Veterinary Public Health in Italy: From Healthy Animals to Healthy Food, Contribution to Improve Economy in Developing Countries.

The role of the veterinarian as a public health officer is intrinsic to the history and the culture of veterinary organization in Italy. The Veterinary service being part of the Health administration since the birth of the Italian State in the XIX Century. In the second half of the last century the birth of the Italian National Health Service confirmed that the function of the Italian veterinary service was to analyze and reduce the risks for the human population connected to the relationship man-animal-environment, animal health, food safety and security. The Italian Veterinary Medicine School curricula, reflected this "model" of veterinarian as well. In the majority of countries in the world, Veterinary Services are organized within the Agriculture Administration with the main function to assure animal health and wellbeing. After the so-called "Mad-cow crisis" the awareness of the direct and essential role of veterinary services in the prevention of human illness has been officially recognized and in the third millennium the old concept of "one health" and "human-animal interface" has gained popularity worldwide.The concept of Veterinary Public Health, has evolved at International level and has incorporated the more than a century old vision of the Italian Veterinary medicine and it is defined as "the sum of the contributions to the physical, mental and social development of people through the knowledge and application of veterinary science" (WHO, Future trends in veterinary public health. Gruppo di lavoro OMS: TE, Italy, 1999, Available from: http://www.who.int/zoonoses/vph/en/ . Last visited 16 Feb 2016, 1999).On the subject of Cooperation, Sustainability and Public Health, the EXPO 2015 event and the activities of international organizations WHO, FAO and World Organization for Animal Health are refocusing at present their worldwide mandate to protect human health and the economy of both the poorest Countries and the developed countries, according to the "new" concept of Veterinary Public Health.Focus of Italian Veterinary Services activity is connected to research, diagnosis and epidemiological analysis of infectious diseases, including zoonosis, food safety as well as food security.

Rift Valley fever in Namibia, 2010.

During May-July 2010 in Namibia, outbreaks of Rift Valley fever were reported to the National Veterinary Service. Analysis of animal specimens confirmed virus circulation on 7 farms. Molecular characterization showed that all outbreaks were caused by a strain of Rift Valley fever virus closely related to virus strains responsible for outbreaks in South Africa during 2009-2010.

Determinants of bluetongue virus virulence in murine models of disease.

Bluetongue is a major infectious disease of ruminants that is caused by bluetongue virus (BTV). In this study, we analyzed virulence and genetic differences of (i) three BTV field strains from Italy maintained at either a low (L strains) or high (H strains) passage number in cell culture and (ii) three South African "reference" wild-type strains and their corresponding live attenuated vaccine strains. The Italian BTV L strains, in general, were lethal for both newborn NIH-Swiss mice inoculated intracerebrally and adult type I interferon receptor-deficient (IFNAR(-/-)) mice, while the virulence of the H strains was attenuated significantly in both experimental models. Similarly, the South African vaccine strains were not pathogenic for IFNAR(-/-) mice, while the corresponding wild-type strains were virulent. Thus, attenuation of the virulence of the BTV strains used in this study is not mediated by the presence of an intact interferon system. No clear distinction in virulence was observed for the South African BTV strains in newborn NIH-Swiss mice. Full genomic sequencing revealed relatively few amino acid substitutions, scattered in several different viral proteins, for the strains found to be attenuated in mice compared to the pathogenic related strains. However, only the genome segments encoding VP1, VP2, and NS2 consistently showed nonsynonymous changes between all virulent and attenuated strain pairs. This study established an experimental platform for investigating the determinants of BTV virulence. Future studies using reverse genetics will allow researchers to precisely map and "weight" the relative influences of the various genome segments and viral proteins on BTV virulence.

West Nile alternative open reading frame (N-NS4B/WARF4) is produced in infected West Nile Virus (WNV) cells and induces humoral response in WNV infected individuals.

