A novel variant of the infectious bronchitis virus resulting from recombination events in Italy and Spain.

Infectious bronchitis is considered to be one of the most devastating diseases in poultry. Control of its spread is typically attempted through biosecurity measures and extensive vaccination. However, the remarkable genetic and antigenic variability of the virus, which originate from both mutations and recombination events, represents an unsolved challenge for this disease. The present study reports on the emergence and spread of recombinant clusters detected in Italy and Spain between 2012 and 2014. A total of 36 Spanish and Italian infectious bronchitis virus (IBV) field strains were investigated and genetically characterized using phylogenetic, molecular, recombination and selection pressure analyses of the complete S1 gene. Based on the partial S1 sequencing, 27 IBV strains originating from Spain and nine from Italy were initially classified as being closely related to the Guandong/Xindadi (XDN) genotype. Phylogenetic analysis of the complete S1 gene revealed that the XDN strains formed a homogeneous clade with the Spanish IBV isolates within the QX genotype, whereas there was higher variability within the Italian strains. Recombination analysis determined that these strains belonged to four groups, which originated from independent recombination events between the QX and 793B IBV genotypes. Our data support the hypothesis of two different scenarios: firstly, in Spain, the large and homogeneous clade probably originated from a single offspring of the recombinant founder, which became dominant and spread throughout the country. Secondly, the nine Italian recombinants, which are characterized by three different recombination patterns, probably represent less fitted strains, because they were less viable with respect to their recombinant parents.

Epidemiological and pathological investigation of fowl aviadenovirus serotypes 8b and 11 isolated from chickens with inclusion body hepatitis in Spain (2011-2013).

Inclusion body hepatitis caused by different fowl aviadenovirus (FAdV) serotypes has been described in several countries in recent years. In Spain, from the spring of 2011 to 2013, an increased number of outbreaks in broiler and broiler breeder flocks from different regions occurred. The objectives of the present work were to carry out the molecular characterization of FAdV strains from Spanish inclusion body hepatitis cases and to study the pathogenicity and viral dynamics of these strains in specific pathogen-free (SPF) chickens. A total of 52 inclusion body hepatitis clinical cases, including 45 from broiler farms and seven from broiler breeder farms, were analysed by conventional polymerase chain reaction and sequencing targeting the FAdV hexon gene. From these, 37 strains were classified as FAdV type 8b, while the remaining 15 were classified as FAdV types 11 (n = 10), 2 (n = 4) and 8a (n = 1). In addition, two different FAdVs belonging to the genotypes 8b and 11 were used for experimental infection. Specific pathogen-free five-day-old birds were inoculated intramuscularly with a high (106.5 tissue culture infective dose (TCID)50/ml) or low (104 TCID50/ml) dose of the above-mentioned FAdVs. No mortality was observed in any of the experimental groups, and only one bird showed evident clinical signs. However, macroscopic and microscopic hepatic lesions, as well as viral DNA, were detected in birds from all infection groups. Inclusion bodies and viral DNA were also detected in the pancreas and in the small and the large intestine in some birds. Long-lasting shedding and transmission to contact birds were confirmed in all infected groups.

Evaluating the efficacy of an avian-specific probiotic to reduce the colonization of Campylobacter jejuni in broiler chickens.

Campylobacteriosis is the most frequent zoonotic disease in humans worldwide, and the contaminated poultry meat by Campylobacter jejuni can be considered one of the important sources of enteric infections in humans. The use of probiotics, which can help to improve the natural defense of animals against pathogenic bacteria, is an alternative and effective approach to antibiotic administration for livestock to reduce bacterial contamination. In vitro experiments showed that Enterococcus faecium, Pediococcus acidilactici, Lactobacillus salivarius, and Lactobacillus reuteri isolated from healthy chicken gut inhibited the growth of C. jejuni. To demonstrate this effect in vivo, 1-d-old broiler chicks received 2 mg/bird per day of a multispecies probiotic product via the drinking water. Controls received no probiotic treatment, and all chicks were infected with C. jejuni orally. Results showed that the cecal colonization by C. jejuni was significantly reduced by probiotic treatment at both 8 and 15 d postchallenge. To confirm this effect, in a second in vivo experiment, 1-d-old broiler chicks received the same dose of the same probiotic via the drinking water and controls received no probiotic, and all chicks were infected with C. jejuni orally. Similarly, probiotic treatment reduced (P=0.001) cecal colonization by C. jejuni at both 8 and 15 d postchallenge. The results of our in vivo experiments conclude that probiotic administration reduced the colonization of C. jejuni in broiler chickens.

Adenoviral overexpression of interleukin-1 receptor antagonist protein increases beta-cell replication in rat pancreatic islets.

