Evaluation of Hematological Profiles and Monocyte Subpopulations in Water Buffalo Calves after Immunization with Two Different IBR Marker Vaccines and Subsequent Infection with Bubaline alphaherpesvirus-1.

Bubaline alphaherpesvirus-1 (BuAHV-1) and Bovine alphaherpesvirus-1 (BoAHV-1) are respiratory viruses that can cause an infection known as "Infectious Bovine Rhinotracheitis" (IBR) in both water buffalo and bovine species. As the main disease control strategy, vaccination can protect animals from clinical disease through the development of specific humoral and cell-mediated immune responses. In the present study, the time-related circulatory kinetics of hematological profile and bubaline monocyte subsets have been investigated in vaccinated buffalo calves after challenge infections with BuAHV-1. Thirteen buffalo calves were selected and grouped into the VAX-1 group, which received an IBR-live-attenuated gE-/tk-deleted marker vaccine; the VAX-2 group, which received an IBR-inactivated gE-deleted marker vaccine; the CNT group, which remained an unvaccinated control. Fifty-five days after the first vaccination, the animals were infected with 5 × 105.00 TCID50/mL of wild-type BuAHV-1 strain via the intranasal route. Whole blood samples were collected at 0, 2, 4, 7, 10, 15, 30, and 63 days post-challenge (PCDs) for the analysis of hematological profiles and the enumeration of monocyte subsets via flow cytometry. The analysis of leukocyte compositions revealed that neutrophils were the main leukocyte population, with a relative increase during the acute infection. On the other hand, a general decrease in the proportion of lymphocytes was observed early in the post-infection, both for the VAX-1 and VAX-2 groups, while in the CNT group, the decrease was observed later at +30 and +63 PCDs. An overall infection-induced increase in blood total monocytes was observed in all groups. The rise was especially marked in the animals vaccinated with an IBR-live-attenuated gE-/tK-deleted marker vaccine (VAX-1 group). A multicolor flow cytometry panel was used to identify the bubaline monocyte subpopulations (classical = cM; intermediate = intM; and non-classical = ncM) and to investigate their variations during BuAHV-1 infection. Our results showed an early increase in cMs followed by a second wave of intMs. This increase was observed mainly after stimulation with live-attenuated viruses in the VAX-1 group compared with the animals vaccinated with the inactivated vaccine or the non-vaccinated animal group. In summary, the present study characterized, for the first time, the hematological profile and distribution of blood monocyte subsets in vaccinated and non-vaccinated water buffalo in response to experimental infection with BuAHV-1. Although not experimentally proven, our results support the hypothesis of a linear developmental relationship between monocyte subsets.

Endogenous and viral microRNAs in nasal secretions of water buffaloes (Bubalus bubalis) after Bubaline alphaherpesvirus 1 (BuHV-1) challenge infection.

Bubaline alphaherpesvirus 1 (BuHV-1) is a pathogen of water buffaloes responsible for economic loss worldwide. MicroRNAs (miRNAs) regulate gene expression produced by alphaherpesviruses and hosts. This study aimed at (a) unravelling the ability of BuHV-1 to produce miRNAs, including hv1-miR-B6, hv1-miR-B8, hv1-miR-B9; (b) measuring the host immune-related miRNAs associated to herpesvirus infection, including miR-210-3p, miR-490-3p, miR-17-5p, miR-148a-3p, miR-338-3p, miR-370-3p, by RT-qPCR; (c) identifying candidate markers of infection by receiver-operating characteristic (ROC) curves; (d) exploiting the biological functions by pathway enrichment analyses. Five water buffaloes BuHV-1 and Bovine alphaherpesvirus 1 (BoHV-1) free were immunized against Infectious Bovine Rhinotracheitis (IBR). Five additional water buffaloes served as negative controls. All animals were challenged with a virulent wild-type (wt) BuHV-1 via the intranasal route 120 days after the first vaccination. Nasal swabs were obtained at days (d) 0, 2, 4, 7, 10, 15, 30, and 63 post-challenge (pc). The animals of both groups shed wt BuHV-1 up to d7 pc. Results demonstrated that (a) miRNAs produced by the host and BuHV-1 could be efficiently quantified in the nasal secretion up to d63 and d15 pc, respectively; b) the levels of host and BuHV-1 miRNAs are different between vaccinated and control buffaloes; c) miR-370-3p discriminated vaccinated and control animals; d) host immune-related miRNAs may modulate genes involved in the cell adhesion pathway of the neuronal and immune system. Overall, the present study provides evidence that miRNAs can be detected in nasal secretions of water buffaloes and that their expression is modulated by BuHV-1.

