Epizootic Hemorrhagic Disease Virus Serotype 8, Italy, 2022.

We describe the detection of epizootic hemorrhagic disease virus (EHDV) serotype 8 in cattle farms in Sardinia and Sicily in October-November 2022. The virus has a direct origin in North Africa; its genome is identical (>99.9% nucleotide sequence identity) to EHDV serotype 8 strains detected in Tunisia in 2021.

Efficacy of an inactivated EHDV-8 vaccine in preventing viraemia and clinical signs in experimentally infected cattle.

Epizootic haemorrhagic disease (EHD), caused by the EHD virus (EHDV), is a vector-borne viral disease transmitted through Culicoides biting midges. EHDV comprises seven serotypes (1, 2, and 4-8), with EHDV-8 having recently emerged and spread in Europe over the last two years. Such event has raised concerns about the significant threat posed by EHDV-8 to livestock industry. In this study, an inactivated vaccine against EHDV-8 (vEHDV8-IZSAM) was developed. Safety and efficacy of the vaccine were evaluated in calves through clinical, serological, and virological monitoring following experimental challenge. The vaccine was proven safe, with only transient fever and localized reactions observed in a few animals, consistent with adjuvanted vaccine side effects. vEHDV8-IZSAM elicited a robust humoral response, as evidenced by the presence of neutralizing antibodies. After challenge with a virulent isolate, viraemia and clinical signs were evidenced in control animals but in none of the vaccinated animals. This study highlights the potential of vEHDV8-IZSAM as a safe and highly effective vaccine against EHDV-8 in cattle. It offers protection from clinical disease and effectively prevents viraemia. With the recent spread of EHDV-8 in European livestock, the use of an inactivated vaccine could be key in protecting animals from clinical disease and thus to mitigate the economic impact of the disease. Further investigations are warranted to assess the duration of the induced immunity and the applicability of this vaccine in real-world settings. Accordingly, joint efforts between public veterinary institutions and pharmaceutical companies are recommended to scale up vaccine production.

A cell-adapted SARS-CoV-2 mutant, showing a deletion in the spike protein spanning the furin cleavage site, has reduced virulence at the lung level in K18-hACE2 mice.

Here we investigated the virulence properties of a unique cell-adapted SARS-CoV-2 mutant showing a ten-amino acid deletion encompassing the furin cleavage site of the spike protein (Δ680SPRAARSVAS689; Δ680-689-B.1) in comparison to its parental strain (wt-B.1) and two Delta variants (AY.122 and AY.21) of concern. After intranasal inoculation, transgenic K18-hACE2 mice were monitored for 14 days for weight change, lethality, and clinical score; oral swabs were daily collected and tested for the presence of N protein subgenomic RNA. At 3 and 7 dpi mice were also sacrificed and organs collected for molecular, histopathological, and immune response profile investigations. The Δ680-689-B.1-infected mice exhibited reduced shedding, lower virulence at the lung level, and milder pulmonary lesions. In the lung, infection with Δ680-689-B.1 was associated with a significant lower expression of some cytokines at 3 dpi (IL-4, IL-27, and IL-28) and 7 dpi (IL-4, IL-27, IL-28, IFN-γ and IL-1α).

Intravenous Infection of Small Ruminants Suggests a Goat-Restricted Host Tropism and Weak Humoral Immune Response for an Atypical Bluetongue Virus Isolate.

Bluetongue virus (BTV) is the etiologic agent of bluetongue (BT), a viral WOAH-listed disease affecting wild and domestic ruminants, primarily sheep. The outermost capsid protein VP2, encoded by S2, is the virion's most variable protein, and the ability of reference sera to neutralize an isolate has so far dictated the differentiation of 24 classical BTV serotypes. Since 2008, additional novel BTV serotypes, often referred to as "atypical" BTVs, have been documented and, currently, the full list includes 36 putative serotypes. In March 2015, a novel atypical BTV strain was detected in the blood of asymptomatic goats in Sardinia (Italy) and named BTV-X ITL2015. The strain re-emerged in the same region in 2021 (BTV-X ITL2021). In this study, we investigated the pathogenicity and kinetics of infection of BTV-X ITL2021 following subcutaneous and intravenous infection of small ruminants. We demonstrated that, in our experimental settings, BTV-X ITL2021 induced a long-lasting viraemia only when administered by the intravenous route in goats, though the animals remained healthy and, apparently, did not develop a neutralizing immune response. Sheep were shown to be refractory to the infection by either route. Our findings suggest a restricted host tropism of BTV-X and point out goats as reservoirs for this virus in the field.

