A rapid, one-step agglutination assay has been developed, based on latex particles sensitized with antibodies against vitellogenin (Vtg), aimed at Atlantic bluefin tuna, Thunnus thynnus (Linnaeus) (ABFT), gender identification. The egg-yolk precursor protein Vtg was used as a gender marker for the assay as it is a female-specific protein synthesized during reproductive maturation. The presence of Vtg in the plasma was revealed in 60-120 s through an agglutination reaction by mixing small volumes of ABFT plasma and an anti-Vtg antibody-latex suspension on a microscope slide. The effectiveness of the present test was restricted to the months of May and June, concomitant with high circulating Vtg levels. Because of its rapidity and ease of performance in the field, the present gender identification assay could be useful for broodstock management in the aquaculture industry as well as in tagging studies on wild populations.

In the poultry industry, control of the red mite D. gallinae primarily relies worldwide on acaricides registered for use in agriculture or for livestock, and those most widely used are carbamates, followed by amidines, pyrethroids and organophosphates. Due to the repeated use of acaricides - sometimes in high concentrations - to control infestation, red mites may become resistant, and acaricides may accumulate in chicken organs and tissues, and also in eggs. To highlight some situations of misuse/abuse of chemicals and of risk to human health, we investigated laying hens, destined to the slaughterhouse, for the presence of acaricide residues in their organs and tissues. We used 45 hens from which we collected a total of 225 samples from the following tissues and organs: skin, fat, liver, muscle, hearth, and kidney. In these samples we analyzed the residual contents of carbaryl and permethrin by LC-MS/MS.Ninety-one (40.4%) samples were positive to carbaryl and four samples (1.7%) were positive to permethrin. Concentrations of carbaryl exceeding the detection limit (0.005 ppm) were registered in the skin and fat of birds from two farms (p<0.01), although these concentrations remained below the maximum residue limit (MRLs) (0.05 ppm) (p<0.01). All organs/tissues of hens from a third farm were significantly more contaminated, with skin and muscle samples exceeding the MRL (0.05 ppm) (p<0.01) of carbaryl in force before its use was banned. Out of 45 chickens tested, 37 (82.2%) were found to be contaminated by carbaryl, and 4 (8.8%) by permethrin. The present study is the first report on the presence of pesticides banned by the EU (carbaryl) or not licensed for use (permethrin) in the organs and tissues of laying hens, which have been treated against red mites, and then slaughtered for human consumption at the end of their life cycle.

