Feasibility of 16S rRNA sequencing for cerebrospinal fluid microbiome analysis in cattle with neurological disorders: a pilot study.

Bacterial infection of the central nervous system (CNS) in cattle requires prompt and adequate antimicrobial treatment. The current gold standard for antemortem etiological diagnosis is cerebrospinal fluid (CSF) culture, which often yields false negative results. CSF has long been considered a sterile district in healthy patients, but this notion has been recently challenged. For this pilot study, we used 16S rRNA gene sequencing to investigate the microbial composition of CSF of cattle presenting with CNS disorders and to compare it between subjects with CNS infections and with CNS disorders of other nature. The study sample was 10 animals: 4 presenting with CNS infectious-inflammatory diseases and 6 with other CNS disorders, based on definitive diagnosis. Since the initial round of a standard 16S rRNA PCR did not yield sufficient genetic material for sequencing in any of the samples, the protocol was modified to increase its sensitivity. Bacterial genetic material was identified in 6 animals and 2 groups were formed: an infectious inflammatory (n = 3) and a noninfectious inflammatory group (n = 3). The most frequently expressed bacterial families were Pseudomonadaceae (44.61%), Moraxellaceae (19.54%), Mycobacteriaceae (11.80%); the genera were Pseudomonas (45.42%), Acinetobacter (19.91%), Mycobacterium (12.01%). There were no detectable differences in the CSF microbial composition of the samples from the two groups. Sequencing of bacterial DNA present in the CSF was possible only after increasing PCR sensitivity. The results of 16S rRNA sequencing showed the presence of a microbial community in the CSF in cattle with neurological disorders. Further studies, in which CSF samples from healthy animals and samples from the environment are included as controls, are needed.

Occurrence of Methicillin-Resistant Coagulase-Negative Staphylococci (MRCoNS) and Methicillin-Resistant Staphylococcus aureus (MRSA) from Pigs and Farm Environment in Northwestern Italy.

Swine farming as a source of methicillin-resistant Staphylococcus aureus (MRSA) has been well documented. Methicillin-resistant coagulase-negative staphylococci (MRCoNS) have been less studied, but their importance as pathogens is increasing. MRCoNS are indeed considered relevant nosocomial pathogens; identifying putative sources of MRCoNS is thus gaining importance to prevent human health hazards. In the present study, we investigated MRSA and MRCoNS in animals and environment in five pigsties in a high farm-density area of northwestern Italy. Farms were three intensive, one intensive with antibiotic-free finishing, and one organic. We tested nasal swabs from 195 animals and 26 environmental samples from three production phases: post-weaning, finishing and female breeders. Phenotypic tests, including MALDI-TOF MS, were used for the identification of Staphylococcus species; PCR and nucleotide sequencing confirmed resistance and bacterial species. MRCoNS were recovered in 64.5% of nasal swabs, in all farms and animal categories, while MRSA was detected only in one post-weaning sample in one farm. The lowest prevalence of MRCoNS was detected in pigs from the organic farm and in the finishing of the antibiotic-free farm. MRCoNS were mainly Staphylococcus sciuri, but we also recovered S. pasteuri, S. haemolyticus, S. cohnii, S. equorum and S. xylosus. Fifteen environmental samples were positive for MRCoNS, which were mainly S. sciuri; no MRSA was found in the farms' environment. The analyses of the mecA gene and the PBP2-a protein highlighted the same mecA fragment in strains of S. aureus, S. sciuri and S. haemolyticus. Our results show the emergence of MRCoNS carrying the mecA gene in swine farms. Moreover, they suggest that this gene might be horizontally transferred from MRCoNS to bacterial species more relevant for human health, such as S. aureus.

16S rRNA Sequencing Analysis of the Gut Microbiota in Broiler Chickens Prophylactically Administered with Antimicrobial Agents.

