Maria Pia Franciosini

Development of a Polymerase Chain Reaction-Based In Vivo Method in the Diagnosis of Subclinical Pigeon Circovirus Infection

Abstract This paper describes a polymerase chain reaction (PCR)-based method performed on blood samples and intestinal content to detect subclinical pigeon circovirus (PiCV) infection in live pigeons. In addition, two sets of primers (primer set 1 and 2), designed in two different regions of the viral genome, were used to provide evidence of possible differences in PCR responses. Blood and intestinal content samples were randomly collected from a total of 50 apparently healthy meat pigeons, aged 1 to 5 wk, which came from central Italy. Samples of primary lymphoid organs were also collected. Results showed a high level of PiCV infection, although clinical signs were not present. The results obtained with the two sets of primers showed that primer set 2 was able to detect a higher number of PCR-positive pigeons (45 of 50 pigeons) than primer set 1 (11 of 50 pigeons). In both cases an increase in positive results with pigeon age indicates that the major direction of transmission is likely horizontal. In these circumstances feces can play an important epidemiologic role, as supported by the consistent circovirus detection in intestinal content. The high sensitivity of this PCR test, which is able to detect very low amounts of viral DNA (5.5 × 10−3 fg of plasmid containing the cloned PiCV genome), makes it suitable for possible application as an epidemiologic tool for identifying virus carriers for subsequent removal from lofts.

Efficacy and safety of an infectious bursal disease virus intermediate vaccine in ovo.

The study was divided into two experiments. In the first experiment, the efficacy of in ovo intermediate vaccine against infectious bursal disease virus (IBDV) was determined by challenge at 21 days of age with virulent IBDV in specific-pathogen-free (SPF) and commercial chickens. This vaccine was able to induce active immunity and to protect SPF chickens to challenge; protection was not complete in commercial chickens, as testified by bursal lesions, bursal index after challenge, and vaccine immunoresponse. In order to detect field and vaccinal viruses, immunoperoxidase staining, enzyme-linked immunosorbent assay, capture, and reverse transcriptase-polymerase chain reaction (RT-PCR) were tested; the RT-PCR was more effective at detecting both kind of viruses. In the second experiment, the immunosuppressive effect of in ovo vaccination was determined by evaluating the immunoresponse against Newcastle disease virus (NDV) vaccination effected at 10 days in both SPF and commercial chickens vaccinated in ovo. The in ovo vaccine causes a reduction of NDV immunoresponse, as testified by lowest geometric mean titer in group I (SPF chickens vaccinated against IBDV in ovo and against NDV at 11 days). In commercial chickens, immunoresponse to NDV vaccination was not influenced by in ovo vaccination.

Detection of Helicobacter spp. in gastric, fecal and saliva samples from swine affected by gastric ulceration.

The aim of this study was to evaluate the presence of Helicobacter (H.) spp. in swine affected by gastric ulceration. Stomachs from 400 regularly slaughtered swine were subjected to gross pathological examination to evaluate the presence of gastric ulcers. Sixty-five samples collected from ulcerated pars esophagea and 15 samples from non-ulcerated pyloric portions were submitted to histopathological and molecular analyses, to detect Helicobacter spp., H. suis and H. pylori by PCR. Feces and saliva swabs were also collected from 25 animals in order to detect in vivo the presence of Helicobacter spp.. Gastric ulcers were detected in 373 cases (93%). The presence of ulcers in association with inflammatory processes was further confirmed by histological examination. Forty-nine percent (32/65) of the ulcerated esophageal portions as well as 53% (8/15) of the non-ulcerated pyloric portions were positive for Helicobacter spp. by PCR. The Helicobacter spp. positive samples were also positive for H. suis, while H. pylori was not detected. These results were confirmed by restriction enzyme analysis. With regard to feces and saliva samples, 15/25 (60%) and 16/25 (64%) were positive for Helicobacter spp. PCR, respectively but all were negative in H. suis and H. pylori specific PCR.

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.

Cloning and expression of pigeon IFN-γ gene

This is the first paper describing the cloning of pigeon IFN-γ gene (PiIFN-γ) and the analysis of the in vitro expressed recombinant protein. The PiIFN-γ gene was identified by RT-PCR as a 498bp, fragment coding for a precursor protein of 165 amino acids instead of 164 amino acids, as observed in the other avian species. The recombinant protein was expressed in vitro by an eukaryotic system and the biological properties of the cytokine were tested using a chicken macrophage cell line. The high degree of amino acid and nucleotide identity, shared with the ChIFN-γ, and the fact that the pigeon protein was functional on chicken cells, indicates a cross-reactivity between pigeon and chicken IFN-γ. The detection of the PiIFN-γ could represent an useful instrument in understanding the role played by this cytokine in immune response related to vaccinations and infectious diseases in the pigeon.

