Diagnosis of dourine in outbreaks in Italy.

Dourine is trypanosomosis that affects equids, it's mainly sexually transmitted. The disease was first eradicated in Italy in the 1940s, but there was then a serious epidemic in the mid-70s. After sporadic reports at the end of the 1990s, in May 2011 it was reported once more. Clinical diagnosis of dourine can be complex, as clinical signs and gross lesions are not always present. Direct laboratory diagnosis is also problematic, given the low number of parasites normally present in infected tissues and the mild, short-lasting parasitaemia. This article describes the epidemiological, clinical and laboratory data enabling confirmation of the suspicion of dourine in Italy in the 2011 epidemic.

A clinical case of dourine in an outbreak in Italy.

In May 2011, dourine was reported in Italy following the declaration of a positive result observed in a stallion undergoing routine testing for stud purposes. Clinical signs, anatomo-histopathological findings and laboratory results that resulted in the confirmation of diagnosis of dourine in a clinically affected mare, which was the likely source of infection in the stallion, are described.

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.

Efficacy of Brucella abortus vaccine strain RB51 compared to the reference vaccine Brucella abortus strain 19 in water buffalo.

Approximately 250,000 water buffalo (Bubalus bubalis) live in the Campania region of southern Italy where the breeding of this species is very popular. Of these animals, almost 150,000 are concentrated in the Caserta province where the prevalence of Brucella abortus in this species represents approximately 20% at herd level. The Italian brucellosis eradication programme provides a slaughter and vaccination strategy for this province. B. abortus strain RB51 (RB51) has become the official vaccine for the prevention of brucellosis in cattle in several countries. The aim of this study was to evaluate the efficacy of RB51 in water buffalo compared to the B. abortus S19 vaccine (S19). The study was performed in accordance with a protocol described in mice. Female buffalo aged five months were inoculated. Five received a RB51 dosage on two occasions that was three times greater than that approved for use in cattle and a booster after one month, five received B. abortus S19 vaccine at the standard dosage and three controls received a phosphate buffer solution. Buffalo were then challenged with a virulent B. abortus strain 544 thirty days post vaccination. Antibodies that developed in the five animals vaccinated with RB51 were not detected by the Rose Bengal test or complement fixation test (CFT) and were also tested by CFT prepared with RB51 antigen. After culling, B. abortus was cultured from the spleen, retropharyngeal and supra-mammary lymph nodes. A statistical evaluation was performed to assess the immunogenicity values obtained in buffalo vaccinated with S19, compared to those obtained in buffalo vaccinated with the RB51 vaccine and in the unvaccinated control group.

Namibia: an example of international cooperation in the study of emerging diseases.

The Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' (IZS A&M) has been engaged for many years in research and studies designed to increase knowledge and expertise when dealing with 'exotic diseases' namely, those diseases which are not present in a country. To achieve these objectives, it is important to create cooperation networks with laboratories and research organisations at national and international levels. The relationship between the IZS A&M with Namibia in particular and, more recently with Botswana, are proving to be very valuable to mutual technical/scientific growth. In 2005, the National Reference Centre for the Study of Exotic Diseases set up its own Virology Laboratory at the Windhoek Central Veterinary Laboratory where the Namibian and IZS A&M personnel, working towards common goals, share diagnostic responsibilities and scientific research. The authors describe the activities involved in this joint project.

Brucella abortus strain RB51 vaccine: immune response after calfhood vaccination and field investigation in Italian cattle population.

