Phylodynamic analysis and evaluation of the balance between anthropic and environmental factors affecting IBV spreading among Italian poultry farms.

Infectious bronchitis virus (IBV) control is mainly based on wide vaccine administration. Although effective, its efficacy is not absolute, the viral circulation is not prevented and some side effects cannot be denied. Despite this, the determinants of IBV epidemiology and the factors affecting its circulation are still largely unknown and poorly investigated. In the present study, 361 IBV QX (the most relevant field genotype in Italy) sequences were obtained between 2012 and 2016 from the two main Italian integrated poultry companies. Several biostatistical and bioinformatics approaches were used to reconstruct the history of the QX genotype in Italy and to assess the effect of different environmental, climatic and social factors on its spreading patterns. Moreover, two structured coalescent models were considered in order to investigate if an actual compartmentalization occurs between the two integrated poultry companies and the role of a third "ghost" deme, representative of minor industrial poultry companies and the rural sector. The obtained results suggest that the integration of the poultry companies is an effective barrier against IBV spreading, since the strains sampled from the two companies formed two essentially-independent clades. Remarkably, the only exceptions were represented by farms located in the high densely populated poultry area of Northern Italy. The inclusion of a third deme in the model revealed the likely role of other poultry companies and rural farms (particularly concentrated in Northern Italy) as sources of strain introduction into one of the major poultry companies, whose farms are mainly located in the high densely populated poultry area of Northern Italy. Accordingly, when the effect of different environmental and urban parameters on IBV geographic spreading was investigated, no factor seems to contribute to IBV dispersal velocity, being poultry population density the only exception. Finally, the different viral population pattern observed in the two companies over the same time period supports the pivotal role of management and control strategies on IBV epidemiology. Overall, the present study results stress the crucial relevance of human action rather than environmental factors, highlighting the direct benefits that could derive from improved management and organization of the poultry sector on a larger scale.

Multilocus sequence typing of Campylobacter jejuni and Campylobacter coli to identify potential sources of colonization in commercial turkey farms.

Poultry are the main reservoir for thermophilic Campylobacter spp., which is the most common causative agent of human bacterial gastroenteritis. The epidemiology of Campylobacter in poultry, particularly in turkeys, is not completely understood. This study aimed at identifying potential sources and transmission routes of thermophilic Campylobacter spp. in commercial turkey farms. C. jejuni and C. coli isolates from breeders (n = 29, 20 C. jejuni and 9 C. coli) and their progeny (n = 51, 18 C. jejuni and 33 C. coli) reared in two different farms for three sequential production cycles were analysed by multilocus sequence typing (MLST). Strains (n = 88, 42 C. jejuni and 46 C. coli) isolated from environmental (i.e. anteroom and in-house overshoes), water (i.e. drinkers and water line), and pest (i.e. flies, Alphitobius diaperinus, and mice) sources were also examined. MLST of C. jejuni and C. coli isolates resulted in 13 and 12 different sequence types (STs) belonging to six and one previously-described clonal complexes (CCs), respectively. Three novel STs were identified. Genetic similarities were detected between isolates from fattening turkeys and the considered environmental, water, and pest sources, and with the breeders to a lesser extent. Source attribution analysis estimated that environmental and water sources accounted for most (∼75%) of fattening turkey isolates and were therefore identified as the most likely sources of flock colonization, followed by pests (∼20%) and breeders (∼5%). These sources may thus be targeted by control measures to mitigate the risk of Campylobacter colonization in commercial turkeys. RESEARCH HIGHLIGHTS High occurrence of C. jejuni and C. coli in commercial turkey flocks. High genetic diversity of C. jejuni and C. coli in commercial turkey flocks. Horizontal transmission responsible for Campylobacter colonization of commercial turkey flocks. Environmental and water sources involved in Campylobacter colonization of commercial turkey flocks. Strategies for prevention and control of Campylobacter colonization of commercial turkey flocks are needed.

Longitudinal field studies of avian metapneumovirus and turkey hemorrhagic enteritis virus in turkeys suffering from colibacillosis associated mortality.

The aim of this study was to evaluate if the exposure to Avian metapneumovirus (aMPV) and/or to Turkey hemorrhagic enteritis virus (THEV) was significant for the induction of episodes of colibacillosis in aMPV and THEV vaccinated turkeys. Colibacillosis-associated mortality was recorded and longitudinal virological studies performed in three consecutive turkey flocks reared in the same farm. aMPV and THEV diagnostic swabs and blood samples were made once a week up to 14 weeks of age. Swabs were processed by molecular techniques for viruses detection and antibody titres were evaluated. Field subtype B aMPVs were detected in all flocks at different ages of life always associated with respiratory signs and increase of colibacillosis-associated mortality. THEV has been consistently detected in all flocks since the 9th week of age. Vaccination with a single dose of the THEV commercial inactivated vaccine available in Italy seems does not protect the birds from the infection. Sequence comparison of the hexon protein of one of the THEV strains detected, and strains isolated worldwide, revealed high similarity between them. These results are consistent with the notion that the hexon protein, being the major antigenic component of the virus, is highly conserved between the strains. Results showed that field aMPV infection is directly correlated to colibacillosis-associated mortality. Less clear appears the role of THEV because the endemicity of aMPV makes difficult to evaluate its role in predisposing colibacillosis in absence of aMPV. It would be interesting to further investigate this issue through experimental trials in secure isolation conditions.

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.

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.

Pathogenicity of a QX strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the Ma5 and 4/91 serotypes.

The aims of this study were firstly to evaluate the pathogenicity of an Italian isolate of the QX strain of infectious bronchitis (IB) virus using 1-day-old female specific pathogen free chicks (layer type) and 1-day-old female commercial broiler type chickens, and secondly to assess the level of protection induced in these birds by a vaccination programme including the IB Massachusetts and 4/91 serotype live attenuated vaccines. Unvaccinated birds showed clinical signs of varying severity, predominantly affecting the upper respiratory tract. Vaccinated birds appeared healthy, with the exception of a very mild conjunctivitis affecting a limited number of the broilers. Vaccination fully protected specific pathogen free birds, since no histopathological lesions were observed, nor was virus detected following challenge. In broilers, replication of the challenge virus was not prevented but was significantly reduced. This study confirms that vaccination at 1 day old and at 14 days of age using the Ma5 and 4/91 IB vaccines may be instrumental in reducing the economic impact of QX IB virus infections in layer and broiler farms.