In poultry production, gut microbiota (GM) plays a pivotal role and influences different host functions related to the efficiency of production performances. Antimicrobial (AM) use is one of the main factors affecting GM composition and functions. Although several studies have focused their attention on the role of AMs as growth promoters in the modulation of GM in broilers, the consequences of higher AM concentrations administered during prophylactic treatments need to be better elucidated. For this purpose, 16S rRNA gene sequencing was performed to evaluate the impact of different prophylactic AM protocols on the composition and diversity of the broiler GM. Diversity analysis has shown that AM treatment significantly affects alpha diversity in ileum and beta diversity in both ileum and caecum. In ileal samples, the Enterobacteriaceae family has been shown to be particularly affected by AM treatments. AMs have been demonstrated to affect GM composition in broiler. These findings indicate that withdrawal periods were not enough for the restoral of the original GM. Further studies are needed for a better elucidation of the negative effects caused by an altered GM in broilers.

Yellow Mealworm Inclusion in Diets for Heavy-Size Broiler Chickens: Implications for Intestinal Microbiota and Mucin Dynamics.

In the present trial, 160 heavy-size male broiler chickens were allocated to 4 dietary treatments (control feed [C] and 5, 10 and 15% TM meal inclusion, respectively, with 5 replicate pens/treatment and 8 birds/pen) to evaluate the influence of TM meal on intestinal microbiota and mucin composition. The broiler chickens fed TM-based diets showed higher beta diversity of their cecal microbiota in comparison with the C birds (p < 0.001). A significant decrease of the relative abundance of Firmicutes phylum and lower Firmicutes:Bacteroidetes ratios (False Discovery Rate [FDR] < 0.05) were also identified in TM15 broiler chickens when compared to the C group. Furthermore, the TM birds showed decreased relative abundance of Clostridium, Coprococcus, L-Ruminococcus and Ruminococcus genera (FDR < 0.05). In relation to the gut mucin composition, higher mucin staining intensity was detected in the intestinal crypts of TM5 birds in comparison with the other TM groups (p < 0.05). In conclusion, dietary TM meal inclusion negatively influenced the cecal microbiota of heavy-size broiler chickens in terms of partial alteration of the physiological microbial population and reduction of the potential beneficial bacteria (with slightly more pronounced effects when testing the 10-15% inclusion levels).

Prevalence of selected cardiotropic pathogens in the myocardium of adult dogs with unexplained myocardial and rhythm disorders or with congenital heart disease.

OBJECTIVE: To determine the prevalence of nucleic acid from selected cardiotropic pathogens in endomyocardial biopsy samples from dogs with unexplained myocardial and rhythm disorders (UMRD) and compare prevalence with that for a group of control dogs with congenital heart disease (CHD). ANIMALS: 47 client-owned dogs. PROCEDURES: Right ventricular endomyocardial biopsy was performed in dogs with UMRD (dilated cardiomyopathy [n = 25], atrioventricular block [6], and nonfamilial ventricular [4] and supraventricular arrhythmias [2]) or CHD (10) that required right ventricular catheterization. Biopsy samples were evaluated histologically, and PCR assays were used for detection of nucleic acid from 12 pathogens. RESULTS: 197 biopsy samples were collected from dogs with UMRD (n = 172) or CHD (25). At least 1 pathogen was detected in 21 of 37 (57%; 95% confidence interval [CI], 41% to 71%) dogs with UMRD, and canine coronavirus was detected in 1 of 10 (10%; 95% CI, 2% to 40%) dogs with CHD. Dogs with UMRD were significantly more likely than dogs with CHD to have pathogens detected in biopsy samples (OR, 11.8; 95% CI, 1.3 to 103.0). The most common pathogens in dogs with UMRD were canine distemper virus, canine coronavirus, canine parvovirus 2, and Bartonella spp. No pathogens were detected in available blood samples from dogs with pathogens detected in biopsy samples. CONCLUSIONS AND CLINICAL RELEVANCE: Detection of nucleic acids from selected cardiotropic pathogens in myocardial tissue from dogs with UMRD suggested a possible association between the 2. Further studies are needed to explore whether this association is causative or clinically important. (J Am Vet Med Assoc 2019;255:1150-1160).

