First National Prevalence in Italian Horse Population and Phylogenesis Highlight a Fourth Sub-Type Candidate of Equine Hepacivirus.

Equine hepacivirus (EqHV, Flaviviridae, hepacivirus) is a small, enveloped RNA virus generally causing sub-clinical hepatitis with occasional fatalities. EqHV is reported in equids worldwide, but for Italy data are limited. To address this, a survey study was set up to estimate prevalence at a national level and among different production categories (equestrian; competition; work and meat; reproduction) and national macro-regions (North, Central, South, and Islands). Data obtained testing 1801 horse serum samples by Real-Time RT PCR were compared within the categories and regions. The NS3 fragment of the PCR-positive samples was sequenced by Sanger protocol for phylogenetic and mutational analysis. The tertiary structure of the NS3 protein was also assessed. The estimated national prevalence was 4.27% [1.97-6.59, 95% CI] and no statistical differences were detected among production categories and macro-regions. The phylogenesis confirmed the distribution in Italy of the three known EqHV subtypes, also suggesting a possible fourth sub-type that, however, requires further confirmation. Mutational profiles that could also affect the NS3 binding affinity to the viral RNA were detected. The present paper demonstrates that EqHV should be included in diagnostic protocols when investigating causes of hepatitis, and in quality control protocols for blood derived products due to its parental transmission.

Pathological and virological insights from an outbreak of European brown hare syndrome in the Italian hare (Lepus corsicanus).

European brown hare syndrome (EBHS) is a highly contagious and fatal viral disease, mainly affecting European brown hares (Lepus europaeus). The etiological agent, EBHS virus (EBHSV), belongs to the Lagovirus genus within the Caliciviridae family. The Italian hare (Lepus corsicanus) is endemic to Central-Southern Italy and Sicily and is classified as a vulnerable species. L. corsicanus is known to be susceptible to EBHS, but virological data available is scarce due to the few cases detected so far. In this study, we describe the occurrence of EBHS in two free-ranging L. corsicanus, found dead in a protected area of Central Italy. The two hares were identified as L. corsicanus using phenotypic criteria and confirmed through mitochondrial DNA analysis. Distinctive EBHS gross lesions were observed at necropsy and confirmed by subsequent histological examination. EBHSV was detected in the livers of the two animals initially using an antigen detection ELISA, followed by an EBHSV-specific reverse transcription-PCR, thus confirming the viral infection as the probable cause of death. The EBHS viruses detected in the two hares were identical, as based on blast analysis performed for the VP60 sequences and showed 98.86% nucleotide identity and 100% amino acid identity with strain EBHSV/GER-BY/EI97.L03477/2019, isolated in Germany in 2019. Phylogenetic analysis places our virus in group B, which includes strains that emerged after the mid-1980s. This study supports previous reports of EBHS in L. corsicanus and further expands the knowledge of the pathological and virological characteristics of the etiological agent. The ability of EBHSV to cause a fatal disease in the Italian hare represents a serious threat to the conservation of this vulnerable species, especially in populations kept in enclosed protected areas.

A double-strain TM (gp45) polypeptide antigen and its application in the serodiagnosis of equine infectious anemia.

Lentiviruses, including equine infectious anemia virus (EIAV), are considered viral quasispecies because of their intrinsic genetic, structural and phenotypic variability. Immunoenzymatic tests (ELISA) for EIAV reported in the literature were obtained mainly by using the capsid protein p26, which is derived almost exclusively from a single strain (Wyoming), and do not reflect the great potential epitopic variability of the EIAV quasispecies. In this investigation, the GenBank database was exploited in a systematic approach to design a set of representative protein antigens useful for EIAV serodiagnosis. The main bioinformatic tools used were clustering, molecular modelling, epitope predictions and aggregative/ solubility predictions. This approach led to the design of two antigenic proteins, i.e. a full sequence p26 capsid protein and a doublestrain polypeptide derived from the gp45 transmembrane protein fused to Maltose Binding Protein (MBP) that were expressed by recombinant DNA technology starting from synthetic genes, and analyzed by circular dichroism (CD) spectroscopy. Both proteins were used in an indirect ELISA test that can address some of the high variability of EIAV. The novel addition of the gp45 double-strain antigen contributed to enhance the diagnostic sensitivity and could be also useful for immunoblotting application.

