Aujeszky’s disease in hunting dogs after the ingestion of wild boar raw meat in Sicily (Italy): clinical, diagnostic and phylogenetic features

Background Aujeszky's disease is caused by Suid Herpes Virus-1 and species belonging to the genus Sus scrofa are the main reservoir hosts. This virus, however, is capable of infecting and causing severe disease, with an almost constant fatal outcome in other species, both domestic and wild (carnivores, monogastric herbivores and ruminants). Moreover, the possibility of transmission to humans has been demonstrated. This study reports and describes the clinical, diagnostic, pathological and phylogenetic aspects of two cases of Aujeszky's disease in two hunting dogs following the ingestion of infected wild boar raw meat. These cases are contextualized in the province of Messina (Sicily), where a high prevalence of Aujeszky's disease has been recorded (average of 12,20% in the period 2010–2019) in farmed pig, and with evidence of spread to other species. A severe outbreak in cattle has recently been reported in these areas. Nevertheless, cases of Aujeszky's disease in dogs are rarely reported and this study represents the first well-documented report in this species in Sicily. Case presentation After a wild boar hunt, two dogs showed neurological symptoms and intense itching unresponsive to therapy. Diagnosis of Aujeszky's disease was made based on clinical suspicion, anamnestic information and confirmed by the isolation of the virus from the brain of both dogs. In addition, molecular typing, sequencing and phylogenetic analysis of the Real-Time PCR products were performed. The sequences studied were placed in the Italian Clade 1 along with the sequences obtained from wild boars and hunting dogs from Italy and France. Conclusions The finding of this disease in non-natural hosts in Sicilian multi-host epidemiological contexts suggests that the risk of inter-species transmission is concrete and that attention should be paid to developing disease control programs in these territories. The data obtained from genome sequencing of the two SuHV-1 isolates contribute to the enrichment of the GenBank with unknown sequences and the phylogenetic analysis implementation.

The Valproic Acid Derivative Valpromide Inhibits Pseudorabies Virus Infection in Swine Epithelial and Mouse Neuroblastoma Cell Lines

Pseudorabies virus (PRV) infection of swine can produce Aujeszky’s disease, which causes neurological, respiratory, and reproductive symptoms, leading to significant economic losses in the swine industry. Although humans are not the natural hosts of PRV, cases of human encephalitis and endophthalmitis caused by PRV infection have been reported between animals and workers. Currently, a lack of specific treatments and the emergence of new PRV strains against which existing vaccines do not protect makes the search for effective antiviral drugs essential. As an alternative to traditional nucleoside analogues such as acyclovir (ACV), we studied the antiviral effect of valpromide (VPD), a compound derived from valproic acid, against PRV infection in the PK15 swine cell line and the neuroblastoma cell line Neuro-2a. First, the cytotoxicity of ACV and VPD in cells was compared, demonstrating that neither compound was cytotoxic at a specific concentration range after 24 h exposure. Furthermore, the lack of direct virucidal effect of VPD outside of an infected cell environment was demonstrated. Finally, VPD was shown to have an antiviral effect on the viral production of two strains of pseudorabies virus (wild type NIA-3 and recombinant PRV-XGF) at the concentrations ranging from 0.5 to 1.5 mM, suggesting that VPD could be a suitable alternative to nucleoside analogues as an antiherpetic drug against Aujeszky’s disease.

Inactivated vaccine against Aujeszky's disease

Background and Aim: The Aujeszky's disease, also known as Pseudorabies, remains one of the most problematic fulminant diseases in domestic animals, affecting the central nervous system. The study aimed to investigate the effect of an inactivated vaccine against Aujeszky's disease based on "Kordai" virus strain. Materials and Methods: To test the inactivation of the "Kordai" strain (grown by the roller method in VNK-21/13 cell culture with an infectious titer of at least 7.5 lg TCD50/ml) which is causative of Aujeszky's disease, next-generation teotropin and propolis preparations were used in concentrations of 0.1%, 0.08%, and 0.04%. Results: As a result of comparative studies on the optimization of parameters for inactivating the "Kordai" virus strain, it was established that teotropin is a more effective inactivant than propolis. At the same time, the optimal final concentration of teotropin for inactivation was 0.1%, along with a reaction medium temperature of 37°C, pH of 7.4-7.6, and duration of inactivation of 14 h. The titer of virus-neutralizing activity (VNA) of antibodies at the pH (neutralization reactions) in vaccinated sheep of 10-12 months of age was 7.5±0.3, Ig TCID50/ml (tissue culture infectious dose 50%), and 3.5±0.3 in the cell culture VNK-21/13 (culture of Syrian hamster kidney cells). Conclusion: To determine colostral immunity in newborn lambs, the method of metabolic status correction was used to vaccinate lambs obtained from immune sheep 4 months after birth. The results showed that lambs obtained from immune sheep had high VNA titers. A sustained immune response in vaccinated animals was obtained after double vaccination.

Aujeszky’s Disease in South-Italian Wild Boars (Sus Scrofa): A Serological Survey

Aujeszky’s disease (AD, pseudorabies) is a viral disease of suids caused by Suid Herpesvirus 1 (SHV-1) also referred as Aujeszky’s disease virus (ADV) or Pseudorabies virus (ADV). Domestic pig and Wild boar (Sus scrofa) are the natural host, but many species can be infected with ADV. The aim of our study was to evaluate seroprevalence of AD in wild boar hunted in the Campania Region, during the 2016–2017 hunting season. A total of 503 serum samples from wild boars hunted in the provinces of Campania Region (Southern Italy) were collected and were tested for antibody against ADV using an AD, blocking ELISA assay. A Seroprevalence of 23.85% (120/503, 95% Confidence Interval (CI): 20.15–27.55) was found. Gender was not significantly associated with of ADV seropositivity (p > 0.05), while the presence of ADV antibodies was statistically associated with age (>36-month, p < 0.0001) and location (Avellino, p = 0.0161). Our prevalence values are like those obtained in 2010 in our laboratory (30.7%), demonstrating a constant circulation of ADV in the area.