In poultry industry, the control of the red mite Dermanyssus galli- nae (De Geer, 1778) (Mesostigmata: Dermanyssidae) (PRM) pri- marily relies worldwide on acaricides; the most widely used are carbamates, followed by amidines, pyrethoids and organophospho- rus. Despite their proved efficacy against D. gallinae, none of these compounds is specifically registered in Italy for use against red mites, except for the very recently labeled organophosphate and spinosad-based products. This means that farmers have always used -and continue to use - acaricides registered for use in agricul- ture or for other farm animal species. In the last few years, some acaricides have become worldwide less effective; thus, to control mite infestations farmers are keen to use chemicals at higher con- centrations, more frequently and repeatedly. This improper use of acaricides to control PRM could lead firstly to the development of acaricide-resistant D. gallinae populations, as previously observed also in Italy (Marangi M et al, 2009, Exp Appl Acarol, 48:11–18), and, more importantly, to the accumulation of acaricides in chick- ens’ organs, in tissues and in eggs. AIM: To highlight some concealed situations of risk for human health, we investigated three farms (denoted A, B and C) in a southern Italian region where D. gallinae populations were found to be significantly tolerant to carbammates and permethrins (Marangi M et al, o.c.), likely due to the high chemical pressure. MATERIALS AND METHODS: Fifteen laying hens at the end of their production cycle and destined to the slaughterhouse were taken from each farm. In the necropsy lab, all 45 hens were euth- anized, and a total of 225 samples were taken from skin, fat, liver, muscle, hearth, and kidney. Feed samples were also collected from each farm. Analysis was carried out using a HPLC coupled to a QQQ Triple Quadrupole Mass Detector for the residual contents of carbaryl and permethrin from all matrices and detection limits and the average pesticide recoveries were performed. Statistical analysis (one-way variance analysis, Duncan’s test and one-tailed t-student test) was applied and the Statistica 6.0 software package was used. RESULTS: Thirty-seven (82.2%) laying hens were positive for car- baryl residues and 4 (8.8%) for permethrin. Ninety-one (40.4%) samples resulted positive for carbaryl (25 skin, 27 fat, 8 liver, 16 muscle, 15 heart and kidney samples), showing a mean concentra- tion of 5 ppm, 0.04 ppm, 0.05 ppm, 0.14 ppm, 0.04 ppm, respec- tively. Four samples (1.7%) resulted positive for permethrin (2 fat and 2 liver samples), with a mean concentration of 0.012 ppm and 0.006 ppm, respectively. On one farm (Farm B), all investigated hens were found to be contaminated by carbaryl, and 80% of their organs and tissues contained residues of the compound, with the highest concentrations in the skin (16 ppm), fat (0.11 ppm) and muscle (0.3 ppm). Concentrations of carbaryl exceeding the detec- tion limit (0.005 ppm) were registered in the skin and fat of birds from two farms (p<0.01), although these concentrations remained below the maximum residue limit (MRLs) (0.05 ppm) (p<0.01). All organs/tissues of hens from a third farm were significantly more contaminated, with skin and muscle samples exceeding the MRL (0.05 ppm) (p<0.01). CONCLUSIONS: This study shows that most laying hens (37 out of 45) from all three investigated farms were contaminated by car- baryl, and that the hens of one farm also contained permethrin. Furthermore, with different accumulation levels among animals - possibly due to differences in factors like management strategies and unpredictable individual predispositions - all organs/tissues were contaminated by carbaryl, with the highest levels found in skin, fat, and muscle. These data are all worrying, because: a) car- baryl was banned by the EU in 2007 (Allegato I, Direttiva 91/414, 1376/07, 07/355); b) no carbaryl-based pro

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.

In poultry industry, the control of the red mite Dermanyssus gallinae primarily relies worldwide on acaricides; the most widely used are carbamates, followed by amidines, pyrethoids and organophosphorus. Despite their proved efficacy against D. gallinae, none of these compounds is specifically registered in Italy for use against red mites, except for the very recently labeled organophosphate and spinosad-based products; moreover, carbaryl is banned since 2007 by the UE. In the last few years, some acaricides have become worldwide less effective; thus, to control mite infestations farmers are keen to use chemicals at higher concentrations and more frequently. This improper use of acaricides to control D. gallinae could lead firstly to the development of acaricide-resistant D. gallinae populations, and, more importantly, to the accumulation of acaricides in chickens' organs, in tissues and in eggs. In the present contribution the authors summarize the situation on dermanyssosis control in Italy, their scientific experiences, and invite vets and farmers to better manage infestation in order to limit the consequences of misuse of chemicals

Beak and Feather Disease Virus (BFDV) is a member of the genus Circovirus and causes the Psittacine Beak and Feather Disease (PBFD) in Psittaciformes. PBFD is a severe disease generally characterized by immunodeficiency and beak and feather disorders. Although Circovirus spp. have been detected in several non-psittacine specie, little is known about the symptoms and the disease associated to this infection in birds other than Psittaciformes. Nevertheless, PBFD has never been described in non-psittacine specie. In this study, we reported an outbreak of Circovirus infection in a flock of Gouldian finches where beak and feather disorders were observed. Sequence analyses on the rep gene of the virus highlighted a strong similarity at nucleotide and amino acid level with the corresponding regions of BFDV

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.

