Direct Fluorescent Antibody Test Research Articles

Retrospective study of laboratory-based surveillance of rabies in wild and domestic animals in the southern United States, 2010-2021.

We performed a retrospective study of all case submissions for the rabies virus (RABV) direct fluorescent antibody test (DFAT) requested of the Tifton Veterinary Diagnostic and Investigational Laboratory (Tifton, GA, USA) between July 2010 and June 2021. Submitted were 792 samples from 23 animal species from 89 counties in Georgia, and 4 neighboring counties in Florida, 1 in South Carolina, and 1 in Alabama. In 13 (1.6%) cases, the DFAT result was inconclusive; 779 (98.4%) cases had a conclusive (positive or negative) test result. Of these 779 cases, 79 (10.1%) tested positive across 10 species. The remaining 700 (89.9%) cases were negative. The main reason for submission for RABV testing was human exposure to a potentially rabid animal in 414 (52.3%) cases. Among the 79 positive cases, 74 (93.7%) involved wildlife; raccoons (51 cases; 68.9%) were the primary host confirmed with RABV infection, followed by skunk and fox (8 cases each; 10.8%), bobcat (5 cases; 6.8%), and bats (2 cases; 2.7%). Only 5 domestic animals (6.3% of the positive cases) tested positive during our study period; one from each of the bovine, canine, caprine, equine, and feline species. Hence, the sylvatic cycle plays the predominant role in circulating RABV infection in our study area.

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.

Comparative performance evaluation of four different methods for diagnosing Giardia infection in dogs and zoonotic assemblages’ identification

Giardia duodenalis (syn. G. intestinalis or G. lamblia) is a parasitic protozoan that infects the upper intestinal tract of a broad range of hosts, including humans and domestic animals. Thus, it has raised concerns about the public health risk due to companion animals. Recently, with the improvement of living standards and increasing contacts between pets and humans, the zoonotic transmission of Giardia has dramatically increased. From a genetic point of view, G. duodenalis should be viewed as a complex species that includes eight different species-specific genetic assemblages. The laboratory diagnosis is mainly based on the finding of microscopic cysts in stool samples by coprological examination. Other methods include the detection of antigens, immunoassays or PCR protocols, which allow the identification of Giardia assemblages. The study aimed to compare the performance of Direct Fluorescence Antibody test (DFA), zinc sulfate flotation technique (ZnSO4), rapid diagnostic test (RDT), end-point PCR amplification (PCR) for the detection of Giardia and to identify the concerning assemblages in a canine population from Central Italy. Direct fluorescence antibody test is the reference standard for laboratory diagnosis of Giardia in fecal samples from dogs, despite the microscopic examination after flotation remains the most useful method in many veterinary diagnostic centers. The present findings demonstrate the high performance of DFA and ZnSO4 in detecting Giardia, while RDT may be useful as alternative or complementary method to the DFA and ZnSO4. PCR performance was low, but it allowed determining Giardia B zoonotic assemblage in 25% of the PCR-positive specimens (15 out of 60), while the remaining PCR-positive isolates belonged to the dog-specific assemblage C. The 26% prevalence of G. duodenalis detected by DFA in owned dogs and the identification of potentially zoonotic assemblages underline the potential risk for public health and indicate frequent cross-species transmission of the parasite between humans and dogs.

Rabies Virus-Neutralizing Antibodies in Free-Ranging Invasive Wild Boars (Sus scrofa) from Brazil.

Rabies, one of the most lethal global zoonoses, affects all mammals. It remains circulating worldwide in sylvatic cycles through terrestrial and airborne reservoirs, and in Brazil, bats are currently the main reservoirs and source of transmission. Wild boars, an important invasive alien species in Brazil, are a proven food source for hematophagous bats and may participate in the Brazilian sylvatic cycle of rabies. We evaluated the presence of this pathogen in hunted wild boars from the São Paulo state using histopathology, the direct fluorescent antibody test (DFA), viral isolation in cell culture (VICC), the rapid fluorescent focus inhibition test (RFFIT), and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results of histopathological, DFA, VICC, and RT-qPCR analysis were negative for all samples; seven serum samples tested positive in the RFFIT, and titers ranged from 0.13 IU/mL to 0.5 IU/mL. The presence of rabies virus-neutralizing antibodies in the studied wild boars suggests the circulation of the virus in these animals. Educative actions directed at hunters should include information on the prevention of this important zoonosis.

