Occurrence and risk factors of Coxiella burnetii in domestic ruminants in Lebanon.

Coxiella burnetii causes diseases in humans (Q fever) and animals, domestic ruminants playing a major role in the epidemiology of the infection. Information on C. burnetii infection in Lebanon is scanty. In order to assess the prevalence of C. burnetii infection in ruminants, a cross-sectional study was undertaken in 2014. A total of 1633 sera from ruminants (865 cattle, 384 sheep and 384 goats) from 429 farms (173 cattle, 128 sheep and 128 goats), in seven provinces of Lebanon were randomly selected and assayed for the presence of antibodies. 39.86% of farms (95% CI: 35.23-44.56) resulted positive. The seroprevalence was 30.63% in Cattle-farms, 46.88% in sheep-farms and 45.31% in goat-farms. Milk samples collected from 282 seropositive animals (86 cows, 93 sheep and 103 goats) from 171 positive farms were tested by a high sensitive Real-Time PCR targeted to the IS1111 transposon of C. burnetii. The overall prevalence in farms was estimated to be 14.04%. Cattle-, sheep- and goat farm prevalence rates were 15.09%, 10% and 17.24%, respectively. The findings of the study show that C. burnetii prevalence in Lebanese domestic ruminants is related to animal species and farming practices. Indeed, the mixed herds with sheep (p < 0.01), the presence of common lambing/kidding areas (p < 0.001) in farms where the use of disinfectants was not a routine practice (p < 0.05) were identified as important risk factors. The results of the study provide baseline information for setting up herd management and public health measures for the prevention and control of Q fever in Lebanon.

Development and Preliminary Evaluation of a New Real-Time RT-PCR Assay For Detection of Peste des petits Ruminants Virus Genome.

A duplex real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for a simple and rapid diagnosis of Peste des petits ruminants (PPR). qRT-PCR primers and TaqMan probe were designed on a conserved region of nucleocapsid protein (Np) of PPR virus (PPRV) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA(®) was used as an internal positive control (IPC) for either RNA isolation or RT-PCR steps. The detection limit of the newly designed duplex real-time RT-PCR (qRT-PCR PPR_Np) was approximately 20 copies/µl with a 95% probability. No amplification signals were recorded when the qRT-PCR PPR_Np was applied to viruses closely related or clinically similar to PPRV- or to PPR-negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of Africa and Middle East. qRT-PCR PPR_Np showed higher sensitivity than the conventional gel-based RT-PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false-negative results caused by the amplification failure, thus improving the accuracy of PPRV detection.

ANALYSIS OF BIOTIC AND ABIOTIC FACTORS INFLUENCING THE OCCURRENCE OF WEST NILE VIRUS INFECTION IN TUNISIA.

Eco-climatic conditions are often associated with the occurrence of West Nile Disease (WND) cases. Among the complex set of biotic and abiotic factors influencing the emergence and spread of this vector-borne disease, two main variables have been considered to have a great influence on the probability of West Nile Virus (WNV) introduction and circulation in Tunisia: the presence of susceptible bird populations and the existence of geographical areas where the environmental and climatic conditions are more favourable to mosquito multiplications. The aim of this study was to identify and classify the climatic and environmental variables possibly associated with the occurrence of WNVhuman cases in Tunisia. The following environmental and climatic variables have been considered: wetlands and humid areas, Normalised Difference Vegetation Index (NDVI), temperatures and elevation. A preliminary analysis for the characterization of main variables associated with areas with a history of WNV human cases in Tunisia between 1997 and 2011 has been made. This preliminary analysis clearly indicates the closeness to marshes ecosystem, where migratory bird populations are located, as an important risk factor for WNV infection. On the contrary the temperature absolute seems to be not a significant factor in Tunisian epidemiological situation. In relation to NDVI values, more complex considerations should be made.

Evidence of West Nile virus lineage 2 circulation in Northern Italy.

A West Nile virus (WNV) strain belonging to lineage 2 was for the first time detected in two pools of Culex pipiens collected in the province of Udine and in tissues of a wild collared dove (Streptopelia decaocto) found dead in the province of Treviso, in North East of Italy. It was molecularly identified by group and WNV lineage specific RT-PCRs and characterized by partial sequencing of the NS3 and NS5 genes. When compared with the sequences of same fragments of NS3 and NS5 of the WNV lineage 2 strain isolated from birds of prey in Hungary (2004), the phylogenetic analysis of these sequences revealed 100% and 99% similarity, respectively. As the Hungarian strain, the NS3 selected sequence differed from the 2010 Greek isolate by one amino-acid located at 249 site which is the site involved in genetic modulation of WNV pathogenicity. The Italian and Hungarian strains have histidine rather than proline at this site. The presence of a lineage 2 strain in regions where the lineage 1 strain is still circulating, creates a new scenario with unpredictable consequences. In this situation comprehensive investigations on the occurrence, ecology, and epidemiology of these different WNV strains circulating in Italy become the highest priority.