Data on the occurrence and epidemiology of Aspergillus spp. in laying hens farms are scant. With the aims of determining levels of airborne contamination in laying hen farms and evaluating the potential risk of infection for workers and animals, 57 air samples from 19 sheds (Group I), 69 from faeces (Group II), 19 from poultry feedstuffs (Group III) and 60 from three anatomical sites (i.e. nostrils, pharynx, ears) of 20 farm workers (Group IV) were cultured. The Aspergillus spp. prevalence in samples ranged from 31.6% (Group III) to 55.5% (Group IV), whereas the highest conidia concentration was retrieved in Group II (1.2 × 10(4) c.f.u. g(-1)) and in Group III (1.9 × 10(3) c.f.u. g(-1)). The mean concentration of airborne Aspergillus spp. conidia was 70 c.f.u. m(-3) with Aspergillus fumigatus (27.3%) being the most frequently detected species, followed by Aspergillus flavus (6.3%). These Aspergillus spp. were also isolated from human nostrils (40%) and ears (35%) (P<0.05) (Group IV). No clinical aspergillosis was diagnosed in hens. The results demonstrate a relationship between the environmental contamination in hen farms and presence of Aspergillus spp. on animals and humans. Even if the concentration of airborne Aspergillus spp. conidia (i.e. 70 c.f.u. m(-3)) herein detected does not trigger clinical disease in hens, it causes human colonization. Correct management of hen farms is necessary to control environmental contamination by Aspergillus spp., and could lead to a significant reduction of animal and human colonization.

Microsporum canis and Trichophyton mentagrophytes are zoophilic dermatophytes which can cause skin infections in animals and humans. The clinical expression of this infection strongly varies depending on host, fungal species as well as enzyme production. No comparative studies are available on the enzymatic activities of M. canis and T. mentagrophytes isolated from breeding rabbits. Thus, the aim of this work was to assess the capability of M. canis and T. mentagrophytes isolated from rabbits both with and without lesions in producing different enzymes. The relationship of dermatophyte enzymatic activities and presence/absence of skin lesions has also been investigated. A total of 260 isolates of T. mentagrophytes and 25 isolates of M. canis sampled both from healthy and lesioned skin of rabbits, as well as from air samples of positive farms were examined. The results showed that T. mentagrophytes and M. canis from rabbits produce different enzymes. However, only elastase and gelatinase were linked to the appearance of lesions in T. mentagrophytes infections, whereas lipase in those by M. canis.

Erysipelotrix rhusiopathiae is the etiologic agent of erysipelas in animals. It is a worldwide diffused disease which affects swine and avian species. Erysipelas in avian is mostly reported in turkey and chicken, although its prevalence is probably underestimated. Even more rarely the disease is reported in wild species, but they could play an important role in disseminating the pathogen. Here we report an unusual focus of erysipelas in a pheasant (Phasianus colchicus) farm. Ten 8-month old birds were brought to our facilities following a history of malaise, sensory impairment and mortality in a pheasant farm. At necropsy, we observed wide alopecic areas, with correspondent thickness of skin. Diffused hemorrhages were evident mainly along the muscles of chest, liver, spleen and epicardium. Catarrhal hemorrhages were also evident in the distal part of gut. Histological observations evidenced necrotic phlogistic foci characterized by fibrotic suppuration from gut and proventricula samples. Thrombotic areas were evident in kidney and spleen samples. In each case, microscopy observations showed bacterial cells and aggregate, also found in the blood. Bacteriological analyses let us isolate and identify E. rhusiopathiae. Following identification, a therapy with -lactamic antimicrobial agents was administered, quick resolving the case. This report confirms the E. rhusiopathiae may circulate in wild birds, such as pheasant. We hypothesize that overcrowding could have promoted the epidemic-like burden of the disease in the farm. Further studies should be carried out to verify the possibility that wild avian species such as pheasant could act as a reservoir for E. rhusiopathiae

Erysipelotrix rhusiopathiae is the etiologic agent of erysipelas in animals. It is a worldwide diffused disease which affects swine and avian species. Erysipelas in avian is mostly reported in turkey and chicken, although its prevalence is probably underestimated. Even more rarely the disease is reported in wild species, but they could play an important role in disseminating the pathogen. Here we report an unusual focus of erysipelas in a pheasant (Phasianus colchicus) farm. Ten 8-month old birds were brought to our facilities following a history of malaise, sensory impairment and mortality in a pheasant farm. At necropsy, we observed wide alopecic areas, with correspondent thickness of skin. Diffused hemorrhages were evident mainly along the muscles of chest, liver, spleen and epicardium. Catarrhal hemorrhages were also evident in the distal part of gut. Histological observations evidenced necrotic phlogistic foci characterized by fibrotic suppuration from gut and proventricula samples. Thrombotic areas were evident in kidney and spleen samples. In each case, microscopy observations showed bacterial cells and aggregate, also found in the blood. Bacteriological analyses let us isolate and identify E. rhusiopathiae. Following identification, a therapy with β-lactamic antimicrobial agents was administered, quick resolving the case. This report confirms the E. rhusiopathiae may circulate in wild birds, such as pheasant. We hypothesize that overcrowding could have promoted the epidemic-like burden of the disease in the farm. Further studies should be carried out to verify the possibility that wild avian species such as pheasant could act as a reservoir for E. rhusiopathiae.

