Direct Fluorescent Antibody Test Research Articles

Optimization of pan-lyssavirus LN34 assay for streamlined rabies diagnostics by real-time RT-PCR

Reliable, validated diagnostic tests are critical for rabies control in animals and prevention in people. We present a performance assessment and updates to the LN34 real-time RT-PCR assay for rabies diagnosis in postmortem animal brain samples. In two U.S. laboratories during 2017 to 2022, routine used of the LN34 assay produced excellent diagnostic sensitivity (99.72% to 100%) and specificity (99.99% to 100%) compared to the direct fluorescence antibody test (DFA). Almost all (>90%) DFA indeterminate results caused by non-specific or atypical fluorescence were negative by LN34 testing, representing up to 111 cases where unnecessary post-exposure prophylaxis could be avoided. LN34 assay original primer sequences showed low sensitivity for some rare lyssaviruses. Increased primer concentration combined with new primer formulation showed improved performance for impacted lyssaviruses with no loss in performance across diverse rabies virus variants from clinical samples. The updated LN34 and internal control assays were combined into a single-well LN34 multiplexed (LN34M) format, run at half reagent volumes. The LN34M assay showed similar detection of rabies virus to the singleplexed assay with simplified assay set-up, lower cost, and improved quality controls.

Skrjabingylus chitwoodorum in a rabies-positive striped skunk in Texas.

We describe here a case of the sinus roundworm, Skrjabingylus chitwoodorum, found incidentally in a rabies-positive striped skunk (Mephitis mephitis) in Texas, USA. Skunks serve as a natural definitive host for this metastrongylid nematode in North America, in which infections result in observable damage to the host cranium, where adult parasites reside. Additionally, skunks are considered the primary reservoir of rabies in Texas. In November 2022, the animal was discovered in northern Texas displaying neurologic signs before euthanasia and submission to the Oklahoma Animal Disease Diagnostic Laboratory for rabies testing. Direct fluorescent antibody testing indicated that the animal was rabies-positive, and, upon tissue collection, numerous adult nematodes were recovered from the cranium and identified as S. chitwoodorum by morphology and amplification of the mitochondrial cytochrome c oxidase subunit I gene. Histologically, we found lymphohistiocytic meningitis in several loci and chronic sinusitis rostral to the cribriform plate. Due to behavioral abnormalities, we additionally tested for Toxoplasma gondii via PCR, but no parasite DNA was detected. Concurrent infection by S. chitwoodorum and rabies virus may contribute to neurologic signs in skunks.

Rabies in free-ranging capybaras (Hydrochoerus hydrochaeris) on Anchieta Island, Ubatuba, Brazil.

Between December 2019 and January 2020, three cases of rabies were reported in free-ranging capybaras on Anchieta Island, Ubatuba-SP, Brazil. This 8.28km² island is located 540m offshore from the mainland. Two of the capybaras exhibited signs of hindlimb paralysis, and one was found dead. Rabies was diagnosed using the direct fluorescent antibody test (dFAT), while RT-qPCR and phylogenetic analysis of nucleotide sequences confirmed the presence of the vampire bat rabies virus (RABV) strain. Although no visible bat bite marks were found on the capybaras, vampire bats are known to inhabit the island. Other wildlife tested negative for rabies during this period, and no further rabies outbreaks have been observed since. Environmental changes and human activities, such as the disturbance of bat roosting sites, may have contributed to the incident. The detection of rabies in capybaras suggests a potential spillover from a vampire bat reservoir. Further investigation is needed to determine whether capybaras act as dead-end hosts or play a role in maintaining the rabies transmission cycle.