An Overview of Anthropogenic Actions as Drivers for Emerging and Re-Emerging Zoonotic Diseases.

Population growth and industrialization have led to a race for greater food and supply productivity. As a result, the occupation and population of forest areas, contact with wildlife and their respective parasites and vectors, the trafficking and consumption of wildlife, the pollution of water sources, and the accumulation of waste occur more frequently. Concurrently, the agricultural and livestock production for human consumption has accelerated, often in a disorderly way, leading to the deforestation of areas that are essential for the planet's climatic and ecological balance. The effects of human actions on other ecosystems such as the marine ecosystem cause equally serious damage, such as the pollution of this habitat, and the reduction of the supply of fish and other animals, causing the coastal population to move to the continent. The sum of these factors leads to an increase in the demands such as housing, basic sanitation, and medical assistance, making these populations underserved and vulnerable to the effects of global warming and to the emergence of emerging and re-emerging diseases. In this article, we discuss the anthropic actions such as climate changes, urbanization, deforestation, the trafficking and eating of wild animals, as well as unsustainable agricultural intensification which are drivers for emerging and re-emerging of zoonotic pathogens such as viral (Ebola virus, hantaviruses, Hendravirus, Nipah virus, rabies, and severe acute respiratory syndrome coronavirus disease-2), bacterial (leptospirosis, Lyme borreliosis, and tuberculosis), parasitic (leishmaniasis) and fungal pathogens, which pose a substantial threat to the global community. Finally, we shed light on the urgent demand for the implementation of the One Health concept as a collaborative global approach to raise awareness and educate people about the science behind and the battle against zoonotic pathogens to mitigate the threat for both humans and animals.

Equine Hepacivirus: A Systematic Review and a Meta-Analysis of Serological and Biomolecular Prevalence and a Phylogenetic Update.

Viral hepatitis has recently assumed relevance for equine veterinary medicine since a variety of new viruses have been discovered. Equine Hepacivirus (EqHV) is an RNA virus belonging to the Flaviviridae family that can cause subclinical hepatitis in horses, occasionally evolving into a chronic disease. EqHV, to date, is considered the closest known relative of human HCV. EqHV has been reported worldwide therefore assessing its features is relevant, considering both the wide use of blood products and transfusions in veterinary therapies and its similitude to HCV. The present review resumes the actual knowledge on EqHV epidemiology, risk factors and immunology, together with potential diagnostics and good practices for prevention. Moreover, adhering to PRISMA guidelines for systematic reviews a meta-analysis of serological and biomolecular prevalence and an updated phylogenetic description is presented as a benchmark for further studies.

Comparison of direct and indirect methods to maximise the detection of Babesia caballi and Theileria equi infections in Central Southern Italy.

Equine piroplasmosis is a disease of equids, caused by tick-borne apicomplexan protozoan pathogens Babesia caballi and Theileria equi, which, according to the World Organisation for Animal Health (OIE), can be diagnosed by enzyme-linked immunosorbent assay (ELISA), immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR). The present study was conducted to evaluate and compare the assays available for the diagnosis of equine piroplasmosis. Data employed were obtained from 1300 blood samples collected between 2012-2014 from asymptomatic and symptomatic equines (horses and donkeys) of central-southern regions of Italy and analyzed by ELISA, IFAT, PCR (one commercial and one from literature) and blood smear microscopic examination. Statistical differences of the proportions of positivity for each parasite and group (asymptomatic and symptomatic) among the methods were verified by the z test to identify the most sensitive. The concordance between each pair of methods - for each parasite and within the groups - and trends in detection of suspect samples of four hypothetical diagnostic algorithms using serological and biomolecular assays were evaluated to identify the most suitable laboratory diagnostic workflow. The results of this study highlighted a lower capacity to detect suspect samples of commercial ELISA for B. caballi in all groups when compared to biomolecular methods and IFAT; and of the commercial PCRs in asymptomatic animals, identifying a PCR from literature and IFAT as the best choice for a combined diagnosis. For T. equi, IFAT detected more suspect samples than ELISA, even if the latter showed good performance and some samples were positive only by the ELISA and PCR, indicating that their simultaneous employment is still advantageous. Host-parasite interaction, amino-acid/genetic diversity and differences in detection limits among the assays could be among the reasons in explaining the present results. In view of further studies, ELISA should be used in combination with PCR, that should regularly be included in the laboratory diagnosis to maximise the detection of early infections and support the evaluation of pharmacological treatment.