Evaluation of an Immunization Protocol Using Bovine Alphaherpesvirus 1 gE-Deleted Marker Vaccines against Bubaline Alphaherpesvirus 1 in Water Buffaloes.

European regulations on the control of infectious diseases provide measures to control Bovine alphaherpesvirus 1 (BoHV-1) infection in both cattle and buffalo. Owing to the reported serological cross-reactivity between BoHV-1 and Bubaline alphaherpesvirus 1 (BuHV-1), we hypothesized a new immunization protocol using BoHV-1 gE-deleted marker vaccines could protect water buffalo against BuHV-1. Five water buffaloes devoid of BoHV-1/BuHV-1-neutralizing antibodies were immunized with two commercial BoHV-1 gE-deleted marker vaccines at 0, 30, 210, and 240 post-vaccination days (PVDs). Five additional water buffaloes were used as controls. At 270 PVD (0 post-challenge days (PCDs), all animals were challenged intranasally with wild-type (wt) BuHV-1. The vaccinated animals produced humoral immunity (HI) as early as PVD 30 whereas, in control animals, antibodies were detected on PCD 10. After challenge infection, HI significantly increased in vaccinated animals compared to that in controls. Real-time PCR for gB revealed viral shedding in vaccinated animals from PCDs 2 to 10. In contrast, positive results were observed from PCDs 2 to 15 in the unvaccinated control group. Although the findings indicated the possible protection capabilities of the tested protocol, these findings did not support its protective roles in water buffaloes against wt-BuHV-1.

Assessment of Different Infectious Bovine Rhinotracheitis Marker Vaccines in Calves.

Three commercially available infectious bovine rhinotracheitis (IBR) live marker vaccines were evaluated for their ability to provide clinical protection to vaccinated calves against wild-type (wt) Bovine alphaherpesvirus-1 (BoHV-1) challenge and their possible effect on wt BoHV-1 latency reactivation following the challenge. On 35 post-vaccination days (PVDs), all animals were challenged with wt BoHV-1. Only the calves in the control group developed severe forms of IBR. The reactivation of latent BoHV-1 was induced by dexamethasone (DMS) treatment on 28 post-challenge days (PCDs). All animals showed IBR clinical signs on three post-DMS treatment days (PDTDs). On PVD 14, all vaccinated animals developed neutralizing antibodies (NAs), whereas in control animals, the NAs appeared post-challenge. The positivity for glycoprotein-B (gB) was detected using real-time polymerase chain reactions in all animals from PCDs 1 to 7. In contrast, the gB-positivity was observed in the immunized calves from PDTDs 3 to 10. Positive expression of gD and gE was observed in nasal swabs of all calves on PDTD 7. These findings suggested that the IBR marker vaccines evaluated in this study protected against wt BoHV-1-induced disease but not against wt BoHV-1-induced latency reactivation, indicating the necessity of developing new products to protect animals from wt BoHV-1-induced latency.

Evaluation of Safety and Efficacy of an Inactivated Marker Vaccine against Bovine alphaherpesvirus 1 (BoHV-1) in Water Buffalo (Bubalus bubalis).