Colibacillosis caused by pathogenic E. coli strains (Avian Pathogenic Escherichia coli) is one of the main causes of economic losses in poultry breeding industry worldwide. This acute infection of birds may be clinically localized or systemic, several of the lesions in visceral organs are observed. Virulence factors in the E. coli genome, described in various studies, have not clarified prevalence of APEC strains, and their importance in the infection pathogenesis is still unknown. Conducted surveys indicate that the presence of virulence genes in E. coli isolates is the starting point of differentiation and characterization of pathogenic E. coli to those intestinal and opportunistic. A total of 158 E. coli strains were examined for the presence of eight virulence genes: iss, tsh, papC, vat, cvaA/B, iucD, astA, irp2, by means of molecular biology technique PCR-multiplex (Ewers et al. 2004). This study identified significant differences of virulence factors among strains isolated from lesions, compared to those from apparently healthy subjects. iss, responsible for increased serum survival in the E. coli bacteria, was the virulence factor with the highest percentage of detection (42.40%). Virulence genes frequency is considered to be an important indicator of the virulence of E. coli strains. Thus the lack of virulence factors in APEC strains resulted in 27.18%, while in AFEC strains resulted in 61.81%. These data obtained from genetic characterization of avian E. coli strains constitute the first report in Albania for colibacillosis infection outbreak in poultry flocks. The presence, appearance and distribution of virulence genes in poultry flocks provide basic information for the control and eradication of the colibacillosis infection.

In questo lavoro, vengono riportate le esperienze cliniche e di diagnostica di laboratorio relative alle infezioni da Circovirus nei volatili d’affezione. Nella maggior parte dei casi di infezione osservati, mancavano le tipiche lesioni alle penne classicamente descritte in caso di infezione da Circovirus ed il piumaggio risultava inalterato. Non sono state mai riscontrate inoltre lesioni a carico del becco. Al contrario, in volatili privi di alterazioni del piumaggio ed in cui Circovirus veniva ricercato per uno stato di sospetta immunodepressione, in quanto si evidenziavano sintomi aspecifici come debolezza e abbattimento ed una maggiore suscettibilità ad infezioni secondarie di natura batterica o parassitaria, la percentuale di positività all’infezione risultava elevata. La diagnosi di laboratorio è stata ottenuta mediante PCR, a partire da penne, feci o, nei soggetti deceduti, campioni d’organo. Tra i campioni analizzati, le penne si sono rivelate particolarmente utili ed attendibili a fini diagnostici permettendo agevolmente la diagnosi in vivo, anche nei casi in cui le lesioni del piumaggio erano completamente assenti. Attualmente, vista la mancata disponibilità di un vaccino commerciale, il controllo dell’infezione è affidato al monitoraggio dei volatili prima di ogni spostamento ed introduzione in un nuovo gruppo. Allo scopo, il test in PCR a partire dalle penne rappresenta un metodo non invasivo per l’animale ma soprattutto efficace ed affidabile anche in assenza di alterazioni esteriori a carico del piumaggio e del becco che inducano a sospettare l’infezione.

