SET-UP AND APPLICATION OF BIOTECHNOLOGICAL METHODS IN AVIAN FIELD

The use of biotechnology in avian field has been increased in the last decade especially on the track of the complete sequencing of chicken genome and on the global threat of avian flu pandemic which alerted the scientific community worldwide. Nevertheless, it is still on the whole a minor and spotty approach in avian field. Matter-of-factly, birds can be roughly divided into two groups on a research point of view: poultry and non-poultry (companion and wild birds). The economic impact of the latter is usually scarce if compared to the former and to other veterinary and human contexts. Thus, routine use of laboratory diagnostic tools including biotechnological methods is usually limited to the more expensive psittacines species (i.e. DNA-based determination of sex, diagnosis of chlamydiosis etc.) or to endangered species. As for poultry, extensive use of biotechnology is performed in vaccine research and genetic selection with a secondary role in avian and comparative pathology. Besides, chicken has historically assumed the role of avian model. Consequently, birds are often considered an uniform class but they are not, and a deep knowledge of the huge diversity which can exist among the several avian species is fundamental to correctly approach and interpret avian research no matter using biotechnology or not. For all these reasons it seemed an unique occasion to match the documented experience and activity in avian field (acquired during a previous doctorate) to biotechnological methods as the subject of this doctoral thesis. After a brief introduction revising fundamental principles of the molecular methods used, this thesis is divided into two parts. The first is focused on avian immunity and in particular on the TIR8-SIGIRR receptor which has been thoroughly investigate in chickens as a model for avian species and with an importance in itself as an ubiquitously diffused food production animal. The second part is made up of some diagnostic challenges arisen during the routine diagnostic activity of the Section of Avian Pathology of the Department of Veterinary Science and Public Health

Epidemiology and genotyping of Candida rugosa strains responsible for persistent intramammary infections in dairy cows

The present study was undertaken during an outbreak of clinical and subclinical mastitis in 14 dairy cows caused by Candida rugosa, in which high somatic cell counts were seen and cases did not respond to antibiotic treatment. Intramammary infection cured spontaneously in 10 cows, whereas 4 cows were culled as a result of persistent infections. Repeated sampling of these cows and biomolecular analysis of the isolates showed that the infections were caused by the same genotype, even over a period of 2 lactations. Random amplification of the genome of C. rugosa milk isolates gave 3 different DNA banding patterns (genotypes G1, G2, and G3). Viable cells of C. rugosa were also isolated from various environmental sources and were present in high concentrations in total mixed ration samples, which could be considered the primary source of diffusion of viable yeast cells in the environment, as demonstrated by genotyping. The proven capacity of these microorganisms to survive in the environment of the cow, such as the total mixed ration, bedding, water, and cow skin, and to cause persistent intramammary infections highlights the importance of mycotic spread in dairy herds

Evidence of Simultaneous Circulation of West Nile and Usutu Viruses in Mosquitoes Sampled in Emilia-Romagna Region (Italy) in 2009

BACKGROUND: In recent years human diseases due to mosquito-borne viruses were increasingly reported in Emilia-Romagna region (Italy), from the chikungunya virus in 2007 to the West Nile virus (WNV) in 2008. An extensive entomological survey was performed in 2009 to establish the presence and distribution of mosquito arboviruses in this region, with particular reference to flaviviruses. METHODOLOGY/PRINCIPAL FINDINGS: From May 6 to October 31, a total of 190,516 mosquitoes were sampled in georeferenced stations, grouped in 1,789 pools according date of collection, location, and species, and analyzed by reverse transcription polymerase chain reaction (RT-PCR) to detect the presence of RNA belong to Flavivirus genus. WNV was detected in 27 mosquito pools, producing sequences similar to those of birds and human strains obtained in 2008 outbreak, pointed out the probable virus overwintering. Isolation of WNV was achieved from one of these pools. Moreover 56 pools of mosquitoes tested positive for Usutu virus (USUV). Most PCR positive pools consisted of Culex pipiens, which also was the most analyzed mosquito species (81.4% of specimens); interestingly, USUV RNA was also found in two Aedes albopictus mosquito pools. Simultaneous circulation of WNV and USUV in the survey area was highlighted by occurrence of 8 mosquito WNV- and USUV-positive pools and by the overlaying of the viruses "hot spots", obtained by kernel density estimation (KDE) analysis. Land use of sampled stations pointed out a higher proportion of WNV-positive Cx. pipiens pool in rural environments respect the provenience of total sampled pool, while the USUV-positive pools were uniformly captured in the different environments. CONCLUSIONS/SIGNIFICANCE: Obtained data highlighting the possible role of Cx. pipiens mosquito as the main vector for WNV and USUV in Northern Italy, and the possible involvement of Ae. albopictus mosquito in USUV cycle. The described mosquito-based surveillance could constitute the foundation for a public health alert system targeting mosquito borne arboviruses

TIR8/SIGIRR is expressed in tissues of adult and embryo chicken

The orphan receptor TIR8/SIGIRR, belongs to the TIR superfamily. TIR8 does not activate NF-kB and IRF3 and negatively modulates the inflammatory responses. It acts as antagonist for the TIR family members and triggers a negative pathway of the TIR receptor system, crucial for dampening inflammation. TIR8 was well characterized in mouse, humans and in other mammals, but it is poorly known in birds. We investigated TIR8 distribution in adult and embryo chicken samples. The pattern of expression of chicken TIR8 in adult was ubiquitary and similar to mammals, but it resulted unique in pancreas, female reproductive tract, heterophils. Different TIR8 isoforms were detected, suggesting the occurrence of post-translational modifications or alternative splicing. Immunohistochemistry revealed TIR8 immunolabeling in the intestine, thymus and oesophageal ganglia. These results demonstrate that TIR8, although evolutionarily conserved, show species-specific peculiarities. We analyzed TIR8 expression in embryos of layer- and broiler-chicken at different incubation time-points. TIR8 was detected since the first stages of development, but it reached a remarkable expression level at day 10. TIR8 was ubiquitously expressed, but the highest expression were found in liver and kidney. This pattern was comparable to those observed in adult chickens and in mammals examined to date. No differences were observed between the two different chicken breeds despite their immunological discrepancies. These are the first findings concerning TIR8 expression in developmental stages and they contribute to better understand the reproductive physiology and transovarian pathogen transmission, together with the recent findings of TLR expression in ovary and embryos of different species

