Rabies Vaccination: Higher Failure Rates in Imported Dogs than in those Vaccinated in Italy.

The current European Union (EU) legislation decrees that pets entering the EU from a rabies-infected third country have to obtain a satisfactory virus-neutralizing antibody level, while those moving within the EU require only rabies vaccination as the risk of moving a rabid pet within the EU is considered negligible. A number of factors driving individual variations in dog vaccine response have been previously reported, including a high rate of vaccine failure in puppies, especially those subject to commercial transport. A total of 21 001 observations collected from dogs (2006-2012) vaccinated in compliance with the current EU regulations were statistically analysed to assess the effect of different risk factors related to rabies vaccine efficacy. Within this framework, we were able to compare the vaccination failure rate in a group of dogs entering the Italian border from EU and non-EU countries to those vaccinated in Italy prior to international travel. Our analysis identified that cross-breeds and two breed categories showed high vaccine success rates, while Beagles and Boxers were the least likely to show a successful response to vaccination (88.82% and 90.32%, respectively). Our analysis revealed diverse performances among the commercially available vaccines, in terms of serological peak windows, and marked differences according to geographical area. Of note, we found a higher vaccine failure rate in imported dogs (13.15%) than in those vaccinated in Italy (5.89%). Our findings suggest that the choice of vaccine may influence the likelihood of an animal achieving a protective serological level and that time from vaccination to sampling should be considered when interpreting serological results. A higher vaccine failure in imported compared to Italian dogs highlights the key role that border controls still have in assessing the full compliance of pet movements with EU legislation to minimize the risk of rabies being reintroduced into a disease-free area.

Serological evidence of H9N2 avian influenza virus exposure among poultry workers from Fars province of Iran.

BACKGROUND: Since the 1990s, influenza A viruses of the H9N2 subtype have been causing infections in the poultry population around the globe. This influenza subtype is widely circulating in poultry and human cases of AI H9N2 have been sporadically reported in countries where this virus is endemic in domestic birds. The wide circulation of H9N2 viruses throughout Europe and Asia along with their ability to cause direct infection in mammals and humans, raises public health concerns. H9N2 AI was reported for the first time in Iran in 1998 and at present it is endemic in poultry. This study was carried out to evaluate the exposure to H9N2 AI viruses among poultry workers from the Fars province. METHODS: 100 poultry workers and 100 healthy individuals with no professional exposure to poultry took part in this study. Serum samples were tested for antibodies against two distinct H9N2 avian influenza viruses, which showed different phylogenetic clustering and important molecular differences, such as at the amino acid (aa) position 226 (Q/L) (H3 numbering), using haemagglutination inhibition (HI) and microneutralization (MN) assays. RESULTS: Results showed that 17 % of the poultry workers were positive for the A/chicken/Iran/10VIR/854-5/2008 virus in MN test and 12 % in HI test using the titer ≥40 as positive cut-off value. Only 2 % of the poultry workers were positive for the A/chicken/Iran/12VIR/9630/1998 virus. Seroprevalence of non exposed individuals for both H9N2 strains was below 3 % by both tests. Statistical analyses models showed that exposure to poultry significantly increases the risk of infection with H9N2 virus. CONCLUSIONS: The results have demonstrated that exposure to avian H9N2 viruses had occurred among poultry workers in the Fars province of Iran. Continuous surveillance programmes should be implemented to monitor the presence of avian influenza infections in humans and to evaluate their potential threat to poultry workers and public health.

Pathologic characterization of genotypes XIV and XVII Newcastle disease viruses and efficacy of classical vaccination on specific pathogen-free birds.