Safety and protective efficacy of the attenuated strain Salmonella gallinarum SGP695AV in chickens. Fowl typhoid, caused by Salmonella enterica subsp. enterica ser. Gallinarum, causes heavy economic loss to the industrial poultry farms due to its strong impact in terms of mortality and morbidity. Furthermore, its management is quite difficult, as the infectious agent may remain in the poultry farms even during sanitary breaks. Therefore, many Countries have implemented strict preventive measures, leading the disease to be eradicated in many Western Countries. Notwithstanding, fowl typhoid is still widely diffused in the Mediterranean Countries, and in Africa, Asia and South America as well. Among the preventive measures needed to control the disease, the vaccination plays a pivotal role. To date, the only available vaccine consists of a rough strain of S. Gallinarum. The aim of this work was to assess the protective efficacy and safety of a live attenuated strain, termed SGP695AV. At T0 and 15 days after (T15), 2x107, 2x109 and 2x1011 UFC of SGP695AV were administered per os to three group of 12 30-day old laying hens, termed A, B and C, respectively. On the other hand, a verified pathogenic S. Gallinarum strain, SG354 were administered per os to another group, termed P, at T0. An equal volume of physiological solution was administered to the group N at T0 and T15. Thirty-seven days after the first administration (T37), 5 randomly chosen chickens from the groups A, B, C and P were humanly sacrificed to evaluate the presence of SGP695AV in livers, spleens and guts. Contemporary, the other animals of groups A, B, C and N were inoculated per os with 1x1013 UFC of SG354. Everyday, each animal was observed to assess its clinical score on the basis of its symptomatology. Serological test by rapid serum agglutination was performed at T0, T15, T37 and 15 days after administration of SG354 (T52). Daily, cloacal swabs were collected to evaluate the fecal excretion of SGP695AV and/or SG354. The data we gathered showed that vaccine did not cause significant adverse effects in chicken. Before T37, Clinical scores was low in A, B and C groups, even when higher doses of the vaccinal strain were administered. Contrarily, the clinical score of the group P between T15 and T37 was up to 200 times higher than those of the groups A, B and C. After infection with SG354, the clinical scores remained low in the three vaccinated groups. The excretion of SGP695AV was low and limited to the first 15 days. The fecal elimination of SG354 after T37 was equally low in the groups A, B and C, while was significantly more consistent in the group N.

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.

Salmonella enterica subsp. enterica serovar Gallinarum (S. Gallinarum) is the causative agent of fowl typhoid, a major threat in poultry which causes mortality and high morbidity. The standard method for detection of S. Gallinarum relies on culture isolation and it requires more than five days for final confirmation. Recently, molecular methods have been developed, but they are not considered as golden standards, also because they do not allow pathogen quantification. Therefore, we developed a real time quantitative PCR (qPCR) which may represent a fast and accurate method that reduce drastically the extent of the analytical process, while being sensitive and specific. In order to grant specificity, a specific probe has been designed to detect the amplification products. The method has been tested in vitro and it has been proved to be effective for detection and absolute quantification of S. Gallinarum. In particular we found high sensitivity and specificity, with accuracy higher than 98%. Therefore, we find that the method may be suitable for diagnostic and other application, such as environmental monitoring.

The poultry red mite, Dermanyssus gallinae, De Geer 1778, is a blood-sucking mite of birds worldwide, including feral pigeons. The mite only visits its host for blood meals, having to do so repeatedly every 2-3 days. The rest of the time, it lives in crevices and cracks in roosting and nesting sites. When the avian host is not available, it can move to and bite humans, causing a non-specific dermatitis with intense itching. From 2001 to 2009, we observed 11 outbreaks of Red Mite Dermatitis (RMD) in city-dwellers in Southern Italy, where all cases were initially misdiagnosed and medical care failed to solve the dermatitis. Mites infested the patients in their dwellings/workplaces, and the source of the infestation was found in abandoned pigeons’ nests in close proximity to the habitations. Cutaneous reactions resulting from contact with D. gallinae are uncharacteristic, and usually remain unrecognized when they arise in atypical contexts such as urban areas. This has led to scarce information being available on this subject. However, feral pigeons are very abundant in cities worldwide, and are well-known source of zoonotic ectoparasites, including red mites. This poses a considerable risk of RMD in city-dwellers due to their contact with this nidicolous arthropod. Physicians and dermatologists should be made more aware of the occurrence of this urban epizoonosis. The management of this skin disorder requires correct identification of the mite and its eradication in the environment with removal of nests and appropriate disinfestation of infested areas.

