Susceptibility of different TMEM154 genotypes in three Italian sheep breeds infected by different SRLV genotypes.

Small ruminant lentiviruses (SRLV) belong to the Retroviridae family and can cause various diseases. One of the most impacting diseases is visna-maedi, a complex disease characterized by long latencies and chronic progressive inflammatory events affecting the nervous system, lungs, mammary gland, and articular joints. A single nucleotide polymorphism (rs408593969, c.103G>A, missense mutation E35K) in the ovine transmembrane protein gene 154 (TMEM154) was identified as protective against small ruminant lentivirus infection in different herds worldwide. However, there is evidence in the scientific literature of a breed-specificity of this protective effect and, furthermore, there are still limited studies regarding the association between the animal genotype and the infecting virus genotype. Thus, the aim of this study was to further investigate the association between the animal genotype for the suggested protective mutation and the infecting virus genotype, in three different sheep breeds reared in northern Italy. The results obtained only partially confirmed the data available in the literature, as the protective effect was confirmed only for SRLV genotype A clusters, while other genotypes (namely B and E) infected AA and GA animals. Further studies with an experimental infection of specific virus genotypes in hosts with specific genotypes are required to confirm the larger number of cases the results obtained in this study.

Genetic Diversity of 17 Autochthonous Italian Chicken Breeds and Their Extinction Risk Status.

The preservation of genetic variability of autochthonous poultry breeds is crucial in global biodiversity. A recent report revealed small breed size and potential risk of extinction of all native Italian poultry breeds; therefore, a correct assessment of their genetic diversity is necessary for a suitable management of their preservation. In this work, we provided an overview of the contribution to poultry biodiversity of some Italian autochthonous breeds reared in conservation centers devoted to local biodiversity preservation. The level of genetic diversity, molecular kinship, inbreeding, contribution to overall genetic diversity, and rate of extinction of each breed were analyzed with a set of 14 microsatellite loci in 17 autochthonous chicken breeds. To evaluate genetic variability, total number (Na), and effective number (Ne) of alleles, observed (Ho) and expected (He) heterozygosity, and F (Wright's inbreeding coefficient) index were surveyed. The contribution of each analyzed breed to genetic diversity of the whole dataset was assessed using MolKin3.0; global genetic diversity and allelic richness contributions were evaluated. All the investigated loci were polymorphic; 209 alleles were identified (94 of which private alleles). The average number of alleles per locus was 3.62, and the effective number of alleles was 2.27. The Ne resulted lower in all breeds due to the presence of low-frequency alleles that can be easily lost by genetic drift, thus reducing the genetic variability of the breeds, and increasing their risk of extinction. The global molecular kinship was 27%, the average breed molecular kinship was 53%, and the mean inbreeding rate 43%, with a self-coancestry of 78%. Wright's statistical analysis showed a 41% excess of homozygous due to breed genetic differences (34%) and to inbreeding within the breed (9%). Genetic variability analysis showed that 11 breeds were in endangered status. The contribution to Italian poultry genetic diversity, estimated as global genetic diversity, and ranged from 30.2 to 98.5%. In conclusion, the investigated breeds maintain a unique genetic pattern and play an important role in global Italian poultry biodiversity, providing a remarkable contribution to genetic variability.

Field application of an indirect gE ELISA on pooled milk samples for the control of IBR in free and marker vaccinated dairy herds.

BACKGROUND: The aim of the present study was to assess the reliability of a new strategy for monitoring the serological response against Bovine Herpesvirus 1 (BoHV1), the causative agent of infectious bovine rhinotracheitis (IBR). Bulk milk samples have already been identified as cost effective diagnostic matrices for monitoring purposes. Nevertheless, most eradication programs are still based on individual standard assays. In a region of northwestern Italy (Piedmont), the voluntary eradication program for IBR has become economically unsustainable. Being the prevalence of infection still high, glycoprotein E-deleted marker vaccines are commonly used but gE blocking ELISAs are less sensitive on bulk milk samples compared to blood serum. RESULTS: A recently developed indirect gE ELISA showed high versatility when applied to a wide range of matrices. In this study, we applied a faster, cost effective system for the concentration of IgG from pooled milk samples. The IgG enriched fractions were tested using a gE indirect ELISA for monitoring purposes in IBR-positive and IBR-marker-vaccinated herds. Official diagnostic tests were used as gold standard. During a 3 years study, a total 250 herds were involved, including more than 34,500 lactating cows. The proposed method showed a very good agreement with official diagnostic protocols and very good diagnostic performances: only 37 positive animals were not detected across the entire study. CONCLUSIONS: The results highlighted the ability of the proposed method to support the surveillance of IBR in the Piedmont region, reducing the costs without affecting the diagnostic performances.