Spatial clusters, temporal behavior, and risk factors analysis of rabies in livestock in Ecuador

Rabies, a globally distributed and highly lethal zoonotic neglected tropical disease, has a significant impact in South America. In Ecuador, animal rabies cases are primarily linked to livestock, and hematophagous bats play a crucial role in disease transmission. This study aims to identify temporal trends, spatial patterns, and risk factors for animal rabies in Ecuador between 2014 and 2019. Epidemiological survey reports from the official Animal Rabies Surveillance Program of the Phyto and Zoosanitary Regulation and Control Agency of Ecuador (AGROCALIDAD) were used. The Animal Rabies Surveillance Program from AGROCALIDAD consists of an official passive surveillance program that receives reports from farmers or individuals (both trained or untrained) who have observed animals with neurological clinical signs and lesions compatible with bat bites, or who have seen or captured bats on their farms or houses. Once this report is made, AGROCALIDAD personnel is sent for field inspection, having to confirm the suspicion of rabies based on farm conditions and compatibility of signs. AGROCALIDAD personnel collect samples from all suspicious animals, which are further processed and analyzed using the Direct Fluorescent Antibody (DFA) test for rabies confirmatory diagnosis. In this case, study data comprised 846 bovine farms (with intra-farm sample sizes ranging from 1 to 16 samples) located in different ecoregions of Ecuador; out of these, 397 (46.93%) farms tested positive for animal rabies, revealing six statistically significant spatial clusters. Among these clusters, three high-risk areas were identified in the southeast of Ecuador. Seasonality was confirmed by the Ljung-Box test for both the number of cases (p < 0.001) and the positivity rate (p < 0.001). The Pacific Coastal lowlands and Sierra regions showed a lower risk of positivity compared to Amazonia (OR = 0.529; 95% CI = 0.318 – 0.883; p = 0.015 and OR = 0.633; 95% CI = 0.410 – 0.977; p = 0.039, respectively). The breeding of non-bovine animal species demonstrated a lower risk of positivity to animal rabies when compared to bovine (OR = 0.145; 95% CI = 0.062 – 0.339; p < 0.001). Similarly, older animals exhibited a lower risk (OR = 0.974; 95% CI = 0.967 – 0.981; p < 0.001). Rainfall during the rainy season was also found to decrease the risk of positivity to animal rabies (OR = 0.996; 95% CI = 0.995 – 0.998; p < 0.001). This study underscores the significance of strengthening the national surveillance program for the prevention and control of animal rabies in Ecuador and other countries facing similar epidemiological, social, and geographical circumstances.

One Health approach to the diagnosis and management of feline rabies: A case report from Ebonyi State, Nigeria

Abstract Rabies, a disease with an almost 100% fatality rate, continues to be endemic and neglected in Nigeria. This case report presents the rare confirmation of feline rabies in July 2022 in Ebonyi State, South Eastern Nigeria. Direct Fluorescent Antibody Test and Reverse-Transcription Polymerase Chain Reaction confirmed that the cat was infected with rabies a few weeks after it was obtained by the owner. The two cat-scratched victims (owner and another in-contact person) opted for unorthodox management with a local traditional healer without seeking conventional medical care. Further outbreak investigations into the incidents involved multiple observational visits and interviews with key informants revealed the existence of risk factors that could have facilitated the transmission of the disease. A one-health approach, which considers the interconnectedness of human, animal, and environmental health, is favourable to achieve sustainable rabies control and was used in outbreak response. Implementing active rabies surveillance, raising awareness about dog and cat vaccination, and discouraging unorthodox management is essential to control rabies within the community. Additionally, improving access to post-exposure prophylaxis is crucial in preventing rabies-related fatalities and achieving zero human rabies deaths by 2030. Information © The Authors 2024

Survey of Common Infectious Diseases in Urban Foxes (Vulpes spp.) in Southeastern Iran.