In this study, we characterised the Salmonella Typhimurium strains responsible for four outbreaks which occurred in distinct rabbit farms (Southern Italy) from 1999 to 2003. Strains were typed by Pulsed Field Gel Electrophoresis (PFGE) and the genetic basis of antimicrobial resistance was established. A major group of clonally related isolates, pulsotype STYMXB.0061, accounted for three of the salmonellosis foci. Strains were resistant to streptomycin, chloramphenicol, tetracycline, ampicillin and sulphonamides encoded respectively by the aadA2, floR, tetG, blaPSE-1, sul1 gene cluster harboured by a Salmonella Genomic Island 1. The clonally related group of isolates included strains phage type DT104, DT12 or undefined type (NT). The fourth salmonellosis focus was caused by a strain pulsotype STYMXB.0147, resistant to sulphonamides (encoded by sul2) and phage type U302. Results provided first molecular characterisation of S. Typhimurium strains isolated from rabbit farms in Italy and highlighted the presence of the pulsotype STYMXB.0061 even before its wide detection among human clinical isolates collected in Italy in the mid 2000s from clinical cases

Beak and Feather Disease Virus (BFDV) is a member of the genus Circovirus and causes the Psittacine Beak and Feather Disease (PBFD) in Psittaciformes. PBFD is a severe disease generally characterized by immunodeficiency and beak and feather disorders. Although Circovirus spp. has been detected in several nonpsittacine species, little is known about the symptoms and the disease associated with this infection in birds other than Psittaciformes. In this study, we reported the identification of Circovirus infection in a flock of Gouldian finches showing beak and feather disorders. Sequence analyses on the rep gene of the virus highlighted a strong similarity at nucleotide and amino acid level with the corresponding regions of BFDV from psittacine species. By contrast, it was more distant to circoviruses identified in finch and canary

Twenty-four Salmonella enterica isolates (13 serovar Enteritidis and 11 Typhimurium) isolated from 5,600 samples from intensive laying hen farms in Italy in 1998-2007 were characterized for antimicrobial resistance genes, pulsotype and phage type. Most of S. Typhimurium strains were pulsotype STYMXB.0147 (81.8%), phage type DT143 and resistant to sulfamethoxazole encoded by sul2. Two multidrug resistant (MDR) strains were identified. One strain, STYMXB.0061, was resistant to ampicillin (A), chloramphenicol (C), streptomycin (S), sulfamethoxazole (Su) and tetracycline (T) encoded by the Salmonella Genomic Island SGI1. The second MDR strain, STYMXB.0110, was resistant to SSuT encoded by sul1 and sul2, aadA1 and tet(C)-flanked by an IS26 element, respectively. The tet(C) gene has been reported to confer low levels of resistance and it has very rarely been detected in S. Typhimurium from poultry. In the current study, the MIC value (32 µg/mL) was consistent with the breakpoint (³16 µg/mL) reported for Enterobacteriaceae. Most of the S. Enteritidis strains were resistant to Su (encoded by sul2). One MDR strain (ANxSSuT) was identified. With the exception of nalidixic acid (Nx), the resistances were respectively encoded by blaTEM, strAB, sul2 and tet(A) harbored by an IncN conjugative plasmid. All isolates were pulsotype SENTXB.0001 with PT14b being the most prevalent identified phage type (57.1%). In Europe, SENTXB.0001 is the predominant PFGE profile from clinical cases and the identification of PT14b has steadily been on the increase since 2001. The findings presented in this study highlight the potential spread of S. Enteritidis phage types PT14b and S. Typhimurium DT143 in a field of particular relevance for zoonoses. Additional, the presence of resistance genes and genetic elements (conjugative plasmid and IS element) underlines the need to assess routinely studies in field, such as poultry farms, relevant fot the public health and suitable for the storage and diffusion of antimicrobial resistance.

Rabbit Hemorrhagic Disease Virus (RHDV) and European Brown Hare Syndrome Virus (EBHSV) are two virus belonging to the Lagovirus genus. They cause a hemorrhagic disease in rabbits and hare, respectively. The high mortality (up to 100%), and the great contagious potential of the viruses make them very dangerous for both wild and domestic animals. The viruses have a number of variants, and recently a new RHDV variant has been widely circulating among rabbits, mainly in France. The variability of viruses may cause some problem in the molecular diagnosis. We set up a RT-PCR based method which could widen the efficacy of the existing molecular techniques, as it could detect simultaneously the variants of RHDV, including the French variants, and the EBHSV, furthermore discriminating the two viral species.