Gut Microbiota and Mucin Composition in Female Broiler Chickens Fed Diets including Yellow Mealworm (Tenebrio molitor, L.).

A total of 160 female broiler chickens were divided into four dietary treatments (control feed [C] and 5, 10 and 15% TM meal inclusion, respectively, with five replicate pens/treatment and eight birds/pen) to investigate the effects of Tenebrio molitor (TM) meal utilization on poultry gut microbiota and mucin composition. The cecal microbiota assessment displayed a shift in the beta diversity in chickens fed TM-based diets. The TM10 and TM15 birds showed a significant decrease in the relative abundance of Firmicutes phylum and lower Firmicutes:Bacteroidetes ratios (False Discovery Rate [FDR] < 0.05), respectively, than the TM5 group. The relative abundance of Clostridium, Alistipes and Sutterella genera significantly increased in TM chickens (FDR < 0.05), while birds fed TM-based diets displayed a significant decrease in the relative abundance of Ruminococcus genus in comparison with the C group (FDR < 0.05). Gut mucin composition evaluation revealed higher mucin staining intensity in the intestinal villi of TM5 birds than the other TM groups, as well as mucin reduction in the intestinal villi of TM10 birds when compared to the C group (p < 0.05). In conclusion, dietary TM meal utilization (especially the 10-15% inclusion levels) may negatively influence either the cecal microbiota or the intestinal mucin dynamics of broiler chickens.

Borrelia burgdorferi sensu lato and spotted fever group rickettsiae in small rodents and attached ticks in the Northern Apennines, Italy.

Ticks and ear biopsies were collected from wild small rodents in 2011 and 2012 in the northern Apennines (Italy), up to 1650 m above sea level. Apodemus spp. (n = 83) and Myodes glareolus (n = 22) were infested by Ixodes ricinus (192 larvae and two nymphs), Dermacentor marginatus (179 larvae and 29 nymphs), and Ixodes trianguliceps (three larvae and two nymphs). We detected several Borrelia burgdorferi sensu lato (s.l.) genospecies (B. afzelii, B. burgdorferi sensu stricto, B. garinii, B. lusitaniae, B. valaisiana) in I. ricinus and skin biopsies. The most common genospecies found in I. ricinus was B. valaisiana, while it was B. lusitaniae in tissues. Spotted fever group (SFG) rickettsiae (Rickettsia monacensis, R. slovaca and R. raoultii) infected I. ricinus, D. marginatus and rodent tissues. Rickettsia slovaca was the Rickettsia species most frequently found in our samples. Coinfections by B. burgdorferi s.l. and SFG rickettsiae indicate an overlap of transmission cycles and potential risk for humans to be infected by multiple pathogens, resulting in more severe symptoms. The findings of B. lusitaniae and R. slovaca in bank voles, and of B. valaisiana in small rodents, open new questions about host-pathogen interactions. In addition, our results highlight the importance of small rodents as data sources for studying tick-borne pathogens.

Modulation of intestinal microbiota, morphology and mucin composition by dietary insect meal inclusion in free-range chickens.