The red fox (Vulpes vulpes) is one of the most common species of wild Canidae and is relatively abundant in Iran. Foxes (Vulpes spp.) transmit many zoonotic diseases, the most important of which are visceral leishmaniasis, rabies, hydatidosis, toxocariasis, and trichinellosis. In this study, visceral leishmaniasis, rabies, ectoparasites, canine gastrointestinal helminths, dermatophytosis, distemper, parvovirus infection, and heartworm infections were evaluated among live-trapped and rescued foxes injured by traffic road accidents referred to the teaching hospital of Kerman, Iran, veterinary faculty. Skin scraping and direct microscopic examination were used to detect ectoparasites and dermatophytosis. Immunochromatography rapid kits were used to detect dirofilariasis, parvovirus infection, and distemper. Necropsy was used to check for gastrointestinal parasites. Rabies and visceral leishmaniosis were screened for with direct fluorescent antibody test and ELISA methods, respectively. Gastrointestinal helminth infections, including Toxocara canis, Taenia taeniaeformis, Dipylidium caninum, Joyeuxiella echinorhyncoids, Toxascaris leonina, Taenia hydatigena, Echinococcus granulosus, Rictolaria spp., Oxynema spp., Macracanthorhynchus hirudinaceus, and Physaloptera spp., were detected. Skin scrapings showed dermatophytosis and various ectoparasites, including Rhipicephalus sanguineus, Ctenocephalides canis and Ctenocephalides felis, and Sarcoptes scabiei, in foxes with dermal lesions. Distemper and parvovirus infection (26.66%) were the common viral diseases, and rabies infection rate was quite high (16.66%). Dirofilariasis and leishmaniasis were detected in 10% of the population. This study showed that urban foxes which often cohabit with humans and domestic animals are carriers of many different pathogens. This interaction may facilitate indirect cross-species transmission of zoonotic disease. Periodic health monitoring and multidisciplinary cooperation for the diagnosis, control, and prevention of these zoonoses is highly recommended.

Epidemiological and viral studies of rabies in Bali, Indonesia.

Rabies has been endemic in Bali since 2009, and cases has recently increased. Unfortunately, there is a lack of available vaccines, which hinders the eradication program. This study aimed to investigate the epidemiological and virological aspects of rabies infection in Bali. A total of 24 brain samples were collected from rabid dogs in all districts of Bali. The samples were tested using the direct fluorescent antibody (DFA) test and polymerase chain reaction (PCR) to confirm the presence of rabies virus in the samples. Samples with the highest virus content were propagated in vivo and then inoculated into BALB/c mice. The brains of dead mice were used to prepare an inoculate cultured in murine neuroblastoma cells. Supernatant-positive viruses representing each district were then reinoculated into eight groups of five BALB/c mice. A brain sample from each dead mouse was tested using DFA and PCR and detected under a fluorescence microscope. All rabies virus-positive samples collected from rabid dogs in all districts of Bali were positive. Rabies virus was detected by DFA test and PCR and was consistently confirmed in the in vivo and in vitro studies. BALB/c mice inoculated with the highest viral dilution (105 cells/mL) of culture supernatant showed typical signs of rabies, indicating that the virus could be properly investigated. This study demonstrated a wide epidemiological distribution of rabies in Bali. The obtained virus can be adapted for in vitro and in vivo studies and can be used to develop a homologous vaccine.

Reemergence of arctic-like rabies virus in a rabies-free area in the Amur river basin