BACKGROUND: West Nile Virus (WNV) is a flavivirus that requires an efficient humoral and cellular host response for the control of neuroinvasive infection. We previously reported the existence of six alternative open reading frame proteins in WNV genome, one of which entitled WARF4 is exclusively restricted to the lineage I of the virus. WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the in vitro production of WARF4 protein following WNV infection of cultured VERO cells and its immunity in WNV infected individuals. RESULTS: We produced a monoclonal antibody against WARF4 protein (MAb 3A12) which detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. MAb 3A12 specificity to WARF4 protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. Western blotting and bioinformatics analyses strongly suggest that the protein could be translated by programmed -1 ribosomal frameshifting process. Since WARF4 is embedded in the NS4B gene, we rename this novel protein N-NS4B/WARF4. Furthermore, serological analysis shows that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals. CONCLUSIONS: N-NS4B/WARF4 is the second Alternative Reading Frame (ARF) protein that has been demonstrated to be produced following WNV infection and might represent a novel tool for a better characterization of immune response in WNV infected individuals. Further serological as well as functional studies are required to characterize the function of the N-NS4B/WARF4 protein. Since the virus might actually make an extensive use of ARFs, it appears important to investigate the novel six ARF putative proteins of WNV.

A nested PCR approach for unambiguous typing of pestiviruses infecting cattle.

An atypical pestivirus ('Hobi'-like pestivirus, putative bovine viral diarrhoea 3, BVDV-3) was identified firstly in contaminated foetal calf serum batches and isolated subsequently from an outbreak of respiratory disease in a cattle herd in Italy. The isolation of the novel pestivirus from animals affected clinically posed concerns about the validity of BVDV eradication programs, considering that 'Hobi'-like pestivirus (BVDV-3) is undetected or mistyped by the molecular diagnostic tools currently employed. In this paper, the development of a nested PCR (nPCR) assay for unambiguous typing of all bovine pestiviruses is reported. The assay consisted of a first-round amplification using an oligonucleotide pair which binds to conserved sequences located in the 5' untranslated region and capsid gene, followed by a heminested PCR using virus-specific forward primers. The assay performances were evaluated analytically, showing good sensitivity and specificity. By analysis of 100 BVDV-positive samples typed using a nPCR assay discriminating ruminant pestiviruses, five samples recognised previously as BVDV-2 were not typed when submitted to the new assay (n=2) or reacted as 'Hobi'-like pestivirus BVDV-3 (n=3). Sequence analysis of the first-round amplification products showed that the untyped strains were border disease viruses, whereas the other three strains were true 'Hobi'-like viruses. The development of a molecular assay able to identify simultaneously all bovine pestiviruses known currently will help warrant biosafety of live vaccines and other biological products and assess the molecular epidemiology of 'Hobi'-like pestivirus, thus leading to the improvement of the eradication programs through unambiguous typing of pestiviruses infecting cattle.

Plasma levels of TNF-α, IFN-γ, IL-4 and IL-10 during a course of experimental contagious bovine pleuropneumonia.

BACKGROUND: Contagious Bovine Pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is widespread in sub-Saharan Africa. The current live vaccine T1/44 has limited efficacy and occasionally leads to severe side effects in the animals. A better understanding of the immune responses triggered by Mycoplasma mycoides subsp. mycoides and their role in disease progression will help to facilitate the design of a rational vaccine. Currently, knowledge of cytokines involved in immunity and immunopathology in CBPP is rather limited. The aim of this study was to characterize the in vivo plasma concentrations of the cytokines TNF-α, IFN-γ, IL-4, IL-10 and the overall role of CD4+ T cells in the development of cytokine levels during a primary infection. Plasma cytokine concentrations in two groups of cattle (CD4+ T cell-depleted and non-depleted cattle) experimentally infected with Mycoplasma mycoides subsp. mycoides were measured and their relationship to the clinical outcomes was investigated. RESULTS: Plasma cytokine concentrations varied between animals in each group. Depletion of CD4+ T cells did not induce significant changes in plasma levels of TNF-α, IL-4, and IL-10, suggesting a minor role of CD4+ T cells in regulation or production of the three cytokines during the time window of depletion (1-2 weeks post depletion). Unexpectedly, the IFN-γ concentrations were slightly, but statistically significantly higher in the depleted group (p < 0.05) between week three and four post infection. Three CD4+ T cell-depleted animals that experienced severe disease, had high levels of TNF-α and IFN-γ. Only one severely diseased non-depleted animal showed a high serum concentration of IL-4 post infection. CONCLUSIONS: Comparison of most severely diseased animals, which had to be euthanized prior to the expected date, versus less severe diseased animals, irrespective of the depletion status, suggested that high TNF-α levels are correlated with more severe pathology in concomitance with high IFN-γ levels.

