Canaries' Microbiota: The Gut Bacterial Communities along One Female Reproductive Cycle.

Investigations of bacterial communities are on the rise both in human and veterinary medicine. Their role in health maintenance and pathogenic mechanisms is in the limelight of infectious, metabolic, and cancer research. Among the most considered, gut bacterial communities take the cake. Their part in animals was assessed mainly to improve animal production, public health, and pet management. In this regard, canaries deserve attention, being a popular pet and source of economic income for bird-keepers, for whom breeding represents a pivotal point. Thus, the present work aimed to follow gut bacterial communities' evolution along on whole reproductive cycle of 12 healthy female canaries. Feces were collected during parental care, molting, and resting phase, and submitted for 16S rRNA sequencing. Data were analyzed and a substantial presence of Lactobacillus aviarius along all the phases, and a relevant shift of microbiota during molting and rest due to an abrupt decrease of the Vermiphilaceae family were detected. Although the meaning of such change is not clear, future research may highlight unforeseen scenarios. Moreover, Lactobacillus aviarius may be deemed for normal bacteria flora restoration in debilitated birds, perhaps improving their health and productivity.

Effect of Vaccination against Glässer's Disease in a Farm Suffering from Polyserositis in Weaned Pigs.

Polyserositis mostly affects 4−8 weeks old piglets and is usually caused by Glaesserella parasuis, and/or Streptococcus suis, and/or Mycoplasma hyorhinis. The present study aimed to investigate the prevalence and etiology of polyserositis in a tricky pig herd. The concurrent effect of vaccination for Glässer's disease was also assessed. A total of 46 sows and 387 piglets were herein investigated, subdivided into three groups based on their immune status (i.e., vaccination of sows and piglets). All the piglets found spontaneously dead between the 2nd and 16th week of age were recorded and necropsied. Whenever polyserositis was diagnosed, biomolecular investigations were carried out to detect the above-mentioned pathogens. Mycoplasma hyorhinis was detected most frequently (n = 23), often as the only causative agent (n = 15), whereas S. suis was observed in 8 cases (6 as the only pathogen). Moreover, Glaesserella parasuis was demonstrated in 6 piglets, always in combination with Mycoplasma hyorhinis and/or Streptococcus suis. Vaccination did not significantly affect mortality rates. Overall, our data indicate that polyserositis is likely caused by an intricate puzzle of pathogens, even when dealing with a small herd and during a short time span. That makes it challenging to achieve the correct diagnosis and to properly manage this health issue.

Inflammatory, Mechanical and Infectious Complications Associated with Peripheral Intravenous Catheters in Dogs and Cats: A Risk Factor Analysis.

The placement of peripheral intravenous catheters (PIVC) is potentially associated with complications that negatively impact healthcare. Our study investigated factors associated with the occurrence of PIVC-related complications in dogs and cats at a Veterinary Teaching Hospital. The second aim was to determine the prevalence of PIVC bacterial colonization. A total of 76 dogs and 40 cats with PIVCs were evaluated for the occurrence of phlebitis and mechanical complications. The devices were removed when they ceased to be functional or when complications occurred, and the content was submitted for bacterial cultures and antimicrobial susceptibility tests. Both multivariable linear regression models and ROC analysis were employed. Complications were recorded in 46.6% of cases, and 20.7% of catheters yielded a positive culture. Among the isolates, 45% were classified as multi-resistant. In dogs, a ≥36-h indwelling time was associated with an increased risk of complications. Male cats seem more prone to developing complications, while the insertion of PIVCs under sedation may represent a protective factor in this species. In conclusion, PIVC-associated complications were frequently observed, and the high rate of positive culture for PIVCs, together with the presence of multi-resistant isolates, is a cause of concern in a hospital setting.

Insights into the Oral Bacterial Microbiota of Sows.

The investigation of bacterial microbiota represents a developing research field in veterinary medicine intended to look for correlations between animal health and the balance within bacterial populations. The aim of the present work was to define the bacterial microbiota of the oral cavity of healthy sows, which had not been thoroughly described so far. In total, 22 samples of oral fluid were collected and analyzed by 16S-rRNA gene sequencing. CLC Genomics Workbench 20.0 (QIAGEN Digital Insights, Aarhus, Denmark) was then used to examine the results. The predominant orders were Lactobacillales, Clostridiales, and Corynebacteriales. Lactobacillaceae, Corynebacteriaceae, Moraxellaceae, Aerococcaceae, and Staphylococcaceae were the most represented families. As regards the most abundant genera, Lactobacillus, Corynebacterium, Acinetobacter, Staphylococcus, Rothia, Aerococcus, and Clostridium can be pointed out as the bacterial core microbiota. Sows were also divided into "gestating" and "lactating" groups, and mild differences were found between pregnant and lactating sows. The data herein described represent an original contribution to the knowledge of the porcine bacterial microbiota. Moreover, the choice of sows as experimental animals was strategic for identifying the adult microbial community. These data provide a basis for further studies on the oral bacterial microbiota of pigs.