Background: A descriptive epidemiological study of the rabies outbreak in the Amur Region of Russia (ARR), which remained rabies-free from 1972 to 2017, was carried out. During 2018-2022, 49 animal rabies cases and one human rabies death were documented in the ARR. This study aimed to detect possible ways of incursion and features of the spatial spread of rabies virus (RABV) in ARR, which was considered rabies-free for 45 years. Methods: After the beginning of the outbreak, 1,450 animal brain samples were tested using a direct fluorescent antibody test. Genetic analysis of RABV isolates was carried out (n = 9). GIS was used to analyze the spatial spread of rabies in ARR and adjacent territories. Results: Rabies has been confirmed in red foxes (Vulpes vulpes ) - 38.8%, raccoon dogs (Nyctereutes procyonoides ) - 10.2%, wolves (Canis lupus ) - 14.3%, dogs - 18.4% and farm animals - 18.3% (n = 49). The first cases were detected south of the ARR, 30 km from the state border between Russia and China. In 2018-2022, the RABV spread within the forest-steppe landscapes of the Zeya-Bureya Plain northwesterly; the maximum distance from the first recorded event was 192.4 km (Me = 77.6). According to a phylogenetic analysis, the isolates from ARR belonged to the Acrtic-like-2 RABV lineage and showed the maximum similarity to the isolates recovered in the province of Heilongjiang (China, 2011, 2018) and the Jewish Autonomous Region (Russia, 1980). Conclusion: Rabies was most likely introduced by wild carnivores from the adjacent territories of Russia or China, located downstream of the Amur River.

Dog-mediated rabies, an increasing threat to families in Kenema district: A call for action

On 5th December 2022, a stray dog attacked and bit five children, aged between 6 to 10 years in the same  community in Kenema District.  The children suffered multiple wounds on their bodies and were rushed to the  hospital for treatment. The dog was killed, head  decapitated and submitted to the Central Veterinary  Laboratory/Teko, in Makeni, Bombali District, for confirmation of rabies. The sample  was confirmed positive  using a direct fluorescent antibody test. The victims of the dog attack were rushed to the Kenema  Regional  Hospital for treatment. Due to the high cost of the rabies post-exposure prophylaxis (PEP) vaccine (rabies  vaccine freeze-dried  from Liaoning Cheng Da Biotechnology Company Limited (SPEEDA®)), the families could  not afford the whole cost of treatment. The  victims were only able to receive the first dose of post-exposure  vaccination which cost Le 400 per dose (US$ 20.5 equivalent). Upon the  intervention of the Ministry of  Agriculture and Food Security, the children received the complete dose. This is just one among many    unreported cases of exposure to dog-mediated rabies within the country.

Evaluation of a rapid immunochromatographic assay for the diagnosis of rabies in regional laboratories of Costa Rica

Rabies is a viral, lethal, and zoonotic disease with worldwide distribution, primarily transmitted through bites from infected dogs and bats. The Biosafety Laboratory (LSE-LANASEVE) of the Animal Health Service of Costa Rica (SENASA) serves as the national reference laboratory for diagnosing rabies in humans and animals. Since regional laboratories lack the equipment for the direct fluorescent antibody test (FAT), we evaluated the rapid immunochromatographic diagnostic test (RIDT) from BioNote, employing FAT as a reference, to improve rabies diagnosis. We analyzed 193 brain tissue samples between 2014 and 2019. Out of these, 174 came from species that RIDT has been validated for: bovines (162), dogs (10), and raccoons (2). The rest were from unvalidated species, including horses (7), humans (1), and others. Among the 174 validated samples, 26 bovine samples were positive for both RIDT and FAT. Reviewing all 193 samples, 28 were positive and 165 negative using both methods. Two horse samples presented inconsistencies, being positive on FAT but negative on RIDT; these were subsequently verified as false negatives by RT-PCR. RIDT exhibited a sensitivity of 94 % (CI95, 83.9-102.3), specificity of 100 %, positive predictive value (PPV) of 100 %, and negative predictive value (NPV) of 99 % (CI95, 97.1-100.5). RIDT has demonstrated reliability in quickly diagnosing rabies for validated species. We advise its application in SENASA's regional laboratories for those particular species. If there's uncertainty, samples should be sent to LSE-LANASEVE for FAT or RT-PCR confirmation.

Severe Neurologic Disease in a Horse Caused by Tick-Borne Encephalitis Virus, Austria, 2021.