Salmonella enterica infections are not common in rabbits, but, whenever they occur, they induce high morbidity and mortality and they also raise concern in terms of public health impact. Moreover, the worldwide spread of multidrug resistant strains complicates the treatment of infection in both rabbits and humans. Despite those premises, knowledge about salmonelloses in rabbits is still poor. In this study four S. Typhimurium strains were isolated from as many rabbit farms, and they were characterized for antimicrobial susceptibility, resistance genes, class 1 integrons and Pulsed-Field Gel Electrophoresis (PFGE). The results showed that a group of strains were circulating in Basilicata, and that it was indistinguishable from human isolates for PFGE profile, multidrug resistance pattern and genetic features, as they harbored the Salmonella Genomic Island 1 (SGI1) which characterized the S. Typhimurium strains which are widespread among humans all over the world. Our data suggest that molecular characterization is a useful tool to promptly recognize Salmonella strains which are potentially harmful to rabbits or humans.

Salmonella enterica subsp. enterica ser Gallinarum is the causative agent of fowl typhoid. The disease represents a major concern among industrial poultry farms, causing heavy economic losses due to its detrimental effects on productivity and animal welfare. The pathogen is highly persistent and its eradication from an infected farm is often difficult. A possible cause of its survival may be the association with parasites, such as the poultry red mite Dermanyssus gallinae. The latter is widespread among European poultry farm, with infestation rates raising up to 90%. This study is aimed to evaluate the association among D. gallinae and S. gallinarum in the Italian intensive poultry farms. Seventy-four samples of mites were collected from 62 intensive poultry farms. Up to 100 mites per sample underwent to homogenization and DNA extraction. The extracted DNA was used as template in a seminested-PCR-based protocol, already proved to be sensitive and specific. Four samples out of 74 (5.41%) resulted positive for the presence of S. gallinarum. Three positive samples came from different laying hen farms, while the other came from a broiler parent farm. Such data confirm the association between D. gallinae and S. gallinarum, once again evidencing the potential vectorial role of the mite for the bacterial pathogen. Furthermore, they suggest that D. gallinae may act as a reservoir of S. gallinarum, as it could be hosted by the mite even during the sanitary breaks, which are carried out in order to eradicate fowl typhoid from an infected poultry farm

In this study, the possibility of the vertical transmission of Circovirus was investigated in a flock of infected Gouldian finches. The detected virus was previously classified as a BFDV strain by sequence analysis. Eggs were collected in the flock for three subsequent reproductive seasons. The DNA virus was found in the eggs only in the first reproductive season. 24,39 % of the tested eggs resulted positive. Circovirus DNA was detected in yolk while all samples of glair tested negative. The findings strongly suggest that the vertical transmission of circovirus is possible. Nevertheless, it seems to occur only in the first year of the outbreak.

Salmonella enterica subsp. enterica serovar Gallinarum (S. Gallinarum) is the causative agent of fowl typhoid, one of the major causes of mortality and morbidity on poultry farms. Even though it has been substantially eradicated in many developed countries, the disease still remains endemic in Central and South America, in Africa and in the Mediterranean countries of Europe. This leads to the routine screening of flocks, mainly by cultivation and serological techniques, which are expensive, as well as time and labour-consuming. Here we describe a simple and specific PCR-based method for detecting S. Gallinarum. It relies on two seminested PCRs which use four pairs of primers designed on the basis of two genomic regions which appear to be exclusive to the pathogen. Furthermore, an internal positive control was devised in order to avoid any false negative results. We performed sensitivity and specificity tests, and our findings showed the cogency of the system and its potential effectiveness even for routine uses.

In this study we evaluated the in vitro variability of flaA in Campylobacter coli strains (Co19 and Co29) isolated from distinct poultry farms. Strains were characterized by flaA PCR-RFLP and subcultured in absence or presence of Salmonella enterica (S. Gallinarum) as a host competitor in poultry. Only transition mutations were detected and no flaA-flaB recombination events were observed. The highest ratio of flaA mutations was detected in the presence of competitor (1x10-6 and 4.8x10-6 for Co19 and Co29, respectively). While, transitions in absence of competitor were observed only in the strain Co29 (5.8x10-7). Data obtained in this investigation represent a preliminary study aimed to evaluate the possible linkage between specific haplotypes and their proneness to acquire point mutations which in turn might help strains to gain a better fitness for survival within and outside hosts.

