Alessandra Piccirillo

Effect of genotype, gender and feed restriction on growth, meat quality and the occurrence of white striping and wooden breast in broiler chickens

Due to their importance for the control of meat quality in broiler chickens, the present study aimed at identifying the factors associated with the occurrence of myopathies and characterizing the meat properties when affected by myopathies. To this aim, a total of 768 broiler chickens were reared until slaughter (46 d) to evaluate the effect of genotype, gender, and feeding regime (ad libitum vs. restricted rate, 80% from 13 to 21 d of age) on performance and meat quality. Standard broilers were heavier (3,270 vs. 3,139 g; P<0.001) and showed lower feed conversion (1.56 vs. 1.61; P<0.001) than the high-yield broilers. Males showed higher final live weight (3,492 vs. 2,845 g) and lower feed conversion (1.54 vs. 1.63) than females (P<0.001). Feed restriction decreased final live weight (3,194 vs. 3,142 g; P<0.01) and feed conversion (1.60 vs. 1.57; P<0.01) compared to ad libitum feeding. At gross examination, feed restriction tended to increase white-striped breasts (69.5 vs. 79.5%; P<0.10), whereas females showed less wooden breasts than males (8.0 vs. 16.3%; P<0.05). White-striped fillets had higher pHu (5.87 vs. 5.83), and lower a* (-0.81 vs. -0.59) and b* color indexes (13.7 vs. 14.5) (P<0.05), whereas wooden breast fillets exhibited higher cooking losses (25.6 vs. 22.1%) and AK-shear force (4.23 vs. 2.84 kg/g) compared with normal fillets (P<0.001). At histological examination, 3.1% of pectoralis major were normal, 26.6% mildly degenerated, 45.3% moderately degenerated, and 25.0% severely degenerated. In conclusion, genotype had a moderate effect on growth without modifying myopathy occurrence. In contrast, gender and feed restriction affected performance, meat quality, and breast abnormalities.

High Prevalence of oqxAB in Escherichia coli Isolates from Domestic and Wild Lagomorphs in Italy

This study aimed to identify and characterize class 1 and 2 integrons and plasmid-mediated quinolones resistance (PMQR) genes in a collection of 113 multidrug resistance (MDR) Escherichia coli isolated from farm and wild lagomorphs between 2006 and 2008 in Northern Italy. Strains were examined for antimicrobial susceptibility by agar disk diffusion method and E-test for colistin (COL); integrons and gene cassettes content by real-time polymerase chain reaction (PCR) and DNA sequencing; PMQR genes by PCR and DNA sequencing; clonal relatedness by multilocus sequence typing; and plasmids by PCR-based replicon typing. Class 1 integrons were detected in 69 isolates (47 farm rabbits, 14 wild rabbits, and 8 wild hares). No class 2 integrons were found. Five different gene cassettes arrays were identified (aadA1, dfrA1-aadA1, orf in682-dhfrA5, orf in682-dfrA5-orfD ins21, and dfrA17-aadA5). Fifteen percent (17/113) of isolates carried oqxAB, no other PMQR determinants. All but one oqxAB-positive E. coli strains were recovered from farm rabbits. Seven out of 17 strains were associated with the predominant ST238 and carried from three to six different plasmid types, such as IncF, IncHI1, IncI1, IncN, IncP, IncX1, IncY, and ColE. COL resistance was identified in 6/113 strains (5.3%). This study provides new insights on the resistance phenotypes and the prevalence and dissemination of oqxAB in E. coli from farm and wild lagomorphs, suggesting that these animals may be reservoir of these genetic determinants in Italy and thus a potential source of PMQR E. coli for humans. PMQR mediated by oqxAB has not been detected in farm and wild lagomorphs before.

Fluoroquinolone resistance and molecular characterization of gyrA and parC quinolone resistance-determining regions in Escherichia coli isolated from poultry

Escherichia coli are a common inhabitant of the gastrointestinal tract of mammals and birds; nevertheless, they may be associated with a variety of severe and invasive infections. Whereas fluoroquinolones (FQ) have been banned in the United States for use in poultry production, the use of these antimicrobials in poultry husbandry is still possible in the European Union, although with some restrictions. The aim of this study was to investigate the FQ resistance of 235 E. coli isolates recovered from chickens and turkeys. Minimum inhibitory concentrations were determined by a microdilution method, whereas mutations in the quinolone resistance-determining regions of the target genes, gyrA and parC, were detected by a PCR-based method. High resistance rates (>60%) were observed for nalidixic acid, flumequine, and difloxacin, whereas resistance to ciprofloxacin, danofloxacin, enrofloxacin, marbofloxacin, and sarafloxacin was less frequently reported (<40%). Sixty-four isolates (27.2%) showed full susceptibility toward the tested FQ, but 57 isolates (24.2%) were resistant to all tested FQ. The remaining 114 E. coli isolates (48.5%) were grouped in 5 different resistance patterns. Isolates resistant only to flumequine or nalidixic acid or both possessed 1 gyrA mutation, whereas isolates with further resistance to enrofloxacin, difloxacin, danofloxacin, and sarafloxacin had in addition 1 or 2 parC substitutions. Two gyrA mutations coupled with 1 substitution in parC were detected in isolates resistant to all tested FQ. The number of mutations and their correlation with the in vitro activity of FQ reflected the currently accepted model, according to which a single gyrA substitution is associated with resistance or decreased susceptibility to older quinolones, whereas further gyrA or parC substitutions are needed for a higher level of resistance.