West Nile transmission in resident birds in Italy.

Migratory birds are considered one of the main sources for West Nile virus (WNV) introduction into European countries. Following the WNV epidemic in the late summer of 1998 in a marshy area of Tuscany (Padule of Fucecchio), an extensive ornithological surveillance programme was carried out in the infected areas from 2006 to 2008. Several species of migratory and resident birds were trapped, sampled and serologically tested. The results of this surveillance programme gave a useful indication of potential sources of WNV re-introduction and spread into Italy. The area under study was also investigated and classified into ecological areas through satellite image processing. In August 2008, the WNV infection re-emerged in Italy in the area surrounding the Po river delta, involving three regions: Lombardy, Emilia Romagna and Veneto. Several surveillance activities were immediately put in place, including the extensive monitoring of wild birds found dead or trapped in the framework of other surveillance programmes. These activities were also prolonged in the 2009, when the virus circulation re-occurred at the border of the area already infected in 2008. The possible epidemiological role of the different species of migratory and resident birds is discussed, in relation to the different ecological patterns identified in the area and their potential ability to introduce, spread and support the endemization of WNV infection.

2009 West Nile disease epidemic in Italy: first evidence of overwintering in Western Europe?

For the second consecutive year a West Nile disease (WND) epidemic has affected Italy causing disease in horses and humans. The infection re-occurred in the same places of the 2008 and moved westerly and southerly involving new areas and regions. The whole genome sequence of the Italian 2009 West Nile disease isolate (WNDV) was compared with those responsible for the 2008 WND outbreaks. The epidemiological findings of the two years of epidemic were compared as well. The high identity between 2008 and 2009 WNV strains (>99%), the earlier virus circulation in 2009 and the re-occurrence of the disease starting from the bordering infected areas reached by the infection in the previous year, strongly support the hypothesis of the overwintering of the virus and the endemisation to local host populations.

A new duplex real-time RT-PCR assay for sensitive and specific detection of African horse sickness virus.

A new real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for a simple and rapid diagnosis of African Horse Sickness (AHS) was developed. Primers and FAM-labeled TaqMan-MGB probes specific for African horse sickness virus (AHSV) were selected from the consensus sequence of the segment 8 of all 9 serotypes of AHSV reference strains. For the determination of the analytical sensitivity, an in vitro transcript (AHS_ns2T7) of the target region was constructed and tested. Furthermore, the AHS_ns2T7 transcript was used either as positive control or as a standard for quantifying target copies. A commercial heterologous Armored RNA was used as an internal positive control (IPC) for both RNA isolation and RT-PCR steps. The qRT-PCR AHS_ns2 was able to amplify the target sequence up to 0.71 copies/reaction. Its flexibility allowed to amplify a wide dynamic range of RNA copies from 1.5 to 0.001fg. Within this range, the Ct values varied from 18 to 38 cycles with SD values always lower than 0.5 confirming their strong and constant linear correlation with the RNA target. Furthermore the newly designed duplex real-time RT-PCR proved to be strictly AHSV-specific as it did not amplify close related viruses.

Antibody response in cattle vaccinated against bluetongue serotype 8 in Italy.

To assess the immunogenicity of Zulvac 8 Bovis (a commercial inactivated vaccine against bluetongue virus serotype 8 - BTV8) under field conditions, 71 cattle vaccinated according to manufacturer schedule in Verona province (Italy) were tested for the presence of BTV8 neutralizing antibodies at 21, 29, 36, 43, 49, 102 and 201 days post-vaccination (dpv). Another group of 528 BTV8 vaccinated cattle in Mantova province (Italy) was also tested once between 113 and 174 dpv. The vaccine was able to elicit an immune response in 69 (97.2%) and 346 (65.5%) animals of the Verona and Mantova groups, respectively.

West Nile virus transmission in 2008 in north-eastern Italy.

After 10 years, West Nile virus (WNV) re-emerged in Italy in August 2008. As on 31 December 2008, the infection affected eight Provinces in three Regions (Emilia Romagna, Veneto, Lombardy), where a total of 794 cases of WNV infection in 251 equine stables were detected on the basis of the clinical signs and as a result of a serological screening in horses living in the area. Only 4.0% (32/794) of the serologically positive animals showed clinical signs, and the 32 clinical cases were reported in 18 different farms. The observed case-fatality rate was 15.6% (5/32). The confirmed clinical cases were detected from end August to mid October. Significant levels of positivity by RT-PCR were also observed in magpies (Pica pica) (9.1%, 95% confidence levels: 6.1-13.4%), carrion crows (Corvus corone) (7.4%, 95% confidence levels: 3.6-14.4%) and rock pigeons (Columba livia) (12.9%, 95% confidence levels: 7.6-21.2%).

