicoides, C. imicola and C. newsteadi s.l., has been observed. The Obsoletus complex density has been affected by the first two treatments. Our results suggest the possibility to control the Culicoides populations with a combination of adulticide and larvicide treatments.

Persistence of Bluetongue virus serotype 1 virulence in sheep blood refrigerated for 10 years.

This paper reports that Bluetongue virus serotype 1 (BTV-1) infected blood collected during the 2006 Sardinia (Italy) epidemic from a ewe with clinical disease and stored at ~ 5°C for 10 years, caused Bluetongue (BT)-like clinical disease and death when inoculated into a susceptible Sarda breed ram. Anatomo-histopathological examination and Real-Time Reverse Transcriptase PCR (Real-Time RT-PCR) confirmed the presence of BTV-1 in several tissues proving that the BTV-1 2006 isolate has maintained its infectivity and virulence.

Novel putative Bluetongue virus in healthy goats from Sardinia, Italy.

In recent years, novel Bluetongue virus (BTV) serotypes have been isolated and/or sequenced by researchers within the field. During Bluetongue surveillance activities, we identified a putative novel BTV serotype in healthy goats from Sardinia, Italy. RNAs purified from blood and serum samples were positive for BTV by a generic real time RT-PCR and c-ELISA, respectively, whereas genotyping and serotyping were unsuccessful. By NGS, the whole genome sequence was obtained from two blood samples (BTV-X ITL2015 strains 34200 and 33531). Overall, Seg 2 of BTV-X ITL2015 shows the highest identity (75.3-75.5% nt/77.4-78.1% aa) with recently isolated BTV-27s from Corsica and with the last discovered BTV XJ1407 from China (75.9% nt /78.2% aa), whereas it is less related with BTV-25 from Switzerland (73.0% nt/75.0% aa) and BTV-26 from Kuwait (62.0% nt/60.5% aa). A specific RT-qPCR targeting Seg 2 of BTV-X ITL2015 was assessed in this study. Considering the Seg 2/VP2 identity of BTV-X ITL2015 with BTV-25, 26, 27s and BTV XJ1407 and that serum of BTV-X ITL2015 infected goats failed to neutralize all tested extant serotypes, we propose the existence of a novel BTV serotype circulating in goats in Sardinia. Isolation was so far unsuccessful thus hampering proper antigenic characterization.