Congenital Oral Squamous Cell Carcinoma in a Suckling Piglet.

A 3-week-old suckling piglet spontaneously died after septicemic colibacillosis. At postmortem examination, bulging and ulcerated lesions were seen, affecting the oral mucosa on the inner surface of the lower lip. After histopathological investigation, the diagnosis of congenital oral squamous cell carcinoma was made. To the best of our knowledge, this is the first case of congenital oral squamous cell carcinoma ever described. A relationship has been shown or suggested between papillomavirus infection and oral squamous cell carcinoma in humans and animals. However, next-generation sequencing study did not demonstrate any papillomavirus sequences in the case reported herein.

Aspergillus Section Fumigati Pneumonia and Oxalate Nephrosis in a Foal.

Equine pulmonary aspergillosis is a rare deep mycosis often due to the hematogenous spread of hyphae after gastrointestinal tract disease. We describe herein the main clinic-pathological findings observed in a foal, which spontaneously died after showing diarrhea and respiratory distress. Necropsy and histopathological investigations allowed to diagnose pulmonary aspergillosis, which likely developed after necrotic typhlitis-colitis. Biomolecular studies identified Aspergillus section Fumigati strain as the causative agent. Notably, severe oxalate nephrosis was concurrently observed. Occasionally, oxalate nephropathy can be a sequela of pulmonary aspergillosis in humans. The present case report suggests that the renal precipitation of oxalates can occur also in horses affected by pulmonary aspergillosis and could likely contribute to the fatal outcome of the disease.

Occurrence, antimicrobial susceptibility, and pathogenic factors of Pseudomonas aeruginosa in canine clinical samples.

Background and Aim: Pseudomonas aeruginosa is a relevant opportunistic and difficult to treat pathogen due to its widespread environmental diffusion, intrinsic resistance to many classes of antimicrobials, high ability to acquire additional resistance mechanisms, and wide range of pathogenic factors. The present study aimed to investigate the prevalence of P. aeruginosa in canine clinical samples, the antimicrobial susceptibility against antipseudomonal antibiotics, and the presence of extracellular pathogenic factors of the isolates, as well as their ability to produce biofilm. Materials and Methods: Overall, 300 clinical specimens from dogs with pyoderma or abscesses (n=58), otitis (n=59), and suspected bladder infection (n=183) were analyzed by standard bacteriological methods. P. aeruginosa isolates were tested for their antimicrobial susceptibility by disk and gradient diffusion methods to determine the minimum inhibitory concentrations. The ability of the isolates to produce biofilm was investigated by a microtiter plate assay, while virulence genes coding for elastase (lasB), exotoxin A (toxA), alkaline protease (aprA), hemolytic phospholipase C (plcH), and exoenzyme S (ExoS) were detected by polymerase chain reaction method. Results: A total of 24 isolates of P. aeruginosa were found in clinical specimens (urine n=3, skin/soft tissue n=6, and ear canal n=15). No resistance was found to ceftazidime, gentamicin, aztreonam, and imipenem (IMI), while low levels of resistance were found to enrofloxacin (ENR) (4.2%) and piperacillin-tazobactam (8.3%). However, 41.7% and 29.2% of the isolates showed intermediate susceptibility to ENR and IMI, respectively. Disk and gradient diffusion methods showed high concordance. The majority of the isolates revealed a weak (33.3%) or intermediate (45.8%) ability to form biofilm, while the strong biofilm producers (20.8%) derived exclusively from the ear canal samples. All isolates (100%) were positive for lasB, aprA, and plcH genes, while exoS and toxA were amplified in 21 (87.5%) and 22 (91.7%) isolates, respectively. Conclusion: In the present study, P. aeruginosa isolates from canine clinical samples were characterized by low levels of antimicrobial resistance against antipseudomonal drugs. However, the high presence of isolates with intermediate susceptibility for some categories of antibiotics, including carbapenems which are not authorized for veterinary use, could represent an early warning signal. Moreover, the presence of isolates with strong ability to produce biofilm represents a challenge for the interpretation of the antimicrobial susceptibility profile. In addition, the high prevalence of the extracellular pathogenic factors was indicative of the potential virulence of the isolates.