Survey of ixodid ticks and two tick-borne pathogens in African buffaloes, Syncerus caffer, from the Caprivi Strip, Namibia.

A capture operation to ascertain health status in free-ranging buffaloes from six different areas in the Caprivi Strip in the northeast corner of Namibia was conducted in October 2009. Basic information on the ticks and tick-borne pathogens normally found in wildlife from this area are scarce. The objective of this study was to assess the host status of African buffaloes, Syncerus caffer, for ixodid ticks and two selected tick-borne pathogens in the Caprivi Strip, a key area bordering Angola, Zambia, Botswana, and Zimbabwe. Four different tick species have been identified among the 233 collected specimens, and, of 95 tested buffaloes, 54 (57%) were positive for Theileria parva, whereas only 3 (3%) showed evidence of being infected with Ehrlichia ruminantium.

An electronic learning course on avian influenza in Italy (2008).

The success of emergency intervention to control contagious animal diseases is dependent on the preparedness of veterinary services. In the framework of avian influenza preparedness, the Italian Ministry of Health, in cooperation with the National Reference Centers for Epidemiology and Avian Influenza, implemented an electronic learning course using new web-based information and communication technologies. The course was designed to train veterinary officers involved in disease outbreak management, laboratory diagnosis, and policy making. The "blended learning model" was applied, involving participants in tutor-supported self-learning, collaborative learning activities, and virtual classes. The course duration was 16 hr spread over a 4-wk period. Six editions were implemented for 705 participants. All participants completed the evaluation assignments, and the drop out rate was very low (only 4%). This project increased the number of professionals receiving high-quality training on AI in Italy, while reducing expenditure and maximizing return on effort.

Confirmation of crayfish plague in Italy: detection of Aphanomyces astaci in white clawed crayfish.

In the summer of 2009, high levels of mortality among white clawed crayfish Austropotamobius pallipes were observed in 3 watercourses of central Italy. PCR and culture methods were used to detect the causative agent of the disease. Two strains of Aphanomyces spp. were isolated and identified by PCR and DNA sequencing as Aphanomyces astaci and A. repetans. This is the first crayfish plague outbreak in Italy to be confirmed by the isolation in culture of a pathogen from Austropotamobius pallipes.

Multispacer sequence typing of Coxiella burnetii from milk and hard tick samples from ruminant farms in Lebanon.

his study was carried out to detect and characterize Coxiella burnetii in ruminant milk samples and in different tick species from seropositive farms in four Lebanese regions. Milk and tick samples were screened for C. burnetii presence by quantitative real-time PCR (qPCR) targeting IS1111 region followed by multispacer sequence typing (MST). The overall positive percentages of 9.6% (27/282) and 95.45% (84/88) for C. burnetii were recorded in ruminant milk and tick samples, respectively. In detail, the C. burnetii DNA was recorded in 52/54 (96.3%) of Rhipicephalus annulatus, 20/21 (95.24%) of Rhipicephalus turanicus, 6/6 (100%) of Hyalomma anatolicum, 5/6 (83.3%) of Rhipicephalus sanguineus and 1/1 of Rhipicephalus bursa. After genotyping of some IS1111-positive samples (17/111), different MST genotypes were identified. Out of 15 positive ticks, 10 were infected with MST2 genotype, 4 were infected with MST7 genotype and 1 was infected with MST57. Moreover, genotypes MST20 and MST58 were found in one cow and one goat milk samples, respectively. The present study confirmed the high genetic diversity of C. burnetii in Lebanon.