To characterize the clinicopathologic features of recently described genotypes of Newcastle disease virus (NDV), 1 representative strain of genotype XIV and 2 of genotype XVII, all isolated from West Africa, were used to infect groups of ten 4-week-old specific pathogen-free chickens. The pathobiology of these 3 strains was compared to a South African NDV strain classified within genotype VII. All chickens infected with the 4 viruses died or were euthanized by day 4 postinfection due to the severity of clinical signs. Gross and histologic lesions in all infected chickens included extensive necrosis of lymphoid tissues (thymus, spleen, bursa of Fabricius, cecal tonsils, gut-associated lymphoid tissue), gastrointestinal necrosis and hemorrhages, and severe hemorrhagic conjunctivitis. Immunohistochemical staining revealed systemic viral distribution, and the most intense staining was in the lymphoid organs. Results demonstrate that the 3 West African strains from the previously uncharacterized genotypes XIV and XVII are typical velogenic viscerotropic NDV strains with lesions similar to the South African strain. Under experimental conditions, QV4 and LaSota NDV vaccine strains successfully protected chickens from morbidity and mortality against the genotype VII and one genotype XVII NDV strain, with no significant differences in the amount of virus shed when 2 vaccine schemes were compared.

Hepatitis E virus genotype 4 in a pig farm, Italy, 2013.

Zoonotic strains of hepatitis E virus (HEV) in Europe have been reported to belong to genotypes 3 and 4. In 2012 and 2013, 57 pig farms in Northern Italy that had previously resulted seropositive for HEV were surveyed for the presence of the virus, with positive samples subsequently genotyped. Hepatitis E RNA was identified in 17/57 (29·8%) seropositive farms. Phylogenetic analysis demonstrated that distinct subtypes of genotype 3 were circulating in the north-east of Italy; as well, for the first time in the Italian swine population, genotype 4 was identified and attributed to subtype d.

Concomitant Infection with Leishmania donovani and L. major in Single Ulcers of Cutaneous Leishmaniasis Patients from Sudan.

In Sudan human leishmaniasis occurs in different clinical forms, that is, visceral (VL), cutaneous (CL), mucocutaneous (ML), and post-kala-azar dermal leishmaniasis (PKDL). Clinical samples from 69 Sudanese patients with different clinical manifestations were subjected to a PCR targeting the cytochrome oxidase II (COII) gene for Leishmania species identification. Mixed infections were suspected due to multiple overlapping peaks presented in some sequences of the COII amplicons. Cloning these amplicons and alignment of sequences from randomly selected clones confirmed the presence of two different Leishmania species, L. donovani and L. major, in three out of five CL patients. Findings were further confirmed by cloning the ITS gene. Regarding other samples no significant genetic variations were found in patients with VL (62 patients), PKDL (one patient), or ML (one patient). The sequences clustered in a single homogeneous group within L. donovani genetic group, with the exception of one sequence clustering with L. infantum genetic group. Findings of this study open discussion on the synergetic/antagonistic interaction between divergent Leishmania species both in mammalian and vector hosts, their clinical implications with respect to parasite fitness and response to treatment, and the route of transmission with respect to vector distribution and or adaptation.

Comparative pathogenicity study of ten different betanodavirus strains in experimentally infected European sea bass, Dicentrarchus labrax (L.).

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a severe pathological condition caused by RNA viruses belonging to the Nodaviridae family, genus Betanodavirus. The disease, described in more than 50 fish species worldwide, is considered as the most serious viral threat affecting marine farmed species in the Mediterranean region, thus representing one of the bottlenecks for further development of the aquaculture industry. To date, four different genotypes have been identified, namely red-spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus and barfin flounder nervous necrosis virus, with the RGNNV genotype appearing as the most widespread in the Mediterranean region, although SJNNV-type strains and reassortant viruses have also been reported. The existence of these genetically different strains could be the reason for the differences in mortality observed in the field. However, very little experimental data are available on the pathogenicity of these viruses in farmed fish. Therefore, in this study, the pathogenicity of 10 isolates has been assessed with an in vivo trial. The investigation was conducted using the European sea bass, the first target fish species for the disease in the Mediterranean basin. Naive fish were challenged by immersion and clinical signs and mortality were recorded for 68 days; furthermore, samples collected at selected time points were analysed to evaluate the development of the infection. Finally, survivors were weighed to estimate the growth reduction. The statistically supported results obtained in this study demonstrated different pathogenicity patterns, underlined the potential risk represented by different strains in the transmission of the infection to highly susceptible species and highlighted the indirect damage caused by a clinical outbreak of VER/VNN.