Recent studies have explored the seropositivity of Bovine alphaherpesvirus 1 (BoHV-1) in water buffaloes, suggesting the urgency for developing strategies to eradicate the virus involving both cattle and water buffaloes. However, in Europe, the glycoprotein E (gE) deleted marker vaccines against BoHV-1 are commercially available only for the cattle industry. This study, for the first time, evaluated the safety and efficacy of a commercial inactivated gE-deleted marker vaccine in water buffalo. Five animals devoid of BoHV-1-neutralizing antibodies were vaccinated via intramuscular route. Five additional animals served as an unvaccinated control group. Sixty days after the first immunization, all animals were experimentally infected with a virulent BoHV-1via intranasal route. A detectable BoHV-1-humoral immune response was observed in the vaccinated group on post-vaccination day 30, whereas the antibodies appeared on post-challenge day 10 in the control group. Moreover, the vaccinated animals neither show viral shedding nor clinical signs compared to the control upon challenge. However, post-challenge, the BoHV-1-specific humoral and cell-mediated immune responses were significantly more increased in vaccinated animals than the control animals. Overall, the present study provides evidence of both the safety and efficacy of an inactivated gE-deleted marker vaccine against BoHV-1 in water buffaloes.

Assessment of Multicolor Flow Cytometry Panels to Study Leukocyte Subset Alterations in Water Buffalo (Bubalus bubalis) During BVDV Acute Infection.

The identification of cross-reactive monoclonal antibodies (mAbs) that recognize orthologous leukocyte differentiation molecules (LDM) in buffaloes has overcome a major impediment limiting research on the immune response to pathogens and development of vaccines. As reported, two pilot trials were conducted to accomplish two objectives: (1) demonstrate that multiparameter flow cytometry can be conducted equally well in buffalo with mAbs directly and indirectly labeled with fluorochromes in research and (2) flow cytometry can be used to compare and extend studies on diseases of economic importance to buffalo using bovine viral diarrhea virus (BVDV) as a model pathogen. Pregnant buffalo cows were infected with BVDV-1 at 81 (trial 1) and 203 (trial 2) days post artificial insemination and flow cytometric evaluations were performed at 0, 3, 4, and 14 days after infection (dpi). Fluorochrome conjugated mAbs were used in trial 1, and fluorochrome conjugated goat isotype specific anti-mouse antibodies were used to label mAbs in trial 2. Flow cytometric analysis revealed a transient lymphopenia occurs during the 1st days following infection similar to lymphopenia reported in cattle. In particular, significant differences were observed between pre- and post-infection absolute values of T lymphocytes (-56%, P < 0.01). CD21+ B lymphocytes (-65%, P = 0.04), and Natural Killer cells (-72%, P < 0.001). No significant differences were observed in monocytes and neutrophil absolute values, or the CD4:CD8 ratio. Animal health status was followed until 15 days after calving. No clinical signs of infection were observed during the evaluation period, however, animals in trial 1 developed complications later the infection. One cow aborted at 57 days post-infection, the second cow developed a prolapse a day after calving and died. These two animals also showed a more pronounced lymphopenia in comparison with animals infected at 203 days of pregnancy (e.g., -77 vs. -22% T lymphocytes at 3 dpi, respectively). The pilot studies have demonstrated that it is possible to use multicolour multiparameter flow cytometry to study the immune response to pathogens affecting the health of buffalo.

Short communication: Milk microbiota profiling on water buffalo with full-length 16S rRNA using nanopore sequencing.

The identification of milk microbial communities in ruminants is relevant for understanding the association between milk microbiota and health status. The most common approach for studying the microbiota is amplifying and sequencing specific hypervariable regions of the 16S rRNA gene using massive sequencing techniques. However, the taxonomic resolution is limited to family and, in some cases, genus level. We aimed to improve taxonomic classification of the water buffalo milk microbiota by amplifying and sequencing the full-length 16S rRNA gene (1,500 bp) using Nanopore sequencing (single-molecule sequencing). When comparing with short-read results, we improved the taxonomic classification, reaching species level. We identified the main microbial agents of subclinical mastitis at the species level that were in accordance with the microbiological culture results. These results confirm the potential of single-molecule sequencing for in-depth analysis of microbial populations in dairy animals.