BACKGROUND: Gut health in poultry depends on the balance between the host, intestinal microbiota, intestinal microscopic features and diet. The effects of insect meal (a promising alternative protein source for poultry feed) on chicken gut morphology have recently been reported, but no data about intestinal microbiota and mucin composition modulation are available. The present study evaluated the effects of dietary Tenebrio molitor (TM) meal inclusion on gut health of free-range chickens by intestinal microbiota, morphology and mucin composition characterization. RESULTS: One hundred forty female medium-growing hybrids were divided into 2 dietary treatments (control feed [C] and 7.5% TM inclusion, with 5 replicate pens/treatment and 14 birds/pen) and slaughtered at 97 days of age (2 birds/pen for a total of 10 chickens/diet). The gut microbiota assessment on cecal content samples by 16S rRNA amplicon based sequencing showed higher alpha (Shannon, P < 0.05) and beta (Adonis and ANOSIM, P < 0.001) diversity in birds fed TM diet than C. In comparison with C group, TM birds displayed significant increase and decrease, respectively, of the relative abundances of Firmicutes and Bacteroidetes phyla, with higher Firmicutes:Bacteroidetes ratios (False Discovery Rate [FDR] < 0.05). The relative abundance of Clostridium, Oscillospira, Ruminococcus, Coprococcus and Sutterella genera was higher in TM chickens than C (FDR < 0.05). On the contrary, TM birds displayed significant decrease of the relative abundance of Bacteroides genus compared to the C group (FDR < 0.05). Gut morphology evaluation by morphometric analysis on small intestine revealed similar villus height, crypt depth and villus height to crypt depth ratio between C and TM birds. Characterization of gut mucin composition by periodic-acid Schiff, Alcian Blue pH 2.5 and high iron diamine staining on small and large intestine showed unaffected mucin staining intensity in TM chickens when compared to C group. CONCLUSIONS: Dietary TM meal inclusion may positively modulate the gut microbiota of the free-range chickens without influencing the intestinal morphology and mucin composition. Since the rapid growth of chickens directly depends on morphological and functional integrity of the digestive tract, the gut health assessment by a post mortem multidisciplinary approach appears to be fundamental.

Pectin-honey hydrogel: Characterization, antimicrobial activity and biocompatibility.

BACKGROUND: Novel pectin-honey hydrogels have been developed and characterized as medical device. Ideally, a wound dressing should maintain optimal fluid affinity, permit moisture evaporation, protect the wound from microbes, and have shape-conformability, biocompatibility, and antibacterial activity. OBJECTIVE: A novel, simple and fast method to produce pectin-honey wound dressings is described. METHODS: The properties of these pectin-honey hydrogels were investigated, including swelling ability, water vapour transmission rate, hydrogen peroxide production, methylglyoxal content and antibacterial activity. Biocompatibility was assessed by proliferation assays using cultured fibroblast cells and by in vivo study with subcutaneous and intraperitoneal implantation in rats. RESULTS: Hydrogel showed a good water vapour transmission rate, fluid uptake and were not cytotoxic for fibroblasts. The hydrogel demonstrated good antibacterial activity toward clinically relevant pathogens, including S. aureus and E. coli. Biocompatibility was confirmed by the measurement of plasma levels of interleukin (IL)1 beta, IL-6, tumour necrosis factor (TNF) alpha, and prostaglandin (PG)E2. No histological changes were observed. CONCLUSIONS: The presence of a natural active component, conformability, and complete resorbability are the main characteristics of this new biocompatible biomaterial that is well tolerated by the body, possibly improves healing, may be used for surgical complications prevention, with a simple and inexpensive production process.

Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.

BACKGROUND: The microbiota of the bovine upper respiratory tract has been recently characterized, but no data for the lower respiratory tract are available. A major health problem in bovine medicine is infectious bronchopneumonia, the most common respiratory syndrome affecting cattle. With this study, we used 16S rRNA gene sequencing to characterize and compare the microbial community composition of the upper and lower respiratory tracts in calves. RESULTS: The microbiota of the upper (nasal swab [NS]) and the lower (trans-tracheal aspiration [TTA]) respiratory tracts of 19 post-weaned Piedmontese calves with (8/19) and without (11/19) clinical signs of respiratory disease, coming from six different farms, was characterized by 16S rRNA gene metabarcoding. A total of 29 phyla (29 in NS, 21 in TTA) and 305 genera (289 in NS, 182 in TTA) were identified. Mycoplasma (60.8%) was the most abundant genus identified in both the NS (27.3%) and TTA (76.7%) samples, followed by Moraxella (16.6%) in the NS and Pasteurella (7.3%) in the TTA samples. Pasteurella multocida (7.3% of total operational taxonomic units [OTUs]) was the most abundant species in the TTA and Psychrobacter sanguinis (1.1% of total OTUs) in the NS samples. Statistically significant differences between the NS and the TTA samples were found for both alpha (Shannon index, observed species, Chao1 index, and Simpson index; P = 0.001) and beta (Adonis; P = 0.001) diversity. Comparison of the NS and TTA samples by farm origin and clinical signs revealed no statistical difference (P > 0.05), except for farm origin for the NS samples when compared by the unweighted UniFrac metric (P = 0.05). CONCLUSIONS: Using 16S rRNA gene sequencing, we characterized the microbiota of the upper and lower respiratory tracts of calves, both healthy individuals and those with clinical signs of respiratory disease. Our results suggest that environmental factors may influence the composition of the upper airway microbiota in cattle. While the two microbial communities (upper and lower airways) differed in microbial composition, they shared several OTUs, suggesting that the lung microbiota may be a self-sustaining, more homogeneous ecosystem, influenced by the upper respiratory tract microbiota.