The Endocannabinoid System in the Mediterranean Mussel Mytilus galloprovincialis: Possible Mediators of the Immune Activity?

Anandamide (AEA) is one of the best characterized members of the endocannabinoid family and its involvement in many pathophysiological processes has been well documented in vertebrates and invertebrates. Here, we report the biochemical and functional characterization of key elements of the endocannabinoid system in hemocytes isolated from the Mediterranean mussel Mytilus galloprovincialis. We also show the effects of exogenous AEA, as well as of capsaicin, on the cell ability to migrate and to activate the respiratory burst, upon in vitro stimulation of phagocytosis. Interestingly, our findings show that both AEA and capsaicin suppress the hemocyte response and that the use of selective antagonists of CB2 and TRPV1 receptors revert their inhibitory effects. Overall, present data support previous evidence on the presence of endocannabinoid signaling in mollusks and advance our knowledge about the evolutionary origins of this endogenous system and its role in the innate response of mollusks.

Exposure to Major Vector-Borne Diseases in Dogs Subjected to Different Preventative Regimens in Endemic Areas of Italy.

Vector-borne diseases (VBDs) are globally widespread arthropod-transmitted diseases with a significant impact on animal and human health. Many drivers have recently spurred the geographic spread of VBDs in dogs. This study has evaluated the exposure to most important VBDs in dogs under different preventative treatments in different regions of Italy, i.e., Veneto, Friuli Venezia-Giulia, Umbria, Giglio Island (Tuscany), Abruzzo and Latium. Serological analyses were performed to detect antibodies against Leishmania infantum, Babesia canis, Anaplasma phagocytophilum/Anaplasma platys, Ehrlichia canis/Ehrlichia ewingii, Borrelia burgdorferi, Rickettsia conorii and the circulating antigen of Dirofilaria immitis. Dogs were categorized according to the treatment schedule usually received, and the association between seropositivity and possible risk factors was statistically evaluated. Overall, 124/242 (51.2%) dogs tested positive for at least one pathogen, while 34 (14.0%) were exposed to two or more pathogens. The most detected seropositivity was against R. conorii, followed by Anaplasma spp., L. infantum, B. canis, and the other pathogens under study. Significant statistical associations were found according to geographical provenance, history of tick infestation, lifestyle and inadequate prophylactic treatments. Random/irregular treatments have been identified as a clear risk factor. These results show that adequate prophylactic treatment protocols are overlooked by dog owners, despite the availability of several effective products, with possible implications in veterinary medicine and on public health.

Vibrio parahaemolyticus control in mussels by a Halobacteriovorax isolated from the Adriatic sea, Italy.

This study evaluated the application of a Halobacteriovorax isolated from water of the Adriatic Sea (Italy) in controlling V. parahaemolyticus in mussels (Mytilus galloprovincialis). Two 72 h laboratory-scale V. parahaemolyticus decontamination experiments of mussels were performed. The test microcosm of experiment 1 was prepared using predator/prey free mussels experimentally contaminated with Halobacteriovorax/V. parahaemolyticus at a ratio of 103 PFU/105 CFU per ml, while that of experiment 2 using mussels naturally harbouring Halobacteriovorax that were experimentally contaminated with 105 CFU per ml of V. parahaemolyticus. For experiment 1, was also tested a control microcosm only contaminated with 105 CFU per ml of V. parahaemolyticus.. Double layer agar plating and pour plate techniques were used to enumerate Halobacteriovorax and V. parahaemolyticus, respectively. 16 S rRNA analysis was used to identify Halobacteriovorax. For both experiments in the test microcosm the concentration of prey remained at the same level as that experimentally added, i.e. 5 log for the entire analysis period. In experiment 1, V. parahaemolyticus counts in mussels were significantly lower in the test microcosm than the control with the maximum difference of 2.2 log at 24 h. Results demonstrate that Halobacteriovorax can modulate V. parahaemolyticus level in the mussels. The public impact of V. parahaemolyticus in bivalves is relevant and current decontamination processes are not always effective. Halobacteriovorax is a suitable candidate in the development of a biological approach to the purification of V. parahaemolyticus in mussels.

Vibrio parahaemolyticus-specific Halobacteriovorax From Seawater of a Mussel Harvesting Area in the Adriatic Sea: Abundance, Diversity, Efficiency and Relationship With the Prey Natural Level.