Comparison of PCR-based methods for the detection of Babesia caballi and Theileria equi in field samples collected in Central Italy.

Equine piroplasmosis (EP) is a disease of equids caused by Theileria equi and Babesia caballi, members of the order Piroplasmida, transmitted by several species of ticks. As the disease is endemic in many countries, a clinical examination or a serological test are required prior to movement of horses to prove freedom from infection and to avoid the introduction of EP with its sanitary and economic impact, especially in areas where it is absent. Currently, numerous diagnostic PCR protocols are available, some of which are recommended by the World Organisation for Animal Health (OIE). In order to adopt this diagnostic method, the Italian National Reference Centre for Equine Diseases (NRC-ED) conducted a preliminary comparison between an end-point PCR, nested PCR, real-time PCR, and commercial real-time PCR, for the detection of T. equi and B. caballi, respectively. One hundred and three field samples, collected during spring-summer 2013 in Latium and Tuscany regions, were employed for the study, and results discordant between detection assays were confirmed by sequencing. The reference assay was defined as that showing the highest sensitivity, and the relative sensitivity (rSe) and specificity (rSp) of the other methods were estimated referring to this assay. Agreement between methods was estimated by calculating the concordance between each pair of methods. Although no statistical differences were detected among PCR-based methods, the non-commercial real-time PCR assays seemed to be the most suitable for detection of T. equi and B. caballi, respectively. An important advantage of direct PCR detection of the pathogen, in comparison to indirect detection using serological methods, is that it allows specific treatment against the causative pathogen species responsible of the infection as well as for the definition of the infectious status of an animal for international movement.

Validation of an immunoblot assay employing an objective reading system and used as a confirmatory test in equine infectious anaemia surveillance programs.

Equine infectious anaemia (EIA) is a blood borne disease that is listed among the notifiable diseases of the World Organisation for Animal Health (OIE). EIA is also regulated by the OIE for the international trading provisions and is generally subject to control programmes. Since 2011, Italy has been conducting a surveillance plan based on a three-tier diagnostic system, using a serological ELISA as screening test, an agar gel immunodiffusion test (AGIDT) as a confirmatory method, and an immunoblot (IB) as an alternative confirmatory assay for discordant results between the first two tests. As for the in-house competitive ELISA (c-ELISA) and the AGIDT, the Italian National Reference Laboratory for EIA (NRL) validated the IB according to the OIE guidelines, employing eight panels containing positive sera, including those from EIA virus (EIAV) proven infected horses, and negative horse, mule and donkey sera collected from different geographical areas. In addition, two international reference image panels were employed for the optimization and the validation of the digital image reading system adopted that allows an impartial measurement of the serum reactivity in the IB assay. The immunological reactivity to EIAV antigens, p26, gp45 and gp90 adsorbed on the IB membrane, determines the serological status of the animal and for EIA, a p26 positive band together with at least one of the other antigen defines a subject as serologically positive for EIAV. For validation, the parameters assessed were threshold values, analytical and diagnostic sensitivity and specificity, repeatability and reproducibility. These parameters were evaluated for each antigen as well as in combination, according to the diagnostic algorithm established above. The validation data defined the IB as having a satisfactory sensitivity, specificity, repeatability and reproducibility for all antigens and species tested. An instrumental recording of the results improves the confidence in using IB as a confirmatory test for EIAV, differently from the AGIDT that is read by an operator. The advantages of using the IB are its higher sensitivity, to that of the AGIDT, which allows an earlier detection of infection that reduces the risk of transmission and therefore the incidence of the EIA, and its higher specificity to that of the ELISA which is based on the discrimination of subjects reacting only against the p26, the antigen used by all ELISAs available, which are not considered as infected by EIAV. In particular, when this assay is used in outbreaks it can detect new cases earlier than the AGIDT, and therefore reduce the restriction period with an economic benefit for the animal owners and the public veterinary sanitary system.