The First Serological Study of Q Fever in Humans in Lebanon.

OBJECTIVE: The aim of this study was to estimate, for the first time, the human seroprevalence of Q fever in Lebanon, by assessing the presence of antibodies against the causative agent, Coxiella burnetii. A total number of 421 serum samples (226 females and 196 males) were collected in February 2015 from hospitals and laboratories dispersed in five Lebanese provinces: Akkar, Bekaa, Mount Lebanon, Nabatieh, and South Lebanon. METHODS: Serial testing approach was used. Samples were first screened for IgG phase II antibodies against C. burnetii by Enzyme Linked Immunosorbent Assay (ELISA) Kit. Then, both positive and inconclusive sera were reexamined by immunofluorescence assay (IFA) test with the aims to confirm and specify the infection status (past or probably acute infection) by detecting IgG (I/II) and IgM (I/II) in human sera. RESULTS: Screening of 421 samples was estimated to be 38.70% (95% CI 34-43.3) positive samples, 5.90% (95% CI 3.7-8.2) suspect samples (as doubtful results), and 55.40% (95% CI 50.7-60.1) negative samples. Furthermore, all positive and suspect samples by ELISA test were retested by immunofluorescence assay test (IFAT), and the prevalence of positive sample was 37% and the infection case was recorded: 23.75% (95% CI 19.7-27.8) samples resulted from past infection, 1.9% (95% CI 0.6-3.2) probably acute infection characterized by several dominance clinical symptoms as: fever, cough, headache, difficulty breathing, and atypical pneumonia, and 0.23% (95% CI 0-0.7) inconclusive sample accompanied by different symptoms as bone metastasis and lung cancer. CONCLUSION: The study records the exposition of 37% of 421 patients to C. burnetii distributed in five Lebanese provinces with the highest seroprevalence in Bekaa and Akkar provinces and the lowest reported in Mount Lebanon. This difference may be due to the presence of high density of livestock production and of major agricultural areas in these two provinces.

A rapid multiplexed chemiluminescent immunoassay for the detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes pathogen bacteria.

A simple and rapid multiplexed sandwich chemiluminescent enzyme immunoassay has been developed for the simultaneous detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes. To achieve the multiplexed detection of the four pathogens, a new polystyrene 96 well microtiter plate format has been designed, in which each main well contains four subwells in the bottom. The monoclonal antibodies specific for each bacteria were separately immobilized in each subwell. When the samples were added to the main wells, the bacteria able to specifically bind to the corresponding monoclonal antibody were captured in one of the four subwells. Subsequently, a mixture of peroxidase-labeled polyclonal antibodies against the four bacteria was added and the peroxidase activity of the bound polyclonal labeled antibodies in each well was measured by an enhanced luminol-based chemiluminescent cocktail using a low-light charge-coupled imaging device. The assay was simple and fast, and the limit of quantification was in the order of 104-105 CFU/mL for all bacterial species. The accuracy of the method, evaluated by comparison of the results with a conventional culturing methodology, was satisfactory, with recovery values ranging from 90 to 120%. This method can be used as a screening test to evaluate the presence of these pathogen bacteria in different foodstuffs.

Laboratory diagnosis of contagious ecthyma: comparison of different PCR protocols with virus isolation in cell culture.