Culicoides species responsible for the transmission of Epizootic Haemorrhagic Disease virus (EHDV) serotype 8 in Italy.

Epizootic haemorrhagic disease (EHD) is a viral disease transmitted by Culicoides biting midges that affects wild and domestic ruminants. The causative agent, EHD virus (EHDV), belongs to the family Sedoreoviridae, genus Orbivirus. The virus has never been reported in Europe until October 2022, when the virus was for the first time detected in Sicily and Sardinia. After the first clinical cases, an intensive entomological field activity was carried out in five EHD affected farms located in Sardinia, with the aim of assessing the EHDV vector competence in European species of Culicoides. EHDV­8 was detected in C. imicola, C. obsoletus/scoticus, C. newsteadi, C. pulicaris ss, and C. bysta. The first 4 species have also been demonstrated to be able to transmit bluetongue virus (BTV). According to these results, it is likely that EHDV­8, sharing the same transmission patterns of BTV, can also spread to Europe.

Characterization of Epizootic Hemorrhagic Disease Virus Serotype 8 in Naturally Infected Barbary Deer (Cervus elaphus barbarus) and Culicoides (Diptera: Ceratopogonidae) in Tunisia.

Epizootic hemorrhagic disease (EHD) is a Culicoides-borne disease of domestic and wild ruminants caused by EHD virus (EHDV). This virus circulates in multiple serotypes. In late September 2021, a novel strain belonging to EHDV-8 was reported in cattle farms in Central-Western Tunisia, and in the fall of 2022, the same virus was also detected in Italy and Spain. In the present study, we described EHDV-8 occurrence in deer and, a preliminary identification of the potential Culicoides species responsible for virus transmission in selected areas of Tunisia. EHDV-8 was identified in deer carcasses found in 2021 and 2022 in the national reserve of El Feidja, Jendouba, Northwestern Tunisia, and isolated on cell culture. Instead, insect vectors were collected in October 2021 only in the areas surrounding the city of Tozeur (Southern Tunisia) where EHDV-8 cases in cattle were confirmed. Morphological identification showed that 95% of them belonged to the Culicoides kingi and Culicoides oxystoma species and both species tested positive for EHDV-8 RNA. C. imicola was not detected in this collection and EHDV-8 RNA was not evidenced in vector pools collected in 2020, prior to official EHDV-8 emergence. EHDV whole genome sequences were also obtained directly from infected biological samples of deer and positive vectors. EHDV-8 sequences obtained from deer and vectors share a nucleotide identity ranging from 99.42 to 100% and amino acid identity from 99.18 to 100% across all genome segments with the EHDV-8/17 TUN2021 reference sequence.

Experimental infection of cattle, sheep, and goats with the newly emerged epizootic hemorrhagic disease virus serotype 8.

Epizootic hemorrhagic disease virus serotype 8 (EHDV-8) emerged in Europe for the first time in late 2022. In this study, we investigated the kinetics of EHDV-8 infection in cattle, sheep, and goats.  Following experimental infection with EHDV-8, four out of five calves displayed fever, while another calf exhibited ulcerative and crusty lesions of the muzzle. RNAemia peaked at day 7 post infection in all calves and remained relatively stable till the end of the study, at 78 days post infection. Infectious virus was isolated up to 21 days post infection in one calf. As far as small ruminants are concerned, one sheep experienced fever and two out of five had consistent RNAemia that lasted until the end of the study. Remarkably, infectious virus was evidenced at day 7 post infection in one sheep. In goats, no RNA was observed. All infected animals seroconverted, and a neutralizing immune response was observed in all species, with calves exhibiting a more robust response than sheep and goats. Our study provides insights into the kinetics of EHDV-8 infection and the host immune responses. We also highlight that sheep may also play a role in EHDV-8 epidemiology. Altogether, the data gathered in this study could have important implications for disease control and prevention strategies, providing crucial information to policy makers to mitigate the impact of this viral disease on livestock.

Reemergence of an atypical bluetongue virus strain in goats, Sardinia, Italy.

