Heat induced epitope retrieval for rabies virus detection by direct fluorescent antibody test in formalin-fixed dog brain tissues

Rabies, a zoonotic encephalitis due to transmission of a lyssavirus, such as rabies virus (RABV), has the highest case fatality of any infectious disease. A global program for the elimination of human rabies caused by dogs is proposed for realization by 2030. Sensitive, specific, and inexpensive diagnostic tests are necessary for enhanced surveillance to detect infection, inform public health and veterinary professionals during risk assessments of exposure, and support overall programmatic goals. Multiple laboratory techniques are used to confirm a suspect case of rabies. One method for the detection of lyssavirus antigens within the brain is the direct rapid immunohistochemical test (dRIT), using light microscopy, and suitable for use under field conditions. Besides dogs, other major RABV reservoirs reside among mammalian mesocarnivores and bats. To date, use of the dRIT has been applied primarily for the diagnosis of RABV in suspect mesocarnivores. The purpose of this study was to assess the usefulness of the dRIT to the diagnosis of rabies in bats, compared to the gold-standard, the direct fluorescent antibody test (DFAT). Brains of 264 suspect bats, consisting of 21 species from Arizona and Texas, were used in the evaluation of the dRIT. The overall sensitivity of the dRIT was 100% (0.969–1.0, 95% CI) and the specificity was 94.6% (0.896–0.976, 95% CI), comparable to the DFAT. This preliminary study demonstrated the utility of the dRIT in the confirmation of RABV infection in bats. Future studies should include additional geographic, lyssavirus, and mammalian species representations for broader application during enhanced rabies surveillance, with incorporation of any potential adjustments to standard protocols, as needed.

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Evaluation of Enzyme Linked Immuno-Sorbent Assay and Rapid Immuno-Diagnostic Test for Rabies Antigen Detection in Archived Dog Brain Tissues

Folia Veterinaria ◽

10.2478/fv-2018-0003 ◽

2018 ◽

Vol 62 (1) ◽

pp. 18-24

Author(s):

G. R. Okoh ◽

H. M. Kazeem ◽

G. S. N. Kia ◽

S. Mailafia

Keyword(s):

Gold Standard ◽

Fluorescent Antibody ◽

Antibody Test ◽

Epidemiological Studies ◽

Antigen Detection ◽

Diagnostic Laboratory ◽

Dog Brain ◽

Kappa Value ◽

Rabies Diagnosis ◽

Brain Tissues

Abstract Rabies urgently requires strengthening of new and existing diagnostic methodology in order to overcome the threat it poses. We evaluated the Enzyme Linked Immuno-Sorbent Assay (ELISA) and the Rapid Immunodiagnostic Test (RIDT) in detecting rabies viral antigens, comparing both tests with the Direct Fluorescent Antibody Test (DFAT) which is the gold standard in rabies diagnosis. Fifty dog brain tissues collected from the archives of the Central Diagnostic Laboratory, National Veterinary Research Institute, Vom, Nigeria, were utilized for this study. ELISA performed better than RIDT and recorded equivalent result with DFAT as compared with RIDT. There was a 96 % agreement between ELISA and DFAT for rabies antigen detection (concordance coefficient 78 % : 95 % C. I. 0.6366 to 0.8654) while there was a 54 % agreement between RIDT and DFAT (concordance coefficient 17 % : 95 % C. I. 0.05138—0.2752). Compared to DFAT, the sensitivities of ELISA and RIDT were 95.5 % and 47.6 %, respectively, and the specificities of ELISA and RIDT were 100 % and 87.5 % respectively. The simple Cohen’s kappa coefficient for ELISA related to the DFAT was found to be 0.834 (95 % C. I. 0.613—1.0). For RIDT, the Kappa value was 0.170 (95 % C. I. 0.003—0.337). The ELISA is as reliable a diagnostic method as the DFAT which is the gold standard for rabies diagnosis. It has an advantage of being able to analyse large number of samples at the same time, making it more suitable for epidemiological studies and for laboratories that cannot perform the DFAT. The unsatisfactory result of RIDT in this study reiterates the need to perform an adequate test validation before it can be used in the laboratory for rabies diagnosis.