Immune response to Brucella abortus strain RB51 vaccine was measured in cattle vaccinated at calfhood. After an increase at day 6 post-vaccination (pv), the antibody level recorded in the 10 vaccinated animals remained constant for two months, and then progressively decreased. All vaccinated animals remained negative from day 162 pv to the end of the study (day 300 pv). Only at days 13 and 14 pv the RB51-CFT showed 100% sensitivity (credibility interval (CI) 76.2%-100%). The results indicate that the possibility to use RB51-CFT for the identification of cattle vaccinated at calfhood with RB51 is limited in time. A field investigation was carried out on 26,975 sera collected on regional basis from the Italian cattle population. The study outcomes indicate that in case of RB51-CFT positive results observed in officially Brucellosis-free (OBF) areas and, in any case, when an illegal use of RB51 vaccine is suspected, the use of the RB51-CFT alone is not sufficient to identify all the vaccinated animals. The design of a more sophisticated diagnostic protocol including an epidemiological investigation, the use of RB51-CFT, and the use of the skin test with RB51 as antigen is deemed more appropriate for the identification of RB51 vaccinated animals.

Quality management for the road transportation of livestock.

Transport can be a significant stress factor for livestock and can result in poor animal welfare and economic losses. Quality management measures are actively employed in fields different from animal welfare and could be applied to improve the welfare of animals and reduce the consequent losses during road transportation and related activities Training and education of staff comprise one possible measure, Web-geographic information system technology used to monitor the true state of transported animals is another innovation that promises major progress. With this technology, behavioural and environmental parameters can be monitored and registered in real time. The resulting data could be useful to control the transport environment and the conduct of staff. Although some parameters cannot be represented through numerical relationships, behavioural and environmental measurements can be used in a risk analysis system to minimise risks of poor welfare during animal transportation. The European Union Joint Research Centre and the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' in Teramo are working on an experimental project to prove the feasibility of a navigation system for long road journeys as referred to in Regulation (EC) 1/2005 of the European Union. Such a system enables the collection of data on transported animals and the verification that welfare requirements are being met at any given moment during the journey.

Use of real-time RT-PCR as a rapid molecular approach for differentiation of field and vaccine strains of bluetongue virus serotypes 2 and 9.

Since 2000 severe, long-lasting epidemics of bluetongue virus (BTV) have been described in Italy, caused by BTV serotypes 2, 4, 9 and 16. Vaccination programs have been applied extensively to control the infection, in spite of concerns about the potential dissemination of attenuated vaccine strains of BTV in susceptible animals. Accordingly, rapid and reliable differentiation between vaccine and field strains is paramount in routine diagnosis of BTV to evaluate the extent of this phenomenon. In the present study, we report the development of two real-time RT-PCR assays able to recognise BTV serotypes 2 and 9, respectively, and we evaluated the use of the assays for discrimination between field and vaccine strains. A total of 65 samples collected in Italy from 2000 to 2006 and diagnosed as positive for either BTV-2 or -9 were analysed by the TaqMan assays. Both the assays were found to be highly sensitive and reproducible, ensuring correct serotype characterisation and prediction of the origin of the strains, as confirmed by characterisation using virus neutralisation and sequencing.

Susceptibility to Guillain-Barré syndrome is associated to polymorphisms of CD1 genes.

Guillain-Barré syndrome (GBS) is the prototype of a postinfectious autoimmune neuropathy. Molecular mimicry between glycolipid antigens expressed by an infective antigen such as Campylobacter jejuni and the human peripheral nerve has been hypothesized to be the causative mechanism of GBS. However, only 1/1000 of C. jejuni enteritis develops GBS. This emphasizes the importance of host-related factors in the development of the disease. HLA studies in GBS failed to show an association or gave conflicting results but MHC class I and II process and present peptides to T lymphocytes making unlikely that the HLA system plays a role in GBS with autoantibodies against self-gangliosides. CD1 molecules are MCH-like glycoproteins specialized in capturing and presenting a variety of glycolipid to antigen-specific T cells. There are five closely linked CD1 genes in humans located in chromosome 1 (named CD1A, B, C, D, and E) all showing limited polymorphism in exon 2 which codifies for the alpha1 domain of CD1 molecules. The nucleotide substitutions in CD1B and CD1C are rare and reported to be silent. In 100 controls and 65 GBS patients (21 with a recent C. jejuni infection and 35 with anti-glycolipid antibodies) we used direct sequencing by polymerase chain reaction to genotype exon 2 of CD1A, CD1D and CD1E genes. CD1D is monomorphic in both controls and patients whereas CD1A and CD1E are biallelic in exon 2. Subjects with CD1E*01/01 genotype are 2.5 times more likely to develop GBS, whereas subjects with CD1A*01/02 or CD1E*01/02 have a reduced relative risk by 3.6 and 2.3 times respectively. The combination of CD1A*01/02 and CD1E*01/02 reduces by 5 times the risk of developing GBS. Although a correlation between CD1E*01/01 genotype and recent C. jejuni infection or presence of antiganglioside antibodies was not found the overall findings indicate that susceptibility to develop GBS is associated with polymorphisms of CD1E and CD1A genes.