The naturally occurring inhibitor of interleukin-1 (IL-1) action, interleukin-1 receptor antagonist protein (IRAP), binds to the type 1 IL-1 receptor but does not initiate IL-1 signal transduction. In this study, we have determined the effects of IL-1beta and IRAP overexpression on adult beta-cell replication and viability. IL-1beta reduced dramatically beta-cell replication in adult rat islets both at 5.5 mM (control: 0.29+/-0.04%; IL-1beta: 0.02+/-0.02%, P<0.05) and 22.2 mM glucose (control: 0.84+/-0.2%; IL-1beta: 0.05+/-0.05%, P<0.05). This effect was completely prevented in islets overexpressing IRAP after adenoviral gene transfer at 5.5 mM (Ad-IL-1Ra+IL-1beta: 0.84+/-0.1%, P<0.05) and 22.2 mM glucose (Ad-IL-1Ra+IL-1beta: 1.22+/-0.2%, P<0.05). Moreover, overexpression of IRAP increased glucose-stimulated beta-cell replication in the absence of IL-1beta exposure (Ad-IL-1Ra: 1.59+/-0.5%, P<0.05). beta-Cell death (TUNEL technique) was increased in IL-1beta-exposed islets but not in Ad-IL-1Ra-infected islets (control: 0.82+/-0.2%; control+IL-1beta: 1.77+/-0.2; IRAP: 0.61+/-0.2%; IRAP+IL-1beta: 0.86+/-0.1%, P<0.05). Comparable results were obtained by flow cytometry. To determine the effect of IRAP overexpression on beta-cell replication in vivo, Ad-IL-1Ra-transduced islets were transplanted into streptozotocin diabetic rats. beta-Cell replication was significantly increased in IRAP-overexpressing islet grafts (0.98+/-0.3%, P<0.05) compared to normal pancreas (0.35+/-0.02%), but not in control islet grafts (0.50+/-0.1%). This study shows that in addition to the effects of IL-1beta on beta-cell viability, this cytokine exerts a deleterious action on beta-cell replication, which can be prevented by IRAP overexpression, and provides support for the potential use of IRAP as a therapeutic tool.

Linear correlation between beta-cell mass and body weight throughout the lifespan in Lewis rats: role of beta-cell hyperplasia and hypertrophy.

We determined the beta-cell replicative rate, beta-cell apoptosis, cross-sectional beta-cell area, and pancreatic beta-cell mass throughout the entire postweaning lifespan (months 1, 3, 7, 10, 15, and 20) of Lewis rats. Beta-cell replication was progressively reduced in the initial months of life but remained stable after month 7 (month 1, 0.99 +/- 0.10%; month 3, 0.24 +/- 0.04%; month 7, 0.12 +/- 0.02%; month 10, 0.14 +/- 0.02%; month 15, 0.10 +/- 0.03%; month 20, 0.13 +/- 0.03%; analysis of variance [ANOVA], P < 0.001). Beta-cell apoptosis was low and did not change significantly from month 1 to 20 of life. Cross-sectional area of individual beta-cells increased progressively in the initial months, remained stable from month 7 to 15, and increased again on month 20. The estimated number of beta-cells per pancreas, calculated as the ratio of total beta-cell mass to individual beta-cell mass, tripled from month 1 to 7 but did not change significantly thereafter. Beta-cell mass increased approximately 8 times from month 1 to 20 (month 1, 2.04 +/- 0.28 mg; month 20, 15.5 +/- 2.32 mg; ANOVA, P < 0.001) and showed a strong and significant linear correlation with body weight (r = 0.98, P < 0.001). In summary, we have shown that beta-cell replication was maintained throughout the lifespan in normal rats, clearly establishing that the beta-cell birth rate does not fall to 0, even in very old rats. Beta-cell mass increased throughout the lifespan, closely matching the increment in total body weight at any time point. This increment was selective for beta-cells, since the growth of the endocrine non-beta-cell mass was limited to the initial months of life. Both beta-cell hypertrophy and hyperplasia contributed to increased beta-cell mass in young animals, but only beta-cell hypertrophy was responsible for the increased beta-cell mass found in old animals. This study provides a global perspective for understanding the dynamics of beta-cell mass in young, adult, and aged animals.

Optimal insulin treatment in syngeneic islet transplantation.

Insulin-induced normoglycemia has shown to have a beneficial effect on the outcome of pancreatic islets transplanted to diabetic recipients. The aim of the study was to identify the insulin treatment that can maximize its beneficial effect on islet transplants. Six groups of streptozotocin diabetic C57Bl/6 mice were transplanted (Tx) with 100 syngeneic islets, an insufficient beta cell mass to restore normoglycemia, and were treated with insulin as follows: group 1 (n = 9): from day 10 before Tx to day 14 after Tx; group 2 (n = 11): from day 6 before Tx to Tx day; group 3 (n = 11): from Tx day to day 6 after Tx; group 4 (n = 7): from Tx day to day 14 after Tx; group 5 (n = 8): from day 10 to day 24 after Tx; group 6 (n = 18): Tx mice were not treated with insulin. Sixty days after Tx, normoglycemia was achieved in 100% of mice in groups 1, 4, and 5, in 73% of mice in group 2, and in only 45% and 33% of mice in groups 3 and 6, respectively (p < 0.01). Intraperitoneal glucose tolerance, determined only in normoglycemic mice, was similar in groups 1, 2, 4, and normal controls. In contrast, normoglycemic mice from groups 3, 5, and 6, exposed to more severe and prolonged hyperglycemia after Tx, showed higher glucose values after glucose injection, suggesting that hyperglycemia had a long-lasting deleterious effect on transplanted beta cell function. The initially transplanted beta cell mass was maintained in the grafts of normoglycemic mice, but was severely reduced in hyperglycemic mice. Transplanted beta cell mass was similar in normoglycemic groups with normal or impaired glucose tolerance, indicating that impaired glucose tolerance was not due to reduced beta cell mass. In summary, the beneficial effect of insulin-induced normoglycemia on transplanted islets was maximal when insulin treatment was maintained the initial 14 days after transplantation. Exposure to sustained hyperglycemia initially after transplantation had a long-lasting deleterious effect on transplanted islets.