Susceptibility of Mediterranean Buffalo (Bubalus bubalis) following Experimental Infection with Lumpy Skin Disease Virus.

Lumpy skin disease (LSD) is a viral disease of cattle and water buffalo characterized by cutaneous nodules, biphasic fever, and lymphadenitis. LSD is endemic in Africa and the Middle East but has spread to different Asian countries in recent years. The disease is well characterized in cattle while little is known about the disease in buffaloes in which no experimental studies have been conducted. Six buffaloes and two cattle were inoculated with an Albanian LSD virus (LSDV) field strain and clinically monitored for 42 days. Only two buffaloes showed fever, skin nodules, and lymphadenitis. All samples collected (blood, swabs, biopsies, and organs) were tested in real-time PCR and were negative. Between day 39 and day 42 after inoculation, anti-LSDV antibodies were detected in three buffaloes by ELISA, but all sera were negative by virus neutralization test (VNT). Cattle showed severe clinical signs, viremia, virus shedding proven by positive real-time PCR results, and seroconversion confirmed by both ELISA and VNT. Clinical findings suggest that susceptibility in buffaloes is limited compared to in cattle once experimentally infected with LSDV. Virological results support the hypothesis of buffalo resistance to LSD and its role as an accidental non-adapted host. This study highlights that the sensitivity of ELISA and VNT may differ between animal species and further studies are needed to investigate the epidemiological role of water buffalo.

Whole-Genome Sequence and Assembly of Eight Africa Horse Sickness Virus Strains Collected in Namibia and South Africa.

In this report, we describe eight complete genome sequences of African horse sickness virus (AHSV) strains belonging to four different serotypes, namely, AHSV-5, AHSV-6, AHSV-8, and AHSV-9. Samples were collected in Namibia and South Africa from infected horses between 2000 and 2011. As expected, phylogenetic analyses of the variable outer capsid protein VP2 genomic sequences of AHSV-6 and AHSV-8 show higher nucleotide identity between the isolated viruses than that of the relevant reference strains. The full-genome sequence of AHSV will provide useful information on its geographical origin, and it will also be instrumental for comparing the distribution of the Namibian isolate with that of global isolates.

Immunogenicity and safety studies of an inactivated vaccine against Rift Valley fever.

Rift Valley fever (RVF) is an emerging transboundary, mosquito-borne, zoonotic viral disease caused by a single serotype of a virus belonging to the Phenuiviridae family (genus Phlebovirus). It is considered an important threat to both agriculture and public health in endemic areas, because the virus, transmitted by different mosquito genera, leads to abortions in susceptible animal hosts especially sheep, goat, cattle, and buffaloes, resulting in severe economic losses. Humans can also acquire the infection, and the major sources are represented by the direct contact with infected animal blood, aerosol, consumption of unpasteurized contaminated milk and the bite of infected mosquitoes. Actually, the EU territory does not seem to be exposed to an imminent risk of RVFV introduction, however, the recent outbreaks in a French overseas department and some cases detected in Turkey, Tunisia and Libya, raised the attention of the EU for a possible risk of introduction of infected vectors. Thus, there is an urgent need to develop new therapeutic and/or preventive drugs, such as vaccines. In our work, we studied the immunogenicity of an inactivated and adjuvanted vaccine produced using a Namibian field strain of RVF virus (RVFV). The vaccine object of this study was formulated with Montanide Pet Gel A, a polymer-based adjuvant that has been previously reported for its promising safety profile and for the capacity to elicit a strong immune response. The produced inactivated vaccine was tested on six sheep and the level of IgM and IgG after the immunization of animals was evaluated by a commercial competitive ELISA, in order to assess the immunogenicity profile of our vaccine and to evaluate its potential use, as an alternative to the attenuated vaccines commercially available, in case of Rift Valley fever epidemic disease on EU territory. Following the administration of the second dose, 35 days after the first one, all animals seroconverted.

Development of a Competitive Enzyme-Linked Immunosorbent Assay Based on Purified Recombinant Viral Protein 7 for Serological Diagnosis of Epizootic Haemorrhagic Disease in Camels.

Epizootic haemorrhagic disease virus (EHDV) is a member of the Orbivirus genus in the Reoviridae family, and it is the etiological agent of an arthropod-transmitted disease that affects domestic and wild ruminants. Due to its significant economic impact, many attempts have been done in order to develop diagnostic immunoassays mainly based on the use of the viral protein 7 (VP7), that is, the immunodominant serogroup-specific antigen. In this work, a recombinant VP7 (recVP7) of EHDV serotype 2 was produced in a baculovirus system, and after purification using ion metal affinity chromatography, we obtained a high yield of recombinant protein characterized by a high degree of purity. We used the purified recVP7 as reagent to develop a competitive enzyme-linked immunoassay (c-ELISA), and we tested the presence of EHDV antibodies in 185 dromedary camel serum samples. The c-ELISA showed good performance parameters in recognising positive sera of naturally EHDV-infected dromedary camels; in particular, our developed test reached 85.7% of sensitivity, 98.1% of specificity, 93% of accuracy, and a high agreement value with results obtained by the commercial ELISA kit (Cohen's kappa value of 0.85) that we adopted as the reference method. This c-ELISA could be a useful screening test to monitor the virus spread in camels that are sentinel animals for endemic areas of disease.