Influence of immunomodulators of viral or bacterial origin and vaccine against Aujeszky’s disease on the proportion of peripheral blood B cells in growing pigs

The consequences of infection by Suid herpesvirus type 1 (SuHV-1) that causes Aujeszky’s disease (AD) are well studied, however, the effects of immunomodulators (IMs) of microbial origin (viral and bacterial) when administered solely or in combination with the attenuated SuHV-1 vaccine are less known. The effects of parenteral administration of IMs, inactivated Parapoxvirus ovis (P. ovis) or a combination of inactivated Propionibacterium granulosum (P. granulosum) and detoxified Escherichia coli lipopolysaccharide (LPS) and attenuated SuHV-1, strain Bartha, on the proportion of peripheral blood CD3- CD21+ B cells were analysed in 30 crossbred, 3-month old pigs using flow cytometry (FCM). Specific antibodies for gE and gB of SuHV- 1 were detected using the enzyme-linked immunosorbent assay (ELISA). Data were compared among six experimental groups: (1) pigs that separately received the vaccine, (2) IM of bacterial origin, (3) IM of viral origin, (4) simultaneous administration of the vaccine and bacterial IM, (5) simultaneous administration of the vaccine and viral IM, and (6) the control group of untreated pigs. Comparison of B cell proportions and the detection of specific antibodies in blood samples of vaccinated pigs on Day 11 of the experiment showed a transient decrease in B cell contents, though this could not be assumed to be related since the control group showed a decrease in B cell proportion on the same day. The results showed that the use of IMs alone or in combination with the attenuated SuHV-1 vaccine did not have a significant impact on the proportion of peripheral blood B cells in growing pigs.

Retrieving Historical Cases of Aujeszky’s Disease in Sicily (Italy): Report of a Natural Outbreak Affecting Sheep, Goats, Dogs, Cats and Foxes and Considerations on Critical Issues and Perspectives in Light of the Recent EU Regulation 429/2016

Aujeszky’s disease is caused by Suid alphaherpesvirus 1, and its main reservoir host is the pig. However, other species are also susceptible. Infection with this virus causes a severe neurological clinical picture named Aujeszky’s disease, usually accompanied by itching and death a few days after the onset of symptoms. This study reports a multi-species outbreak of Aujeszky’s disease that occurred in Sicily, which led to the death of 2 goats, 15 sheep, 2 dogs, 2 cats and 2 foxes. The diagnosis was made by culture, indirect immunofluorescence on brain samples and confirmed by biological test on rabbits. This study reports the first cases of Aujeszky’s disease in Italy in cats, goat and sheep. The finding of Aujeszky’s disease in several species in Sicily suggests a potential epizootic risk. In such areas where a multi-host system is recognised, an analysis of the risk factors should be carried out in order to develop targeted strategies for the control and eradication of the disease. The critical issues that hinder the control of Aujeszky’s disease in the studied territory and perspectives for eradication in the light of EU regulation 429/2016 are also discussed.

Studies of biological properties of continuous suspension ВНК-21/SUSP/ARRIAH cell line

The results of the studies of cytomorphological, karyological, cultural properties of continuous suspension ВНК-21/SUSP/ARRIAH subline of newborn Syrian hamster kidney cells intended for foot-and-mouth disease, rabies, bovine parainfluenza-3, Aujeszky’s disease virus reproduction, as well as for production of diagnostic veterinary biologicals are presented. When cultured in suspension, BHK-21/SUSP/ARRIAH cell subline undergoes selection towards hypoploidy: modal class is represented by cells with 41 chromosomes (32–40% of cells); the share of cells containing 40–42 chromosomes is 78–80%; the share of polyploids averages around 1%; the range of variation in the number of chromosomes is from 36 to 54. BHK-21/SUSP/ARRIAH cell subline cultured in suspension with cell seeding concentration of 0.6–0.8 million cells/cm3 demonstrates growth rate of 6.67–11.00 and 96–99% cell viability. After 48 hours, G1-phase (diploid-2n) cells prevail in the cell population of the new subline (30.0–75.0% of cells); cells that undergo preparation for mitosis (S-phase) and mitosis (G2+M-phase) account for 3.0 to 20.0% of the entire population; the number of meganucleated and multinucleated cells (>4n) at the beginning and at the end of the logarithmic phase increases to 2%. BHK-21/SUSP/ARRIAH cells recover rapidly after cryopreservation and demonstrate 95–99% viability and growth rate of 3.36–5.88 at passages 1 to 3 and 6.85–10.95 at passages 4 to 12. Continuous suspension BHK-21/SUSP/ARRIAH cell line ensures virus accumulation at the following titres: FMD virus – 7.30– 8.00 lg TCID50/cm3, rabies virus – 7.25–8.00 lg CCID50/cm3, bovine parainflunza-3 virus – at least 6.00 lg TCID50/cm3, Aujeszky’s disease virus – 7.50–7.80 lg TCID50/cm3.