Detection of environmental contamination with feline and canine parvoviruses: new perspectives and challenges.

AIMS: To develop a protocol for environmental sampling to detect parvoviruses of dogs and cats in the environment. METHODS AND RESULTS: Environmental contamination was carried out using different dilutions of parvovirus-contaminated materials; further field samplings were performed in areas in which clinical cases of parvovirus infections were present. Sterile cotton swabs and sponges for microbial surface sampling were used. Viruses were detected in these samples with different methods: conventional PCR, nested PCR and real-time PCR, detecting viral DNA; virus isolation, detecting infectious virus; and a commercial rapid enzyme immunoassay, detecting viral antigen. No substantial differences were observed in the two sampling methods, although the sponge was more convenient for sampling rough surfaces. Molecular assays were the most sensitive methods, identifying even very low amounts of viral DNA (up to 10 copies of viral DNA/10 µl of sample). Virus isolation and the rapid test detected the viruses only at the highest viral concentrations, both in the experimental setting and field conditions. CONCLUSIONS: Environmental sampling and molecular protocols were effective in detecting environmental contamination with parvoviruses. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol will be useful to identify possible sources of infection and to assess the efficacy of disinfection protocols in the environment.

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.

AIMS: Genetic diversity and antimicrobial resistance of Campylobacter coli and Campylobacter jejuni were investigated along the broiler chicken production chain in central Italy. METHODS AND RESULTS: Campylobacter sp. isolated from cloacal swabs in farms (n = 116) and from the neck skin of chilled and eviscerated carcasses at slaughter (n = 24) were identified as C. coli (n = 99) and C. jejuni (n = 41) by multiplex PCR. Characterization by single amplified fragment length polymorphism (s-AFLP) revealed a specific genotype of Campylobacter for each farm. Minimal inhibitory concentration showed high prevalence of fluoroquinolones (70%), tetracycline (70%) and erythromycin (30%) resistance among C. coli isolates. Campylobacter jejuni isolates showed lower prevalence of fluoroquinolone (39%) and tetracycline (10%) resistance, and all isolates were susceptible to erythromycin. The S-AFLP types of the C. coli and C. jejuni isolates were associated with their antimicrobial resistance profiles (P < 0·001). CONCLUSIONS: The genetic diversity detected in Campylobacter isolates suggested that a specific genotype was harboured in each farm. A considerable number of C. coli isolates were resistant to erythromycin. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter coli was detected more frequently than C. jejuni in contrast to common findings for poultry. The high prevalence of 30% resistance to erythromycin in C. coli strains isolated from poultry is worrisome, as this is the first antibiotic of choice to treat human campylobacteriosis.

Gut complex carbohydrates and intestinal microflora in broiler chickens fed with oregano (Origanum vulgare L.) aqueous extract and vitamin E.

One hundred and seventy one-day-old female broiler chicks were randomly divided into three groups fed with different dietary treatments: basal control diet (C); C supplemented (2 g/kg) with an oregano aqueous extract (O); C supplemented (150 mg/kg) with vitamin E (E). Growth performance was evaluated at 21 (T1) and 42 days (T2). On the same days, morphological, histochemical and microbiological analyses were performed. The O group showed the highest (p < 0.01) body weight at T1, while no differences were observed at T2. Light microscopic observation and conventional histochemistry showed no differences with regard to the two sampling times, whereas significant differences emerged among the treatments. The O treatment generally enhanced goblet cell reactivity more than both the C and E treatments. Coliform count was lower in the ileum tract of the O group at both T1 and T2 (p < 0.05) and increased with age in all groups. Escherichia coli showed the lowest values in the caecum of the O group (p < 0.001) at both sampling times. Enterococci, lactobacilli and staphylococci populations showed no differences among the different experimental groups in the caecum. In the ileum, the O group did not exhibit the sharp decline (p < 0.001) in the lactic acid bacteria population observed in the other two experimental groups. In conclusion, oregano aqueous extract supplementation seemed to elicit the best response among treatments, enabling better growth performance, enhancing both the quantity and quality of glycoconjugates involved in indirect defence actions and significantly reducing both the coliform and E. coli counts.

Effects of dietary Lactobacillus acidophilus and Bacillus subtilis on laying performance, egg quality, blood biochemistry and immune response of organic laying hens.