Evaluation of intestinal bacterial flora of conventional and organic broilers using culture-based methods

Abstract The major bacteria colonizing the intestinal tract (ileum and caecum) of organic (O) and conventional (C) chickens were counted, isolated and identified by conventional methods. Chickens were obtained from 7 conventional and 7 organic chicken farms (n=203). Intestinal sampling was performed at different ages, every 10 days, starting at 20 days until 40 and from 20 days to 80 days of age, respectively, for conventional and organic birds. Statistical analysis was performed on two separate data sets (40 days of age and all ages).The comparison of C vs O systems was analyzed with univariate and multivariate procedures. There were large differences in bacterial counts in relation to the portion of intestine, the rearing system and the farms. In the ileum of conventional birds Enterobacteria were higher than in organic birds (7.03 vs 6.09 CFUxlog/g; P<0.05), whereas the contrary was observed for Lactobacilli (6.75 vs 7.07 CFUxlog/g; P<0.05). With respect to the other microflora, the effect of farm probably masked possible differences. The effect of rearing system was more visible in the caecum than in the ileum: Enterobacteria levels were higher in C than in O chickens (7.42 vs 7.05 CFUxlog/g; P<0.01), whereas Enterococci (7.65 vs 6.55 CFUxlog/g; P<0.05), Lactobacilli (7.85 vs 7.31 CFUxlog/g; P<0.05) and total aerobia (8.12 vs 7.66 CFUxlog/g; P<0.01) counts were higher in organic chickens. Multivariate analysis of caecum microflora showed the possibility of discriminating the rearing system. In the ileum of conventional birds Enterobacteria and total aerobia increased with age, while Lactobacilli decreased. In the O system, Enterobacteria, Lactobacilli and total anaerobia showed a similar trend, whereas total aerobia and Enterococci showed the opposite trend. A similar situation was observed in the caecum. Further investigations are necessary to better assess the role and effect of the enteric flora on the productive performance and on the health status of reared chickens.

Genetic diversity and antimicrobial resistance profiles of Campylobacter coli and Campylobacter jejuni isolated from broiler chicken in farms and at time of slaughter in central Italy

Genetic diversity and antimicrobial resistance of Campylobacter coli and Campylobacter jejuni were investigated along the broiler chicken production chain in central Italy.

Effects of oregano (Origanum vulgareL.) and rosemary (Rosmarinus officinalis L.) aqueous extracts on broiler performance, immune function and intestinal microbial population

ABSTRACT A 57-day study was performed to determine the effects of two aqueous extracts (AEs) on broiler performance, immune function and intestinal microflora. Four groups of 75 one-day-old female broilers (Ross308) received one of the following treatments: (1) a standard commercial feed (C); (2) C supplemented with 2 g/kg rosemary AE (R); (3) C supplemented with 2 g/kg oregano AE (O); (4) C supplemented with 1 g/kg oregano AE + 1 g/kg rosemary AE (OR). Individual body weight, average daily gain, average daily feed intake and feed conversion efficiency were determined at 1, 11, 22, 36 and 57 days. Sample collections for IgG titration and intestinal microflora examination were performed at 22 and 57 days. The addition of oregano AE alone or in combination with rosemary AEs improved body weight up to 36 days of age (P < .01). A time effect was recorded for total serum IgG in all groups (P < .001) and the percentage increase of the value was positively (P < .05) influenced by the AE supplementation. Lactobacilli raised (P < .001) in ileum and cecum of all groups supplemented with AEs. Staphylococcus spp. population was constantly lower in both intestinal tracts of the AE supplemented groups. On the basis of our results, AEs could improve broiler performance and immune function and contribute to a balanced gut microflora, essential for the digestion process and protection against enteropathogenic organisms.

Evidence for a primate origin of zoonotic Helicobacter suis colonizing domesticated pigs

Helicobacter suis is the second most prevalent Helicobacter species in the stomach of humans suffering from gastric disease. This bacterium mainly inhabits the stomach of domesticated pigs, in which it causes gastric disease, but it appears to be absent in wild boars. Interestingly, it also colonizes the stomach of asymptomatic rhesus and cynomolgus monkeys. The origin of modern human-, pig- or non-human primate-associated H. suis strains in these respective host populations was hitherto unknown. Here we show that H. suis in pigs possibly originates from non-human primates. Our data suggest that a host jump from macaques to pigs happened between 100 000 and 15 000 years ago and that pig domestication has had a significant impact on the spread of H. suis in the pig population, from where this pathogen occasionally infects humans. Thus, in contrast to our expectations, H. suis appears to have evolved in its main host in a completely different way than its close relative Helicobacter pylori in humans.

Investigation on intestinal bacterial flora and Salmonella spp. presence in organic and conventional chickens

Abstract The aim of this study was to evaluate the possible differences in the intestinal microflora composition among the different rearing systems (conventional vs organic) and the Salmonella diffusion using bacteriological techniques. The results showed that the differences between the two groups at the same age, expressed by the bacterial count, are not conclusive in showing an influence of the rearing systems. Salmonella Hadar was isolated once in caeca of conventional and once in caeca of organic ones. Though the results are preliminary and referred to a well defined geographic area in Central Italy, Salmonella detection does not seem to be common in conventional and organic chicken farms.