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Re-assessment of direct fluorescent antibody negative brain tissues with a real-time PCR assay to detect the presence of raccoon rabies virus RNA

Journal of Virological Methods ◽

10.1016/j.jviromet.2011.04.009 ◽

2011 ◽

Vol 174 (1-2) ◽

pp. 110-116 ◽

Cited By ~ 13

Author(s):

Annamaria G. Szanto ◽

Susan A. Nadin-Davis ◽

Richard C. Rosatte ◽

Bradley N. White

Keyword(s):

Real Time ◽

Rabies Virus ◽

Real Time Pcr ◽

Fluorescent Antibody ◽

Pcr Assay ◽

Virus Rna ◽

Direct Fluorescent Antibody ◽

Raccoon Rabies Virus ◽

Brain Tissues

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A case study of rabies diagnosis from formalin-fixed brain material : short communication

Journal of the South African Veterinary Association ◽

10.4102/jsava.v82i4.83 ◽

2011 ◽

Vol 82 (4) ◽

Cited By ~ 5

Author(s):

J. Coertse ◽

L. H. Nel ◽

C. T. Sabeta ◽

J. Weyer ◽

A. Grobler ◽

...

Keyword(s):

South Africa ◽

Rabies Virus ◽

Fluorescent Antibody ◽

Domestic Dog ◽

Health Priorities ◽

Fluorescent Antibody Technique ◽

Rt Pcr ◽

Antibody Technique ◽

Formalin Fixed ◽

Brain Tissues

Rabies is caused by several Lyssavirus species, a group of negative sense RNA viruses. Although rabies is preventable, it is often neglected particularly in developing countries in the face of many competing public and veterinary health priorities. Epidemiological information based on laboratory-based surveillance data is critical to adequately strategise control and prevention plans. In this regard the fluorescent antibody test for rabies virus antigen in brain tissues is still considered the basic requirement for laboratory confirmation of animal cases. Occasionally brain tissues from suspected rabid animals are still submitted in formalin, although this has been discouraged for a number of years. Immunohistochemical testing or a modified fluorescent antibody technique can be performed on such samples. However, this method is cumbersome and cannot distinguish between different Lyssavirus species. Owing to RNA degradation in formalin-fixed tissues, conventional RT-PCR methodologies have also been proven to be unreliable. This report is concerned with a rabies case in a domestic dog from an area in South Africa where rabies is not common. Typing of the virus involved was therefore important, but the only available sample was submitted as a formalin-fixed specimen. A real-time RT-PCR method was therefore applied and it was possible to confirmrabies and obtain phylogenetic information that indicated a close relationship between this virus and the canid rabies virus variants from another province (KwaZulu-Natal) in South Africa.

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Performance evaluation of the polyclonal anti-rabies virus ribonucleoprotein IgG antibodies produced in-house for use in direct fluorescent antibody test

Journal of Virological Methods ◽

10.1016/j.jviromet.2020.113879 ◽

2020 ◽

Vol 280 ◽

pp. 113879

Author(s):

Gabriela Hidaka da Silva ◽

Jaqueline Helena Santos da Silva ◽

Keila Iamamoto ◽

Tamires Santos de Arruda ◽

Iana Suly Santos Katz ◽

...

Keyword(s):

Performance Evaluation ◽

Rabies Virus ◽

Fluorescent Antibody ◽

Antibody Test ◽

Igg Antibodies ◽

Direct Fluorescent Antibody

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An outbreak of sheep rabies in Shanxi province, China

Epidemiology and Infection ◽

10.1017/s0950268811001348 ◽

2011 ◽

Vol 139 (10) ◽

pp. 1453-1456 ◽

Cited By ~ 8

Author(s):

Y. ZHU ◽

G. ZHANG ◽

M. SHAO ◽

Y. LEI ◽

Y. JIANG ◽

...

Keyword(s):

Rabies Virus ◽

Clinical Presentation ◽

Fluorescent Antibody ◽

Antibody Test ◽

Shanxi Province ◽

N Gene ◽

Gene Fragment ◽

Rt Pcr ◽

Dog Brain

SUMMARYThis study describes an outbreak of rabies in a flock of 110 sheep in Yangqu county, Shanxi province, China, in 2010. The flock, housed in a cave being used as a sheepfold, was attacked by a rabid dog, which resulted in 36 deaths over the following 2 days from the crushing and trampling caused by panic, and some 15 further deaths from rabies 2 weeks later. Rabies in the sheep was confirmed by the fluorescent antibody test (FAT) and RT–PCR. Rabies virus was isolated from the index dog brain and its N gene was partially sequenced (nucleotides 77-880). Results showed that the canine isolate (SXTYD01) was rabies virus with the N gene fragment 100% identical to that of the virus isolated from rabid sheep. This is the first confirmed sheep rabies outbreak in China. The clinical presentation of the disease is also described.