Efficacy of a modified-live virus vaccine in pigs experimentally infected with a highly pathogenic porcine reproductive and respiratory syndrome virus type 1 (HP-PRRSV-1).

PRRS is one of the main viral diseases in pig production, causing huge economic losses to the swine industry worldwide. The virus shows an intrinsic genomic instability and is able to change continuously, with the emergence of new strains, with different pathogenicity patterns. Commercially available vaccines only partially prevent or counteract the disease and the correlated losses. Moreover, the emergence of highly virulent and pathogenetic isolates represents a particular concern for PRRS control and diagnosis. The purpose of this study was to evaluate the efficacy of a modified-live virus (MLV) PRRSV-1 commercial vaccine in reducing the severity of the disease and minimizing losses upon challenge with a highly pathogenic PRRSV-1.1 Italian isolate (PRRSV-1_PR40/2014). Four different groups were compared: C (unvaccinated-uninfected), VAC-C (vaccinated-uninfected), PR40 (unvaccinated-infected) and VAC-PR40 (vaccinated-infected). The tested vaccine provided partial, but statistically significant clinical, virological and pathological protection after challenge under experimental conditions. In particular, vaccinated animals showed reduced viremia in terms of duration and magnitude, reduced respiratory signs and pathological lesions. Vaccination was able to trigger adaptive immunity able to respond efficiently also against the HP PR40 isolate. Vaccinated animals showed higher average daily weight gain, even during the viremic period, compared to non-vaccinated challenged pigs.

Genetic diversity of Theileria equi and Babesia caballi infecting horses of Central-Southern Italy and preliminary results of its correlation with clinical and serological status.

Babesia caballi and Theileria equi are tick-borne pathogens causing equine piroplasmosis infecting the Equidae family in which they cause significant sanitary and economic losses. Furthermore, equine piroplasmosis is included in the World Animal Health Organization (OIE) notifiable diseases list with possible movement restrictions for positive horses. Thirty-nine EDTA and whole-blood samples collected during 2013 and 2014 from symptomatic and asymptomatic horses of Central-Southern Italy were included in the present study either because of their strongly positive results in Real Time (RT) PCRs targeting the 18S rRNA gene specific for each piroplasm and/or due to their serological ELISA/18S rRNA RT-PCR discordant results. A nested PCR amplifying the hypervariable V4 region of the 18S rRNA gene of both piroplasms was performed on all samples. T. equi positive samples were also analysed with a PCR targeting the equi merozoite antigen 1-gene (EMA-1). The sequences obtained were thirty for T. equi, 25 of which were for the hypervariable V4 region of the 18S rRNA gene and 13 for the EMA-1 gene, with eight samples positive for both targets, while only six 18S rRNA gene sequences were retrieved for B. caballi. The phylogenetic analysis results are as follows: T. equi sequences of the 18S rRNA gene clustered in three different phylogenetic groups, respectively in the A (15), B (9) and C (1) while those of B. caballi in the A (1), B1 (3) and B2 (2) groups. T. equi sequences for EMA-1 gene clustered in A (11) and in B (2). This analysis confirms that both T. equi and B. caballi sequences present a genetic heterogeneity independently of their geographical location, similar to that reported by other authors. Statistical associations were evaluated between phylogenetic groups of T. equi 18S rRNA gene and each of the following variables, using Fisher's exact test: clinical signs, serological ELISA/18S rRNA RT-PCR discordant results and T. equi EMA-1 negativity. The different groups were found to be statistically related to the presence of signs (less present in group B samples), to ELISA negativity/18S rRNA RT-PCR positivity (more seronegative samples in group B). No statistical analysis was performed for the B. caballi as the number of sequences available was insufficient and for the EMA -1 sequences which almost all grouped in the same cluster. The results here obtained provide additional information about T. equi and B. caballi sequences, which could also be used to verify the performance of serological and molecular diagnostic methods.

Evaluation of immune responses in mice and sheep inoculated with a live attenuated Brucella melitensis REV1 vaccine produced in bioreactor.