A new polymerase chain reaction (PCR) assay for rapid diagnosis of contagious ecthyma was designed and applied to 21 clinical samples from Greece. This assay, which detects a highly conserved gene from the parapox genome, was evaluated for its sensitivity and specificity in order to be considered as a useful diagnostic tool. A comparative study with two published PCR protocols one using primers PPP1-PPP3, PPP1-PPP4 which targets putative virion envelope gene B2L and the other using VIR1-VIR2 primers which amplifies ORF virus interferon resistant (VIR) gene, as well as cell culture virus neutralization assay was carried out. All samples tested were amplified successfully with the PCR protocol established in the laboratory. The combination of primers PPP1-PPP3 and PPP1-PPP4 in a semi-nested PCR gave a positive result in 20 of 21 samples while primers VIR1-VIR2 failed to amplify successfully 7 of 21 samples. The diagnostic value of parapox viral DNA amplification was also compared with the results of virus isolation by cell culture and was positive in three samples that the virus isolation was obtained.

Phylogenetic correlation of Greek and Italian orf virus isolates based on VIR gene.

Thirteen orf virus isolates obtained during the time period between 1995 and 2004 from crusted scab lesions of nine sheep and four goats from different geographical areas of Greece and Italy with suspected contagious ecthyma infection were analyzed. DNA of all isolates was successfully amplified by PCR with the primers 045F-045R and identified them as parapox virus. Partial DNA sequence of orf virus interferon resistant (VIR) gene, phylogenetic analysis of the available isolates and amino acid comparison of the interferon resistance protein encoded by this genomic region was carried out. According to the results of the present report a precise characterisation of the genomic region studied might provide evidence for the genetic variation and movement of the circulating orf virus strains.

Expression of bioactive recombinant bovine interferon-gamma using baculovirus.

The precise role of bovine interferon-gamma (BoIFN-gamma) in disease and therapy is still poorly defined. Clearly it is involved in defence against parasites, bacteria, viruses and possibly tumor cells. This paper reports the expression of BoIFN-gamma in a baculovirus system to generate a fully functional recombinant protein. Bovine interferon-gamma cDNA was cloned from mitogen stimulated peripheral blood mononuclear cell (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The cDNA open reading frame (ORF) encoding for a putative 166 amino acid protein (22KDa) was cloned and expressed into baculovirus transfer vector pBlueBac 4.5/V5 His. This vector was co-transfected with Autografa californica multiple nuclear polyhedrosis virus (AcMNPV) DNA into Spodoptera frugiperda cells (Sf9) and the recombinant virus, named AcBoIFN-gamma, was then recovered. Recombinant BoIFN-gamma (rBoIFN-gamma His) was accumulated in the serum-free medium of AcBoIFN-gamma-infected cells. The nickel affinity spin column purified rBoIFN-gamma His was shown to be a glycosylated 20-22 KDa protein as confirmed by SDS-PAGE glycan determination and showed antiviral activity in vitro against the bovine viral diarrhoea-mucosal disease virus (BVD/MD). The production of this bioactive rBoIFN-gamma His will allow us to explore this cytokine as a potential vaccine adjuvant or therapeutic agent for bovine diseases.

A promising new ELISA diagnostic test for cattle babesiosis based on Babesia bigemina Apical Membrane Antigen-1.

Babesiosis due to Babesia bigemina is a relevant tick-borne disease, affecting cattle worldwide. Many surface proteins of the pathogen including the Apical Membrane Antigen 1 (AMA-1) - have been analysed for vaccine and diagnostic purposes. This study focused on B. bigemina AMA-1 and on its use for the assessment of diagnostic tests. After bioinformatic analyses, AMA-1 codifying region was amplified and cloned into an expression vector used to induce protein synthesis in Escherichia coli cells. AMA-1 was purified by affinity chromatography and used to set up the best condition for an ELISA protocol. Bovine field sera positive to B. bigemina were used to evaluate the presence of anti-AMA-1 antibodies. In order to verify the assay specificity, sera positive to Babesia bovis or to the piroplasm Theileria annulata were also included. Significant differences were obtained between sera negative to both B. bigemina and B. bovis and samples positive to B. bigemina, to B. bovis or to both pathogens. No significant reaction was observed with T. annulata positive sera. The results showed that AMA-1 protein is suitable to be used as antigen in diagnostic assays for babesiosis diagnosis in cattle, as it does not show any cross reaction with anti-T. annulata antibodies.

