Evaluation of LN34 Pan-Lyssavirus RT-qPCR assay for rabies diagnosis in Brazil.

Rabies, a fatal zoonotic viral disease affecting mammals, including humans, remains a significant global health concern, particularly in low-income countries. The disease, primarily transmitted through infected animal saliva, prompts urgent diagnosis for timely post-exposure prophylaxis (PEP). The gold standard diagnostic test, direct fluorescent antibody test (dFAT), while sensitive, suffers from limitations such as subjective interpretation and high costs. As a confirmatory technique, the LN34 Pan-Lyssavirus RT-qPCR assay has emerged as a promising tool for universal Lyssavirus detection. This study evaluated its performance using 130 rabies virus isolates representing eleven Brazilian variants and 303 clinical samples from surveillance operations. The LN34 assay demonstrated 100% sensitivity and 98% specificity compared to dFAT. Additionally, it detected all samples, including those missed by dFAT, indicating superior sensitivity. The assay's specificity was confirmed through Sanger nucleotide sequencing, with only a minimal false-positive rate. Comparative analysis revealed higher accuracy and concordance with dFAT than traditional rabies tissue culture infection tests (RTCIT). False-negative RTCIT results were attributed to low viral load or suboptimal sampling. These findings underscore the LN34 assay's utility as a confirmatory technique, enhancing rabies surveillance and control in Brazil. Its widespread adoption could significantly improve diagnostic sensitivity, crucial for effective PEP and public health interventions.

Comparison of five different laboratory techniques for the rabies diagnosis in clinically suspected cattle in Brazil.

The direct-fluorescent antibody test (dFAT) is considered the "gold standard" assay to diagnose rabies. However, it is crucial to develop molecular techniques, such as RT-PCR and RT-qPCR, since many laboratories lack the needed supplies for performing complementary methods (viral isolation, for example). For this purpose, diagnostic techniques must be specific and sensitive to guarantee accuracy. This present investigation aimed to detect rabies virus (RABV) in 126 clinically suspected cattle in Brazil using different diagnostic tests [dFAT, mouse inoculation test (MIT), immunohistochemistry (IHC), RT-PCR and RT-qPCR] and to compare those results obtained under routine laboratory conditions. The results of the present investigation demonstrate that the molecular techniques are more sensitive and may detect low viral load, even though the non-homogeneous viral distribution caused a false-negative result in dFAT. We also observed a usual alteration in antigens distribution among regions of the central nervous system (CNS). By both dFAT and IHC assays, the most reliable CNS structures were thalamus and midbrain. Although this investigation demonstrated diagnostic sensitivity and specificity close to 100 % in all laboratory techniques employed, a dFAT auxiliary test is required for bovine specimens, such as molecular techniques, when there are poor sampling conditions (low viral load combined with unavailability of brainstem structures).

Large protein as a potential target for use in rabies diagnostics.

Rabies is a zoonotic viral disease that remains a serious threat to public health worldwide. The rabies lyssavirus (RABV) genome encodes five structural proteins, multifunctional and significant for pathogenicity. The large protein (L) presents well-conserved genomic regions, which may be a good alternative to generate informative datasets for development of new methods for rabies diagnosis. This paper describes the development of a technique for the identification of L protein in several RABV strains from different hosts, demonstrating that MS-based proteomics is a potential method for antigen identification and a good alternative for rabies diagnosis.

Large protein as a potential target for use in rabies diagnostics

Rabies is a zoonotic viral disease that remains a serious threat to public health worldwide. The rabies lyssavirus (RABV) genome encodes five structural proteins, multifunctional and significant for pathogenicity. The large protein (L) presents well-conserved genomic regions, which may be a good alternative to generate informative datasets for development of new methods for rabies diagnosis. This paper describes the development of a technique for the identification of L protein in several RABV strains from different hosts, demonstrating that MS-based proteomics is a potential method for antigen identification and a good alternative for rabies diagnosis.