This research aimed to study the abundance and molecular diversity of Vibrio parahaemolyticus-specific Halobacteriovorax strains isolated from seawater of the Adriatic Sea and the relationship between predator and prey abundances. Moreover, predator efficiency of the Halobacteriovorax isolates toward V. parahaemolyticus and Vibrio cholerae non-O1/O139 strains was tested. V. parahaemolyticus NCTC 10885 was used as primary host for the isolation of Halobacteriovorax from seawater by the plaque assay. Molecular identification was performed by PCR detection of a fragment of the 16S rRNA gene of the Halobacteriovoraceae family members. Moreover, 700 bp PCR products were sequenced and compared between them and to clones described for other sampling sites. Vibrio counts were performed on TCBS agar from 100 ml of filtered water samples and presumptive colonies were confirmed by standard methods. Predatory efficiency of Halobacteriovorax isolates was tested by monitoring abilities of 3-day enrichments to form clear lytic halos on a lawn of Vibrio preys, by the plaque assay. Out of 12 seawater samples monthly collected from June 2017 to May 2018, 10 were positive for V. parahaemolyticus specific Halobacteriovorax with counts ranging from 4 to 1.4 × 103 PFU per 7.5 ml. No significant relationship was found between Halobacteriovorax and Vibrio abundances. The 16SrRNA sequences of our Halobacteriovorax strains, one for each positive sample, were divided into three lineages. Within the lineages, some sequences had 100% similarity. Sequence similarity between lineages was always <94.5% suggesting that they may therefore well belong to three different species. All Halobacteriovorax isolates had the ability to prey all tested Vibrio strains. Additional research is necessary to assess whether stable strains of Halobacteriovorax are present in the Adriatic Sea and to understand the mechanisms by which Halobacteriovorax may modulate the abundance of V. parahaemolyticus and other vibrios in a complex marine ecosystem.

Polymorphonuclear cells and reactive oxygen species in contagious bovine pleuropneumonia: New insight from in vitro investigations.

Reactive oxygen species (ROS) are suggested to play a role in the pathogenesis of contagious bovine pleuropneumonia, a severe respiratory disorder caused by Mycoplasma mycoides subsp. mycoides (Mmm). The present study investigated the generation of ROS by different strains of Mmm, as well as their effect on the oxidative response of bovine neutrophils. The production of ROS was indirectly measured using a luminol-based chemiluminescence assay. Our results confirm that Mmm can produce ROS via the metabolism of glycerol, significant differences existing between African and European strains. Mmm was capable of adhering to the external surface of neutrophils. Interestingly, Mmm enhanced the respiratory burst of bovine neutrophils. This activity was particularly pronounced with the African field strain and in presence of glycerol. Taken together, our data argue in favour of a major role for neutrophils as the main source of ROS in contagious bovine pleuropneumonia.

Microbial Diseases of Bivalve Mollusks: Infections, Immunology and Antimicrobial Defense.

A variety of bivalve mollusks (phylum Mollusca, class Bivalvia) constitute a prominent commodity in fisheries and aquacultures, but are also crucial in order to preserve our ecosystem's complexity and function. Bivalve mollusks, such as clams, mussels, oysters and scallops, are relevant bred species, and their global farming maintains a high incremental annual growth rate, representing a considerable proportion of the overall fishery activities. Bivalve mollusks are filter feeders; therefore by filtering a great quantity of water, they may bioaccumulate in their tissues a high number of microorganisms that can be considered infectious for humans and higher vertebrates. Moreover, since some pathogens are also able to infect bivalve mollusks, they are a threat for the entire mollusk farming industry. In consideration of the leading role in aquaculture and the growing financial importance of bivalve farming, much interest has been recently devoted to investigate the pathogenesis of infectious diseases of these mollusks in order to be prepared for public health emergencies and to avoid dreadful income losses. Several bacterial and viral pathogens will be described herein. Despite the minor complexity of the organization of the immune system of bivalves, compared to mammalian immune systems, a precise description of the different mechanisms that induce its activation and functioning is still missing. In the present review, a substantial consideration will be devoted in outlining the immune responses of bivalves and their repertoire of immune cells. Finally, we will focus on the description of antimicrobial peptides that have been identified and characterized in bivalve mollusks. Their structural and antimicrobial features are also of great interest for the biotechnology sector as antimicrobial templates to combat the increasing antibiotic-resistance of different pathogenic bacteria that plague the human population all over the world.

Computational modeling of immune system of the fish for a more effective vaccination in aquaculture.