Evidence of co-infection with Mycobacterium bovis and tick-borne pathogens in a naturally infected sheep flock.

Ticks are responsible for the transmission of pathogens of veterinary importance, including those affecting sheep. The current study was designed to investigate co-infections with tick-borne and other pathogens in a naturally infected sheep flock with poor health condition using serology and PCR. Infection with Anaplasma ovis was detected by serology and PCR in 56% of the animals. The presence of Rickettsia spp. of the Spotted Fever Group (SFG) was detected by PCR and sequence analysis in 31% of the animals. All the animals were negative for Anaplasma phagocytophilum either by serology or PCR. Twelve sheep were randomly selected for anatomopathological studies. Five of these animals presented lesions consistent with Mycobacterium tuberculosis complex (MTBC) infection and spoligotyping confirmed infection with Mycobacterium bovis spoligotype SB0339. Co-infection with tick-borne pathogens and MTBC could contribute to the poor health condition observed in these animals but other uncontrolled factors may also be responsible. The differential expression of immune response genes supported previous findings in ruminants and suggested that infection with tick-borne pathogens and M. bovis may results in unique gene expression patterns in sheep. The results underline the need for further research into the possible role of sheep in the epidemiology of animal tuberculosis.

The 2011 West Nile disease outbreak in Sardinia region, Italy.

In 2011, strains of West Nile Virus (WNV) belonging to lineage 1 spread for the first time in Sardinia region (Italy). In contrast to previous WNV Italian incursion, the strains were found in Culex modestus and, more surprisingly, they were able to cause severe clinical signs in the affected birds. Based on the partial sequence of the NS3 encoding gene, the Sardinian WNV strains demonstrated a high similarity with the other WNV strains recently detected in the Mediterranean Basin. Nonetheless, the 2011 Sardinian sequences were grouped in a distinct sub-cluster. Both the NS3-249P and NS3-249T genotypes were detected in the Sardinian outbreaks confirming that the co-circulation of different genotypes in the affected population might be common for WNV as for many RNA viruses. No association, however, was observed between virulence and viral genotype.

A molecular survey of Rickettsia felis in fleas from cats and dogs in Sicily (Southern Italy).

Rickettsia felis, the agent of flea-borne spotted fever, has a cosmopolitan distribution. Its pathogenic role in humans has been demonstrated through molecular and serologic tests in several cases. The cat flea (Ctenocephalides felis) is considered the main reservoir and the biological vector. The aim of this study was to assess the presence and occurrence of R. felis in fleas collected from dogs and cats in various sites of Palermo (Sicily). Between August and October 2012, 134 fleas were collected from 42 animals: 37 fleas from 13 dogs and 97 fleas from 29 cats. Two species of fleas were identified: 132 Ctenocephalides felis (98.51%) collected on all animals and only two C. canis (1.49%) on one dog. Out of 132 C. felis, 34 (25.76%), 12 from dogs (32.43%) and 22 (22.68%) from cats, were positive for R. felis DNA by a polymerase chain reaction (PCR), confirmed by sequencing. The only two C. canis fleas were negative. About half of examined animals (47.62%, 20/42) were infested with at least one infected flea; in particular 46.15% of dogs (6/13) and 48.28% of cats (14/29). It seems that in the Palermo district there is a peri-domestic cycle, with a relatively high prevalence of R. felis infection in the cat flea, an insect widely diffused in home environments and which can frequently bite humans. The results also suggest that R. felis should be considered in the human differential diagnosis of any spotted-like fever or febrile illness without a clear source of infection in Sicily, especially if the patient is known to have been exposed to flea bites.