Davide Giovanardi

Continued use of IBV 793B vaccine needs reassessment after its withdrawal led to the genotype's disappearance

Abstract Over a period of almost two years, broilers chickens on several hundred Italian farms, were monitored for infectious bronchitis virus. Detections were genotyped using a hypervariable region of the gene coding for the S1 segment of the spike protein. A range of genotypes were detected which comprised QX, Q1, Mass, D274 and 793B. Sequences of 793B viruses detected in chickens, vaccinated with either of the two commonly used 793B type vaccines were almost identical to sequences of one or other of these vaccines. This strong indication of vaccine association led to the withdrawal of live 793B vaccine use on all of the farms of the study. Except for one sample collected soon after 793B vaccination ceased, it was no longer possible to detect 793B vaccine on these farms. It appears that field 793B strains have disappeared from the region of Italy tested thus obviating any need for current vaccine protection against 793B.

Class 1 and class 2 integrons in avian pathogenic Escherichia coli from poultry in Italy

The aim of this study was to investigate the occurrence of class 1 and 2 integrons in avian pathogenic Escherichia coli (APEC) from poultry in northern Italy. Strains were tested for phenotypic resistance to aminoglycosides and sulphonamides, and the association between the presence of integrons and the resistance to these antimicrobials was evaluated. A total of 299 isolates (158 from turkeys, 110 from broilers, and 31 from layer hens) were collected from 200 industrial farms. Antimicrobial susceptibility test by the disk diffusion method was performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. All strains were screened for the presence of class 1 and 2 integrons by PCR and sequencing. About 55% of APEC contained integrons (class 1, 49.8%; class 2, 10.4%). Different variants of the aadA (5 variants) and the dfrA (4 variants) genes, encoding for streptomycin and trimethoprim resistance respectively, were detected in integron-positive isolates. Less common gene cassettes, such as sat, estX, and orfF, were also identified. Fifteen and 4 gene cassette arrays were found among class 1 and 2 integrons, respectively. High levels of resistance were observed for triple sulphonamides (79.3%), streptomycin (67.2%), and sulfamethoxazole combined with trimethoprim (62.2%), whereas resistance against gentamycin (16.7%), kanamycin (14.7%), and apramycin 3.0%) was low. Integron positivity was significantly higher in isolates phenotypically resistant to aminoglycosides (63.6% vs. 37.8%, P<0.001) and sulfonamides (64.1% vs. 21.1%, P<0.001) than in susceptible ones. Integron-borne aminoglycoside and sulfonamide resistance in APEC represents a concern for the poultry industry in Italy, since they are among the most commonly used antimicrobials in poultry therapy.

Rapid detection of subtype B avian metapneumoviruses using RT-PCR restriction endonuclease digestion indicates field circulation of vaccine-derived viruses in older turkeys

Live vaccines predominantly control avian metapneumovirus (aMPV) infection in poultry flocks, but vaccine virus can be found for extended periods after application. The most frequently used aMPV vaccine in Italy, VCO3 subtype B, was shown to contain a unique Tru9I restriction endonuclease site within the amplicons produced by a commonly used aMPV diagnostic reverse transcriptase (RT)-nested polymerase chain reaction (PCR). Analysis of European and database logged subtype B aMPV sequences confirmed that the sequence occurred only in the VC03 vaccine. A subsequent RT-PCR restriction endonuclease study of field samples, collected from turkeys between 2007 and 2012, detected subtype B vaccine-derived strains in 12 of 90 samples tested that were collected from birds under 12 weeks of age.

