Degenerate PCR method for identification of an antiapoptotic gene in BHV-1

To investigate on the hypothetical presence of an antiapoptotic gene, we utilized the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers) strategy amplifying unknown sequences from a background of genomic (bovine herpesvirus type-1) BHV-1 DNA. An alignment of carboxyl-terminal domains belonging to three proteins encoded by gamma 34.5, MyD116 and GADD34 genes, was carried out to design degenerate PCR primers in highly conserved regions. This allowed the amplification of a 110 bp fragment. This fragment was subjected to automatic sequencing and DNA sequence analysis revealed that its position resided between the nt 14363 and the nt 14438 in bovine herpesvirus type-1 (BHV-1) Cooper strain sharing an identity of 86% (UL14). Transient transfections showed that ULI 4 protein is efficient in protecting MDBK and K562 cells from sorbitol induced apoptosis. The protein's anti-apoptotic function may derive from its heat shock protein-like properties

Phage therapy in veterinary medicine

To overcome the obstacle of antimicrobial resistance, researchers are investigating the use of phage therapy as an alternative and/or supplementation to antibiotics to treat and prevent infections both in humans and in animals. In the first part of this review, we describe the unique biological characteristics of bacteriophages and the crucial aspects influencing the success of phage therapy. However, despite their efficacy and safety, there is still no specific legislation that regulates their use. In the second part of this review, we describe the comprehensive research done in the past and recent years to address the use of phage therapy for the treatment and prevention of bacterial disease affecting domestic animals as an alternative to antibiotic treatments. While in farm animals, phage therapy efficacy perspectives have been widely studied in vitro and in vivo, especially for zoonoses and diseases linked to economic losses (such as mastitis), in pets, studies are still few and rather recent

Comparison of fluorescence polarization assay with Rose Bengal (RB) test and complement fixation tests for the diagnosis of buffalo (Bubalus bubalis) brucellosis in a high-prevalence area

The fluorescence polarisation assay (FPA) was evaluated for the serological diagnosis of bovine brucellosis in buffalo (Bubalus Bubalis) in southern Italy. This assay uses O-polysaccharide prepared from Brucella Abortus lipopolysaccharide conjugated with fluorescein isothiocyanate as a tracer. It has many methodological advantages over older, more established tests and can be performed in short time. To measure the fluorescence polarization, a Tecan GENios Pro (Prionics) fluorescence-polarization analyzer was used with the procedure described by Nielsen et al. 1996. A cut-off value of 117 millipolarization (mP) units was used for testing 912 buffalo sera from Campania Region (526 positive sera and 386 negative sera according to the complement fixation test; CFT). All samples were tested with the Rose Bengal plate (RB). Sensitivity and specificity (Sn) for RB were 84.0% and 87.8% and for FPA were 92.6% and 88.9 percent. The FPA gave a kappa coefficient of agreement with respect to CFT of 0.755, while RB (relative to the CFT) gave coefficients of 0,715. Finally, ROC analysis suggested a cut-off value which did not agree with the one recommended in the test procedure for cow

Occurrence and Antimicrobial Susceptibility Profiles of Streptococcus equi subsp. zooepidemicus Strains Isolated from Mares with Fertility Problems

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus), is a β-hemolytic Streptococcus belonging to the Lancefield group C; it is a rare human pathogen, but in horses, it is frequently associated with endometritis. This study aimed to isolate S. zooepidemicus strains, associated with bacterial endometritis in mares, and to define their antimicrobial resistance profile. Twenty-three isolates were recovered from one hundred ninety-six equine uterine swabs (11.7%). Bacterial identification was carried out by Api 20 Strep and confirmed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), while antimicrobial susceptibility testing was performed by disk diffusion method on Muller Hinton agar plates. The antibiotic resistance profiles of the isolates revealed a high percentage of resistance to amikacin (95.6%), ampicillin (73.9%) and tetracycline (69.6%), while ceftiofur and ceftriaxone were highly effective with 82.6% and 78.3% of the isolates inhibited, respectively. An intriguing value of resistance to penicillin (34.8%), which represents the first-choice antibiotic in equine S. zooepidemicus infections, was observed. Furthermore, a high prevalence of multidrug-resistant strains (82.6%) was recorded. Continuous surveillance of this potential zoonotic pathogen and an appropriate antimicrobial stewardship program with the promotion of correct use of antimicrobials, after a proper diagnosis, are needed to allow an effective therapy

Serological and molecular survey of Q fever in the dog population of the Campania region, southern Italy

: Q fever is a re-emerging zoonosis whose epidemiological cycle in ruminants is well defined, while the role of other species (including pets) is still debated. In this study, the serological and molecular prevalence of Coxiella burnetii in a sample of dogs in the Campania region, southern Italy was evaluated. A seroprevalence of 5.97 % (16/268) was observed using a commercial multispecies ELISA, compared to only 2.7 % (5/197) at the molecular level. No risk factors correlated with higher levels of exposure except for the size of the animal (small dogs showed significantly higher seroprevalence). Positive samples were further evaluated for reactivity to phase I and II antigens using IFA and phase-specific ELISAs (for specific IgG detection). Two animals showed antibodies against both phases of infection, suggesting that Coxiella burnetii seroconversion in dogs follows similar dynamics to those observed in ruminants. One of the five samples that showed positive results in real-time PCR was confirmed at the PCR endpoint and showed similarity with other Coxiella spp. strains detected in tick and dog samples when sequenced. In this study, we demonstrated exposure to Coxiella burnetii for different categories of dogs in southern Italy, including pet dogs living indoors. Since reports of transmission of infection from pets to humans have been described in both rural and urban areas, careful surveillance of these species is also necessary. In the lack of additional information, comprehending the risk to humans requires monitoring of wild and domestic animal populations