Rabies virus circulation in a highly diverse bat assemblage from a high-risk area for zoonoses outbreaks in the Brazilian Amazon

Bats are the second most diverse order of mammals and play a central role in ecosystem dynamics. They are also important reservoirs of potentially zoonotic microorganisms, of which rabies virus is the most lethal among the bat-transmitted zoonotic pathogens. Importantly, recent outbreaks of human rabies have been reported from the Brazilian Amazon. Here we present a survey of bat species and rabies virus (RABV) circulation in a bat assemblage in the Marajó region, northern Brazil. Using data from mist-net captures and bioacoustic sampling, 56 bat species were recorded along the Jacundá River basin over a 10-day expedition in November 2022. For the investigation of RABV, we used the direct fluorescent antibody test (DFAT) and the rapid fluorescent focus inhibition test (RFFIT). In total, 159 bat individuals from 22 species were investigated for RABV. Five adults of the common vampire bat, Desmodus rotundus, showed RABV-specific antibodies in serum samples. Additionally, we report on local residents with injuries caused by D. rotundus bites and the occurrence of colonies of non-hematophagous bats from different species roosting inside human residences. This scenario raises concerns about the risks of new cases of human rabies and other zoonotic diseases associated with bats in the region and highlights the need for epidemiological surveillance and mitigation measures to prevent outbreaks of emerging infectious diseases.

Incidence of human rabies following bite or exposure to laboratory confirmed rabid animals

Background: Rabies is one of the important endemic fatal zoonotic viral disease afflicting humans and animals in Punjab, India. The present study investigated the incidence of rabies in humans bitten/exposed to laboratory confirmed rabid animals, as well as incidence after use of vaccine or rabies immunoglobulin (RIG) and clinico-epidemiological studies. Methods: A study was conducted during August 2021 to September 2022 on forty (40) rabies suspected animals presented to diagnose rabies by direct fluorescent antibody test (dFAT) at rabies diagnostic laboratory (RDL), Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Punjab, India. A detailed questionnaire was prepared for obtaining information about exposure/bite of humans by rabid animals, death of humans, demographic and epidemiological information of victims. Results: Out of total forty (40) suspected rabies cases, 30(75%) were found positive for rabies by dFAT. Laboratory confirmed rabies (LCR) incidence was 60.80% and 21.73% in stray and pet dogs, respectively. All pet dogs were vaccinated but no stray dog was vaccinated. The LCR incidence in buffaloes and cattle was 77.77% and 100%, respectively. Further in humans exposed to rabid animals (59), males were at more risk than females. The human rabies incidence was 3.38% (2/59). Highest incidence of dog bites in adult males on lower limb was observed from urban stray dogs (60.80%) followed by children. Post exposure vaccination was given to 98.3% humans exposed to rabid animals. Human’s rabies in two cases was due to no vaccination or RIG. Conclusions: Vaccination is an important step in controlling rabies in India. There is a need for integrated and comprehensive management of street dogs and bite management.

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.

Clinical features of atypical presentations of mucocutaneous herpes simplex virus infection observed in immunosuppressed individuals. Part II: the knife-cut sign.

The knife-cut sign is a distinctive manifestation of herpes simplex virus (HSV) type 1 or HSV type 2 infection that has been described in at least 10 immunocompromised patients. It appears as an extremely painful linear erosion or fissure in an intertriginous area such as the body folds beneath the breast, or within the abdomen, or in the inguinal region. Also, concurrent HSV infection at other mucocutaneous sites, or viscera, or both have been observed. The patients had medical conditions (at least 9 patients) and/or immunosuppressive drug therapy (6 patients). The diagnosis of HSV infection was confirmed by viral culture (8 patients), biopsy (4 patients), direct fluorescence antibody testing (3 patients), immunohistochemistry staining (2 patients), polymerase chain reaction (2 patients), or Western blot serologic assay (1 patient). Knife-cut sign-associated HSV infection is potentially fatal; three patients died. However, clinical improvement or complete healing occurred in the patients who received oral valacyclovir (1 patient), or intravenous acyclovir (2 patients), or intravenous acyclovir followed by foscarnet (1 patient). In summary, HSV infection associated with a positive the knife-cut sign is a potentially fatal variant of HSV infection that occurs in the intertriginous areas of immunocompromised patients and usually requires intravenous antiviral therapy.

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.