MOTIVATION: A computational model equipped with the main immunological features of the sea bass (Dicentrarchus labrax L.) immune system was used to predict more effective vaccination in fish. The performance of the model was evaluated by using the results of two in vivo vaccinations trials against L. anguillarum and P. damselae. RESULTS: Tests were performed to select the appropriate doses of vaccine and infectious bacteria to set up the model. Simulation outputs were compared with the specific antibody production and the expression of BcR and TcR gene transcripts in spleen. The model has shown a good ability to be used in sea bass and could be implemented for different routes of vaccine administration even with more than two pathogens. The model confirms the suitability of in silico methods to optimize vaccine doses and the immune response to them. This model could be applied to other species to optimize the design of new vaccination treatments of fish in aquaculture. AVAILABILITY AND IMPLEMENTATION: The method is available at http://www.iac.cnr.it/∼filippo/c-immsim/. CONTACT: nromano@unitus.it. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genetic diversity of Arcobacter isolated from bivalves of Adriatic and their interactions with Mytilus galloprovincialis hemocytes.

The human food-borne pathogens Arcobacter butzleri and A. cryaerophilus have been frequently isolated from the intestinal tracts and fecal samples of different farm animals and, after excretion, these microorganisms can contaminate the environment, including the aquatic one. In this regard, A. butzleri and A. cryaerophilus have been detected in seawater and bivalves of coastal areas which are affected by fecal contamination. The capability of bivalve hemocytes to interact with bacteria has been proposed as the main factor inversely conditioning their persistence in the bivalve. In this study, 12 strains of Arcobacter spp. were isolated between January and May 2013 from bivalves of Central Adriatic Sea of Italy in order to examine their genetic diversity as well as in vitro interactions with bivalve components of the immune response, such as hemocytes. Of these, seven isolates were A. butzleri and five A. cryaerophilus, and were genetically different. All strains showed ability to induce spreading and respiratory burst of Mytilus galloprovincialis hemocytes. Overall, our data demonstrate the high genetic diversity of these microorganisms circulating in the marine study area. Moreover, the Arcobacter-bivalve interaction suggests that they do not have a potential to persist in the tissues of M. galloprovincialis.

Innovative vaccination protocol against vibriosis in Dicentrarchus labrax (L.) juveniles: improvement of immune parameters and protection to challenge.

The effect of vaccination on immune parameters of European sea bass, Dicentrarchus labrax, is not fully established, as well as surveyed throughout rearing till the commercial size. Furthermore, available information on the possible role of booster treatments is scarce. Sea bass juveniles were vaccinated against Listonella anguillarum using a commercial bivalent formulation administered by immersion (priming: 95 dph; booster: 165 dph) or by immersion (priming: 95 dph; booster: 165 dph) and subsequent i.p. injection (booster: 233 dph). Serum specific IgM and numbers of IgM(+) cells in head kidney and spleen evidenced B-cell responses mainly after the immersion booster, accompanied by increased TcR-ß transcripts and leucocyte respiratory burst. Immune enhancement was confirmed by the protection towards i.p. challenges with a virulent strain. RPS accounted for >70% in fish immersion-boosted and near 100% in fish further boosted i.p. Differently from usual farm practices, this innovative vaccination protocol proved to be highly effective. Booster treatments are therefore strongly recommended.

Electrochemical genosensors for the detection of Bonamia parasite. Selection of single strand-DNA (ssDNA) probes by simulation of the secondary structure folding.

A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, and bioinformatics data, derived from the simulation of the secondary structure folding and prediction of hybridisation reaction, was carried out in order to rationalize the selection of ssDNA probes for the detection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis. Six ssDNA probes (from 11 to 25 bases in length, 2 thiolated and 4 biotinylated) were selected within different regions of B. ostreae and B. exitiosa PCR amplicons (300 and 304 bases, respectively) with the aim to discriminate between these parasite species. ssDNA amplicons and probes were analyzed separately using the "Mfold Web Server" simulating the secondary structure folding behaviour. The hybridisation of amplicon-probe was predicted by means of "Dinamelt Web Server". The results were evaluated considering the number of hydrogen bonds broken and formed in the simulated folding and hybridisation process, variance in gaps for each sequence and number of available bases. In the experimental part, thermally denatured PCR products were captured at the sensor interface via sandwich hybridisation with surface-tethered probes (thiolated probes) and biotinylated signalling probes. A convergence between analytical signals and simulated results was observed, indicating the possibility to use bioinformatic data for ssDNA probes selection to be incorporated in genosensors.