As evidenced by sero-epidemiological studies, infections of horses with the tick-borne encephalitis virus (TBEV) occur frequently in TBEV-endemic areas. However, there are only very few reports of clinical cases. A possible underreporting may be due to a variety of diagnostic challenges. In this study, ELISA and neutralization tests were applied to serum samples. Brain tissue samples were investigated for the presence of nucleic acids of TBEV, Equid alphaherpesvirus 1, Borna disease virus 1, West Nile and Usutu viruses, rustrela virus, as well as Eastern, Western, and Venezuelan equine encephalitis viruses with RT-qPCR, RT-PCR, and qPCR, respectively. TBEV-specific amplification products were subjected to Sanger sequencing. In addition, a direct fluorescent antibody test for rabies was performed. Clinical and patho-histological findings are reported. Using specific RT-qPCR and RT-PCR assays, TBEV nucleic acids were demonstrated in brain tissue samples. Sequencing revealed the Western (formerly Central) European subtype of TBEV as the etiological agent. A high titer of TBEV-specific neutralizing antibodies was found in the serum. RNAscope in situ hybridization revealed TBEV RNA confined to neuronal cell bodies and processes. No other pathogens or nucleic acids thereof could be detected. Diagnostic procedures need to be carried out early after the onset of neurological signs to allow for a final etiological diagnosis of acute TBEV infections in horses.

Molecular characterization of rabies virus in trade dogs from Plateau state, Nigeria

Rabies is one of the most dreaded diseases known to mankind and it is endemic in Nigeria. This study was aimed at the detection and molecular  characterisation of the rabies virus antigen in trade dogs in Plateau State. A cross-sectional study was performed to determine the prevalence of rabies  antigen in the brain tissues of slaughtered dogs, from February to June 2018 using a direct fluorescent antibody test and mouse inoculation technique.  Direct Fluorescent Antibody Test, Polymerase Chain Reaction (PCR), sequencing and phylogenetic analysis of the identified rabies virus were also  undertaken. Phylogenetic analysis was done using molecular evolutionary genetic analysis (MEGA) 10 to determine their evolutionary relationship with  other rabies virus sequences deposited in Genbank. Results indicated, out of 450 dog brain samples tested 14(3.1%) were positive for rabies virus  antigen. Zoographic data obtained showed 66.3% of dogs were female, and the dogs were kept mainly for security (n=54) and breeding (n=28). Fourteen  positive samples were used to inoculate 70 adult mice, only 8 samples were positive and 2 samples were also positive by RT PCR. Phylogenetic analysis of  the nucleotide sequences showed that the two rabies virus sequences in this study, sequence MZ148311 had very close identity with EU038106 (99.8%  homology) and sequence MZ148312 had perfect identity with EU038087 (100% homology) was most closely related to lyssavirus that has been reported  to be circulating previously in Plateau and Nigeria, belonging to the Africa 2 lineage. 

Rabies virus infection is associated with variations in calbindin D-28K and calretinin mRNA expression levels in mouse brain tissue.

Rabies virus (RABV) infection leads to a fatal neurological outcome in humans and animals and is associated with major alterations in cellular gene expression. In this study, we describe the effects of RABV infection on the mRNA expression levels of two genes, encoding the Ca2+-binding proteins (Ca-BPs) calbindin D-28K (Calb1) and calretinin (Calb2), in the brains of BALB/c mice. Sixty 4-week-old mice were divided into two test groups and one control group. Mice were inoculated intramuscularly with either a street rabies virus (SRV) strain or a challenge virus standard (CVS-11) strain and sacrificed at 3-day intervals up to day 18 postinfection. A direct fluorescent antibody test (DFAT) was used to verify the presence of RABV antigen in brain tissues, and real-time quantitative PCR (RT-PCR) was used to assess gene expression. Infection with both RABV strains resulted in significant (p < 0.05) increases in Calb1 and Calb2 expression in the test animals when compared with the controls at various time points in the study. Correlation analysis indicated very weak insignificant (p > 0.05) negative and positive relationships, respectively, between Calb1 expression (r = -0.04) and Calb2 expression (r = 0.08) with viral load (CVS-11 strain). Insignificant (p > 0.05) relationships were also observed Calb1 expression (r = -0.28) and Calb2 expression (r = 0.06) and viral load for the SRV strain.The observed alterations in Calb1 and Calb2 expression in this study indicate possible impairments in neuronal Ca2+ buffering and Ca2+ homeostasis as a result of RABV infection and, consequently, possible involvement of calbindin-D28K and calretinin in the neuropathogenesis of rabies.