A versatile and sensitive lateral flow immunoassay for the rapid diagnosis of visceral leishmaniasis.

Visceral leishmaniasis (VL) is a zoonotic infectious disease with a severe impact on humans and animals. Infection is transmitted by phlebotomine sand flies, and several domestic and wild mammals act as reservoirs for the infection, so the prompt detection of infected hosts is crucial to preventing and controlling the spread of the disease and its transmission to humans. A rapid and portable tool for VL diagnosis based on the lateral flow immunoassay (LFIA) technology is described herein. The device exploits a highly specific chimeric recombinant antigen as the recognition element for capturing anti-leishmanial antibodies, and protein A labelled with gold nanoparticles as the signal reporter. The LFIA shows excellent diagnostic sensitivity (98.4%), specificity (98.9%), and agreement with serological reference methods for diagnosing canine VL. The long-term stability of the LFIA device was confirmed based on six months of storage at room temperature or 4 °C, and the qualitative response of the device was not affected by limited thermal stress. The use of the broadly specific protein A means that the LFIA can be readily adapted to diagnose VL in dogs (the main reservoir for human infection) and other mammals, thus further assisting efforts to control the spread of VL. Graphical abstract A rapid and portable diagnostic tool for visceral leishmaniasis (VL) based on lateral flow immunoassay (LFIA) technology. The presence of anti-leishmanial antibodies is revealed through the binding of these antibodies to a highly specific chimeric antigen. Employing a broadly specific signal reporter (protein A labelled with gold nanoparticles) enables the LFIA to be easily adapted to diagnose VL in different animals.

Pestivirus infection in cattle dairy farms: E2 glycoprotein ELISA reveals the presence of bovine viral diarrhea virus type 2 in northwestern Italy.

BACKGROUND: Bovine viral diarrhea virus (BVDV) types 1 and 2 are members of the Pestivirus genus of the Flaviviridae family. This genus also includes the HoBi-like virus, tentatively classified as BVDV type 3. BVDV-1 is widely distributed in Italy despite the extensive use of BVDV-1-based vaccines, while BVDV-2 and HoBi-like Pestivirus have been detected occasionally. Monitoring the occurrence of sporadic or atypical pestiviruses is a useful approach to evaluate the need for additional vaccine strains that can be used in BVDV control programs. RESULTS: In this study we developed a multiwell antibody ELISA based on the recombinant E2 protein of the three bovine pestiviruses. We evaluated the assay's applicability for surveillance purposes using pooled milk samples, each prepared from a maximum of 35 lactating cows and collected from 176 dairy herds. As expected, the majority of the pooled samples reacted to a greater extent against the BVDV-1 E2 antigen. All three milk pools from a single farm reacted to the BVDV-2 antigen, however. Further analysis using spot tests, antigen detection, and sequence analysis of the 5'-UTR region confirmed the presence of five persistently infected calves carrying a BVDV-2a strain. CONCLUSIONS: This study highlights for the first time that sporadic circulation of BVDV-2 can be predicted by immunoenzymatic methods in the absence of specific vaccination.

Development and validation of an indirect ELISA as a confirmatory test for surveillance of infectious bovine rhinotracheitis in vaccinated herds.

BACKGROUND: Bovine herpesvirus 1 (BoHV1) is a member of the viral subfamily of Alphaherpesvirinae that infects various species, including cattle, sheep, and goats. The virus causes infectious bovine rhinotracheitis (IBR), which is included in a European list of diseases that may require control and eradication programs. The lack of confirmatory tests affects the validity of diagnostic tools, especially those used for vaccinated herds. In this study, we report the development and validation of an indirect enzyme-linked immunosorbent assay (ELISA) based on BoHV1 glycoprotein E, which was expressed as a secreted recombinant antigen in a mammalian cell system. The performance of the new rec-gE ELISA was compared with that of commercially available indirect and/or blocking ELISAs. RESULTS: The sample set included blood sera from animals from IBR-positive farms, IBR-free farms, and marker-vaccinated farms. The indirect ELISA proposed in this study is based on antibody reactivity against BoHV1 gE, and showed high sensitivity and specificity (98.41 and 99.76 %, respectively). CONCLUSIONS: The ELISA performed well, in terms of both its diagnostic sensitivity and specificity, and as a confirmatory methodology, and therefore should improve the diagnostic protocols used for IBR surveillance.