How Behavioral Changes Can Indicate Serious Cerebral Pathology: A Case Report of Concomitant Olfactory Neuroblastoma and Distemper Virus Encephalitis in a Swiss Shepherd Dog.

Behavioral alterations in dogs are not easy to understand and cure. The situation is more difficult when an encephalitis due to Canine Distemper Virus (CDV) and a concomitant olfactory neuroblastoma are present. This case report deals with the story of a 5-year-old Swiss shepherd dog with behavioral changes, seizures, epistaxis and ataxia. Following clinical and laboratory exams, a suspected diagnosis of CDV infection was hypothesized, and a therapy based on Ω-interferon was administered. Every supporting therapy failed and the worsening of the clinical conditions led to the euthanasia of the patient. A neoformation in the right frontal lobe was found post mortem. Histopathology and immunohistochemistry investigation showed a non-suppurative demyelinating encephalitis, suggestive of CDV infection, and a desmoplastic epithelioid olfactory neuroblastoma. To the best of authors' knowledge, this is the first clinical pathological report of a non-suppurative encephalitis due to CDV infection and olfactory neuroblastoma in a dog.

Cleaning with a wet sterile gauze significantly reduces contamination of sutures, instruments, and surgical gloves in an ex-vivo pelvic flexure enterotomy model in horses.

The objective of this study was to investigate whether cleaning surgical materials used to close pelvic flexure enterotomies with a wet sterile gauze will reduce contamination and whether the use of a full thickness appositional suture pattern (F) or a partial thickness inverting (or Cushing) suture pattern (C) would make a difference in the level of contamination. Large colon specimens were assigned to group F or C and divided into subgroups N and G. In group G, a wet sterile gauze was passed over the suture material, another over the instruments, and another over the gloves. In group N, no treatment was applied. The bacterial concentration was measured by optical density (OD) at 24 h. The OD of subgroup CG was lower than that of subgroup CN (P = 0.019). The OD of subgroup FG was lower than that of subgroup FN (P = 0.02). The OD of subgroups CG, CN, FG, and FN was lower than that of the negative control (P < 0.003, P < 0.001, P < 0.001, and P < 0.00). The use of a sterile wet gauze significantly reduced contamination of suture materials. A partial thickness inverting suture pattern did not produce less contamination than a full thickness appositional suture pattern.

