Molecular detection, histopathological analysis, and immunohistochemical characterization of equine infectious anemia virus in naturally infected equids.

Equine infectious anemia (EIA), a disease caused by equine infectious anemia virus (EIAV), is considered an obstacle to the development of the horse industry. There is no treatment or vaccine available for EIA, and its pathogenesis, as well as the immune response against the virus, is not fully understood. Therefore, an immunohistochemistry assay was developed for the detection of viral antigens in tissues of equids naturally infected with EIAV. Sections of organs of six equids from Apodi-RN, Brazil, that tested positive for EIA by serological tests (ELISA and AGID) were fixed in 10% formalin solution and embedded in paraffin. Immunohistochemistry was performed using a polyclonal anti-EIAV antibody. EIAV antigens were observed in red spleen pulp cells and hepatic sinusoids, as well as bronchiolar and alveolar epithelial cells of the lungs and proximal and distal tubules of the kidneys. The presence of EIAV in the spleen and liver was expected due to viral tropism by macrophages, which are abundantly present in these organs. However, EIAV was also found in lung and kidney epithelial cells, indicating that the virus infects cell types other than macrophages. In conclusion, the immunohistochemical assay standardized in this study was able to detect EIAV antigens in spleen, liver, kidney and lung cells from naturally infected EIAV equids. Immunostaining observed in the spleen confirms viral tropism by mononuclear phagocytes; however, the presence of EIAV in lung and kidney epithelial cells indicates that virus may be eliminated in urine and/or oronasal secretions, suggesting new routes for viral excretion.

Enzyme-linked immunosorbent assay and agar gel immunodiffusion assay for diagnosis of equine infectious anemia employing p26 protein fused to the maltose-binding protein.

A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.

Equine infectious anemia virus in naturally infected horses from the Brazilian Pantanal.

Equine infectious anemia (EIA) has a worldwide distribution, and is widespread in Brazil. The Brazilian Pantanal presents with high prevalence comprising equine performance and indirectly the livestock industry, since the horses are used for cattle management. Although EIA is routinely diagnosed by the agar gel immunodiffusion test (AGID), this serological assay has some limitations, so PCR-based detection methods have the potential to overcome these limitations and act as complementary tests to those currently used. Considering the limited number of equine infectious anemia virus (EIAV) sequences which are available in public databases and the great genome variability, studies of EIAV detection and characterization molecular remain important. In this study we detected EIAV proviral DNA from 23 peripheral blood mononuclear cell (PBMCs) samples of naturally infected horses from Brazilian Pantanal using a semi-nested-PCR (sn-PCR). The serological profile of the animals was also evaluated by AGID and ELISA for gp90 and p26. Furthermore, the EIAV PCR amplified DNA was sequenced and phylogenetically analyzed. Here we describe the first EIAV sequences of the 5' LTR of the tat gene in naturally infected horses from Brazil, which presented with 91% similarity to EIAV reference sequences. The Brazilian EIAV sequences also presented variable nucleotide similarities among themselves, ranging from 93,5% to 100%. Phylogenetic analysis showed that Brazilian EIAV sequences grouped in a separate clade relative to other reference sequences. Thus this molecular detection and characterization may provide information about EIAV circulation in Brazilian territories and improve phylogenetic inferences.

Health and epidemiological approaches of Trypanosoma evansi and equine infectious anemia virus in naturally infected horses at southern Pantanal.

Equine infectious anemia virus (EIAV) and Trypanossoma evansi are endemic in Brazilian Pantanal Biome, an important area for livestock production. In this sense, we evaluated the epidemiological single and co-infection effects of T. evansi and EIAV in naturally infected horses in the southern Pantanal wetland by serological tests and hematological assays. Both higher seroprevalence and heath poor condition of the sampled animals were associated with differences in horse management between farms. We found that the negative animals for both infectious agents (NN) represented the major group in F1 (37%), and the smallest group in F2 (19%). Furthermore, we recorded higher EIAV seroprevalence (56%) in F2, compared to F1 (38%). We observed that T. evansi infection was mostly related to young horses, as seen by their higher seroprevalence, ranging from 70.7% in the beginning of the rainy season to 81% in the end of flood period, in comparison with the values of 42% and 68%, respectively, in working animals. on the other hand, working animals showed a higher seroprevalence for EIAV (48%) in both seasons than young horses. We observed that the management of working horses could be a risk factor of EIAV infection. On the other hand, as T. evansi is maintained in the study region by many species of wild mammals, the mechanical transmission through blood-sucking vectors ensures the infection to horses since early. Our results showed that single or co-infection by EIAV and T. evansi caused different degree of anemia in the infected animals. Moreover, the health of horses in Brazilian Pantanal is also influenced by differences in horse management and environmental circumstances.

Antibodies and PMBC from EIAV infected carrier horses recognize gp45 and p26 synthetic peptides.

Equine infectious anemia virus (EIAV) is a lentivirus causing a persistent infection in horses characterized by recurrent febrile episodes and high levels of viremia associated with a novel antigenic strain of the virus. The virus contains two envelope glycoproteins, gp90 and gp45, and four internal proteins, p26, p15, p11 and p9. Considering that the most infected horses are able to restrict EIAV replication to very low levels and that gp45 and p26 contain highly conserved epitopes among lentiviruses, it would be necessary to identify those conserved epitopes stimulating cellular and humoral responses. The aims of this study were to determine if the synthetic peptides identified as gp45 (aa 523-547) and p26 (aa 318-346) representing two highly conserved and immunodominant regions of EIA virus are recognized by PBMC and antibodies to EIAV adult mixed-breed naturally infected carrier horses, and if these peptides are able to induce immune responses in mice. Antibodies from 100% of carrier horses, evaluated by ELISA, recognized both peptides; PBMC from 80% of carrier horses, evaluated by lymphoproliferation assay, recognized, at least, one peptide. Furthermore, immunization with 100 microg of each peptide elicited humoral and cellular responses in BALB/c mice, antibodies appeared at 48 or 63 days of immunization with gp45 or p26, respectively. Although the kinetics of gp45- and p26-specific antibody responses were similar, percentage of positivity was higher for gp45. The lymphoproliferation assay, evaluated by BrdU uptake, was higher in mice immunized with gp45 or p26 than in the control group (P<0.05). Based on our findings, we consider that both peptides could be included in an effective vaccine design to induce long-term immunological memory.

Otimizaçäo da produçäo e estabilizaçäo do antígeno do vírus da anemia infecciosa equina, para uso diagnóstico / Improvement of production and stabilization of equine infectious anemia virus antigen, for diagnosis use

Foi produzido antígeno do vírus da anemia infecciosa equina em células de derme equina (ED) cronicamente infectadas. O rendimento da produçäo foi comparado entre células lisadas e näo lisadas no final do período de incubaçäo. Maiores quantidades de antígeno foram obtidas quando as células foram lisadas. Esta lise foi induzidas através de ciclos de congelamento ou metabólica, devido a acidez do meio. O antígeno produzido foi tratado com fenilmetilsulfonilfluoreto, um inibidor de proteinase, garantindo maior estabilidade em diferentes temperaturas de armazenamento

[Immunodiffusion serologic study of equine infectious anemia in the Province of Buenos Aires, Argentina]. / Estudio serológico por inmunodifusión de la anemia infecciosa equina en la Provincia de Buenos Aires, Argentina.

Twenty seven per cent of 238 serum samples obtained from horses with clinical diagnosis were positive for the immunodifusion test, while 17% of the 452 sera obtained from asintomatic horses were positive. Twenty one per cent of the 870 sera studied were positive.