Alternative methods to reduce the animal use in quality controls of inactivated BTV8 Bluetongue vaccines.

The acceptance of serology data instead of challenge for market release of new batches of commercial vaccine is under evaluation by regulatory agencies in order to reduce the use of animals and costs for manufacturers. In this study two vaccines for Bluetongue virus serotype 8 were submitted to quality controls required by the European Pharmacopoeia and tested on sheep in comparison with a commercial inactivated vaccine. Body temperature, antibody titres and viraemia of vaccinated and controls sheep were recorded. In addition IL4 and IFNγ in sera and supernatant derived from in vitro stimulation of blood cells were also quantified using two commercial ELISA kit. The outer-capsid protein VP2 contained in vaccine formulations was quantified using a home-made capture-ELISA. Results obtained indicates that in-lab evaluation of cell-mediated and humoral immune response are useful parameters to predict the efficacy of BTV inactivated vaccines avoiding the challenge phase required to release new batches of vaccines with proven clinical efficacy and safety. The correlation observed between serology data and VP2 protein concentration of final product could be useful in-process control to predict if a new vaccine batch of BTV must be discarded or released to the market.

Production of monoclonal antibodies binding to the VP7 protein of African horse sickness virus.

Monoclonal antibodies (MAbs) against AHSV were produced by immunising BALB/c mice with AHSV serotype 9 and six clones able to recognize specifically the VP7-AHSV with a strong reactivity were selected. The specificity of the MAbs was assessed in i-ELISA against a commercial VP7-AHSV and in immunoblot against a home-made VP7-AHSV, expressed by a Baculovirus expression system; potential cross-reactions with related orbiviruses (Bluetongue virus and Epizootic Haemorrhagic Disease virus) were investigated as well. One of the six MAbs selected, MAb 7F11E14, was tested in direct immunofluorescence and reacted with all nine AHSV serotypes, but didn't cross-react with BTV and EHDV. MAb 7F11E14 was also used to develop a competitive ELISA and was able to detect AHSV antibodies in the sera of AHS infected animals.

Evaluation of veterinary autogenous vaccines safety by an MTT in-vitro cytotoxicity assay.

In Italy, veterinary autogenous vaccines manufacturing is regulated by the legislative decree of the Ministry of Health, March 17th, 1994, n. 287. The production is performed by the network of the 'Istituti Zooprofilattici Sperimentali' (IZSs), public health institutes scattered all over the Italian territory. The aim of this research was to evaluate the feasibility of an in vitro method to test the abnormal toxicity of autogenous bacterial vaccines as an alternative to animal models routinely employed. For this purpose, the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise (IZSAM) in partnership with the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER), evaluated the toxicity of 49 batches of autogenous bacterial vaccines, previously shown to be safe in guinea pigs and mice, on animal model, by means of the methyl tetrazolium (MTT) assay. All vaccines showed cytotoxic effects when tested 1:2 diluted and undiluted; overall, all vaccines lost toxicity at 1:128 dilution. As expected, these findings suggest a different susceptibility of this assay  compared to the laboratory animal model. On the other hand, these results do not clarify which components of the vaccines are responsible for the cytotoxic effect. Overall, more experiments are warranted in order to standardize the MTT assay which could be coupled with the trials in laboratory animals.

Type C/D botulism in the waterfowl in an urban park in Italy.

This report describes an outbreak of botulism occurred among a free-living population of mallards (Anas platyrhynchos) and geese (Anser anser) in an urban park. Mortality rate among investigated population was 86,8% (118 dead out of 136). Twenty-seven carcasses were collected for macroscopic examination and screened for microbiological, virological, toxicological investigations. A sick mallard was captured and neurological symptoms were observed. No causative agent of viral avian diseases was found in the examined animals and screening for environmental neurotoxic substances proved negative as well. In contrast, microbiological cultures from specimens tested positive for botulinum toxin-producing clostridia. Blood serum and fecal extract of the sick mallard proved positive for botulinum neurotoxin in the standard mouse protection test using reference Clostridium botulinum type C antitoxin. Gene content of cultured strains showed a mosaic composition of bont/C and bont/D sequences, defining them as type C/D chimeric organisms.

Preliminary results on innocuity and immunogenicity of an inactivated vaccine against Peste des petits ruminants.

