Crossbreeding impacts on caecal bacterial count and antibody titres in chickens.

1. A crossbreeding experiment was conducted using 20 sires and 120 dams from each Fayoumi (F) and Rhode Island Red (R) breeds. The F1 chicks of ½F½R and their reciprocal ½R½F crosses were used to estimate direct, maternal and heterotic effects.2. The caecal bacterial counts of Salmonella typhimurium and Enterococcus faecium and the IgA, IgG and IgM antibody titres were determined. Chicks of each genetic group (120 birds) were divided into three groups (40 birds per group) and chicks of the first group were assigned as the control group, while the second group was inoculated with S. typhimurium and E. faecium at 7 and 10 d of age (106 cfu/chick) and the third was inoculated with S. typhimurium at 10 d of age (106 cfu/chick).3. The generalised least square procedure was used to estimate cross-breeding effects. The S. typhimurium counts in the R breed was highly significant, followed by ½F½R cross, ½R½F cross, and F breed. The differences among genetic groups in E. faecium counts and caecal pH was not significant. The estimates of IgA, IgG, and IgM antibody titres were highly significant in the F breed, followed by ½R½F cross, ½F½R cross, and the R breed.4. The estimates of direct additive effects were highly significant F breed for the counts of S. typhimurium and antibody titres of IgA, IgG, and IgM. The maternal effects were highly significant F breed for S. typhimurium, E. faecium count, and antibody titres of IgA, IgG, and IgM. Estimates of direct heterosis were significant for S. typhimurium count and IgA antibody titre but not significant for E. faecium count, caecal pH, and antibody titres of IgG and IgM.5. The results showed that the Fayoumi breed could be used in breeding programmes to improve immunity traits against S. typhimurium in chickens.

Polymorphic characterisation of gallinacin candidate genes and their molecular associations with growth and immunity traits in chickens.

1.Four gallinacin (GAL) genes were assessed by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) to characterise these genes in Fayoumi (F) and Rhode Island Red (R) breeds and their crosses of Rhode Island Red × Fayoumi (½R½F) and Fayoumi × Rhode Island Red (½F½R).2.Genes examined were GAL2, GAL3, GAL4 and GAL5. The molecular associations between the SNPs of the gallinacin genes and body weight, caecal bacterial count and the serum antibody titres of IgA, IgG and IgM were determined. In the R breed, the frequency of TC genotype was higher than TT and CC genotypes for the GAL3 gene. The GG genotype frequency was higher than AA and AG genotypes for the GAL4 gene in the other genetic groups, and the CA genotype frequency was higher than CC and AA genotypes in crosses for the GAL5 gene.3.In all populations, the frequency of the C allele was higher than the T allele for the GAL3 gene, the G allele was higher than the A allele for the GAL4 gene and the C allele was higher than the A allele for the GAL5 gene. The observed heterozygosity in R, ½R½F and ½F½R was 0.476, 0.375 and 0.158 for the GAL3 gene, 0.458, 0.615 and 0.250 for the GAL4 gene and 0.053, 0.792 and 0.739 for the GAL5 gene, while the expected heterozygosities were 0.490, 0.430 and 0.145 for the GAL3 gene, 0.430, 0.348 and 0.219 for the GAL4 gene and 0.229, 0.478 and 0.496 for the GAL5 gene, respectively.4.On a molecular level, the genotype TT was significantly higher for body weight than TC and CC genotypes in the GAL3 gene. Birds with the GG genotype had a significantly lower Salmonella typhimurium count than birds with AA genotype in the GAL4 gene. Birds with the genotype AA had higher significant body weights than those with CC and CA genotypes in the GAL5 gene.5.The results indicated that the GAL3, GAL4 and GAL5 genes are potential candidates for selection programmes to improve S. typhimurium resistance and body weight in chickens.

Circulation dynamics of Salmonella enterica subsp. enterica ser. Gallinarum biovar Gallinarum in a poultry farm infested by Dermanyssus gallinae.