Swine influenza in sub-saharan Africa--current knowledge and emerging insights.

Pigs have been associated with several episodes of influenza outbreaks in the past and are considered to play a significant role in the ecology of influenza virus. The recent 2009 pandemic influenza A/H1N1 virus originated from swine and not only did it cause widespread infection in humans, but was also transmitted back to swine in Asia, Europe and America. What may be the prevailing situation in Africa, particularly in sub-Saharan Africa, with respect to the circulation of classical swine or pandemic influenza? The ecology of influenza viruses, as well as the epidemiology of human or animal influenza, is poorly understood in the region. In particular, little is known about swine influenza in Africa despite the relevance of this production in the continent and the widespread pig husbandry operations in urban and rural areas. In this review, the gap in the knowledge of classical and pandemic swine influenza is attributed to negligence of disease surveillance, as well as to the economic and public health impact that the disease may cause in sub-Saharan Africa. However, emerging serological and virological evidence of swine influenza virus in some countries in the region underscores the importance of integrated surveillance to better understand the circulation and epidemiology of swine influenza, a disease of global economic and public health importance.

Fatal case of human rabies imported to Italy from India highlights the importance of adequate post-exposure prophylaxis, October 2011.

In October 2011, an Indian man resident in Italy was admitted to a hospital in Mantua, Italy with symptoms of acute encephalitis. Due to a recent history of bite by a suspected rabid dog in India, where he had received incomplete post-exposure treatment, rabies was suspected. The patient died after 22 days of intensive care treatment and rabies was confirmed post mortem. This report stresses the need of appropriate post-exposure prophylaxis in rabies-endemic countries.

An SYBR Green-based real-time RT-PCR assay for the detection of H5 hemagglutinin subtype avian influenza virus.

Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulted in the need of novel sensitive and specific molecular assays. In this study, an SYBR Green-based real-time reverse transcription-PCR (RRT-PCR) assay was developed for the detection of H5 subtype AI virus. Sequence analysis of the Mexican lineage H5N2 isolates (subgroup B) revealed several mismatches in the primer/hydrolysis probe set reported in the commonly used RRT-PCR assay for the detection of H5 North American lineage. The present assay was designed to circumvent the challenge that these viruses represent for the specific detection of H5 subtype AI viruses. This RRT-PCR assay successfully detected a range of different H5 subtype AI strains from both Eurasian and North American lineages representing different avian H5 HA clades from diverse geographical locations. The sensitivity of the present method was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection of 10(0) 50% egg infectious dose (EID50)/mL and 4.2 gene copies/µl. The linear ranges of the assay span within 10(6)-10(0) EID50/mL and 10(6)-10(0) gene copies/µl. The results obtained from this method were directly compared with those of the H5 RRT-PCR assay recommended by the OIE. The comparison was performed with 110 tracheal and cloacal swabs from various bird species collected during field and laboratory investigations in Eurasia and Africa in 2006 and 2008 and showed 100% agreement. This assay is recommended as an alternative method, also allowing a 'double check' approach detection, to be use mainly in outbreak scenarios with higher risk of poultry infections by Central American/Caribbean H5 AI viruses.

Understanding the ecological drivers of avian influenza virus infection in wildfowl: a continental-scale study across Africa.