Approved medication of water with enrofloxacin to treat turkey colibacillosis: Assessment of efficacy using a PK/PD approach

The efficacy of administering enrofloxacin at 10mg/kg in medicated water to turkeys was evaluated by applying a PK/PD approach to the kinetic parameters obtained after oral pulsed administration and to the MIC values of avian pathogenic Escherichia coli (APEC) strains isolated from commercial turkey flocks. The kinetic parameters of enrofloxacin were evaluated in 10 healthy and 10 colisepticemic turkeys that received the drug dissolved in medicated water at 89 μg/mL and 71 μg/mL, respectively, for 10h/day for 5 days. Blood samples were collected for 24h from all turkeys on the last day of treatment, and the serum was analysed by HPLC with fluorimetric detection. The mean AUC (7374.53±1067.64 h ng/mL and 7656.95±1460.61 h ng/mL) and C(max) values (673.09±186.18 ng/mL and 543.50±68.75 ng/mL) obtained for healthy and sick turkeys were not significantly different. High-level resistance was observed in 30.3% of strains, 40.5% exhibited intermediate resistance, and only 29.2% were susceptible; the MIC(50) and MIC(90) values were 1mg/L and 32 mg/L, respectively. The PK/PD parameters C(max)/MIC(50) (0.67 and 0.54 for healthy and sick turkeys, respectively) and AUC/MIC(50) (7.37 and 7.66) were lower than the efficacy breakpoints reported for fluoroquinolones. These results indicate that authorised dosage of enrofloxacin used in pulsed medicated water administration could be ineffective against more than the 70% of circulating APEC strains, indicating the need to test the drug susceptibility of APEC prior to administering the drug and adopting a more convenient medication schedule for mass treatment.

A molecular epidemiology study based on VP2 gene sequences reveals that a new genotype of infectious bursal disease virus is dominantly prevalent in Italy

ABSTRACT A distinctive infectious bursal disease (IBD) virus genotype (ITA) was detected in IBD-live vaccinated broilers in Italy without clinical signs of IBD. It was isolated in specific-pathogen-free eggs and molecularly characterized in the hypervariable region of the virus protein (VP) 2. Phylogenetic analysis showed that ITA strains clustered separately from other homologous reference sequences of IBDVs, either classical or very virulent, retrieved from GenBank or previously reported in Italy, and from vaccine strains. The new genotype shows peculiar molecular characteristics in key positions of the VP2 hypervariable region, which affect charged or potentially glycosylated amino acids virtually associated with important changes in virus properties. Characterization of 41 IBDV strains detected in Italy between 2013 and 2014 showed that ITA is emergent in densely populated poultry areas of Italy, being 68% of the IBDV detections made during routine diagnostic activity over a two-year period, in spite of the immunity induced by large-scale vaccination. Four very virulent strains (DV86) and one classical strain (HPR2), together with eight vaccine strains, were also detected. The currently available epidemiological and clinical data do not allow the degree of pathogenicity of the ITA genotype to be defined. Only in vivo experimental pathogenicity studies conducted in secure isolation conditions, through the evaluation of clinical signs and macro/microscopic lesions, will clarify conclusively the virulence of the new Italian genotype.

Genetic diversity of Escherichia coli isolates of animal and environmental origins from an integrated poultry production chain

Escherichia coli is a normal inhabitant of the intestinal tract of chickens, but when an imbalance in the gut microbiota occurs, E. coli may overgrow and cause extraintestinal infections. The aims of this study were to assess the distribution and spread of E. coli isolates with specific phylogenetic groups and antimicrobial resistance characters among asymptomatic breeder flocks and their broiler progenies with early symptoms of colibacillosis. Broiler flocks were treated with lincospectin during the first week of life and sampled at one, 21 and 42 days. The majority of the 363 E. coli isolates belonged to phylogenetic group A (53.17%), followed by groups D (23.14%), B1 (19.28%) and B2 (4.41%). In broilers, group A was the most represented in birds of 21 and 42 days of age whereas group B1 was the most represented phylogroup in one-day old chicks. More than 90.00% of the isolates were resistant to one or more antimicrobials. Along the life-time of broilers, no differences were found on the occurrence of resistant isolates except for the number of E. coli with elevated MIC to spectinomycin, which increased significantly after the lincospectin treatment. According to XbaI-macrorestriction analysis, a high genetic diversity among E. coli isolates was underlined. Four antimicrobial resistant E. coli isolates of phylogroups A, B1 and D collected from breeders showed similar PFGE patterns to five isolates collected from the respective broiler progenies suggesting a potential spread of these isolates from breeders to broilers.