Expression of TIR8 receptor in chicken tissues

The orphan receptor TIR8, also known as SIGIRR, belongs to the TLR/IL-1R (TIR) superfamily and plays an important role in the immune response. The signalling pathways of the receptors belonging to the TIR family are tightly regulated at multiple levels and through different mechanisms. TIR8 negatively modulates innate immunity and inflammatory responses in the areas where it is primarily expressed (gastrointestinal tract, kidney and lung). TIR8 has been well characterized in mouse, humans and in other Mammalian species, but it is still poorly known in chicken. Given the importance of gastrointestinal diseases in chicken, the aim of our study was to investigate the distribution of TIR8 in a wide panel of non-pathologic tissues and organs. TIR8 expression was analyzed in chicken samples at both levels of transcript mRNA and translated protein. The pattern of expression of TIR8 (ubiquitous) was similar to Mammals for some tissues (high levels in kidney and gastrointestinal tract), but it resulted unique for other tissues. High expression was detected in liver, pancreas and female reproductive tract. Interestingly, the receptor was highly expressed also in heterophils, the most common granulocytes of birds. Few isoforms of chicken TIR8 were detected by Western blot, suggesting the occurrence of different post-translational processing in different organs. Immunohistochemistry revealed TIR8 immunolabelling in chicken intestine and thymus. These results demonstrate that the receptor, although evolutionarily conserved, show species-specific peculiarities

Molecular biological characterization of avian poxvirus strains isolated from different avian species

Fifteen strains of Avipoxvirus from different avian species were isolated and molecular biologically characterized. Most strains did not produce evident pocks on the chorioallantoic membranes of commercial and specific-pathogen free embryonated chicken eggs where, on the contrary, microscopic signs of viral growth were always detected. Polymerase chain reaction of highly conserved P4b gene was positive for all cases confirming to be a reliable diagnostic method for Avipoxvirus. Sequencing of these amplicons confirmed most strains clustered either with Fowlpox virus or with Canarypox virus whereas a possible new clade could be hypothesized for one strain from Japanese quail. Classification of Avipoxvirus strains by amplification of the newly identified locus fpv140 revealed major limitations as only five samples were positive. These results underline the importance to undertake similar studies on higher numbers of Avipoxvirus isolates and on wider genomic regions of this large viral group

Unusual avian mycobacteriosis in commercial brown layers

An unusual outbreak of avian mycobacteriosis in 46-week-old commercial brown layers is described. Reduced feed intake, gradual drop in egg production and increased mortality rate were observed in the flock housed in Northern Italy. 8 birds were humanely euthanatized and necropsied. The birds were in poor body condition, with pale, shrivelled combs, and principally presented prominent orbital lesions. Grossly, multiple, variable-sized nodular lesions were found in the intestinal wall, liver, spleen, lungs, bone marrow and conjunctiva. Histologically, both visceral and orbital lesions displayed granulomatous nature with variable numbers of acid fast bacilli. Polymerase chain reaction confirmed Mycobacterium avium as the causative agent. The peculiarity of this outbreak of mycobacteriosis regards type and age of the affected birds and the uncommon involvement of the soft orbital tissues

TIR8 expression in tissues of adult and embryo chicken

The orphan receptor TIR8/SIGIRR, belongs to the TIR superfamily and plays an important role in the inflammatory responses. TIR8 does not activate NF-kB and IRF3 and negatively modulates the inflammatory responses. It acts as antagonist for the TIR family members and triggers a negative pathway of the TIR receptor system, crucial for dampening inflammation in the gastrointestinal tract (GI) and in other districts. TIR8 was well characterized in mouse, humans and in other mammals, but it is poorly known in birds. We investigated TIR8 distribution in adult and embryo chicken samples at both mRNA and protein levels. The pattern of expression of chicken TIR8 in adult was ubiquitary and similar to mammals (high levels in kidney and GI), but it resulted unique in other tissues (pancreas, female reproductive tract, heterophils). Different protein isoforms of TIR8 were detected, suggesting the occurrence of different post-translational modifications or alternative splicing. Immunohistochemistry revealed TIR8 immunolabeling in the intestine, thymus and oesophageal ganglia. These results demonstrate that TIR8, although evolutionarily conserved, show species-specific peculiarities. We also analyzed TIR8 expression in embryos of two chicken breeds (layer- and broiler-type) at different time-points of incubation. TIR8 was detected since the first stages of chicken development (day 1), but it reached a remarkable expression level at day 10. This receptor was ubiquitously expressed, but the highest expression levels were found in liver and kidney. This pattern was comparable to those observed in adult chickens and in mammals examined to date. No expression differences were observed between the two different chicken breeds despite their marked immunological discrepancies. These are the first findings concerning TIR8 expression in developmental stages and they contribute to better understand the reproductive physiology and transovarian pathogen transmission, together with the recent findings of TLR expression in ovary and embryos of different species