Biotechnology advances: a perspective on the diagnosis and research of Rabies Virus.

Rabies is a widespread zoonotic disease responsible for approximately 55,000 human deaths/year. The direct fluorescent antibody test (DFAT) and the mouse inoculation test (MIT) used for rabies diagnosis, have high sensitivity and specificity, but are expensive and time-consuming. These disadvantages and the identification of new strains of the virus encourage the use of new techniques that are rapid, sensitive, specific and economical for the detection and research of the Rabies Virus (RABV). Real-time RT-PCR, phylogeographic analysis, proteomic assays and DNA recombinant technology have been used in research laboratories. Together, these techniques are effective on samples with low virus titers in the study of molecular epidemiology or in the identification of new disease markers, thus improving the performance of biological assays. In this context, modern advances in molecular technology are now beginning to complement more traditional approaches and promise to revolutionize the diagnosis of rabies. This brief review presents some of the recent molecular tools used for RABV analysis, with emphasis on rabies diagnosis and research.

Suplementação de vacas leiteiras com homeopatia: células somáticas do leite, cortisol e imunidade / Supplementation of dairy cows with homeopathy: milk somatic cells, cortisol and immunity

Avaliou-se o efeito da suplementação de uma combinação homeopática sobre a contagem de células somáticas do leite (CCS), o teor sanguíneo de cortisol e a resposta de anticorpos neutralizantes antivírus da raiva de vacas leiteiras. Trinta e duas vacas Holandesas em lactação foram blocadas em pares e aleatoriamente alocadas a um de dois tratamentos por 63 dias, posterior a um período de padronização de 14 dias. A CCS mensurada no final da padronização ajustou os valores semanais de CCS no modelo de análise estatística. Os tratamentos foram: 150 gramas de uma combinação homeopática (Hypothalamus, 10-30; Colibacilinum, 10-30; Streptococus Beta Hemolyticum, 10-60; Streptococus Uberis, 10-60; Phytolacca, 10-60; Calcium Phosphoricum, 10-30; Natrum Muriaticum, 10-60; Urtica Urens, 10-30; Silicea Terra, 10-400) em veículo mineral, ou 150 gramas do mesmo veículo mineral (controle). A homeopatia tendeu a aumentar a CCS de 124 para 222 x1.000 células mL-1 (P=0,09) e a CCS linearizada (P=0,08). Não foram detectados efeitos de tratamento sobre a concentração sérica de cortisol após estresse induzido por aspiração percutânea do saco ventral do rúmen (P=0,59) ou sobre o título de anticorpos neutralizantes em resposta à vacinação antivírus da raiva (P=0,40). A suplementação com homeopatia tendeu a aumentar a CCS de vacas com baixa CCS.

The effect of supplementing a homeopathic combination on milk somatic cell count (SCC), blood cortisol content and the antibody response to rabies vaccination of dairy cows was evaluated. Thirty-two lactating Holstein cows were paired blocked and randomly assigned to one of two treatments for 63 days, following a 14-day standardization period. The SCC measured at the end of standardization period adjusted weekly SCC values in the statistical analysis model. Treatments were: 150 grams of a homeopathic combination (Hypothalamus, 10-30; Colibacilinum, 10-30; Streptococcus Beta Hemolyticum, 10-60, Streptococcus Uberis, 10-60; Phytolacca, 10-60; Calcium Phosphoricum, 10-30; Natrum Muriaticum, 10-60; Urtica Urens, 10-30, Silicea Terra, 10-400) in mineral vehicle, or 150 grams of the same mineral vehicle (Control). Homeopathy tended to increase SCC from 124 to 222 x1,000 cells mL-1 (P=0.09) and linear SCC (P=0.08). There were no detectable treatment effects upon serum cortisol concentration following stress induced by percutaneous aspiration of the ventral rumen (P=0.59) and upon serum antibody title in response to rabies vaccination (P=0.40). The supplementation with homeopathy tented to increase the SCC of low SCC cows.