The objective of this study was to evaluate the effects of two different probiotic micro-organisms on the performance, egg quality and blood parameters of organically reared hens. A total of 900 16-week-old Hy-Line layer hybrids were randomly assigned to three groups of 300 birds each. The control (CTR) group was fed a corn-soya bean cake-based diet; the L group was fed the same diet supplemented with 0.1% Lactobacillus acidophilus, while the B group was fed the same diet supplemented with 0.05% Bacillus subtilis. Data were recorded at the beginning (weeks 5 and 6: T1) and at the end (weeks 19 and 20: T2) of the experiment, and no differences in hen performance were recorded between dietary groups or sampling times. All of the investigated clinical chemistry parameters, except GGT, were affected by diet (p < 0.05), with the best results recorded for the probiotic-treated groups. The immune-response values showed higher blood bactericidal activity in the B and L groups at T2 (p < 0.05) and a lower lysozime concentration in the B group at T1. Higher antibody production against Newcastle disease virus was observed in the L group compared to the CTR (p = 0.013). No differences in oxidative status were recorded, and no effects of diet on egg quality were observed. Among the physical egg characteristics, only the Roche scale colour was affected by diet (p < 0.05): the egg yolk was paler in the L group. The age of the hen was the most relevant factor affecting physical egg characteristics. The chemical parameters of the egg were almost unaffected by supplementation with probiotics except for the lipid content, which decreased with the L diet (p < 0.05). Both probiotic inclusions had beneficial effects on hen metabolism and welfare, and L. acidophilus induced the best immune response.

Microbiological and parasitological survey of zoonotic agents in apparently healthy feral pigeons.

Microbiological and parasitological investigation was carried out on a colony of feral pigeons, located in a green area near the main hospital of a Central Italy city. One hundred pigeons were submitted to clinical examination. Cloacal swabs, grouped in pool of 4 samples, were analyzed to detect the presence of Coxiella burnetii, Chlamydia psittaci, Chlamydophila spp. using a biomolecular procedure, while individual cloacal samples were examined for Salmonella spp., Campylobacter spp., and yeasts by means of a specific culture media. An ELISA test was used to determine the presence of Giardia spp., and Cryptosporidium spp. coproantigens. Individual serological samples were also tested with the modified agglutination test (MAT) in order to detect antibodies against Toxoplasma gondii. The pigeons did not show any clinical signs. The cloacal pools proved to be negative for C. burnetii DNA while three pools were positive for C. psittaci or Chlamydophila spp. DNAs. Salmonella spp. was not detected. C. jejuni and C. coli were found in 13% and 4% of the samples, respectively. No Giardia spp. and Cryptosporidium spp. were detected. Thirty-three out of 100 samples (33%) were positive for yeast colonies. The seroprevalence for T. gondii was 8%. Although with moderate incidence, potentially zoonotic agents were present thus highlighting the need for sanitary surveillance on feral pigeon colonies.

Are Italian-Polish veterinarians and breeders prepared to control an outbreak of Brucella canis infection in dogs?

Brucella canis infection is one of the most important causes of infertility in dogs and is a zoonosis for which no effective treatment or vaccines exist. It is not a mandatory notifiable disease. Following an increase of cases in Europe and worldwide, an investigation was performed to evaluate how much Italian and Polish veterinarians and breeders know about canine brucellosis and understand their perceptions of this infection. For this reason, two questionnaires were prepared, in Italian and Polish. Eighteen Italian and Polish veterinarians, specialists in canine reproduction, responded to the first survey and 44.4% of them affirmed having diagnosed canine brucellosis at least once in their clinical practice, and different perceptions emerged regarding the infection in the two countries. The second survey was completed by 145 Italian and Polish breeders; the disease was completely unknown to 22.8% of them, whereas 2.1% had diagnosed infection by B. canis in their kennels. In conclusion, knowledge of B. canis infection differs between these countries, with extremes ranging from diagnosed cases to complete underestimation of the presence of the problem. However, based on international data and reporting of a recent large outbreak in Italy, awareness of this contagious infectious disease and its management must be increased.

Influence of different rearing systems on natural immune parameters in broiler turkeys.