Peste des petits ruminants (PPR) virus belongs to the family Paramyxoviridae and represents a major threat to small livestock industry. In recent years, outbreaks of PPR have occurred in Turkey and North Africa. In endemic areas, disease prevention is accomplished using live­attenuated vaccines. However, the use of live vaccines in non­endemic regions, such as Europe, is not approved by Veterinary Authorities. In these regions inactivated vaccines are then the only viable alternative. In this study an inactivated vaccine (iPPRVac) was formulated with either a water­in­oil emulsion (ISA 71 VG) or with delta inulin adjuvant, alone (AFSA1) or combined with a TLR9 agonist oligonucleotide (AFSA2). These formulations were then tested for immunogenicity on rats. The iPPRV formulation with AFSA2 adjuvant induced 100% seroconversion in rats after 2 injections and was subsequently evaluated in goats. Five goats were immunised twice subcutaneously, 36 days apart with iPPRVac + AFSA2. The immunised goats all seroconverted to PPR by day 9 and remained seropositive until the end of the experimental period (133 days). These data indicate that the rat model is useful in predicting vaccine responses in goats and that inactivated vaccine, when formulated with a delta inulin adjuvant, represents a promising alternative to live attenuated vaccines for PPR vaccination campaigns in non­endemic areas.

Production of Monoclonal Antibodies in Serum-free Media.

Four hybridoma cell lines (C4B, 10C2G5, 6C5F4C7, 2D10G11) were adapted to grow in serum-free conditions. Cell proliferation, viability, and antibody production in Nutridoma SP and Ex-Cell HSF 610 media were evaluated and results compared with those obtained using DMEM containing 10% fetal bovine serum (control medium). Clone C4B showed the best IgG productivity in control medium, but viability and number of cells per milliliter were similar for the three media. For clone 10C2G5, the highest values of cell viability were obtained with both control medium and Nutridoma SP; no significant differences in IgG yields and number of cells/mL were observed among the three media. Clone 6C5F4C7 provided no significant differences when grown in the two serum-free media and in control medium. Clone 2D10G11 showed the best IgG productivity in control medium and in Ex-Cell HSF 610, even if Ex-Cell HSF 610 provided the lowest number of cells/mL; no significant differences among the three media were obtained for viability. Purified antibodies produced from each hybridoma cell line grown in serum-free media showed a higher degree of purity than those produced by the same cell lines grown in control medium. Purified MAbs were also titrated by ELISA to test MAb-antigen affinity.

Virus and host factors affecting the clinical outcome of bluetongue virus infection.

UNLABELLED: Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species, breed, age, BTV serotypes, and strains within a serotype affect the clinical course of bluetongue. Results obtained indicate that in small ruminants, there is a marked difference in the susceptibility to clinical disease induced by BTV at the host species level but less so at the breed level. No major differences in virulence were found between divergent serotypes (BTV-8 and BTV-2). However, we observed striking differences in virulence between closely related strains of the same serotype collected toward the beginning and the end of the European BTV-8 outbreak. As observed previously, differences in disease severity were also observed when animals were infected with either blood from a BTV-infected animal or from the same virus isolated in cell culture. Interestingly, with the exception of two silent mutations, full viral genome sequencing showed identical consensus sequences of the virus before and after cell culture isolation. However, deep sequencing analysis revealed a marked decrease in the genetic diversity of the viral population after passaging in mammalian cells. In contrast, passaging in Culicoides cells increased the overall number of low-frequency variants compared to virus never passaged in cell culture. Thus, Culicoides might be a source of new viral variants, and viral population diversity can be another factor influencing BTV virulence. IMPORTANCE: Bluetongue is one of the major infectious diseases of ruminants. It is caused by an arbovirus known as bluetongue virus (BTV). The clinical outcome of BTV infection is extremely variable. We show that there are clear links between the severity of bluetongue and the mammalian host species infected, while at the breed level differences were less evident. No differences were observed in the virulence of two different BTV serotypes (BTV-8 and BTV-2). In contrast, we show that the European BTV-8 strain isolated at the beginning of the bluetongue outbreak in 2006 was more virulent than a strain isolated toward the end of the outbreak. In addition, we show that there is a link between the variability of the BTV population as a whole and virulence, and our data also suggest that Culicoides cells might function as an "incubator" of viral variants.

Inactivated and adjuvanted vaccine for the control of the African horse sickness virus serotype 9 infection: evaluation of efficacy in horses and guinea-pig model.