Antimicrobial resistance and class 1 and 2 integrons in Escherichia coli from meat turkeys in Northern Italy

This study is aimed at determining the antimicrobial resistance (AMR) and the presence of class 1 and 2 integrons in 48 avian pathogenic Escherichia coli (APEC) strains isolated from meat turkeys during three sequential production cycles. Thirty avian faecal E. coli (AFEC) strains from the first cycle were also analysed. Strains were tested for AMR against 25 antimicrobials by disk diffusion test and were screened for the presence of integrons and associated gene cassettes by polymerase chain reaction followed by sequencing. Genetic relatedness of isolates was established by pulsed-field gel electrophoresis. High levels of resistance were detected to tetracyclines, penicillins and sulphonamides in APEC and AFEC. Resistance to aminoglycosides, fluoroquinolones, cephalosporins and phenicols was variable, based on the antimicrobial drug and the isolate (APEC vs. AFEC). Full susceptibility to colistin was detected. Multidrug resistance of up to seven antimicrobial classes was exhibited by APEC (93.8%) and AFEC (100%). Nearly 44% of strains tested positive for class 1 and/or class 2 integrons containing the dfrA, aadA and sat2 genes, alone or in combination, coding for streptomycin/spectinomycin, trimethoprim and streptothricin resistance, respectively. The estX and orfF genes of unknown function were also detected. A significant association was found between the presence of integrons and the resistance to aminoglycosides and potentiated sulphonamides. The results of this study showed that AMR, multidrug resistance and class 1 and 2 integrons are widespread among pathogenic and commensal E. coli from Italian turkeys. More attention should be addressed to limit the use of antimicrobials in turkeys and the AMR of turkey E. coli.

Characterization and antimicrobial resistance analysis of avian pathogenic Escherichia coli isolated from Italian turkey flocks

This study investigated the occurrence of avian pathogenic Escherichia coli (APEC) in a finishing turkey commercial farm, carrying out longitudinal surveys involving 3 consecutive flocks. The diversity and the distribution of the E. coli strains detected during colisepticemia outbreaks were examined. The strains were isolated, serogrouped, assessed for the presence of virulence-associated genes, typed by random amplification of polymorphic DNA (RAPD), and antimicrobial resistance analysis was then carried out. Escherichia coli O78 and O2 were predominantly found. Moreover, based on the somatic antigens used in the study, strains were recovered that were nontypeable. On one occasion, an E. coli O111 strain was found in turkeys. The E. coli isolates differed in terms of antibiotic resistance and RAPD profile. All strains possessed the virulence genes that enabled them to be considered APEC. Strains not only differed between flocks, but also within the same flock. These findings point out the importance of addressing colibacillosis therapy on the basis of a sensitivity test.

Virulence - associated genes in Avian Pathogenic Escherichia coli of turkey

Abstract 50 Escherichia coli (APEC-Avian Pathogenic Escherichia coli) strains and 15 E. coli (AFEC-Avian Faecal Escherichia coli) from turkeys affected by colibacillosis and from healthy turkeys were tested for the presence of eight different virulence-associated genes. Besides, APEC were serotyped. O78 has been the most detected serotyped. The presence of the tested virulence genes was prevalently related to the APEC isolates. With reference to serogroup, all the tested O78 resulted iss and irp2 positive. Besides, tsh e cva/cvi were respectively present in 88.9 and 83.3% of O78. Nevertheless, the finding of a not typeable strains equipped with all the eight tested virulence genes among the APEC isolates suggest the importance of a careful and complete characterisation of the isolate to evaluate the real potential pathogenic attitude of the bacterium.

In vitro evaluation of live attenuated vaccines against Salmonella enteritidis: cell-mediated immune response

Abstract The use of Salmonella enteritidis (SE) live attenuated vaccines is one of the major tool to reduce this infection in commercial poultry. In this work, techniques, evaluating the presence and the expression of some cytokines, were studied to improve the knowledge of the cellular-mediated immune response following SE vaccination. This study demonstrated that SE vaccination enhances the production of INF-γ, IL-8, iNOs, while downregulates IL-1β. Between these immunologic parameters, the evaluation of INF-γ seems to be the most significant and easy test to plan and optimize SE vaccination programs.