Dermanyssus gallinae (Mesostigmata: Dermanyssidae, De Geer, 1778) is an ectoparasite of poultry, suspected to play a role as a vector of Salmonella enterica subsp. enterica ser. Gallinarum. Despite an association between them being reported, the actual dynamics in field remain unclear. Therefore, the present study aimed to confirm the interactions among mites, pathogen and chickens. The study was carried out in an industrial poultry farm infested by D. gallinae, during an outbreak of fowl typhoid. The presence of S. Gallinarum in mites was assessed and quantified by a semi-nested polymerase chain reaction (PCR) and real-time PCR, respectively, in mites collected during two subsequent productive cycles and the sanitary break. The anti-group D Salmonella antibodies were quantified by an enzyme-linked immunosorbent assay. During the outbreak and the sanitary break, S. Gallinarum was constantly present in mites. In the second cycle, scattered positivity was observed, although hens did not exhibit signs of fowl typhoid, as a result of the vaccination with BIO-VAC SGP695 (Fatro, Ozzano Emilia Bo, Italy). The data strongly suggest that D. gallinae acts as reservoir of S. Gallinarum, thus allowing the pathogen to persist in farms. Furthermore, the present study has highlighted the interactions among D. gallinae, S. Gallinarum and hens with respect to enhancing the mite-mediated circulation of S. Gallinarum in an infested poultry farm.

Efficacy of a novel neem oil formulation (RP03™) to control the poultry red mite Dermanyssus gallinae.

Dermanyssus gallinae (Mesostigmata: Dermanyssidae) is the most harmful ectoparasite of laying hens, represents an occupational hazard for poultry workers, and a growing threat to medical science per se. There is increasing demand for alternative products, including plant-derived acaricides, with which to control the mite. The present study investigated the efficacy of neem oil against D. gallinae on a heavily infested commercial laying hen farm. A novel formulation of 20% neem oil, diluted from a 2400-p.p.m. azadirachtin-concentrated stock (RP03™), was administered by nebulization three times in 1 week. Using corrugated cardboard traps, mite density was monitored before, during and after treatment and results were statistically analysed. Mite populations in the treated block showed 94.65%, 99.64% and 99.80% reductions after the first, second and third product administrations, respectively. The rate of reduction of the mite population was significantly higher in the treated block (P < 0.001) compared with the control and buffer blocks. The results suggest the strong bioactivity of neem, and specifically of the patented neem-based formulation RP03™, against D. gallinae. The treatment was most effective in the 10 days following the first application and its effects persisted for over 2 months. Further studies will aim to overcome observed side effects of treatment represented by an oily layer on equipment and eggs.

Molecular characterization and phylogenetic inferences of Dermanyssus gallinae isolates in Italy within an European framework.

In order to investigate the genetic relationships between Dermanyssus gallinae (Metastigmata: Dermanyssidae) (de Geer) isolates from poultry farms in Italy and other European countries, phylogenetic analysis was performed using a portion of the cytochrome c oxidase subunit 1 (cox1) gene of the mitochondrial DNA and the internal transcribed spacers (ITS1+5.8S+ITS2) of the ribosomal DNA. A total of 360 cox1 sequences and 360 ITS+ sequences were obtained from mites collected on 24 different poultry farms in 10 different regions of Northern and Southern Italy. Phylogenetic analysis of the cox1 sequences resulted in the clustering of two groups (A and B), whereas phylogenetic analysis of the ITS+ resulted in largely unresolved clusters. Knowledge of the genetic make-up of mite populations within countries, together with comparative analyses of D. gallinae isolates from different countries, will provide better understanding of the population dynamics of D. gallinae. This will also allow the identification of genetic markers of emerging acaricide resistance and the development of alternative strategies for the prevention and treatment of infestations.

In field evaluation of impact on clinical signs of an inactivated autogenous vaccine against Pasteurella multocida in rabbits.