African horse sickness (AHS) is a non-contagious viral disease of solipeds transmitted by Culicoides. The disease is endemic in most African countries. Past experience has shown that Italy is a country exposed to emerging infectious diseases endemic to Africa; an incursion of AHS virus together with the widespread presence of Culicoides vectors could be the cause of a serious epidemic emergency. A live attenuated vaccine containing seven of the nine viral serotypes, serotype 5 and 9 are excluded, is commercially available from Onderstepoort Biological Products. However, the use of live vaccines is a matter of endless disputes, and therefore inactivated or recombinant alternative products have been investigated over the years. Since research on AHS is hampered by the use of horses to evaluate vaccine potency, in a previous experiment serological response to serotypes 5 and 9 was assayed in guinea-pigs and horses. A durable and comparable serological response was observed in the two animal species. In the present study antibody response in horses and guinea-pigs, immunised with the inactivated-adjuvanted vaccine formulated with serotype 9, was tested over a period of 12 months. When immunity was challenged, horses were protected from infection and disease. Antibody response in horses and guinea-pigs compared favourably.

Immunogenicity of two adjuvant formulations of an inactivated African horse sickness vaccine in guinea-pigs and target animals.

Monovalent, inactivated and adjuvanted vaccines against African horse sickness, prepared with serotypes 5 and 9, were tested on guinea-pigs to select the formulation that offered the greatest immunity. The final formulation of the vaccines took into account the immune response in the guinea-pig and the inflammatory properties of two types of adjuvant previously tested on target animals. A pilot study was subsequently conducted on horses using a vaccine prepared with serotype 9. The vaccine stimulated neutralising antibodies from the first administration and, after the booster dose, 28 days later; high antibody levels were recorded for at least 10 months. The guinea-pig appears to be a useful laboratory model for the evaluation of the antigenic properties of African horse sickness vaccines.

An ELISA for the evaluation of gamma interferon production in cattle vaccinated with Brucella abortus strain RB51.

The results of an enzyme-linked immunosorbent assay (ELISA) implemented for the detection of gamma interferon (gamma interferon) production in cattle vaccinated with Brucella abortus strain RB51 are presented. A purified protein fraction derived from RB51 (RB51 brucellin) has been used as antigenic stimulus for whole blood. The test was evaluated for 300 days in ten heifers vaccinated at calfhood with 10 x 10(9) colony-forming units of RB51 and in five control heifers. All animals came from officially brucellosis-free herds. Vaccinated animals started to give positive results from day 17 post vaccination (pv) until day 239 pv. All vaccinated animals gave a positive reaction at least once (with a stimulation index exceeding 2.5). Nevertheless, if sampling on day 20 pv is excluded (90% of vaccinated animals gave positive results), the sensitivity of the test varies from 20% to 70%, with a 40% average. A stimulation index over 2.5 was also recorded in three control animals. The results suggest that the gamma-interferon test is not suitable for the detection of cattle vaccinated with RB51, either at the individual or at the herd level.

Brucella abortus strain RB51 vaccine: immune response after calfhood vaccination and field investigation in Italian cattle population.

Immune response to Brucella abortus strain RB51 vaccine was measured in cattle vaccinated at calfhood. After an increase at day 6 post-vaccination (pv), the antibody level recorded in the 10 vaccinated animals remained constant for two months, and then progressively decreased. All vaccinated animals remained negative from day 162 pv to the end of the study (day 300 pv). Only at days 13 and 14 pv the RB51-CFT showed 100% sensitivity (credibility interval (CI) 76.2%-100%). The results indicate that the possibility to use RB51-CFT for the identification of cattle vaccinated at calfhood with RB51 is limited in time. A field investigation was carried out on 26,975 sera collected on regional basis from the Italian cattle population. The study outcomes indicate that in case of RB51-CFT positive results observed in officially Brucellosis-free (OBF) areas and, in any case, when an illegal use of RB51 vaccine is suspected, the use of the RB51-CFT alone is not sufficient to identify all the vaccinated animals. The design of a more sophisticated diagnostic protocol including an epidemiological investigation, the use of RB51-CFT, and the use of the skin test with RB51 as antigen is deemed more appropriate for the identification of RB51 vaccinated animals.

Infection of lung epithelial cells and induction of pulmonary adenocarcinoma is not the most common outcome of naturally occurring JSRV infection during the commercial lifespan of sheep.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). In this study, we followed over a 31-month period the natural transmission of JSRV in adult sheep and in their offspring. We established groups derived from flocks with either a high or low incidence of OPA and monitored virus transmission, clinical disease and macroscopic/microscopic lung lesions at necropsy. Results obtained show that (i) JSRV infection can occur perinatally or in the first few months of life in lambs and in adult sheep; (ii) only a minority of JSRV-infected animals develop clinical disease during their commercial lifespan; and (iii) JSRV is more readily detectable in peripheral blood leucocytes and lymphoid organs than in the lungs. These data support a model of opportunistic JSRV infection and tumorigenic conversion of type II pneumocytes/Clara cells in the lungs, while lymphoreticular cells serve as the principal virus reservoir.