Effect of age on the occurrence of muscle fiber degeneration associated with myopathies in broiler chickens submitted to feed restriction

&NA; To evaluate muscle fiber degeneration (MFD) associated with white striping and wooden breast, pectoralis major of 192 broilers differing for genotype (standard vs. high breast yield), gender, and feeding regime (ad libitum vs. restricted rate 80% from 13 to 21 d of age) were sampled at 14, 21, 28, 35, and 46 d of age for histological analyses by hematoxylin and eosin (H&E) staining to evaluate tissue morphology, Massons trichrome to identify collagen presence, and Oil red and Nile blue for lipid presence. Microvessels (diameter ≤15 &mgr;m), nuclei positive to anti‐cleaved lamin A and monoclonal proliferating cell nuclear antigen (PCNA) antisera were counted to assess apoptotic and regenerative processes, respectively. Significant differences were found according to feeding system, age, and their interactions. The frequency of chickens with MFD was higher with ad libitum than restricted feeding (75.0% vs. 62.5%; P = 0.01) and increased with age (18.8%, 28.1%, 75.1%, 96.9%, and 96.9% at 14, 21, 28, 35, and 46 d). However, at 14 d a similar frequency (18.8%) was found in all broilers; at 21 d, MFD occurred more in broilers fed ad libitum than in those under restriction (50.0% vs. 6.3%; P < 0.01); at 28 d differences were reduced (87.5% vs. 62.5%; P = 0.10) to disappear by 35 (100% and 93.8%) and 46 d (96.9% and 96.9%). The number of microvessels decreased with age (20.7 to 9.46; P < 0.001) and the number of nuclei positive to the anti‐cleaved lamin A antibody increased. At histology, MFD at 46 d corresponded to loss of typical cross striations, massive necrotic process, degenerating fibers surrounded by inflammatory cells, scattered fibers in an abundant collagen‐rich connective tissue, numerous adipose cells; necrotic fibers showed a high percentage of apoptotic nuclei, and regenerating fibers appeared positive to anti‐PCNA antibody. In conclusion, MFD soon occurred after 2 wk of growth and increased dramatically within 28 d. Early feed restriction reduced MFD as long as animals were restricted, but no residual effect was recorded after re‐alimentation.

Campylobacter geochelonis sp. nov. isolated from the western Hermann's tortoise (Testudo hermanni hermanni)

During a screening study to determine the presence of species of the genus Campylobacter in reptiles, three putative strains (RC7, RC11 and RC20T) were isolated from different individuals of the western Hermanns tortoise (Testudo hermanni hermanni). Initially, these isolates were characterized as representing Campylobacterfetus subsp. fetus by multiplex PCR and partial 16S rRNA gene sequence analysis. Further whole- genome characterization revealed considerable differences compared to other Campylobacter species. A polyphasic study was then undertaken to determine the exact taxonomic position of the isolates. The three strains were characterized by conventional phenotypic tests and whole genome sequencing. We generated robust phylogenies that showed a distinct clade containing only these strains using the 16S rRNA and atpA genes and a set of 40 universal proteins. Our phylogenetic analysis demonstrates their designation as representing a novel species and this was further confirmed using whole- genome average nucleotide identity within the genus Campylobacter (~80 %). Compared to most Campylobacter species, these strains hydrolysed hippurate, and grew well at 25 °C but not at 42 °C. Phenotypic and genetic analyses demonstrate that the three Campylobacter strains isolated from the western Hermanns tortoise represent a novel species within the genus Campylobacter, for which the name Campylobactergeochelonis sp. nov. is proposed, with RC20T (=DSM 102159T=LMG 29375T) as the type strain.

Molecular characterization and genotypic antimicrobial resistance analysis of Campylobacter jejuni and Campylobacter coli isolated from broiler flocks in northern Italy