Office spirometry can improve the diagnosis of obstructive airway disease in primary care setting.

INTRODUCTION: Spirometry may reveal pre-clinical abnormal airway function in asymptomatic subjects and allow a better definition of severity in clinically diagnosed asthma and COPD. The hypothesis of this study was that telespirometry might increase the diagnostic accuracy of asthma and COPD. METHODS: In the Italian "Alliance" study, 638 general practitioners (GPs) were trained to perform telespirometry and were asked to enroll the following categories of subjects: (a) current or ex-smokers without respiratory symptoms; (b) subjects with respiratory symptoms but without a pre-existing diagnosis of asthma or COPD; (c) subjects with a pre-existing clinical diagnosis of asthma; and (d) subjects with a pre-existing clinical diagnosis of COPD. Subjects completed a case report form (CRF) and performed telespirometry in the GP's office. Traces were sent by telephone to a Telespirometry Central Office, where they were interpreted by a pulmonary specialist, according to appropriately defined criteria. The results were returned in real time to the GP. RESULTS: Overall, 9312 subjects were recruited and 7262 (78%) performed an acceptable telespirometric examination and the CRF. In the asymptomatic group, 340/1437 (24%) of the telespirometries were abnormal (147 with moderate-to-severe airway obstruction, i.e. FEV(1) <80% of predicted). Among symptomatic subjects, 1433/3725 (38%) had abnormal telespirometries (682 with moderate-to-severe obstruction). Of the asthmatic subjects, 336/1285 (26%) had moderate-to-severe airway obstruction, while telespirometry was normal in 184/815 (23%) of the COPD group. CONCLUSION: Telespirometry, performed in a GP's office, can aid the diagnosis of obstructive airway diseases and could help GPs to better manage airway obstruction.

Split-screen synchronized display. A useful video-EEG technique for studying paroxysmal phenomena.

Video-EEG is an indispensable and widely used technique for studying paroxysmal phenomena. We present a method of processing video recordings to compare the sequence of ictal manifestations of different episodes. Five presentations of two seizures recorded in each of five patients are shown, in which apparently different ictal features are strongly superimposed and stereotyped. Split-screen synchronized display is a simple and valid technique for studying and presenting particular semeiological aspects of epileptic seizures. (Published with videosequences).

Randomized, double-blind study of prulifloxacin versus ciprofloxacin in patients with acute exacerbations of chronic bronchitis.

BACKGROUND: Recently the role of bacteria in acute exacerbations of chronic bronchitis (AECB) as well as antibiotic treatment with selected drugs, especially fluoroquinolones, have been better defined. OBJECTIVE: To assess the efficacy and safety in patients with AECB of prulifloxacin in comparison with ciprofloxacin. METHODS: AECB was defined according to the guidelines for the evaluation of new anti-infective drugs for the treatment of respiratory tract infections (1992). 235 patients took part in the trial; 117 (88 males and 29 females, mean age 64.8 years) received 600 mg prulifloxacin once daily and 118 (91 males and 27 females, mean age 64.5 years) 500 mg ciprofloxacin twice a day, for a duration of 10 days. The study design was randomized, multicenter, double-blind, double-dummy. Efficacy evaluations were performed by comparing pretreatment and posttreatment assessments. The clinical response was determined by 4-point rating scores on cough, dyspnea, and expectoration (volume and appearance). The microbiological response was assessed on sputum specimen. RESULTS: Clinical success was observed in 84.7 and 85% of patients in the prulifloxacin and ciprofloxacin groups, respectively. The 95% confidence interval proved the equivalence of treatments. Both drugs successfully eradicated the most commonly isolated strains, including Haemophilus influenzae, Streptococcus pneumoniae, Klebsiella pneumoniae and Pseudomonas aeruginosa. Both treatments were well tolerated. Adverse drug reactions were always of mild or moderate intensity. CONCLUSION: The study showed that a 10-day course of prulifloxacin is as effective and safe as ciprofloxacin in the treatment of patients with AECB.