Comparison of Rabies Cases Received by the Shomal Pasteur Institute in Northern Iran: A 2-Year Study.

The rabies virus, which belongs to the genus Lyssavirus, the family Rhabdoviridae, is the causative agent of rabies, a contagious, deadly, and progressive neurological infection. This illness is commonly distributed worldwide and affects all warm-blooded animals. Regarding the zoonotic aspects of rabies, the prevalence of rabies was investigated in this study. Over 2 years, 188 samples were examined via the direct fluorescent antibody test (DFAT) and mouse inoculation test (MIT) techniques by using brain tissue samples. Our findings showed that 73.94% of samples were rabies positive. The highest number of samples belonged to cows and dogs, respectively. The positivity rate in cows was 71.88%, followed by dogs with a 57.78% infection rate. These findings suggested that despite the heavy monitoring protocols in Iran, rabies is still a prevalent disease, and it is advised that vaccinations and screening programs should be carried out more frequently with heavier observation.

Diagnostic sensitivity and specificity of immunohistochemistry for the detection of rabies virus in domestic and wild animals in South Africa.

We estimated the diagnostic sensitivity (DSe) and specificity (DSp) of an immunohistochemistry (IHC) protocol compared to the direct fluorescent antibody test (DFAT), which is the gold standard test for rabies diagnosis. We obtained brain samples from 199 domestic and wild animal cases (100 DFAT-negative, 99 DFAT-positive), by convenience sampling from 2 government-accredited rabies virus (RABV) testing laboratories in South Africa, between February 2015 and August 2017. Tissues that had been stored at 4-8°C for several days to weeks at the 2 accredited laboratories were formalin-fixed and paraffin-embedded. Nighty-eight cases tested IHC-positive using a polyclonal anti-RABV nucleoprotein antibody and a polymer detection system. The overall DSe and DSp for the RABV IHC test were 98% (95% CI: 93-100%) and 99% (95% CI: 95-100%), respectively. Domestic dogs accounted for 41 of 98 RABV IHC-positive cases, with the remainder in 4 domestic cats, 25 livestock, and 28 wildlife. Herpestidae species, including 7 meerkats and 9 other mongoose species, were the most frequently infected wild carnivores, followed by 11 jackals. Three cases in domestic dogs had discordant test results; 2 cases were IHC-/DFAT+ and 1 case was IHC+/DFAT-. Considering the implications of a false-negative rabies diagnosis, participating in regular inter-laboratory comparisons is vital, and a secondary or confirmatory method, such as IHC, should be performed on all submitted specimens, particularly negative cases with human contact history.

Epitope mapping of diagnostic anti-rabies monoclonal antibodies provide structural insight into rabies variant identification in infected wildlife.

Rabies virus is a member of the lyssavirus family and causes over 60,000 deaths a year. There are currently five strains of rabies virus circulating throughout the continental United States. In order to better control the spread of rabies virus, the Center of Disease Control monitors the prevalence and host range of different rabies virus variants (RVV) throughout the United States. This surveillance relies on a panel of monoclonal antibodies that are known to differentially bind to the nucleoprotein of RVV in a direct fluorescent antibody test (DFA). While these tests are known to accurately distinguish RVV, it is unknown where these antibodies bind to the rabies nucleoprotein. To better understand the fundamental principles of this assay, the epitopes of three monoclonal antibodies from the current panel, which are suspected to have conformational epitopes, were determined using hydrogen-deuterium mass spectrometry. We determined these antibodies bind to distinct surface-exposed epitopes of the nucleoprotein, one of which makes contacts with multiple subunits of the 11-mer ring that is formed by the nucleoprotein. After determining the epitopes, a multiple sequence alignment was examined to look at the conservation of the amino acids in each epitope to determine how these antibodies distinguish between RVV. By determining epitopes, we can now predict how viral mutations could change the specificity and/or sensitivity of a rabies DFA. The results of this work can be used to help further develop rapid immunoassays for more economical RVV diagnostics in the field, in regions that may lack access to microscopy-based DFA diagnostics.