The Brucella melitensis REV1 vaccine is the most widely employed vaccine for prophylaxis against brucellosis in sheep and goats. The objective of vaccination is disease control in herds or preventing infection in farms. In this study, we produced REV1 vaccine with a protocol, based on the use of liquid medium in a bioreactor, that resulted efficient, safe, relatively fast, and cost-effective. The live attenuated vaccine produced was tested in mice and sheep to investigate its immunogenicity and efficacy. Seventy-two female BALB/c mice were obtained and subdivided in 2 groups, one was stimulated with 1 × 106 colony-forming units (CFUs) of B. melitensis while the other with physiological solution alone and acting as control group. Furthermore, 25 sheep were subdivided into 5 groups: four were inoculated with a B. melitensis dose, ranging from 0.6 × 109 and 3.2 × 109 CFUs and the other was the control group. In addition, a serological diagnosis was performed for sheep by rapid serum agglutination and the complement-fixation test. Immunocompetent cells from both experiment were collected at different times post vaccination and immunostained to evaluate innate and adaptive-immune responses. In mice flow cytometry was used to detect macrophages, T lymphocytes, dendritic cells, memory cells, naïve cells, natural killer cells, major histocompatibility complex type II, B lymphocytes, regulatory T lymphocytes, T helper lymphocytes, cytotoxic T lymphocytes and recently activated CD4+ and CD8+ lymphocytes. In sheep, macrophages, T helper cells, cytotoxic T lymphocytes, regulatory T lymphocytes, dendritic cells, memory cells and naïve lymphocytes, by the same method, were analyzed. The results showed, both in mice and sheep, that the live, attenuated REV1 vaccine stimulated all immunocompetent cells tested, with a balanced innate and adaptive response. In the sheep experiment, the administered vaccine dose was very important because, at the lower doses, immunological tolerance tended to disappear, while, at the highest dose, the immunological tolerance remained active for a long period. In our experimental conditions, the optimal vaccine dose for sheep was 3.2 × 109 CFUs, although a good immune response was found using a dose of 1.6 × 109 CFUs. The vaccine produced in this study could be extensively employed in developing countries to control the brucellosis in sheep and goats.

A highly pathogenic porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) strongly modulates cellular innate and adaptive immune subsets upon experimental infection.

Highly pathogenic (HP) PRRSV isolates have been discovered within both PRRSV-1 and PRRSV-2 genotypes and investigated in recent years especially for their ability to cause extremely severe disease in conventional pig herds. The exacerbation of general and respiratory clinical signs has been attributed not only to an efficient replication (virulence) but also to the ability to dysregulate viral recognition and induce mechanisms of immune evasion or immune enhancement of humoral and cellular anti-viral responses differently from non-HP PRRSV isolates in terms of intensity and temporal onset. Thus, the understanding of the immunopathogenesis of HP PRRSV is a major concern for the study of virus biology and development of efficacious vaccines. The present study aims at addressing the modulation of relevant immune cell subsets by flow cytometry in the blood of 4-week-old pigs experimentally infected with the recently discovered PR40/2014 HP PRRSV-1.1 strain phenotypically characterized in Canelli et al. (2017) compared to pigs infected with a non-HP PRRSV isolate (PR11/2014) and uninfected controls. PR40 infected animals showed an early and marked reduction of pro-inflammatory CD172α+ CD14+CD16+ and CD14+CD163+ monocytes and TCRγδ+CD8α+/CD8α- lymphocytes when pigs were most infected, possibly due to a recruitment sustaining an acute inflammatory response in target tissues. The prolonged increased CD3+CD16+ NKT cell levels may sustain peripheral inflammation and/or the anti-viral response. The late reduction (potential depletion) of γ/δ T lymphocytes and CD3+CD4+CD8α- naïve Th lymphocytes paralleled with the delayed increase of CD3+CD4+CD8α+ memory and CD3+CD4-CD8α/ß + cytotoxic T lymphocytes. In addition, PR40 infection showed an early depletion of activated CD4+CD25+ T lymphocytes and Tregs together with an intense and lasting depletion of CD21+ B lymphocytes. Overall, these features demonstrate that the more severe clinical signs observed upon infection with the HP PR40 strain are sustained by remarkable changes in the peripheral blood distribution of immune cells and provide further insights into the immune regulation/immunopathogenesis induced by PRRSV-1 subtype 1 European isolates.