The aim of this study was to determine serological values of lysozyme, hemolytic complement levels (alternative pathway), and bactericidal activity of serum in turkeys kept in different rearing systems (industrial, backyard, and experimental). Results showed that the values for serum bactericidal activity and hemolytic complement levels increased with age, and their values were higher in experimental and in industrial turkeys than in turkeys reared in backyard. Lysozyme concentration showed a similar pattern; its value was higher in the industrial and experimental groups than in the backyard group. Data obtained suggest that rearing system can have an influence on the natural immune parameters considered; experimental and industrial groups showed a similar trend, differentiated from that observed in the backyard group. In the backyard group, the values observed may suggest that hybrid turkeys, selected for high production, have difficulty with being reared outside where predators (foxes and weasels) and weather conditions could be responsible for a stress situation.

Sequence comparison of pigeon circoviruses.

The genome sequences of eight pigeon circoviruses (PiCV) were determined and compared with four previously published sequences. The viruses compared were from the USA, five European countries, China and Australia and included PiCVs from racing, feral, ornamental and meat pigeons and a Senegal dove (Streptopelia senegalensis). The 12 PiCV genomes, ranging from 2032 to 2040 nucleotides in length, displayed similar organizations. Pairwise comparisons showed that the genome nucleotide sequence identities ranged from 85.1% to 97.8% and that the amino acid identities of the putative replication associated (Rep) and putative capsid (Cap) proteins displayed ranges of 91.5-99.1% and 73.0-99.3%, respectively. Comparative analyses identified conserved nucleotide sequences within the Rep gene and 3' intergenic regions, which would be suitable for diagnostic PCR primers, and variable amino acid sequences within the capsid proteins, which should be considered when selecting virus isolates for vaccine development.

Occurrence and antimicrobial susceptibility of Campylobacter spp. on fresh and refrigerated chicken meat products in Central Italy.

This study investigated the presence and the level of Campylobacter spp. contamination in 41 thigh samples (with skin) and 37 skinless breast samples collected at the end of slaughter (T1) and after 10 day period at refrigeration temperature (4°C) (T2), corresponding to their commercial shelf life. The isolates were phenotypically classified as Campylobacter spp. and successively identified by conventional multiplex PCR. The antimicrobial susceptibility of the isolates from fresh thigh and breast samples was also determined via the microdilution method (MIC) in Eucamp microtitre plates with known scalar concentrations of: gentamicin (GEN), streptomycin (ST), ciprofloxacin (CIP), tetracycline (TET), erythromycin (ERY), and nalidixic acid (NA). A greater percentage of positivity for Campylobacter spp. (P < 0.001) was observed in thighs and C.jejuni appeared to be the most common species identified at this level (P < 0.001) followed from its association with C.coli. There was a global reduction of Campylobacter spp. in both thigh and breast samples at T2 (P < 0.001) showing that the refrigeration was able to reduce Campylobacter count. The prevalence of resistance to CIP, TET, NA, and ERY was evidenced for C.jejuni and C. coli. The co (TET-NA, CIP-NA) and multiple resistant (CIP-TET-NA, CIP-TET- NA-ERY) isolates came from the thigh products. It should be highlighted the presence of Campylobacter spp. isolates resistant to ST occurred in breast samples, responsible for the ST-CIP co-resistance and ST-CIP-TE multi-resistance profiles, higher in breast than in thigh products (P > 0.001). The presence of Campylobacter isolates resistant to ST can be further investigated since it is used for therapeutic treatment of several bacterial diseases in humans.

Effects of two different probiotics on microflora, morphology, and morphometry of gut in organic laying hens.

The current study investigated the effects of Lactobacillus acidophilus and Bacillus subtilis, used as probiotics, on the microflora, morphology, and morphometry of the gut in organic laying hens. The birds (180 Hy-Line laying hens) were divided into 3 homogenous groups and received a pre-deposition diet from 16 to 20 wk of age and a deposition diet for the remaining 7 months of the experiment. The control group ( CTR: ) was fed a corn-soybean cake-based diet, the second group ( L: ) received the same diet supplemented with 0.1% of L. acidophilus while in the third group ( B: ) the basal diet was supplemented with 0.05% of B. subtilis At 18 wk of age ( T1: ) and at 5 ( T2: ) and 7 months ( T3: ) from the beginning of deposition, 9 subjects per group were humanely killed for microbiological, morphological and morphometric analyses of the intestinal tract. The 2 probiotic-supplemented diets increased Lactobacillus spp. and Bifidobacterium spp. counts compared with the CTR diet. The lowest viable counts of E. coli, coliforms and staphylococci were observed in the L group (P < 0.001). Clostridium spp. decreased (P < 0.001) in both L and B subjects. The probiotic supplementation appeared to affect the intestinal microbial population, promoting the presence of beneficial bacteria such as Lactobacillus spp. and Bifidobacterium spp. and reducing potential harmful bacteria such as E. coli, clostridia and staphylococci. Morphological and morphometric analyses did not reveal substantial differences among groups. At T3, the plasma cell infiltrate in the villi of the CTR hens was more severe than that observed in the L and B groups (P = 0.009).