L'objectif de la présente étude était d'examiner si le nettoyage du matériel chirurgical utilisé pour fermer les entérotomies de la courbure pelvienne avec une gaze stérile mouillée réduisait la contamination et si l'utilisation d'un patron de suture d'apposition de la pleine épaisseur (F) ou d'un patron de suture inversé d'une épaisseur partielle (ou Cushing) (C) faisait une différence dans le degré de contamination. Des spécimens du gros côlon ont été assignés au groupe F ou C dans les sous-groupes N et G. Dans le groupe G, une gaze stérile mouillée a été passée par-dessus le matériel de suture, une autre par-dessus les instruments, et une autre par-dessus les gants. Dans le groupe N, aucun traitement ne fut effectué. Les concentrations bactériennes ont été mesurées par densité optique (DO) à 24 h. La DO du sous-groupe CG était inférieure à celle du sous-groupe CN (P = 0,019). La DO du sous-groupe FG était inférieure à celle du sous-groupe FN (P = 0,02). Les DO des sous-groupes CG, CN, FG, et FN étaient inférieures à celles des témoins négatifs (P < 0,003, P < 0,001, P < 0,001, et P < 0,00). L'utilisation d'une gaze stérile mouillée a réduit de manière significative la contamination de matériel de suture. Un patron de suture inversé avec épaisseur partielle n'a pas entrainé moins de contamination qu'un patron de suture par apposition avec pleine épaisseur.(Traduit par Docteur Serge Messier).

Investigation into Usutu and West Nile viruses in ticks from wild birds in Northwestern Italy, 2012-2014.

To assess the potential role of ticks as carriers of West Nile virus (WNV) and Usutu virus (USUV), we tested 1721 ticks from 379 wild birds in Northwestern Italy between 2012 and 2014. Ticks were analyzed in pools using a pan-flavivirus real-time RT-PCR and positive pools were subjected to RT-PCR for USUV and WNV genome detection. All the tested samples resulted negative, suggesting that Ixodes spp. ticks, at least in our study area, are not competent vectors and not even exploitable sentinels for USUV and WNV.

Pro-inflammatory cytokines and structural biomarkers are effective to categorize osteoarthritis phenotype and progression in Standardbred racehorses over five years of racing career.

BACKGROUND: Joint impact injuries initiate a progressive articular damage finally leading to post-traumatic osteoarthritis (PTOA). Racehorses represent an ideal, naturally available, animal model of the disease. Standardbred racehorses developing traumatic osteoarthritis of the fetlock joint during the first year of their career were enrolled in our study. Age-matched controls were contemporarily included. Biomarker levels of equine osteoarthritis were measured in serum and synovial fluid (SF) at baseline, and repeated yearly over the next 4 years of training (from T1 to T4). The effect of time and disease on the biomarker concentrations were analysed, and their relationship with clinical and radiographic parameters were assessed. We hypothesized that the kinetics of pro-inflammatory cytokines and structural biomarkers of joint disease would demonstrate progression of degenerative joint status during post-traumatic osteoarthritis and clarify the effect of early joint trauma. RESULTS: The concentrations of IL1-ß, IL-6, TNF-α in the SF of PTOA group peaked at T0, decreased at T1, and then progressively increased with time, reaching levels higher than those observed at baseline starting from T3. CTXII and COMP levels were similar in PTOA and control horses at baseline, and increased in serum and synovial fluid of PTOA horses starting from T2 (serum and synovial CTXII, and serum COMP) or T3 (synovial COMP). The percentual change of TNF-α in the SF of the affected joints independently contributed to explaining the radiological changes at T3 vs T2 and T4 vs T3. CONCLUSIONS: Temporal changes of selected biomarkers in STBRs with an acute episode of traumatic fetlock OA demonstrated that long-term increased concentrations of inflammatory cytokines, type II collagen fragments and COMP, in the SF and serum, are related to PTOA. Based on the observed decrease in inflammatory merkers at T1, we hypothesize that the progression of PTOA could be effectively modulated by proper treatment strategies. Annual variations of synovial concentration of TNF-α can reliably predict radiographic progression of PTOA.

Evaluating the feeding preferences of West Nile virus mosquito vectors using bird-baited traps.