Dermanyssus gallinae infestation is a major threat for industrial poultry farms. The mite is worldwide diffused, and its prevalence is very high: it is reported that more than 80% of poultry farms are infested in some European Countries. The infestation leads to detriment of animal welfare and economic losses, as it negatively affects the egg and meat production. It may also contribute to the diffusion of infectious diseases as it has been widely recognized as a vector of a number of pathogens. Remarkably, D. gallinae could be also considered an occupational hazard, as it may infest the poultry farm workers. Currently, the control of D. gallinae infestation is performed by using specific drugs such as carbamates, organophosphates, pyrethroids or amidines (amitraz), but most of them can not be used in presence of live animals. Furthermore, in field and in vitro reports have been assessing the increase in acaricide resistances. In the light of these premises, we evaluated the susceptibility of field populations of D. gallinae against three acaricide drugs, namely phoxim (an organophosphate), Amitraz and -Cyalothrin (a pyrethroid). A total of 68 mite populations were collected from Italian industrial poultry farms for a four-year period (2008-2012), and their susceptibility to the selected drugs have been tested at field-use concentration (1X) and at lower (1/2X and 1/4X) and higher (2X and 4X) concentrations. The data showed that -Cyalothrin exhibits a lower efficacy if compared with the other two drugs. By comparing the effectiveness of the drugs by years, we found that resistance to Amitraz is constantly increasing since 2008 to 2012, while effectiveness of Phoxim has been decreasing since 2009 to 2010, while it has been increasing for the last two years. The mite resistance to -Cyalothrin has been decreasing since 2009 to 2011, but in 2012 its efficacy has significantly collapsed. Such trends remained the same independently by the concentrations. Interestingly, higher concentrations did not provide a significant increase of drug efficacy. On aggregate, our data show an increment in mite resistances against the most used acaricides, even if the trends are different for each drug. To avoid a further decrease in drug effectiveness, it should be advisable a turn over of drugs usually employed against D. gallinae in field. The use of drugs at different concentration than those recommended by manufacturers. Finally, the understanding of the genetic bases of the acaricide resistance may greatly help to struggle the insurgence and diffusion of resistant D. gallinae strains.

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.

The red mite, Dermanyssus gallinae (Acarina: Mesostigmata), is a temporary blood-sucking ectoparasite of poultry and other avian species, with a worldwide distribution. It can occasionally bite mammals, including humans, and cause dermatitis. Medical textbooks frequently cite itching and cutaneous lesions resulting from red-mite bites as a normal occurrence in subjects working in close conjunction with poultry. In fact, D. gallinae is one of the most common pests and a major cause of economic loss in the poultry industry, with a farm prevalence of up to 90%.1 In the literature, red-mite dermatitis (RMD) is generally regarded as an urban hygiene issue.

Domestic and wild birds may act as carriers of human pathogenic fungi, although the role of laying hens in spreading yeasts has never been investigated. We evaluated the presence of yeasts in the cloaca (Group I, n = 364), feces (Group II, n = 96), and eggs (Group III, n = 270) of laying hens. The occurrence and the population size of yeasts on the eggshell, as well as in the yolks and albumens, were assessed at the oviposition time and during storage of eggs at 22 ± 1°C and 4 ± 1°C. A statistically higher prevalence and population size of yeasts were recorded in Group I (49.7% and 1.3 × 104 cfu/ml) and II (63.8% and 2.8 × 105 cfu/ml) than in Group III (20.7% and 19.9 cfu/ml). Candida catenulata and Candida albicans were the most frequent species isolated. Candida famata and Trichosporon asteroides were isolated only from the eggshells, whereas Candida catenulata was also isolated from yolks and albumens. During storage, the yeast population size on the shell decreased (from 37.5 to 8.5 cfu/ml) in eggs at 22 ± 1°C and increased (from 4.6 to 35.3 cfu/ml) at 4 ± 1°C. The laying hens harbor potentially pathogenic yeasts in their gastrointestinal tract and are prone to disseminating them in the environment through the feces and eggs. Eggshell contamination might occur during the passage through the cloaca or following deposition whereas yolk and albumen contamination might depend on yeast density on eggshell.