Re-emergence of West Nile virus in Italy.

In August 2008, West Nile disease re-emerged in Italy. The infection is affecting the North Eastern regions and, as of November 2008, has caused 33 clinical cases and five fatalities in horses. Until now, no deaths have been reported in birds. Mosquitoes, blood, serum and tissue samples, from horses and birds, within and around the outbreak area, have been collected and tested by various methods both serologically and virologically. West Nile virus strains have been isolated from blood samples of one horse and one donkey and from pools of brain, kidneys, heart and spleen of a pigeon and three magpies. When compared to the strain isolated during the 1998 Tuscany outbreak, the 255 bp sequence of the genome region coding for the envelope (E) protein of the isolated WNV strains, exhibited a 98.8% and 100% similarity at nucleotide and amino-acid level respectively.

Experimental infection of goats with an unusual strain of Mycoplasma mycoides subsp. capri isolated in Jordan.

Goats were infected experimentally with a mycoplasma (the "Irbid" strain) isolated previously from a goat with contagious agalactia in northern Jordan. The strain was unusual in that, although it had been identified by molecular methods as Mycoplasma mycoides subsp. mycoides LC/Mycoplasma mycoides subsp. capri, it showed no inhibition of growth by any of the hyperimmune rabbit antisera conventionally used to speciate members of the Mycoplasma mycoides cluster. Animals were infected either intratracheally or by aerosol and placed "in-contact" with other goats. After 2 weeks, those infected intratracheally became febrile, showing a nasal discharge and slight conjunctivitis, followed a week later by respiratory distress and polyarthritis; lesions seen at necropsy included coagulative necrotic pneumonia, fibrinous pleurisy with pleural exudate, and inflammatory exudates, necrosis and fibrosis in the joints. Animals infected by aerosol showed much milder clinical signs, including nasal discharge and occasional swollen joints. In the "in-contact" goats, seroconversion was first seen after 7 weeks, accompanied by coughing and laboured respiration; lesions in this group consisted of fibrinous pneumonia with focal areas of necrosis and abundant pleural exudate.

First human case of Usutu virus neuroinvasive infection, Italy, August-September 2009.

We report the first worldwide case of Usutu virus (USUV) neuroinvasive infection in a patient with diffuse large B cell lymphoma who presented with fever and neurological symptoms and was diagnosed with meningoencephalitits. The cerebrospinal fluid was positive for USUV, and USUV was also demonstrated in serum and plasma samples by RT-PCR and sequencing. Partial sequences of the premembrane and NS5 regions of the viral genome were similar to the USUV Vienna and Budapest isolates.

Phylogenetic analysis of West Nile virus isolated in Italy in 2008.

In Italy the first occurrence of West Nile virus (WNV) infection was reported in Tuscany region during the late summer of 1998. In August 2008, the WNV infection re-emerged in Italy, in areas surrounding the Po river delta, and involving three regions Lombardy, Emilia Romagna and Veneto. WNV was isolated from blood and organs samples of one horse, one donkey, one pigeon (Columba livia) and three magpies (Pica pica). The phylogenetic analysis of the isolates, conducted on 255 bp in the region coding for the E protein, indicates that these isolates belong to the lineage I among the European strains. According to the analysis, both the 1998 and 2008 Italian strains as well as isolates from Romania, Russia, Senegal and Kenya fell in the same sub-cluster.

Time-resolved fluoroimmunoassay for quantitative determination of ampicillin in cow milk samples with different fat contents.

In this paper, we have reported an immunoassay with time-resolved revelation system for ampicillin in raw milk samples. Immunological methods appear to be a promising approach in the analysis of beta-lactam compounds, because they do not need previous sample pre-treatments. In fact, beta-lactam ring is not very stable in extensive sample pre-treatment procedures requested in conventional analytical techniques. Specimens were collected from lactating cows bred in various conditions and assayed for the fat contents. Ampicillin was assayed in samples with different fat concentrations. The assay was performed using ampicillin-specific polyclonal antibody raised in rabbit; the immunogen was synthesized using bovine thyroglobulin conjugated to ampicillin by glutaraldehyde reaction; as fluorescent marker we used goat anti-rabbit IgG conjugated with a chelating molecule complexed with Eu(3+). Bovine serum albumin (BSA) conjugated with ampicillin was synthesized and used to prepare a solid phase on polystyrene microtiter plates. The use of a lanthanide chelate as label allowed to achieve 1 ng mL(-1) sensitivity, which is four times more sensitive than limits requested from European Community. Fat contents did not affect the assay performance.

Laboratory tests for evaluating the level of attenuation of bluetongue virus.