Bluetongue virus (BTV) is the etiologic agent of bluetongue, a WOAH (founded as Office International des Épizooties, OIE)-notifiable economically important disease of ruminants. BTV is transmitted by Culicoides biting midges and 24 different "classical" serotypes have been reported to date. In recent years, several putative novel BTV serotypes, often referred to as "atypical" BTVs, have been documented. These are characterized by unusual biological characteristics, most notably avirulence and vector-independent transmission. Here, we describe the recurrence of such an atypical virus strain BTV-X ITL2021 detected in goats six years after its first discovery in Sardinia, Italy. Combined serological and genome analysis results clearly suggest that the two strains belong to the same BTV serotype. However, unlike the 2015 strain, BTV-X ITL2021 was successfully isolated in BSR cell-culture allowing further serological characterization. Lastly, seropositivity for BTV-X ITL2021 was detected by virus-neutralization in approximately 74% of animals tested, suggesting that this atypical BTV serotype has been circulating undetected in asymptomatic animals for years.

Epizootic Haemorrhagic Disease Virus Serotype 8 in Tunisia, 2021.

Epizootic haemorrhagic disease (EHD) is a Culicoides-borne viral disease caused by the epizootic haemorrhagic disease virus (EHDV) associated with clinical manifestations in domestic and wild ruminants, primarily white-tailed deer (Odocoileus virginianus) and cattle (Bos taurus). In late September 2021, EHDV was reported in cattle farms in central/western Tunisia. It rapidly spread throughout the country with more than 200 confirmed outbreaks. We applied a combination of classical and molecular techniques to characterize the causative virus as a member of the serotype EHDV-8. This is the first evidence of EHDV- 8 circulation since 1982 when the prototype EHDV-8 strain was isolated in Australia. This work highlights the urgent need for vaccines for a range of EHDV serotypes.

Uterine contractility is strongly influenced by steroids and glucose metabolism: an in vitro study on bovine myometrium.

BACKGROUND: Energetic substrates and hormonal phase are important for uterine contractions. Etomoxir a muscle carnitine palmitoyltransferase-1 inhibitor, able to diverge uterine metabolic pathways towards glycolysis, facilitates glucose utilisation. The aim of this study was to evaluate its effect on uterine contractility in different hormonal situations. METHODS: Uterine samples were collected from 60 cows during follicular phase, luteal phase and pregnancy. The cows were slaughtered at a local abattoir. Longitudinal strips were mounted vertically in a 30-ml organ bath connected to an isometric force transducer. Contractions were recorded with an ink-writing polygraph. After the equilibration period, etomoxir was added to the organ bath at different concentrations. The amplitude and frequency of contractions were registered before and after addition of etomoxir. RESULTS: In 17 strips from pregnant cows, etomoxir increased the amplitude (p < 0.05) of contractions but not the frequency in comparison with basal conditions. In 15 strips from cows in the luteal phase, etomoxir increased the amplitude (p < 0.05) and frequency of contractions (p < 0.05 at 5 µM and p < 0.01 at 8 and 10 µM). In 18 strips from cows in the follicular phase, etomoxir increased the frequency of contractions but not the amplitude (p < 0.01 at 5 µM and p < 0.05 at 8 and 10 µM). CONCLUSIONS: This study confirms the importance of glucose for uterine contractility and, moreover, it underlines different patterns of contraction with regard to the hormonal status.

In vitro activity of human chorionic gonadotropin (hCG) on myometrium contractility.

LH/hCG receptor has been found in extragonadal tissues in human and animals. The myometrium presents such receptors but their functional role is still not clear. Aim of our study was to test the activity of human chorionic gonadotropin (hCG) on bovine uterine contractility. Uterine strips from cows both during follicular and luteal phases were mounted in an organ bath and then exposed to increased doses of hCG. The amplitude of the myometrium contractions were significantly decreased in the follicular and luteal phase but the frequency was not affected. These findings prove a relaxing effect of hCG in the bovine uterus, as already shown in the sow and human, and its possible functional role in modulating uterine contractility.

Update on anesthesia and the immune response in newborns delivered by cesarian section.

In infants delivered by cesarian section, the immune system could be influenced by anesthetic drugs that may cross the placenta and reach the fetal circulation. Factors that determine placental transfer of anesthetics and their distribution into and ultimate removal from fetal tissue include physiochemical properties of the compound; the anatomic features of the maternal circulation, placenta, and fetus; and the hemodynamic and pharmacokinetic events that occur within them. Because the anesthetic agents can interfere with many neonatal function, the aim of this manuscript is to review the interference of regional or general anesthesia on neonatal immune response.

Pentavalent Disabled Infectious Single Animal (DISA)/DIVA Vaccine Provides Protection in Sheep and Cattle against Different Serotypes of Bluetongue Virus.