Characterization of caprine herpesvirus 1 (CpHV1) glycoprotein E and glycoprotein I ectodomains expressed in mammalian cells.

Caprine herpesvirus 1 (CpHV1) is a member of ruminant alphaherpesviruses antigenically related to the prototype bovine herpesvirus 1 (BoHV1). Although cross reactivity between the two viruses involves many structural glycoproteins, the use of two competitive BoHV1 ELISAs detecting anti gB and gE antibodies has been proposed for CpHV1 infection, resulting mainly in a gB+/gE- reactivity and leading to suppose that CpHV1 gE may represent an useful target for the development of specific diagnostic test. Since CpHV1 gE gene has been only partially characterized so far, in this study the genome fragment of the short unique unit (Us) encompassing gI and gE gene was amplified and sequenced. Gene fragments encoding the ectodomain of both glycoproteins were subcloned into pSECTag2/Hygro and expressed in HEK293T cells as secreted form in serum free medium. Due to the lack of specific monoclonal antibodies (Mabs), the same recombinant glycoproteins were obtained from BoHV1 and used as positive control with a panel of specific gE and gI Mabs as well as in some ELISA assays. Results clearly indicate that the ectodomain of CpHV1 gE, immobilized on solid face in an indirect ELISA format, represents a sensitive and specific marker of infection, when compared with neutralization test, with absence of very low degree of cross-reactivity with BoHV1 gE counterpart, while the use of CpHV1 gI-ELISA or a combination of gE/gI complex did not significantly improve the sensitivity of the assay. In addition, in the rare event in which cross species barrier occurs for both viruses from their natural host to other species, the use of both BoHV1 and CpHV1 gE in a comparative assay may be proposed.

Small ruminant lentivirus genotype B and E interaction: evidences on the role of Roccaverano strain on reducing proviral load of the challenging CAEV strain.

Live attenuated vaccines provide the most consistent protective immunity in experimental models of lentivirus infections. In this study we tested the hypothesis that animals infected with a naturally attenuated small ruminant lentivirus field strain of genotype E may control a challenge infection with a virulent strain of the caprine arthritis encephalitis virus (CAEV-CO). Within genotype E, Roccaverano strain has been described as attenuated since decreased arthritic pathological indexes were recorded in Roccaverano-infected animals compared to animals of the same breed infected with genotype B strains. Moreover, under natural conditions, animals double-infected with genotypes B and E appear less prone to develop SRLV-related disease, leading to a putative protective role of Roccaverano strain. Here we present evidence that goats experimentally infected with the avirulent genotype E SRLV-Roccaverano strain control the proviral load of a pathogenic challenge virus (CAEV-CO strain) more efficiently than naïve animals and appear to limit the spread of histological lesions to the contralateral joints.

Immunological parameters in goats experimentally infected with SRLV genotype E, strain Roccaverano.

Genotype E of small ruminant lentivirus has been recently described in goats from different breeds in Italy. Genotype E infection may differ from known genotypes since deletions of dUTPase and VPR proteins have been confirmed in different independent areas and goat breed, and play a key role on virus replication and pathogenesis. In particular, genotype E Roccaverano strain has been described as low pathogenic since does not lead to clinical symptoms in goats. In contrast, classical CAEV infected goats of the same area and breed presented arthritis. In this study, we have used intratracheal and intra-bone marrow routes to establish genotype E persistent infections. Humoral and cellular immune responses elicited in the host against genotype E and genotype B derived antigens were evaluated until 200 days post-inoculation. Compared to genotype B antigen, seroconversion against genotype E GAG P16-25 antigen was detected at 2-3 weeks after inoculation, significantly earlier and at higher titres. Interestingly, antibody avidity did not increase in the course of the experiment neither against P16-25 nor against SU5, both derived from genotype E. T cell proliferation against P25-GST fusion protein antigens derived from genotype E was firstly detected at 15 days post-inoculation and was maintained throughout time until week 20 post-infection, while T cell proliferation against the genotype B P25 was not produced by the end of the experiment at 20 weeks post-inoculation. The strength of reaction was also higher when using P25 E as stimulator antigen. In contrast with antibody and T cell proliferation, cytotoxic-T-lymphocyte (CTL) activity in the circulating lymphocytes (effector cells) using blood-derived macrophages (BDM) as target cells, was not strain specific being surprisingly higher against genotype B infected antigen presenting cells (APCs). This is the first study reporting experimentally induced immunological changes in SRLV genotype E infection and indicates that CTL activity may be the adaptive immune response able to induce protection against heterologous infection.