In Italy, the use of autogenous inactivated vaccines prepared with the bacterial strains isolated from affected animals is authorized by the Ministry of Health in farms where bacterial diseases occur frequently. The autogenous vaccine performed using Pasteurella multocida is frequently used in rabbit farms, but the feedback of its application is not available. Therefore, the aim of this study is to give information about the impact on the clinical signs of a bivalent autogenous vaccine in rabbits of a genetic centre. The vaccine was prepared using two P. multocida strains belonging to serogroups A and F, equipped with virulence genes and responsible for cyclical outbreak of pasteurellosis in the farm. The vaccine was administered with a first injection, followed by another one after 15 days, then another one four months after the first injection, and then continuing with a further injection every six months to all rabbits. Clinical conditions and mortality rates were monitored for two years after the first vaccination. The improvement in clinical condition and the decrease of the mortality rate were significant especially in the first year post-vaccine. In addition, the number of animals removed due to the disease decreased greatly. Based on the finding of P. multocida strains belonging to serogroup D and serogroup A equipped with different virulence-gene patterns from those previously found, we suggest that the vaccine was unable to prevent the introduction and spreading of new strains among the rabbits.

Virulence-associated genes in avian pathogenic Escherichia coli from laying hens in Apulia, Southern Italy.

1. Escherichia coli isolated from lesions (Avian Pathogenic E. coli - APEC) of layer hens affected by colibacillosis and from intestinal contents of clinically-healthy birds (Avian Faecal E. coli - AFEC) were serotyped. All the isolates were investigated for the presence of virulence genes to determine which genes were more closely related to those from lesions. 2. A number of different serogroups were detected, O78 being predominant among the isolates from colibacillosis. 3. E. coli isolated from lesions were not linked to a specific pathotype (set of common virulence genes). 4. The presence of the virulence genes, with the exception of astA, was associated more generally with APEC strains. 5. Statistically, genes such as cva/cvi, tsh, iss, irp2 and iucD were more related to isolates from colibacillosis. 6. It is suggested that the detection of these genes in a rapid and inexpensive test for field practitioners could provide useful information about the potential virulence of E. coli isolated in commercial layer flocks.

Chlamydia psittaci infection in canaries heavily infested by Dermanyssus gallinae.

Dermanyssus gallinae is a haematophagous ectoparasite responsible for anemia, weight loss, dermatitis and a decrease in egg production. Dermanyssus gallinae may play a role in the modulation of the host immune system, maybe predisposing the host to some bacterial infections such as chlamydiosis. This is an important zoonosis. Humans are exposed to Chlamydia psittaci through inhalation of the agent dispersed from the infected birds. In this study, a syndrome observed in an aviary of canaries was investigated. A heavy infestation by D. gallinae was reported. Simultaneously, a C. psittaci infection was molecularly confirmed in the canaries. Combined therapy was applied successfully. The association of C. psittaci with the examined mites has been confirmed. Therefore, we think that D. gallinae have played a role in the spreading of C. psittaci infection among the canaries. Moreover, D. gallinae could have played an important role predisposing the canaries to the development of chlamydiosis, by inducing anemia and debilitation. The control of mites in the aviaries may represent a crucial step for the prevention of important infection such as chlamydiosis in birds and humans.

Epidemiology and risk factors for dermatophytoses in rabbit farms.

While dermatophytoses of several animal species have been extensively investigated, information on their occurrence and epidemiology in rabbits is limited. We carried out a study from October 2006 to February 2007 of 23 rabbit farms in Apulia and Basilicata regions (southern Italy) in order to investigate the occurrence and risk factors associated with dermatophytoses in breeding rabbits. Dermatophytes were isolated from 86.9% (20/23) of the farms and from 51.8% (420/810) of the animals sampled. Trichophyton mentagrophytes (47.9%) and Microsporum canis (3.2%) were isolated from diseased (71.7%) and healthy (48.4%) animals as well from the surrounding environment (7.5%). The occurrence of lesions, the age of rabbits, and farm management (e.g., temperature, humidity and methods and frequency of disinfection practices) were identified as the most significant risk factors (P < 0.05) for the occurrence of dermatophytes. Animals in fattening and finishing stages were the most frequently infected (i.e., 58.2 and 61.6% respectively). Dermatophyte prevalence was significantly (P < 0.05) higher in areas with higher temperature (>20°C) and relative humidity ranging from 62-65%. The results of the present investigation suggest that zoonotic dermathophytes are present in rabbit farms and highlight the importance of correct management procedures for the control of the infections.