Bluetongue (BT) is a midge-borne OIE-notifiable disease of ruminants caused by the bluetongue virus (BTV). There are at least 29 BTV serotypes as determined by serum neutralization tests and genetic analyses of genome segment 2 encoding serotype immunodominant VP2 protein. Large parts of the world are endemic for multiple serotypes. The most effective control measure of BT is vaccination. Conventionally live-attenuated and inactivated BT vaccines are available but have their specific pros and cons and are not DIVA compatible. The prototype Disabled Infectious Single Animal (DISA)/DIVA vaccine based on knockout of NS3/NS3a protein of live-attenuated BTV, shortly named DISA8, fulfills all criteria for modern veterinary vaccines of sheep. Recently, DISA8 with an internal in-frame deletion of 72 amino acid codons in NS3/NS3a showed a similar ideal vaccine profile in cattle. Here, the DISA/DIVA vaccine platform was applied for other serotypes, and pentavalent DISA/DIVA vaccine for "European" serotypes 1, 2, 3, 4, 8 was studied in sheep and cattle. Protection was demonstrated for two serotypes, and neutralization Ab titers indicate protection against other included serotypes. The DISA/DIVA vaccine platform is flexible in use and generates monovalent and multivalent DISA vaccines to combat specific field situations with respect to Bluetongue.

First seroprevalence investigation of epizootic haemorrhagic disease virus in Libya.

Background: Epizootic haemorrhagic disease (EHD) is a vector-borne viral disease of domestic and wild ruminants. Epizootic haemorrhagic disease virus (EHDV) is transmitted by Culicoides spp. EHDV is a member of the Orbivirus genus within the Reoviridae family. It shares many morphological and structural characteristics with other members of the genus, such as the bluetongue virus, African horse sickness virus, and equine encephalosis virus. Aims: The purpose of our study was to investigate the epidemiological situation of EHDV in Libya in order to gain some knowledge about the presence of this virus in the country. Methods: In this study, we investigated the seroprevalence of EHDV in Libya, testing 855 blood samples collected during 2015. The samples were collected from domestic ruminants (cattle, sheep, and goats) originating from 11 provinces of Libya. Sera were tested by competitive enzyme-linked immunosorbent assays and positive samples confirmed by serum neutralization test. Results: The overall seroprevalence of EHDV was estimated to be 4% (95% confidence intervals = 2.8%-5.4%). Small ruminant seroprevalence was significantly (p = 0.016) higher than that found in cattle. Neutralizing antibodies against EHDV-6 were detected in a sheep from the western region of Libya. Conclusion: This study suggests that EHDV has circulated or is circulating in Libya, and sheep could play an important role in the epidemiology of EHDV, and the virus may still be circulating in North Africa.

Neutralization of SARS-CoV-2 Variants by Serum from BNT162b2 Vaccine Recipients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved rapidly, leading to viral lineages characterized by multiple mutations in the spike protein, which could potentially confer to the virus the ability to avoid the vaccine-induced immune response, making the vaccines less effective or ineffective. Here, we initially evaluated the neutralization capabilities in vitro by serum neutralization (SN) of six serum samples collected from recipients of the BNT162b2 vaccine against 11 SARS-CoV-2 isolates belonging to the major SARS-CoV-2 lineages that had been circulating in Italy. Then, we considered 30 additional serum samples by SN assay against the dominant B.1.617.2 (Delta) variant. A B.1 lineage isolate was used as a reference. In the first analysis, significant differences when compared with the reference strain (p > 0.05) were not evidenced; instead, when the panel of 30 sera was tested against the B.1.617.2 (Delta) variant, a significant (p = 0.0015) 2.38-fold reduction in neutralizing titres compared with the reference after the first vaccine dose was demonstrated. After the second vaccine dose, the reduction was not significant (p = 0.1835). This study highlights that the BNT162b2 vaccine stimulates a humoral response able to neutralize all tested SARS-CoV-2 variants, thus suggesting a prominent role in mitigating the impact of the SARS-CoV-2 pandemic in real-world conditions. Long-term follow-up is currently ongoing.

Peritoneal adhesions in human and veterinary medicine: from pathogenesis to therapy. A review.

Any peritoneal inflammatory process consequent to infections or surgical injuries may induce abdominal adhesion formation. Peritoneal adhesions are connective laciniae that develop among abdomino-pelvic organs that limit physiologic visceral motion. Consequently, fertility may be impaired, and intestinal obstruction and pelvic pain may develop, mainly in subjects that had undergone gynaecological surgery. This review illustrates the pathogenic steps of adhesiogenesis and the therapeutic scenario that evolved over the years to tackle the threat of peritoneal adhesions, both in domestic animals and in women.