Small ruminant lentivirus genotype E is widespread in Sarda goat.

The highly divergent SRLV genotype E has recently been characterized in Italy as a low pathogenic caprine lentivirus in the Roccaverano breed. The availability of a genotype specific diagnostic test based on a comparative assay, using a combination of genotype specific recombinant antigens allows a wide serosurvey in other goat populations. The island of Sardinia still has the highest small ruminant population of any Italian region and crossbreeding has been limited to goats, mainly with the Maltese breed. A serological survey was carried out on sheep flocks and goat herds, using individual sera as well as a bulk milk-adapted procedure. Genotype E was identified in more than 50% of goat herds and none of the sheep flocks thus supporting the idea that this genotype is specifically associated with the goat species. The full-length proviral sequence of a Sardinian isolate revealed and confirmed the deletion of dUTPase subunit and the absence of both vpr gene and the 71bp repeat of the LTR. Genetic similarity of this isolate with the prototype strain Roccaverano was not more than 84%, supporting the designation of two subtypes within genotype E. Nevertheless, in vitro properties of the Sardinian strain were different from those of the Roccaverano strain in terms of ability to infect synovial membrane and produce syncitia. Remarkable differences in the HV1 and HV2 of the env gene were recorded, with the Sardinian isolate displaying sequence motif more similar to arthritic strains. Data presented suggest diffusion of genotype E is wider than previously thought.

Development of specific diagnostic test for small ruminant lentivirus genotype E.

Small ruminant lentivirus (SRLV) belonging to the highly divergent genotype E has recently been identified in the Italian goat breed Roccaverano. In this report we have developed a specific serological test based on recombinant matrix/capsid antigen fusion protein. Performance has been evaluated and compared with a similar test based on genotype B antigen. Herds under study were selected according to the infectious status characterized by blood PCR and sequencing. Results clearly showed that B and E based recombinant ELISA only detected homologous infection and an apparent cross-reactivity was recorded in a herd in which co-infection was present. Three commercially available ELISAs showed different abilities in detecting genotype E infection, being the whole virus-based immunoassay the best choice. Genotype E-recombinant antigen was not detected in ELISA by three commercially available Mabs known to be cross-reactive among CAEV and MVV capsid antigens, further supporting the high divergence of the E genotype from others. Finally, a SRLV-free herd according to commercial ELISA testing, was analysed in the same area where genotype E was identified and few animals belonging to Roccaverano breed were found slightly reactive with the E antigens. Our results suggest that the prevalence of genotype E in other small ruminant populations may be conveniently estimated using a comparative assay based on a combination of genotype specific recombinant antigens and may highlight a wider space in which SRLVs evolve.

Characterisation of Mycoplasma capricolum P60 surface lipoprotein and its evaluation in a recombinant ELISA.

This paper reports the identification and characterisation of a 60kDa surface protein antigen (P60) of Mycoplasma capricolum subspecies capricolum (Mcc), and describes its diagnostic application. Genomic localization and presence in P60 of conserved functional domains suggested a structural and functional relationship with the immunodominant antigen P48 of Mycoplasma agalactiae, a basic membrane protein. A rP60-ELISA was developed, and it resulted in a high specificity for Mcc infections after evaluation with 125 goat sera. The comparison with an existent ELISA based on whole Mcc cell lysates showed that the two assays have comparable sensitivities, but the rP60-ELISA has the significant advantage of a greater specificity. Results indicate that P60 is a potential marker of Mcc infection, especially useful in areas where the presence of M. capricolum subspecies capripenumoniae is also reported.

Genetic characterization of small ruminant lentivirus in Italian mixed flocks: evidence for a novel genotype circulating in a local goat population.