Validation of an indirect ELISA employing a chimeric recombinant gag and env peptide for the serological diagnosis of equine infectious anemia.

The National Reference Center for equine infectious anemia (EIA) validated a commercial ELISA (Eradikit® EIAV Indirect ELISA, In3diagnostic®, Turin, Italy) employing a chimeric recombinant gag and env peptide for the detection of EIA virus antibodies, following the guidelines of the World Organization for Animal Health. The validation parameters evaluated were: analytical sensitivity (Se) and specificity (Sp); diagnostic Se and Sp; precision, based on repeatability and reproducibility through the estimation of the standard deviation (SD) and the coefficient of variation (CV); accuracy, estimated from a multiple K and relative Sp and Se with respect to those of the agar gel immunodiffusion test (AGIDT). Positive and negative predictive values were also defined. The assay showed a high specificity and a limit of detection of 1.43 log10 major than AGIDT. Diagnostic Se was 100% and Sp was 99.3%, while SD values ranged from 1.58 to 5.01 with a CV between 2.8% and 28.8%. Multiple K was 0.98 and relative Se and Sp were respectively 99.1% and 100%. The assay proved to be robust and to possess a high sensitivity in detecting first antibodies produced at onset of infection as well as high analytic and diagnostic Se and Sp values, confirming it as a serological assay fit for purpose within EIA surveillance programs.

Evaluation of six serological ELISA kits available in Italy as screening tests for equine infectious anaemia surveillance.

BACKGROUND: ELISAs are known to have a higher diagnostic sensitivity than the agar gel immunodiffusion (AGID) when employed for serological diagnosis of equine infectious anaemia (EIA). For this purpose, an "in-house" and five commercial ELISAs available in Italy were assessed by the National Reference Centre for EIA for their analytic specificity (Sp); precocity, defined as capability of detecting first antibodies produced during a new infection; precision based on repeatability and reproducibility, estimated from the coefficient of variation (CV); accuracy, estimated from multiple K and relative Sp and sensitivity (Se). Two serum panels, positive for non-equine retroviruses and the most frequent equine viruses, were employed to measure analytic Sp. ELISA precocity was also compared to that of one "in-house" and three commercial AGID kits, employing a panel of sera, collected weekly from horses infected with modified EIA viruses. Precision and accuracy were defined using results of a panel containing positive and negative sera examined in an inter-laboratory trial with the participation of the ten Official Laboratories. Furthermore, a questionnaire was used to assess the appropriateness of each kit for routine use. RESULTS: Analytic Sp was 100%, while the 75th percentile of CVs for positive sera varied from 0.4% to 12.73% for repeatability and from 1.6% to 44.87% for reproducibility. Although CV of the negative serum was constantly high, its outcome was unaltered. Relative Se ranged from 98.2% to 100%, relative Sp was constantly 100% and multiple K ranged from 0.95 to 1. Precocity differed among the assays: three kits detected 4.8% and 42.9% positive samples on 21 days post infection (dpi), all assays detected positive samples on 28 dpi, between 47.6% and 95.2%. Precocity of ELISAs was superior to that of the AGIDs except for two assays. In view of the feedback obtained from the questionnaires, all kits were considered appropriate for routine use. CONCLUSION: All ELISAs having high Se and precocity are preferable as a screening test in EIA surveillance programmes to the AGID tests examined. These two tests can be incorporated in a serial diagnostic pathway to improve the efficacy of a surveillance plan.

Evolution of equine infectious anaemia in naturally infected mules with different serological reactivity patterns prior and after immune suppression.