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Detection of rabies virus antigen in brain tissue of dogs slaughtered for human consumption in Taraba State, Nigeria

Sokoto Journal of Veterinary Sciences ◽

10.4314/sokjvs.v17i4.2 ◽

2020 ◽

Vol 17 (4) ◽

pp. 9-13

Author(s):

A.B. Tirmidhi ◽

H.M. Kazeem ◽

A. Jibril ◽

B.M. Jahun ◽

O. Orakpoghenor

Keyword(s):

Public Health ◽

Local Government ◽

Brain Tissue ◽

Rabies Virus ◽

Fluorescent Antibody ◽

Antibody Test ◽

Virus Antigen ◽

Human Consumption ◽

Safe Handling ◽

Direct Fluorescent Antibody

Rabies as an ancient zoonosis constitutes a threat to public health by causing over 59,000 annual human mortalities worldwide. The aim of this study was to detect rabies virus in brain tissue of dogs slaughtered for human consumption in Taraba State, Nigeria. A total of 150 dogs comprising 136 adults and 14 puppies consisting of 82 males and 68 females was sampled from slaughter points in five Local Government Areas. Brain samples were collected from each dog in labeled sterile sample bottles and screened for rabies virus antigen using direct fluorescent antibody test (DFAT). Results showed that 3 out of the 150 (2%) brain samples screened were positive for rabies virus; out of which 2 were from Unguwan Kasa (14.3%) and 1 was from Quarter Five (7.1%). This therefore suggests the presence of rabies virus in dogs slaughtered for human consumption in Taraba State, Nigeria and their role as reservoirs of the virus. Therefore, there is need for awareness education on safe handling of dog meat to minimize the risk for butchers/meat handlers. Keywords: Brain samples, dogs, Prevalence, Rabies, Taraba State

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Clarifying Indeterminate Results on the Rabies Direct Fluorescent Antibody Test Using Real-Time Reverse Transcriptase Polymerase Chain Reaction

Public Health Reports ◽

10.1177/0033354918810776 ◽

2018 ◽

Vol 134 (1) ◽

pp. 57-62 ◽

Cited By ~ 4

Author(s):

Kim Appler ◽

Scott Brunt ◽

Jodie A. Jarvis ◽

April D. Davis

Keyword(s):

Real Time ◽

Rabies Virus ◽

Fluorescent Antibody ◽

Antibody Test ◽

The United States ◽

Rt Pcr ◽

Chain Reaction ◽

Virus Variant ◽

Direct Fluorescent Antibody ◽

Polymerase Chain

Objectives: Each year, rabies virus infection results in the death of more than 50 000 persons worldwide. In the United States, the Centers for Disease Control and Prevention (CDC) reported 23 human rabies cases from May 1, 2008, through October 1, 2017. Although rabies testing in the United States is highly reliable, some specimens submitted to rabies laboratories do not have adequate tissues or may be substantially decomposed. In these instances, the specimen may be considered unsatisfactory for testing or produce indeterminate results using the gold standard direct fluorescent antibody test. The objective of this study was to evaluate the number of unsatisfactory samples or samples with indeterminate results that were positive for rabies virus after additional testing using real-time reverse transcriptase polymerase chain reaction (RT-PCR). Methods: In 2016, we retested all unsatisfactory specimens or specimens with indeterminate results using real-time RT-PCR. We further typed any sample that was real-time RT-PCR positive to identify the infecting rabies virus variant. Results: Of 210 retested unsatisfactory specimens or specimens with indeterminate results, 9 (4.3%) were positive for rabies. In each case, the animal was infected with a homologous rabies virus variant. Conclusion: These results confirm the recommendation by CDC and state public health laboratories that indeterminate results should be considered positive and justify the prompt treatment of exposed persons through an animal that is suspected to have rabies.

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