In this study, characterization of the gag gene of small ruminant lentiviruses was carried out in Italian mixed flocks. The nearly complete gag gene was amplified and sequenced. Within genotype A, subtype A1 and a novel subtype, A8, were found in goats, and another novel subtype, A9, was found in both sheep and goats. Subtype B1 was found in both host species and subtype B2 was identified only in sheep. A novel, highly divergent sequence was obtained from goats in two epidemiologically related flocks and is proposed to represent a novel genotype, E. Major epitopes of matrix and capsid antigen were highly divergent, suggesting that serological identification of animals infected with genotype E may have been missed by using currently available diagnostic tests. A recombinant subunit ELISA, based on genotype E-specific epitopes, was developed and a third independent flock carrying this genotype was identified, based on serology.

Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes.

Two groups of sheep were experimentally infected by intratracheal route with two small ruminant lentivirus (SRLV) isolates belonging to different genotypes (It-561 genotype A3 and It-Pi1 genotype B2). Seroconversion was evaluated using recombinant homologous and heterologous matrix protein/capsid antigen fusion protein. Results clearly indicate that seroconversion against homologous antigen was detected well in advance as regards heterologous antigen in both groups, although the advantage of using homologous antigen was less evident in detecting seroconversion against the caprine arthritis encephalitis virus (CAEV)-like strain, compared with the maedi-visna virus (MVV)-like infection. Commercially available ELISAs detect CAEV-like seroconversion earlier than MVV-like infection suggesting a closer relationship between CAEV-like isolate and the antigen used in the latter ELISA tests. Seven recombinant subunits developed from matrix protein and capsid antigen of strain K1514 (prototype A1) were used to better define the antibody response in sheep infected with It-561 isolate. Two animals clearly reacted against type specific epitopes in the early stage of infection. This study highlights the relative insensitivity of gag encoded cross-reacting epitopes during the early stage of infection and suggests the development of novel diagnostic tests based on both genotype specific antigens.

Molecular and antigenic characterization of a Mycoplasma bovis strain causing an outbreak of infectious keratoconjunctivitis.

An unusually high incidence of infectious keratoconjunctivitis followed by pneumonia and arthritis was observed in beef calves of a managed herd. No Moraxella spp. or bacteria other than Mycoplasma spp. were obtained from conjunctival and nasal swabs. A strategy was designed for characterization of bovine mycoplasmas at species and strain level on the basis of a combination of molecular tools and the immunoblotting method. The strategy made it possible to rapidly assign the bacterium responsible for this outbreak to one of the phylogenetic clusters of bovine mycoplasmas delineated in this study and then to identify it as Mycoplasma bovis. The strain, designated Sar 1, showed a 100% 16S rDNA sequence identity with two European strains (120/81 and MC3386) isolated in Germany and Ireland, respectively, and hosts a vsp gene analog to the vspA, vsp422-4, and vsp422-8 genes of the M. bovis reference strain PG45T and of the field strain 422. The use of a cross-reactive rabbit serum developed against the Mycoplasma agalactiae immunodominant antigen P48 confirmed the molecular findings. The immunological response of calves against M. bovis was also investigated. This is the first report on the occurrence of M. bovis on the Island of Sardinia (Italy).

Genetic heterogeneity of small ruminant lentiviruses involves immunodominant epitope of capsid antigen and affects sensitivity of single-strain-based immunoassay.

The pol and gag gene fragments of small ruminant lentivirus field isolates collected in the last decade in Italy were amplified, sequenced, and analyzed. Phylogenetic analysis revealed that the majority of ovine isolates form a distinct cluster more similar to caprine lentivirus prototypes than to the visna virus prototype. These findings confirm and extend those reported by Leroux et al. (Arch. Virol., 142:1125-1137, 1997). Moreover, we observed that a variable region of Gag, included in the fragment analyzed, corresponded to one of the three major capsid antigen epitopes, which suggests that the antibody response to this epitope may be type specific. To test this hypothesis, two recombinant peptides, derived from the Icelandic prototype K1514 and this novel genotype, were expressed and used in an enzyme-linked immunosorbent assay to screen a panel of ovine and caprine sera collected from different geographical locations in Italy. Several sera reacted in a type-specific manner, indicating that in a diagnostic setting the combination of at least these two type-specific peptides is necessary to cover a wide range of infections. Additionally, these results support the hypothesis of cross-species transmission based on the phylogenetic analysis described above. This has implications for the control and eradication of small ruminant lentivirus infections.