Fire-spotting modelling in operational wildfire simulators based on Cellular Automata: A comparison study

One crucial mechanism in the spread of wildfires is the so-called fire-spotting: a random phenomenon that occurs when embers are transported over large distances. Fire-spotting speeds up the rate of spread and starts new ignitions that can jeopardise firefighting operations. Unfortunately, operational fire-spread simulators may not account for spotting events, thus overlooking the harmful consequences associated with this phenomenon. In this work, three fire spotting parametrisations are integrated in the operational wildfire simulator PROPAGATOR based on Cellular Automata (CA). RandomFront, a physics-based parametrisation of fire-spotting, is tested for the first time in the context of CA simulators. RandomFront is compared with other two parametrisations already adopted in CA based simulators, those by Alexandridis and co-authors and by Perryman and collaborators. A wildfire occurred in the summer of 2021 in the municipality of Campomarino (Molise, Italy), and where spotting effects were clearly reported, is used as a case study. This case study, featuring evident airborne transport of firebrands, paves the way for a framework for comparing parameterised spotting models used in operational scenarios. RandomFront produced a more complex burning probability pattern than the other parametrisations and it predicted a higher probability of burning in the zone mainly affected by the fire-spotting

Safety of B. abortus rough mutant strain RB51 administration in Buffalo cows

The objective of this study was to determine if B. abortus rough mutant strain RB51 is eliminated in Buffalo milk. Five milk buffaloes were inoculated with the triple of the recommended calfhood dose (3.0 – 10.2 x 1010 cfu/ml) of B. abortus RB51 strain by subcutaneous route in the right axillary region. Milk samples were taken aseptically on a daily basis for the first 30 days and weekly for the second and third months. The samples were inoculated on Brucella Medium Base (BMB) (Oxoid) and Rifampin Brucellae Medium (RBM) and incubated under 10% CO2 at 37°C for 10 days. The suspicious colonies were recultured in BMB and RBM. PCR analysis was also performed on milk samples. There were no isolations of bacteria with characteristics of Brucella from any of the milk samples collected during 90 days of the study. However Brucella RB51 DNA was detected on day 2 and 3 post vaccination in one buffalo cow and on day 21 post vaccination in another buffalo cow. It was concluded that the strain used at this dose wasn't eliminated by milk in Buffaloes inoculated during lactation, however PCR positive results underline the necessity of milk pasteurization in order to minimize food-chain exposure

Simultaneous detection of enteropathogenic viruses in buffalos faeces using multiplex reverse transcription-polymerase chain reaction (mRT-PCR)

A multiplex reverse transcription- polymerase chain reaction (mRT-PCR) assay that detects Bovine Viral Diarrhoea Virus, Bovine Coronavirus, and Group A Rotaviruses in infected cell-culture fluids and clinical faecal samples is described. One hundred twenty faecal samples from buffalo calves with acute gastroenteritis were tested. The mRT-PCR was validated against simplex RT-PCR with published primers for Pestivirus, Coronavirus and Rotavirus. The multiplex RT-PCR was equally sensitive and specific in detecting viral infections compared with simplex RT-PCR. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. This mRT-PCR may be a sensitive and rapid assay for surveillance of buffalo enteric viruses in field specimens. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of acute gastroenteritis

Apoptosis is mediated by FeHV-1 through the intrinsic pathway and interacts with the autophagic process

Background: Although FeHV-1 is a primary feline pathogen, little is known about its interactions with host cells. Its relationship with several cellular pathways has recently been described, whereas its interplay with the apoptotic process, unlike other herpesviruses, has not yet been clarified. The aim of this work was to evaluate whether FeHV-1 induces apoptosis in its permissive cells, as well as the pathway involved and the effects of induction and inhibition of apoptosis on viral replication. Methods: Monolayers of CRFK cells were infected at different times with different viral doses. A cytofluorimetric approach allowed the quantification of cells in early and late apoptosis. All infections and related controls were also subjected to Western blot analysis to assess the expression of apoptotic markers (caspase 3-8-9, Bcl-2, Bcl-xL, NF-κB). An inhibitor (Z-VAD-FMK) and an inducer (ionomycin) were used to evaluate the role of apoptosis in viral replication. Finally, the expression of autophagy markers during the apoptosis inhibition/induction and the expression of apoptosis markers during autophagy inhibition/induction were evaluated to highlight any crosstalk between the two pathways. Results: FeHV-1 triggered apoptosis in a time- and dose-dependent manner. Caspase 3 cleavage was evident 48 h after infection, indicating the completeness of the process at this stage. While caspase 8 was not involved, caspase 9 cleavage started 24 h post-infection. The expression of other mitochondrial damage markers also changed, suggesting that apoptosis was induced via the intrinsic pathway. NF- κB was up-regulated at 12 h, followed by a gradual decrease in levels up to 72 h. The effects of apoptosis inhibitors and inducers on viral replication and autophagy were also investigated. Inhibition of caspases resulted in an increase in viral glycoprotein expression, higher titers, and enhanced autophagy, whereas induction of apoptosis resulted in a decrease in viral protein expression, lower viral titer, and attenuated autophagy. On the other hand, the induction of autophagy reduced the cleavage of caspase 3. Conclusions: In this study, we established how FeHV-1 induces the apoptotic process, contributing to the understanding of the relationship between FeHV-1 and this pathway