Evaluation of the poultry red mite, Dermanyssus gallinae (Acari: Dermanyssidae) susceptibility to some acaricides in field populations from Italy.

Red mite field populations from seven naturally infested Italian caged laying poultry farms were investigated for their susceptibility to acaricide formulations available on the market, containing amitraz, carbaryl and permethrin. A minimum of 3,000 mites of all stages were collected from each farm and were tested with five acaricide concentrations (5, 10, 20, 50, 100%) plus an untreated control (0%). Field red mite populations were found to be tolerant even with the highest concentrations with carbaryl and permethrin for six (86%) and three (42%) of the investigated farms, respectively (P < 0.05). Furthermore, six (86%) of the investigated farms showed a red mite population susceptible to amitraz at any concentration. Out of the seven field populations tested with amitraz, one population is becoming less tolerant whereas another was the most tolerant to carbaryl and permethrin at any concentration. Data show that the lack of effectiveness of some acaricides is spreading in Europe and call for the adoption of alternative management strategies to avoid development of resistance.

Phylogenetic relationship between Dermanyssus gallinae populations in European countries based on mitochondrial COI gene sequences.

Phylogenetic analysis of Dermanyssus gallinae mites originating from UK, France and Italy was performed using partial amplification of the mitochondrial COI gene. Results showed that UK samples reveal the greatest variation and diversity and are linked to one of the French populations highlighting North-South genetic transitions in European red mite populations. Intra-farm variations between mite samples highlighted the diversity between national populations and possibly its origin from the different chemical strategies used in each country.

Polyomavirus Infection in Gouldian Finches (Erythrura gouldiae) and Other Pet Birds of the Family Estrildidae.

A syndrome characterized by apathy, diarrhoea and high mortality of nestlings was observed in a flock of pet birds of the family Estrildidae. Enlargement of the liver, pulmonary congestion and urate accretions in the kidney were observed. Microscopically, there was glomerular atrophy, oedema and congestion of the lungs and necrosis and fibrosis of the liver. Cowdry type B intranuclear inclusion bodies were detected in the tissues. Polyomavirus was detected by polymerase chain reaction. The entire genome of the virus was amplified and sequenced, revealing 99 % identity to the sequence of finch polyomavirus isolated from the Eurasian bullfinch (family Fringillidae).

Lapine rotaviruses of the genotype P[22] are widespread in Italian rabbitries.

An epidemiological survey was carried out to investigate the distribution of the VP7 and VP4 specificities of lapine rotaviruses (LRVs) in rabbitries from different geographical regions of Italy. Almost all the strains were characterized as P[22],G3, confirming the presence of the newly-recognized rotavirus P[22] VP4 allele in Italian rabbits. Only one P[14],G3 LRV strain was identified and two samples contained a mixed (P[14] + [22],G3) rotavirus infection. All the LRV strains analyzed exhibited a genogroup I VP6 specificity and a long dsRNA electropherotype. However, one of the P[14],G3 strains possessed a super-short pattern. Altogether, these data highlight the epidemiological relevance of the P[22] LRVs in Italian rabbitries.

Genotyping Campylobacter jejuni strains isolated from the gut and oviduct of laying hens.

