Novel duplex RT-qPCR for animal rabies surveillance.

Rabies is a lethal zoonosis affecting mammals worldwide. Diagnosis of rabies follows international standard protocols, primarily relying on direct immunofluorescence (DI) followed by mouse inoculation test (MIT). WHO recommends molecular biology techniques such as RT-qPCR for replacing MIT to diagnose rabies in animal samples. Recently, a real-time PCR protocol that detects all rabies virus variants identified worldwide was validated. This assay is a pan-Lyssavirus TaqMan quantitative RT-PCR called LN34. A modified LN34 assay protocol was tested at the Paraná State Reference Laboratory (Lacen/PR) using animal samples previously tested by DI and MIT, the gold standard (GS). This method has been changed to a RT-qPCR duplex format to better fit the diagnostic routine. The new assay was called duplex LN34 and ß-actin RT-qPCR. All the 88 samples evaluated using the GS test, modified pan-Lyssavirus TaqMan RT-qPCR and duplex LN34 and ß-actin RT-qPCR showed 100% agreement with each other. This novel duplex RT-qPCR protocol has shown adequate diagnostic performance and may be used in research and surveillance purposes, replacing the standard MIT and ending mice use for rabies diagnosis.

EXPOSURE OF WILD BOAR (SUS SCROFA) TO THE COMMON VAMPIRE BAT AND LACK OF IMMUNE PROTECTION TO RABIES VIRUS IN BRAZILIAN HUNTERS.

Rabies virus is recognized as one of the most fatal zoonotic agents affecting all mammals. Wild boars (Sus scrofa), classified as a large-size exotic invasive species in Brazil with nationwide hunting permitted, may serve as an extra blood source for the common vampire bat (Desmodus rotundus). Our aim was to document wild boar exposure to vampire bats to determine the seroprevalence of rabies virus antibodies in wild boars and to determine the immune status of hunters in southern and central-western Brazilian regions. Serum samples were collected from 80 wild boars and 49 hunters from natural and degraded areas of the Atlantic Forest biome of southern Brazil and in degraded areas of the Cerrado biome of central-western Brazil. The rabies-modified rapid fluorescent focus inhibition test was performed to detect the presence of rabies virus neutralizing antibodies in wild boars and considered seropositive when ≥0.10 IU/mL. The simplified fluorescence inhibition microtest was used for samples from hunters with a titer of ≥0.50 IU/mL and considered indicative of seroconversion. While 11% (9/80) of wild boars had serum titers for rabies exposure (≥0.10 IU/mL), 88% (43/49) of corresponding hunters lacked immune protective titers (<0.50 IU/mL). Wild boars showed serum titers for rabies likely due to contact with contaminated saliva of vampire bats or from infected carcass consumption. Additionally, Brazilian wild boars can be exposed to rabies and may play an important role in the sylvatic rabies cycle by providing a blood supply for vampire bats, highlighting the possibility of direct transmission of rabies virus to hunting dogs and hunters. These findings suggested hunters are a potential risk group for contracting rabies, and the World Health Organization may consider adding this occupation to their recommendations of who should receive the pre-exposure rabies vaccination.

Comparative analysis of Mouse Inoculation Test and Virus Isolation in Cell Culture for rabies diagnosis in animals of Parana, Brazil.

INTRODUCTION: Rabies is an acute zoonotic disease, caused by a rhabdovirus that can affect all mammals, and is commonly transmitted by the bite of a rabid animal. The definitive diagnosis is laboratorial, by the Fluorescent Antibody Test (FAT) as a quick test and Mouse Inoculation Test (MIT) as a confirmatory test (gold standard). Studies conducted over the past three decades indicate that MIT and Virus Isolation in Cell Culture (VICC) can provide the same effectiveness, the latter being considered superior in bioethics and animal welfare. The aim of this study was to compare VICC with MIT, in terms of accuracy, biosafety and occupational health, supply and equipment costs, bioethics and animal welfare, in a Brazilian public health lab. METHODS: We utilized 400 samples of animal neurological tissue to compare the performance of VICC against MIT. The variables analyzed were accuracy, biosafety and occupational health, time spent in performing the tests, supply and equipment costs, bioethics and animal welfare evaluation. RESULTS: Both VICC and MIT had almost the same accuracy (99.8%), although VICC presented fewer risks regarding biosafety and mental health of the technicians, and reduced time between inoculation and obtaining the results (approximately 22 days less). In addition, VICC presented lower supply costs (86.5% less), equipment costs (32.6% less), and the advantage of not using animals. CONCLUSIONS: These results confirm that VICC can replace MIT, offering the same accuracy and better features regarding cost, results, biosafety and occupational health, and bioethics and animal welfare.

Comparative analysis of Mouse Inoculation Test and Virus Isolation in Cell Culture for rabies diagnosis in animals of Parana, Brazil

Abstract INTRODUCTION: Rabies is an acute zoonotic disease, caused by a rhabdovirus that can affect all mammals, and is commonly transmitted by the bite of a rabid animal. The definitive diagnosis is laboratorial, by the Fluorescent Antibody Test (FAT) as a quick test and Mouse Inoculation Test (MIT) as a confirmatory test (gold standard). Studies conducted over the past three decades indicate that MIT and Virus Isolation in Cell Culture (VICC) can provide the same effectiveness, the latter being considered superior in bioethics and animal welfare. The aim of this study was to compare VICC with MIT, in terms of accuracy, biosafety and occupational health, supply and equipment costs, bioethics and animal welfare, in a Brazilian public health lab. METHODS: We utilized 400 samples of animal neurological tissue to compare the performance of VICC against MIT. The variables analyzed were accuracy, biosafety and occupational health, time spent in performing the tests, supply and equipment costs, bioethics and animal welfare evaluation. RESULTS: Both VICC and MIT had almost the same accuracy (99.8%), although VICC presented fewer risks regarding biosafety and mental health of the technicians, and reduced time between inoculation and obtaining the results (approximately 22 days less). In addition, VICC presented lower supply costs (86.5% less), equipment costs (32.6% less), and the advantage of not using animals. CONCLUSIONS: These results confirm that VICC can replace MIT, offering the same accuracy and better features regarding cost, results, biosafety and occupational health, and bioethics and animal welfare.