One of the most important steps when preparing a live attenuated vaccine is the assessment of the level of attenuation in target animals. It is costly and time consuming as it requires, on each occasion, a large number of susceptible animals and contained accommodation. This study assessed the consistency of the bovine foetal aorta endothelial (BFA) cell line and newborn mice for evaluating the attenuation level of BTV4, BTV9 and BTV16 Italian field isolates. Following serial passages in BHK(21c13) or Vero cell cultures, BTV attenuated clones demonstrated a reduced replication capability in the BFA cells compared to the homologous virulent strains. Similarly, following intracerebral inoculation, the attenuated clones were completely innocuous to newborn mice contrary to the homologous virulent strains which killed all animals within 10 days. Vaccines produced with the BTV9 or BTV4 attenuated clones were safe, immunogenic and capable of preventing clinical symptoms and viraemia in sheep following challenge with homologous virulent virus. The two assays may be valuable indicators of the gradual changes occurring in the BTV population leading to virus attenuation, they can predict the safety of a BTV attenuated vaccine and, in turn, reduce the number of sheep and cattle required to assess the level of attenuation attained.

Serological evidence of USUTU virus occurrence in north-eastern Italy.

In the recent years, USUTU virus (USUV), a flavivirus of the Japanese encephalitis virus complex, has been reported in Central Europe. As part of a systematic surveillance programme to monitor possible entrance and/or circulation of vector-borne viruses, since 2001, sentinel-chicken flocks were placed throughout the Italian territory nearby areas considered at risk of virus introduction. They have been periodically checked for the presence of antibodies against flaviviruses by indirect ELISA, plaque reduction neutralization test for USUTU, West Nile and tick-borne encephalitis viruses. In July 2007, a sentinel chicken in a flock of 20 animals located within the Ravenna province seroconverted to USUV reaching neutralizing titres up to 1:5120. A second chicken seroconverted to the same virus 2 months later. Although no virus was rescued from these animals and from wild or farm birds sampled in the area, these results still provided evidence of the circulation of USUV in north-eastern Italy.

Clinical, humoral and IFNgamma responses of cattle to infection with Mycoplasma mycoides var. mycoides small colony and attempts to condition the pathogenesis of the infection.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides var. mycoides small colony (MmmSC), is one of the most important diseases of cattle in Africa. The role of innate or acquired cell mediated and humoral immunity in conferring protection against MmmSC infection has not yet been elucidated. On the other hand, the pathological lesions caused by the aetiological agent have been considered indicative of an immunopathological process. In this study ten naïve cattle were exposed to in-contact infection with animals infected by intubation with a strain of MmmSC. Clinical signs, antibody response, IFNgamma release and pathological changes at necropsy were analysed and compared with the events following in-contact infection of an equal number of animals kept under daily treatment with cyclosporine for the entire observation period of 84 days. Cyclosporine is a suppressor of the immune response related to the T-cell system. Under the conditions of the experiment, cyclosporine appeared to condition the pathogenesis of CBPP by delaying the events that follow infection, bringing further support to the possibility that the immune response may have an impact on the disease outcome.

Development and validation of a competitive ELISA kit for the serological diagnosis of ovine, caprine and bovine brucellosis.

A competitive ELISA (Brucella-Ab c-ELISA) was standardized and validated for the detection of Brucella antibodies in cattle, sheep and goat sera using a monoclonal antibody (MAb 4B5A) produced against Brucella melitensis biotype 2. The specificity and sensitivity of the assay were 100% to a 67.5% cut-off point (B/Bo%). When compared with an indirect ELISA, the Brucella-Ab c-ELISA did not demonstrate cross-reactions when testing positive sera for antibodies to some Enterobacteriaceae. A comparison was made between the Brucella-Ab c-ELISA and the complement fixation and Rose Bengal tests. Results demonstrated that the Brucella-Ab c-ELISA is a valuable tool for the serological diagnosis of bovine and ovine/caprine brucellosis.

Studies on an inactivated vaccine against rabies virus in domestic animals.

An inactivated vaccine against rabies virus was prepared from the attenuated ATCC PV-12 viral rabbit Pasteur strain. The virus was grown on Baby Hamster Kidney (BHK21) cells, and the supernatant was purified by filtration and inactivated with beta-propriolactone. The inactivated product was checked according to the NHI and European Pharmacopoeia methods. Part of the product was then lyophilised and the other part was adjuvanted with Al(OH)3. Both parts were used to vaccinate and boost groups of horses, cattle and sheep at different intervals. Their immunogenicity was compared with a similar commercial product. Blood samples were collected on a regular basis and the antibody titre was determined by the Fluorescence Antibody Virus Neutralisation (FAVN) test. No significant differences were found between species after both inoculations even though the immune response increased in intensity and duration after the booster dose in all the animals tested and was stronger and lasted longer with the adjuvanted aliquot.