Campylobacter jejuni frequently colonizes the avian intestine. Recent evidence suggests that this organism can also colonize the oviduct of laying hens. However, the source and role of this colonization are unknown. Isolates from the ceca, cloacae, and oviducts of 11 laying hens in three intensive egg-producing flocks were genotyped by Fla typing with the restriction fragment length polymorphism of the polymerase chain reaction product of the flaA and flaB genes (fla typing) and pulsed-field gel electrophoresis (PFGE). A diversity in fla types and PFGE types was observed within and between flocks. Individual birds could be colonized by different genotypes at various intestinal and oviduct sites. However, the oviduct of individual birds appeared to be colonized by only one genotype at the time of sampling. In two birds, matching isolates investigated from the intestinal and reproductive tracts were genotypically identical but different from those oviduct isolates found in other birds in the same flock. Interestingly, not all cecal isolates appeared to be equally able to colonize the oviduct. These results suggest that oviduct colonization may result from ascending infection via the cloaca and that some strains of C. jejuni may be better adapted than others to oviduct colonization.

Detection of the new emerging rabbit haemorrhagic disease type 2 virus (RHDV2) in Sicily from rabbit (Oryctolagus cuniculus) and Italian hare (Lepus corsicanus).

Rabbit haemorrhagic disease virus (RHDV), a member of the genus Lagovirus, causes rabbit haemorrhagic disease (RHD), a fatal hepatitis of rabbits, not previously reported in hares. Recently, a new RHDV-related virus emerged, called RHDV2. This lagovirus can cause RHD in rabbits and disease and mortality in Lepus capensis (Cape hare). Here we describe a case of RHDV2 infection in another hare species, Lepus corsicanus, during a concurrent RHD outbreak in a group of wild rabbits. The same RHDV2 strain infected rabbits and a hare, also causing a RHD-like syndrome in the latter. Our findings confirmed the capability of RHDV2 to infect hosts other than rabbits and improve the knowledge about the epidemiology and the host range of this new lagovirus.

Characterization of Dermanyssus gallinae (Acarina: Dermanissydae) by sequence analysis of the ribosomal internal transcribed spacer regions.

In the present work mites previously identified as Dermanyssus gallinae De Geer (Acari, Mesostigmata) using morphological keys were investigated by molecular tools. The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from mites were amplified and sequenced to examine the level of sequence variations and to explore the feasibility of using this region in the identification of this mite. Conserved primers located at the 3'end of 18S and at the 5'start of 28S rRNA genes were used first, and amplified fragments were sequenced. Sequence analyses showed no variation in 5.8S and ITS2 region while slight intraspecific variations involving substitutions as well as deletions concentrated in the ITS1 region. Based on the sequence analyses a nested PCR of the ITS2 region followed by RFLP analyses has been set up in the attempt to provide a rapid molecular diagnostic tool of D. gallinae.

Phospholipase activity of yeasts from wild birds and possible implications for human disease.

Over the last decades, reports on yeast infections in humans have increased especially with respect to immunocompromised individuals. Phospholipases are enzymes which may be associated with pathogenic processes caused by opportunistic yeasts. Phospholipase activity (ph.a.) was investigated in 163 isolates of 13 species of yeasts. A total of 133 isolates were obtained through the screening of a total of 768 cloacae of wild birds (Group I: 182 birds of prey; Group II: 165 passeriformes and Group III: 421 other wild migratory birds), while 30 isolates were recovered from the droppings of birds housed in 32 distinct aviaries (Group IV). Phospholipase production was evaluated and quantified at 2 and 5 day pre-incubation (Pr.t) and incubation times (I.t). Isolates from cloacae (48.1%) and excreta (73.3%) produced ph.a. with the highest values registered after 5 days of I.t. Candida albicans, C. tropicalis, C. glabrata, C. lusitaniae, C. pelliculosa, Cryptococcus albidus, C. laurentii, Trichosporon beigelii, and Saccharomyces cerevisiae displayed the highest ph.a. after 2 days of Pr.t while Candida famata, C. guilliermondii and Cryptococcus neoformans after 5 days of Pr.t. Ph.a. was never found in Rhodotorula rubra isolates recovered from the cloacae of wild birds. Isolates (73.3%) from bird droppings showed a higher ph.a. than those from cloacae thus indicating that wild birds not only act as carriers but may also spread phospholipase-producing yeasts in the environment.