Despite considerable effort for surveillance of wild birds for avian influenza viruses (AIVs), empirical investigations of ecological drivers of AIV prevalence in wild birds are still scarce. Here we used a continental-scale dataset, collected in tropical wetlands of 15 African countries, to test the relative roles of a range of ecological factors on patterns of AIV prevalence in wildfowl. Seasonal and geographical variations in prevalence were positively related to the local density of the wildfowl community and to the wintering period of Eurasian migratory birds in Africa. The predominant influence of wildfowl density with no influence of climatic conditions suggests, in contrast to temperate regions, a predominant role for inter-individual transmission rather than transmission via long-lived virus persisting in the environment. Higher prevalences were found in Anas species than in non-Anas species even when we account for differences in their foraging behaviour (primarily dabbling or not) or their geographical origin (Eurasian or Afro-tropical), suggesting the existence of intrinsic differences between wildfowl taxonomic groups in receptivity to infection. Birds were found infected as often in oropharyngeal as in cloacal samples, but rarely for both types of sample concurrently, indicating that both respiratory and digestive tracts may be important for AIV replication.

Recurrent rotavirus diarrhoea outbreaks in a stud farm, in Italy.

A total of 47 stool samples were collected at the same stud farm from young foals with rotavirus diarrhoea and from their stud mares. Illness involved foals during three consecutive winter seasons. Infection in the farm appeared firstly in January-February 2008. After vanishing in the warm seasons, cases reappeared in March 2009 and 2010. Determination of the rotavirus G- and P-types was carried out using nested RT-PCR in samples collected in 2009 and 2010. A total of 19 of 47 samples resulted positive for rotavirus. The G type was determined in 19/47 samples, whereas the P genotype was determined in 17/47 samples. All equine strains presented a G14 VP7 in combination with a P[12] VP4, suggesting persistence of the same viral strain in the stud farm, during at least two consecutive winter periods. Sequence analysis of the genes encoding the outer capsid rotavirus proteins VP7 and VP4 revealed that the virus had a close relationship between strains recently isolated in the rest of Europe.

Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens.

Betanodaviruses are the causal agents of viral encephalo-retinopathy, an infectious disease affecting more than 40 marine fish species, characterized by high morbidity and mortality. Because of its severe impact, robust diagnostic tools are required. The aim of this work was to develop and validate a real-time TaqMan PCR assay to detect betanodaviruses in clinical specimens by amplifying a conserved region of the RNA2 strand. The method proved to be specific and sensitive, being capable of detecting as low as 10 TCID(50)/ml. For clinical validation, samples from 100 marine fish were collected during a natural outbreak of disease and tested by three distinct laboratory methods, namely real-time TaqMan PCR, RT-seminested PCR and virus isolation. The results indicated optimal agreement between tests. The assay that was developed is capable of detecting members of all of the betanodavirus genetic groups currently described and can be considered a valid alternative to the time-consuming and contamination-prone nested PCR.

Emergence of a new genetic lineage of Newcastle disease virus in West and Central Africa--implications for diagnosis and control.

Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which affect many species of birds and may cause severe economic losses in the poultry sector. The disease has been officially and unofficially reported in many African countries and still remains the main poultry disease in commercial and rural chickens of Africa. Unfortunately, virological and epidemiological information concerning ND strains circulating in the Western and Central regions of Africa is extremely scarce. In the present study, sequence analysis, pathotyping and detailed genetic characterization of virulent ND strains detected in rural poultry in West and Central Africa revealed the circulation of a new genetic lineage, distinguishable from the lineages described in the Eastern and Southern parts of the continent. Several mismatches were observed in the segment of the matrix gene targeted by the primers and probe designed for the molecular detection of APMV-1, which were responsible for the false negative results in the diagnostic test conducted. Furthermore, deduced amino acid sequences of the two major antigens eliciting a protective immune response (F and HN glycoprotein) revealed protein similarities <90% if compared to some common vaccine strains. Distinct mutations located in the neutralizing epitopes were revealed, indicating the need for detailed assessment of the efficacy of the current vaccines and vaccination practices in Africa. The present investigation provides important information on the epidemiology, diagnosis and control of NDV in Africa and highlights the importance of supporting surveillance in developing countries for transboundary animal diseases.

The first specific detection of a highly pathogenic avian influenza virus (H5N1) in Ivory Coast.