Development of a polymerase chain reaction-based in vivo method in the diagnosis of subclinical pigeon circovirus infection.

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 x 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.

Atrophy of the primary lymphoid organs of meat pigeons in Italy associated with circoviruslike particles in the bursa of Fabricius.

During a survey effected in a meat pigeon slaughterhouse of central Italy, atrophy of primary lymphoid organs (bursa of Fabricius and thymus) and hypoplasia of bone marrow were observed. Histologic, ultrastructural, and hematologic examinations were performed on a total of 80 randomly selected 30-day-old meat pigeons. By histologic studies, lymphocytic depletion and necrosis with cyst formations in the bursa of Fabricius were detected in all subjects that showed thymus and bursa atrophy at necropsy. Basophilic intranuclear inclusions were also observed in bursal cells. After ultrastructural studies, these inclusions were proved to be viral particles resembling circoviruslike particles in morphology and size. Severe lymphocytic depletion of the bursa was plausibly associated with the presence of the viral particles.

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.

Diagnosis of duck circovirus infections by conventional and real-time polymerase chain reaction tests.

Development of the first conventional and real-time polymerase chain reaction (PCR) tests for the diagnoses of duck circovirus (DuCV) infections is described. Both tests amplified a 230 bp fragment specific to the DuCV Rep gene. Although both tests had the same detection limit (13 x 10(3) target DNA/ml) when the target DNA was diluted in water, the detection limit of the real-time test (13 x 10(4) target DNA/ml) was 10-fold less than the conventional test (13 x 10(5) target DNA/ml) when the amplifications were performed in the presence of cellular DNA. Using the conventional PCR test, DuCV DNA was detected in 85 (84%) of 101 bursa of Fabricius samples from dead or sick ducks, aged between 1 and 12 weeks, and in samples from 35 (94%) of 37 flocks. Application of the SYBR Green-based real-time PCR test to 54 selected bursa of Fabricius samples indicated that more samples were positive by real-time PCR than by conventional PCR, allowed the numbers of genome copies to be estimated and showed that some bursa of Fabricius samples contained over 10(13) genome copies/g tissue. Although DuCV infections were detected in birds aged from 1 to 12 weeks, higher virus DNA levels were detected in ducks aged older than 5 weeks than in ducks younger than 5 weeks. An in situ hybridization method for the detection of DuCV in histological samples was also developed. Additional work is required to determine the clinicopathological significance of DuCV infections.

Use of an experimental chicks model for paratuberculosis enteritis (Johne's disease).

Developments in the diagnosis and treatment of paratuberculosis is constrained by the lack of an experimental animal model. To investigate this problem conventional chicks immunodepressed by a Cyclophosphamide injection and concurrent inoculation of Infectious Bursal Disease Virus (IBDV) were infected with Mycobacterium paratuberculosis and kept for 4 months. The immunodepressed chicks eliminated mycobacteria with their faces from the first month until the third month and developed typical intestinal lesions of mycobacterial infection characterized by aggregation of macrophages with monocytes and lymphocytes. Diarrhoea was absent. The number of lymphocytes decreased by about 80%. The serological tests carried out with Complement Fixation test were negative. For the positive bacteriology and typical granulomatous lesions, the conventionally reared chicks proved to be a useful laboratory model for reproduction of Johne's disease.

Naturally occurring nephroblastomas in light meat broilers.

Twenty-seven cases of nephroblastoma were observed in 54 culled light meat broilers during a 1-year period. The tumours were generally unilateral and varied in size. Metastases were not observed. Histopathological studies showed epithelial and mesenchymal structures at different stages of development. Ultramicroscopy and immunohistochemistry findings revealed viral particles in intercellular spaces and in the lumen of the tubules not morphologically unlike avian leukosis/sarcoma viruses.