Experimental axonopathy induced by immunization with Campylobacter jejuni lipopolysaccharide from a patient with Guillain-Barré syndrome.

New Zealand white rabbits were immunized with a lipopolysaccharide (LPS) extracted from a Campylobacter jejuni HS:19 strain isolated from a GBS patient expressing GM1 and GD1a-like epitopes, Freund's adjuvant (group I) and Freund's adjuvant plus keyhole lympet hemocyanin (KLH) (group II). Both groups showed high titers of anti-LPS and anti-GM1 and lower titers of anti-GD1b and anti-GD1a antibodies. Weakness and axonal degeneration in sciatic nerves was detected in 1/11 of group I and 6/7 of group II. This model replicates, at least in part, the pathogenetic process hypothesized in the human axonal GBS with antiganglioside antibodies post C. jejuni infection and indicates that KLH plays an additional role in neuropathy induction.

Differentiation of Italian field and South African vaccine strains of bluetongue virus serotype 2 using real-time PCR.

The current outbreaks of bluetongue (BT) disease in sheep in the central parts of the Mediterranean basin are being combated by extensive vaccination to control further spread of the virus and to suppress its long-term maintenance in the field. To be able to monitor the success of this campaign, and to be able to identify new foci of the disease, it is necessary to harness diagnostic methods, both rapid and sensitive, for differentiating reliably field from vaccine strains of bluetongue virus (BTV). A new method is described for their differentiation using fluorescence resonance energy transfer (FRET) probes with real-time PCR. The method is based on the principle that the melting temperature of a DNA duplex gives information about the sequence, and allows even double-base alterations in the amplicon to be identified. The RT-PCR, the generation of melting curves, and fluorescence detection were all performed using the LightCycler system (Roche Diagnostics, Mannheim, Germany).

Model proficiency testing scheme for serological diagnosis of Brucellosis: interlaboratory study.

A model interlaboratory testing scheme was developed by the Italian National Reference Laboratory for Brucellosis. This scheme was planned for both qualitative (Rose Bengal Plate Test; RBPT) and quantitative (Complement Fixation Test; CFT) serological tests and involved a total of 42 laboratories. In the preparation of this scheme, reference was made to general protocols and guidelines and to methods reported in the literature, which were applicable to analytical chemistry laboratories. Six field sera from naturally infected animals, one positive serum at a titer below the European Union (EU) positivity threshold, and 5 sera positive at titers between 20 and 851 International Units of Complement Fixation Test (IUCFT)/mL plus one negative serum were used to produce a panel of test sera. To evaluate laboratory performances in the quantitative test for each tested sample examined, z-scores based on robust summary statistics (the median and normalized interquartile range) were used. To evaluate overall laboratory performance, 2 types of combined z-scores were used: Rescaled Sum of Scores and Sum of Squared Scores. In the case of the qualitative test (RBPT), results were analyzed by a Bayesian approach. A Beta distribution, based on the result of each laboratory, was calculated and used to estimate the probability of each laboratory giving a correct result and its uncertainty.