The Virology Laboratory of the Central Laboratory of Animal Diseases in Ivory Coast at Bingerville received samples of wild and domestic avian species between February and December 2006. An RT-PCR technique was used to test for avian influenza (AI) and highly pathogenic AI subtype viruses. Among 2125 samples, 16 were type A positive; of which, 12 were later confirmed to be H5N1. Fifteen of these 16 type A positive samples were inoculated into the chorioallantoic cavity of 11-day-old embryonated hens' eggs for virus isolation. Eight produced virus with hemagglutination titres from 1/64 to 1/512. The 4/16 M-RT-PCR positive samples, which were H5N1 negative, were shown to be H7 subtype negative. The diagnostic efficiency of the laboratory for the surveillance of H5N1 in Ivory Coast was demonstrated. The positive cases of H5N1 were from a sparrowhawk (Accipter nisus); live market poultry and in free-range poultry, where the mortality rate was approximately 20% (2/10) and 96.7% (29/30) respectively. Currently, investigations into intensive poultry farms have proved negative for H5N1. No human cases have been reported this time.

Prevalence, disease associations and risk factors for colonization with intestinal spirochaetes (Brachyspira spp.) in flocks of laying hens in north-eastern Italy.

The present study investigated the occurrence of anaerobic intestinal spirochaetes of the genus Brachyspira in laying hen flocks in Treviso province, north-eastern Italy, with respect to prevalence, spirochaete species present, disease associations and risk factors for colonization. A total of 450 faecal samples from 45 sheds on 29 laying hen farms were cultured for intestinal spirochaetes. Nineteen sheds on 12 farms contained chickens with symptoms consistent with avian intestinal spirochaetosis, including reduced egg production, wet litter and/or pasty vents. Spirochaetes were isolated from 157 (34.8%) samples from 21 (72.4%) farms, and from 32 (71.1%) sheds. From these positive samples, 189 spirochaetal isolates were speciated using three polymerase chain reaction assays and a restriction fragment polymorphism analysis of 16S rDNA polymerase chain reaction products. Overall, 52 (27.5%) isolates were identified as pathogenic Brachyspira intermedia, 26 (13.8%) as pathogenic Brachyspira pilosicoli, 93 (49.7%) as non-pathogenic (Brachyspira innocens/Brachyspira murdochii), and 18 (9.6%) were unidentified. Faeces from 14 sheds (31%) on 10 farms (34.5%) contained B. intermedia and/or B. pilosicoli, and disease consistent with avian intestinal spirochaetosis was observed in nine of these sheds on seven farms. There was a significant association (P=0.042) between the presence of spirochaetes and using deep pits rather than conveyor belts for manure disposal. Sheds housing chickens >40 weeks of age were significantly more likely to contain spirochaetes (P=0.048) and pathogenic species (P=007) than sheds housing younger chickens. A significant association (P=0.02) was found between infection with pathogenic spirochaetes and reduced egg production.

New perspectives in avian influenza diagnosis.

Recent outbreaks of avian influenza (AI) occurring in Europe, in the Americas, in Asia and in Africa have provided field evidence of how challenging the control of this infection can be, particularly in densely populated poultry areas or in areas where free-range rural village poultry and backyard flocks are present. In these areas, laboratory testing is mainly applied to trace viral circulation in a given area or in a susceptible population to implement an early warning system, rather than to diagnose the presence of the virus in a diseased flock or animal. This implies the use of rapid, sensitive and possibly cost-effective laboratory tests adaptable to very high throughputs. As a consequence, new diagnostic approaches and technologies have been increasingly developed and applied. Molecular biology and biotechnology are providing important and precious contributions to the field of AI diagnosis, making extremely rapid, specific and sensitive techniques available. However, the use of some of these technologies is still limited, due to their costs and to the requirement of advanced technical and scientific expertise. Therefore, more conventional and well-established techniques, should not be abandoned but rather reconsidered and improved or modified.