BACKGROUND: The total contact rates (TCRs) between mosquito vectors and their potential hosts have a serious impact on disease transmission dynamics. Culex pipiens (sensu stricto) (s.s.) is considered the main vector of the West Nile Virus (WNV) in Europe and birds are the reservoir hosts. The results of our previous study showed that WNV seroreactors are significantly more prevalent among raptors compared to a range of other wild avian groups. The current study aims to assess the role of bird type (raptor vs others) and bird size on mosquito feeding preferences in a free-choice experiment using bird-baited traps. METHODS: From July to September 2014, a battery of six bird-baited traps was operated in twelve mosquito capture sessions. Eight bird species, belonging to five different orders, including raptors, were used. After each session, the trapped mosquitoes were collected and identified using standard keys. Two sets of independent generalized linear mixed models (GLMM) were used to assess mosquito vector feeding preferences (MFp) among different bird species and types. RESULTS: A total of 304 mosquitoes belonging to seven taxa were collected, C. pipiens being by far the most abundant (84.2 % of the total mosquito catch). Most C. pipiens were engorged (83.59 %). The selected model showed that 25.6 % of the observed variability of MFp is explained by the interaction between bird size and bird type, with C. pipiens preferring to feed on large birds, especially raptors. The proportion of engorged mosquitoes was 1.9-fold higher in large (22.88 %; range 0-42 %) than in medium-sized raptors (11.71 %; range 0-33 %), and was nearly the same in medium-sized (9.08 %; range 0-26 %) and large (8.5 %; 6-24 %) non-raptor species. CONCLUSION: Culex pipiens showed an obvious preference for large raptors, which concurs with the higher seroprevalence to WNV in our previous study. The appreciable feeding by C. pipiens on large raptors makes them useful alternative sentinels to poultry for WNV surveillance. Thus, wildlife parks and rehabilitation centers can contribute to surveillance efforts to a greater extent.

Presence of host-seeking Ixodes ricinus and their infection with Borrelia burgdorferi sensu lato in the Northern Apennines, Italy.

Host-seeking ticks were collected in the Northern Apennines, Italy, by dragging at 35 sites, at altitudes ranging from 680 and 1670 m above sea level (asl), from April to November, in 2010 and 2011. Ixodes ricinus (4431 larvae, 597 nymphs and 12 adults) and Haemaphysalis punctata (11,209 larvae, 313 nymphs, and 25 adults) were the most abundant species, followed by Haemaphysalis sulcata (20 larvae, five nymphs, and 13 adults), Dermacentor marginatus (42 larvae and two adults) and Ixodes hexagonus (one nymph). Greatest numbers of ticks were collected at locations characterised by southern exposure and limestone substratum, at altitudes <1400 m asl; I. ricinus was most abundant in Turkey oak (Quercus cerris) wood, whereas H. punctata was mostly collected in hop hornbeam (Ostrya carpinifolia) wood and on exposed rocks. Ixodes ricinus was also found up to 1670 m asl, in high stand beech (Fagus sylvatica) wood. The overall prevalence of Borrelia burgdorferi sensu lato (sl) in 294 host-seeking I. ricinus nymphs was 8.5 %. Borrelia garinii was the most frequently identified genospecies (64.0 % of positive nymphs), followed by B. valaisiana, B. burgdorferi sensu stricto, B. afzelii, and B. lusitaniae. Based upon the comparison with the results of previous studies at the same location, these research findings suggest the recent invasion of the study area by the tick vector and the agents of Lyme borreliosis.

Further circulation of West Nile and Usutu viruses in wild birds in Italy.

Usutu virus (USUV) and West Nile virus (WNV) are emerging pathogens that can cause neurological disease in humans. From March 2012 to June 2013, a sero-survey on wild birds was carried out to investigate the circulation of both viruses in Northwest (NW) Italy. Samples belonging to 47 different bird species have been collected using a volunteer based network and a wildlife rehabilitation center. Four of 297 serum samples had neutralizing antibodies against USUV (P=1.34%, IC 95% 0.36-3.4), while 10 of 233 samples tested positive for WNV (P=4.29%, IC 95% 2.07-7.75). Neutralizing antibodies for WNV were significantly more prevalent (p<0.001) in trans-Saharan migrants (P=21%, IC 95% 9.55-37.3) than in resident and short-distance birds, but no migratory habit-related differences were found for USUV. Antibodies in resident bird species suggest that both viruses are circulating in NW Italy.