Information on equine infectious anaemia (EIA) in mules, including those with an equivocal reaction in agar gel immunodiffusion test (AGIDT), is scarce. For this, a study was conducted to evaluate the clinical, viral loads and pathological findings of two groups of naturally infected asymptomatic mules, respectively with a negative/equivocal and positive AGIDT reactivity, which were subjected to pharmacological immune suppression (IS). A non-infected control was included in the study that remained negative during the observation period. Throughout the whole study, even repeated episodes of recrudescence of EIA were observed in 9 infected mules, independently from their AGIDT reactivity. These events were generally characterised by mild, transient alterations, typical of the EIA acute form represented by hyperthermia and thrombocytopenia, in concomitance with viral RNA (vRNA) peaks that were higher in the Post-IS period, reaching values similar to those of horses during the clinical acute phase of EIA. Total tissue viral nucleic acid loads were greatest in animals with the major vRNA activity and in particular in those with negative/equivocal AGIDT reactivity. vRNA replication levels were around 10-1000 times lower than those reported in horses, with the animals still presenting typical alterations of EIA reactivation. Macroscopic lesions were absent in all the infected animals while histological alterations were characterised by lymphomonocyte infiltrates and moderate hemosiderosis in the cytoplasm of macrophages. On the basis of the above results, even mules with an equivocal/negative AGIDT reaction may act as EIAV reservoirs. Moreover, such animals could escape detection due to the low AGIDT sensitivity and therefore contribute to the maintenance and spread of the infection.

Validation according to OIE criteria of a monoclonal, recombinant p26-based, serologic competitive enzyme-linked immunosorbent assay as screening method in surveillance programs for the detection of Equine infectious anemia virus antibodies.

The Italian National Reference Center for equine infectious anemia (CRAIE; Rome, Italy) developed and validated a monoclonal, recombinant p26-based competitive enzyme-linked immunosorbent assay (cELISA) for the detection of EIA virus antibodies employing the 2010 criteria of the World Organization for Animal Health (OIE). The following parameters were evaluated: cutoff values, repeatability, reproducibility, concordance, analytical sensitivity (Se), absolute analytical specificity (Sp), and diagnostic Se and Sp. Positive and negative predictive values were also defined in relation to the estimated prevalence. When the cELISA was used as a screening test for 96,468 samples in the Italian EIA surveillance program, 17% more EIA cases were detected than by the agar gel immunodiffusion test, and the apparent diagnostic Sp estimated from these samples was 99.8%, which was more than the diagnostic Sp (80.2%) estimated from validation. The high Se and Sp of the cELISA confirm its fit for purpose as a screening test.

Babesia caballi and Theileria equi infections in horses in Central-Southern Italy: Sero-molecular survey and associated risk factors.

Babesia caballi and Theileria equi are tick-borne pathogens, etiological agents of equine piroplasmosis that affect different species of Equidae causing relevantly important direct and indirect losses. A field study was conducted to evaluate the distribution of the equine piroplasms in an area of Central-Southern Italy and to identify correlated risk factors. Serum samples of 673 asymptomatic horses were collected during spring-summer of 2013 to estimate the seroprevalence of the parasites within the study area using T. equi and B. caballi Antibody test kit (VMRD(®), Inc, Pullman, WA, USA). The 273 seropositive samples were subsequently tested by real time PCR to verify the presence of the genome of the piroplasms, indicative of the carrier status of the subjects. The variables chosen to identify which were the risk factors associated with the serological and PCR-positivity for each of the equine piroplasms were the following: gender, age, breed, access to pasture, altitude, land cover, climatic zone, soil type and province location (coastal/inland). The resulting overall seroprevalence for T. equi was 39.8% (268/673) and for B. caballi was 8.9% (60/673) while 70.3% of the PCR tested samples (185/263) were positive for T. equi and 10.3% (27/263) for B. caballi. The univariate and multiple logistic regression models were used to assess the association of the risk factors with the different outcomes. The risk factors found to be associated with T. equi seropositivity were gender, age, breed, access to pasture, land cover, soil type and province location, while those associated with PCR-positivity were age, soil type and province location. As the number of B. caballi seropositive subjects was limited, the multiple logistic regression model was performed only for the PCR-positive status, identifying climatic zone and soil type as the sole risk factors. In the study area, a major diffusion of T. equi, in terms of seroprevalence and PCR-positivity was present when compared to that of B. caballi, probably related to the cumulative effect of the life-long infection of the former protozoan. The identification of risk factors relative to each piroplasm infection, specific to a study area, is important in the development and improvement of tailored control and prevention programmes aimed at containing health and economic consequences.