Genetic variability and genotypic antimicrobial resistance (AMR) of Campylobacter jejuni and Campylobacter coli from commercial broiler farms were investigated in this study. Campylobacter isolates were genetically characterized by random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) and flaA-SVR and flaB-SVR sequence-based typing. Eight RAPD types were identified in C. jejuni and three in C. coli, while 16 fla profiles were detected among all isolates. Further, 13 flaA-SVR and 13 flaB-SVR alleles were identified. Both typing methods detected a high level of genetic diversity, but fla-SVR typing showed a higher discriminatory power. Indeed, Simpsons index of fla typing (D=0.920) was higher than that of RAPD typing (D=0.814). Moreover, the association of flaA-SVR and flaB-SVR sequence analysis showed a higher discriminatory power compared with the sequence analysis of single loci. Isolates were also analysed by the mismatch amplification mutation assay PCR test and the detection of cmeB gene to determine the occurrence of genetic determinants of AMR to macrolides and fluoroquinolones and multidrug resistance. The A2074C and A2075G mutations in the 23S rRNA gene, the C257T mutation in the gyrA gene, and the cmeB gene were higher in C. coli (19.0%, 67.0%, 100.0% and 100.0%, respectively) than in C. jejuni (0.0%, 3.1%, 48.3% and 48.3%, respectively). This study showed a high degree of genetic diversity and a high prevalence of genetic determinants of macrolide resistance, fluoroquinolone resistance and multidrug resistance among C. jejuni and C. coli isolates from Italian commercial broiler farms.

A Longitudinal Study on Thermophilic Campylobacter spp. in Commercial Turkey Flocks in Northern Italy: Occurrence and Genetic Diversity

SUMMARY. Poultry are recognized as a main reservoir of thermophilic campylobacters, but few studies have been carried out on commercial meat turkeys. This study was aimed at assessing the occurrence of thermophilic Campylobacter spp., their genetic diversity, and the trend of the infection during the whole production cycle of three turkey flocks from different farms in Northern Italy. Flocks were monitored from the time of housing 1-day-old poults to slaughter time by collecting samples (meconium and cloacal swabs) at weekly intervals up to the recovery of Campylobacter spp. and then twice a month. A conventional culture method and a multiplex PCR assay were used for Campylobacter detection and identification. A subset of isolates was genetically characterized by random amplified polymorphic DNA-PCR (RAPD-PCR) and flagellin gene A short variable region (flaA-SVR) sequencing. Although at different times, all flocks became colonized by Campylobacter jejuni or Campylobacter coli (or both) that persisted throughout the entire production cycle. Overall, nine RAPD types and 14 flaA-SVR types were detected with differences in their distribution among flocks and sampling times. Moreover, changes in the Campylobacter genotypes colonizing turkeys were observed over time within each flock. These findings suggest that Italian commercial turkeys might be widely colonized by different genotypes of C. jejuni and C. coli and also suggest that differences in the distribution and epidemiologic dynamics of these microorganisms might occur among flocks.

Epidemic of infectious laryngotracheitis in Italy: characterization of virus isolates by PCR-restriction fragment length polymorphism and sequence analysis.

Abstract Between May 2007 and October 2008, 34 outbreaks of mild to moderate forms of infectious laryngotracheitis (ILT) occurred in commercial broiler flocks in Italy. Affected birds showed watery eyes, conjunctivitis, nasal discharge, reduction of feed and water consumption, and gasping with expectoration of blood-stained mucus. The mortality rate was <10%. Gross lesions consisted of conjunctivitis, excess of mucus, blood, or presence of diphtheritic membranes in trachea. A real-time PCR assay was performed to confirm the presence of ILT virus (ILTV) DNA in tracheal tissue homogenates. Twenty-three ILTV isolates were propagated on the chorion-allantoic membrane of embryonated chicken eggs showing typical plaques. PCR combined with restriction fragment length polymorphism and gene sequencing of isolates showed a high genetic correlation between field strains and chicken embryo origin vaccines.

Serological survey for influenza type A viruses in domestic dogs (Canis lupus familiaris) and cats (Felis catus) in North-Eastern Italy.

To ascertain the potential transmission of influenza A viruses to dogs and cats, a serological survey was carried out in North‐eastern Italy. In a 4‐year period, 637 serum samples were screened using a Mab‐based competitive ELISA for anti‐nucleoprotein A (NPA) antibody detection of influenza viruses. No evidence of anti‐NPA antibodies was observed.

Salmonella Typhimurium phage type DT160 infection in two Moluccan cockatoos (Cacatua moluccensis): clinical presentation and pathology.

Abstract This paper reports on two fatal cases of Salmonella Typhimurium phage type DT160 infection in Moluccan cockatoos (Cacatua moluccensis) from a zoological collection in Italy. No previous clinical signs were observed in birds before death, except for anorexia and mild diarrhea in one bird. At post mortem, necrotic foci surrounded by a hyperemic halo were observed in lungs, heart, liver, spleen, kidneys, and intestine. Microscopically, heterophils and macrophages with rare lymphocyte infiltration associated with gram-negative, rod-shaped bacteria aggregates were detected in necrotic foci. Bacteriology confirmed the presence of Salmonella Typhimurium phage type DT160 in the tissues of birds. The source of Salmonella Typhimurium in these birds remains unknown, but the authors emphasize the need to better control salmonella infections in these avian species because they are important zoonotic agents and responsible for disease in animals and humans. This is the first documentation of Salmonella Typhimurium phage type DT160 infection in Moluccan cockatoos.