Molecular analysis and associated pathology of beak and feather disease virus isolated in Italy from young Congo African grey parrots (Psittacus erithacus) with an "atypical peracute form" of the disease.

This study is the first report on the genetic and pathogenic characterization of beak and feather disease virus (BFDV) occurring in Italy. Twenty BFDV strains isolated in Italy from juvenile Congo African grey parrots (Psittacus erithacus) were investigated. Seventeen strains showed an "atypical peracute form" (aPF) of the disease, and three a chronic form (CF). The birds with aPF had been weaned, were independent as far as food and protection were concerned and apparently were without lesions. The gene coding for the putative coat protein was amplified in all isolates while the BFDV genome was sequenced completely in 10 samples, eight of them belonging to aPF affected birds and two from CF of the disease. All full genomes clustered into the J strain of BFDV, where two new subtypes were identified. Recombination analyses showed evidence of genetic exchanges in two BFDV genomes. In addition, a correlation between viral isolate and origin of the breeding material was shown, while an association between the genetic features of the virus and the clinical form was not observed. Histologically, apoptosis was detected frequently in aPF samples and sporadically in CF samples. Interestingly, BFDV antigens were detected in the nuclei and cytoplasm of such apoptotic cells. The data presented here support the hypothesis that, in the absence of a defined BFDV genetic variant accountable for a specific clinical form of psittacine beak and feather disease, differences in the apoptotic rate between aPF and CF are strictly host related.

Small ruminant lentiviruses in Jordan: evaluation of sheep and goat serological response using recombinant and peptide antigens.

Small ruminant lentiviruses infect sheep and goats worldwide, causing chronic progressive diseases and relevant economic losses. Disease eradication and prevention is mostly based on serological testing. The goal of this research was to investigate the presence of the small ruminant lentiviruses (SRLVs) in Jordan and to characterize the serological response in sheep and goat populations. A panel of sera were collected from flocks located in Northern Jordan and Jordan Valley. The samples were tested using three ELISA assays: a commercially available ELISA based on p25 recombinant protein and transmembrane peptide derived from British maedi-visna virus (MVV) EV1 strain, an ELISA based on P16-P25 recombinant protein derived from two Italian strains representative of MVV- and caprine arthritis encephalitis virus (CAEV)-like SRLVs, and an ELISA based on SU5 peptide from the same two Italian isolates. The results indicate that both MVV- and CAEV-like strains are present in Jordan and that the majority of the viruses circulating among sheep and goat populations belong to the MVV-like genotype.

Development of specific diagnostic test for small ruminant lentivirus genotype E.

Small ruminant lentivirus (SRLV) belonging to the highly divergent genotype E has recently been identified in the Italian goat breed Roccaverano. In this report we have developed a specific serological test based on recombinant matrix/capsid antigen fusion protein. Performance has been evaluated and compared with a similar test based on genotype B antigen. Herds under study were selected according to the infectious status characterized by blood PCR and sequencing. Results clearly showed that B and E based recombinant ELISA only detected homologous infection and an apparent cross-reactivity was recorded in a herd in which co-infection was present. Three commercially available ELISAs showed different abilities in detecting genotype E infection, being the whole virus-based immunoassay the best choice. Genotype E-recombinant antigen was not detected in ELISA by three commercially available Mabs known to be cross-reactive among CAEV and MVV capsid antigens, further supporting the high divergence of the E genotype from others. Finally, a SRLV-free herd according to commercial ELISA testing, was analysed in the same area where genotype E was identified and few animals belonging to Roccaverano breed were found slightly reactive with the E antigens. Our results suggest that the prevalence of genotype E in other small ruminant populations may be conveniently estimated using a comparative assay based on a combination of genotype specific recombinant antigens and may highlight a wider space in which SRLVs evolve.