Serum levels of reactive oxygen species (ROS) in the bitch.

The aim of this study was to determine the serum concentrations of reactive oxygen species (ROS) during the different phases of the estrous cycle in the bitch, in order to establish their physiological values. 56 healthy mixed-breed bitches were enrolled at this purpose and divided into 4 groups, standing on the different phases of the estrus cycle. Blood samples were collected in all groups and serum ROS concentrations were determined. Proestral concentrations were statistically higher than anestral ones, and statistically lower than those found in estrus (p<0.001). The highest concentrations of ROS were detected at estrus, that is, in the peri-ovulatory period. This sharp increase in ROS concentrations is related to the acute inflammatory process underlying ovulation and to the increase in immune and metabolic activities, cytological changes and myometrial contractility promoted by the high levels of estrogens. In diestrus, the mean concentration of ROS decreases. This reduction did not show any statistically significant difference with the mean value observed in proestrus. In this phase, in fact, the high concentrations of progesterone, exerting an antioxidant and immunodepressive effect, justify the lower mean concentration of ROS detected. In anestrus, the lowest concentrations of ROS were observed, for the reduced metabolic and endocrine activity occurring in this phase of the estrous cycle. In conclusion our results establish the physiologic levels of ROS during the estrous cycle in the bitch and reflect the endocrine morphologic and metabolic changes occurring during it.

Thyreotropic effect of human Chorionic Gonadotropin (hCG) in mares at estrus.

The beta-subunits of thyroid stimulating hormone (TSH) and human chorionic gonadotropin (hCG) share a high homology, just like the ectodomains of their receptors, do. As a consequence, hCG was shown to exert a thyrotropic action in humans and hamsters. This study aimed to investigate whether hCG, used to induce ovulation, displays a thyrotropic effect in the equine species too. Forty mares at estrus were divided in two groups; 20 were intravenously treated with sterile saline solution (NaCl 0.9%) (group A); 20 were intravenously treated with 4000 I.U. of hCG (group B). All the mares were artificially inseminated 24 hours after the administration of the drugs and underwent blood collection at estrus detection (T1), 2, 6, 24 hours later (T2, T3, T4), and 3 and 6 days later (T5, T6), in order to detect serum fT3 and fT4 concentrations. fT3 concentrations gave rise to similar and constant trends in the two groups, without any statistically significant difference, whereas fT4 concentrations declined in both groups, remaining statistically higher in group B than in group A for 48 hours (p < 0.05), thus accounting for a thyrotropic effect of hCG. Pregnancy rate was 70% in group B and 40% in group A, respectively. The higher success in the outcome of pregnancy may result from the immunomodulating and steroidogenic effects of hCG and from the higher levels of thyroid hormones observed in the hCG treated group.

Follicular development in pregnant cows after the administration of equine chorionic gonadotropin (eCG): a new insight.

The follicular development in the cow occurs in a wave-like pattern, and it takes place also during pregnancy. In the cow, Equine Chorionic Gonadotropin (eCG) is used for superovulation, but a decrease in total fertility has been reported, likely because of its immunogenic properties in species other than equine. In this regard, immune response has been implicated in follicular growth, ovulation, and placental development. So, aims of our study are to test the safety of eCG administered during pregnancy and characterize the ovarian activity, the quality of oocytes, the hormonal status, and interleukin levels in eCG-treated pregnant cows.

Reactive oxygen species (ROS): involvement in bovine follicular cysts etiopathogenesis.

Ovulation is compared to an acute inflammatory process during which vasoactive agents, prostanoids, leukotrienes and Reactive Oxygen Species (ROS) develop. The aim of this study was to evaluate the levels of ROS in cystic and follicular fluid, in order to establish their involvement in the etiopathogenesis of Cystic Ovarian Follicle (COF) in dairy cows. The study was conducted in 30 healthy cows (group C) and 30 cows affected by COF (group COF). The fluid of follicular cysts and of preovulatory follicles was drawn by means of ultrasound guided aspiration from the cows of both groups. The fluid obtained was analyzed by a photometric analytical system to detect ROS level. ROS concentration was statistically lower in the cystic fluid than in the follicular one (62.4 +/- 13.36 U.Carr vs. 84.89 +/- 26.99 U.Carr) (p<0.05), thus suggesting that an alteration of the cascade responsible for ROS production may be implicated in the complex etipathogenesis of COF.