Current status and molecular epidemiology of rabies virus from different hosts and regions in Malawi

Although rabies is endemic in Malawi, there have been no studies in which rabies virus was systematically investigated and characterized in multiple animal hosts in that country. In order to provide molecular epidemiological data on rabies virus in Malawi, 683 suspected rabies case reports from 2008 to 2021 were examined, and 46 (dog = 40, cow = 5, and cat = 1) viable rabies-positive brain samples archived at the Central Veterinary Laboratory (CVL), Lilongwe, Malawi, were analyzed genetically. The results showed an increase in the submission of brain samples from 2008 to 2010, with the highest number of submissions observed in 2020. Of the 683 case reports analyzed for the period under review, 38.1% (260/683) (CI: 34.44 – 41.84) were confirmed by direct fluorescent antibody test. Among the confirmed cases, 65.4% (170/260) (CI: 59.23 – 71.09) were canine rabies. Further, phylogenetic analysis revealed that sequences from different animal hosts clustered together within the Africa 1b lineage, suggesting that the strains circulating in livestock are similar to those in domestic dogs. This finding supports the hypothesis that canine rabies is spilling over to livestock and emphasizes the need for further studies to provide data for effective control of rabies in Malawi.

Immunohistochemical localisation of Glutamate ammonia ligase (GLUL) in cerebrum in different clinical forms of canine rabies

Rabies, one of the earliest known diseases reported in the history of mankind, is a fatal acute viral disease of the central nervous system. Despite numerous studies on etiology of rabies, its exact mechanism of neuropathogenesis remains unidentified. Glutamate ammonia ligase (GLUL) is a protein present in brain which play an important role in the pathogenesis of many neurological conditions. GLUL was identified to be over expressed in brain of human rabies. Cerebral cortex is the part of brain primarily responsible for coordination of movements and behaviour in animals and humans. Hence the work was designed to study the immunohistochemical localisation of GLUL in cerebrum in rabid carcasses in order to elucidate its role in the pathology of this deadly viral infection. The carcasses of 219 rabies suspected dogs that were brought to the Department of Veterinary Pathology, CVAS, Mannuthy, for necropsy between January 2021 and August 2022 formed the materials for the study. One hundred and thirty-three cases were found positive for rabies using the gold standard test for rabies - Direct Fluorescent Antibody Test (dFAT) which was further confirmed by polymerase chain reaction targeting N gene with amplicon size 533 bp. Among these 133 confirmed cases, 30 selected samples (frontal, occipital, temporal, parietal lobes of cerebrum) were further processed for histopathological and immunohistochemical (IHC) studies. The IHC signals of GLUL obtained were compared with the clinical form of rabies. Immunohistochemical staining revealed localisation of GLUL in glial cells with different staining intensity in encephalitic and paralytic form of rabies. The study concluded that the IHC signals for GLUL were stronger for furious form of rabies and weak for dumb form of the disease.

Um ensaio RT-rtPCR para detecção de vírus da raiva em espécimes bovinos

ABSTRACT: Bovine rabies is endemic in most Brazilian States, including Rio Grande do Sul (RS), which has faced an unprecedented rabies outbreak between 2011 and 2018. We described a real-time reverse transcription quantitative PCR (RT-rtPCR) for detection of rabies virus (RABV) in bovine samples. The primers were designed targeting a highly conserved region of the nucleoprotein (N) gene of RABV obtained from cattle. The detection limit corresponded to 13 DNA copies and the intra- and inter-run repeatability was adequate (CV&lt;9%) in all dilutions tested. Amplification of other pathogens associated with neurological disease in cattle or cross-contamination was not observed. Brain samples from cattle suspicious of rabies (n=21) were tested in triplicate by the RT-rtPCR and by the gold-standard direct fluorescent antibody test (DFAT), resulting in 100% of sensitivity and specificity of the RT-rtPCR. Testing of additional 41 bovine brain samples submitted to the routine DFAT testing yielded 37 (90.2%) concordant results (30 positive/7 negative) and 4 (9.7%) inconclusive in DFAT and RT-rtPCR positive. These results showed a good concordance between the tests and a higher sensitivity of the RT-rtPCR. This assay represents an alternative for RABV detection, either as a confirmatory test or for large-scale diagnosis in endemic regions.