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Introduction: Bluetongue is an arthropod-borne viral disease of ruminants, which is caused by bluetongue virus (BTV) that exists in more than 30 different serotypes. The disease is endemic in Pakistan. However, little is known about circulating BTV serotypes in the country. This study reports the serotypes of BTV in North-Western Pakistan. Methods: A total of 758 competitive ELISA-positive serum samples were tested using serum neutralization tests against the 28 BTV serotypes (BTV-1 to BTV-27 and BTV-X). The test samples originated from cattle (n = 296), buffalo (n = 80), sheep (n = 136), and goats (n = 246). Results: Neutralizing antibodies against one or more of the 28 BTV serotypes were detected in 59.1% of the positive samples. Antibodies against BTV-26 had the highest (56.5%) prevalence, whereas, those against BTV-19 had the lowest (0.9%) prevalence. Species-wise, neutralizing antibodies against all the 28 serotypes of BTV were found in cattle, whereas antibodies against 24, 21, and 19 different serotypes of BTV were detected in goats, sheep, and buffalo, respectively. Neutralizing antibodies against 19 different BTV serotypes were detected for the first time in Pakistan. It was also the first time that neutralizing antibodies against atypical serotypes (i.e., BTV-25, BTV-27, and BTV-X) were found in cattle. Conclusion: Bluetongue in Pakistan has a complex epidemiology, as evidenced by the detection of antibodies against a large number of BTV serotypes. Findings of the current study may be helpful in selecting appropriate vaccines for control of the disease in the country.

From wild dogs (Lycaon pictus) in the Serengeti to tigers (Panthera tigris altaica) in the Russian Far East, canine distemper virus (CDV) has been repeatedly identified as a threat to wild carnivores. Between 2020 and 2022, six Indian leopards (P. pardus fusca) presented to Nepali authorities with fatal neurological disease, consistent with CDV. Here, we report the findings of a serosurvey of wild felids from Nepal. A total of 48 serum samples were tested, comprising 28 Bengal tigers (P. t. tigris) and 20 Indian leopards. Neutralizing antibodies were identified in three tigers and six leopards, equating to seroprevalences of 11% (CI: 2.8–29.3%, n = 28) and 30% (CI: 12.8–54.3%, n = 20), respectively. More than one-third of seropositive animals were symptomatic, and three died within a week of being sampled. The predation of domestic dogs (Canis lupus familiaris) has been posited as a potential route of infection. A comparison of existing diet studies revealed that while leopards in Nepal frequently predate on dogs, tigers do not, potentially supporting this hypothesis. However, further work, including molecular analyses, would be needed to confirm this.

A serum neutralization test (SNT) is an essential method for the serological diagnosis of pestivirus infections, including classical swine fever, because of the cross reactivity of antibodies against pestiviruses and the non-quantitative properties of antibodies in an enzyme-linked immunosorbent assay. In conventional SNTs, an immunoperoxidase assay or observation of cytopathic effect after incubation for 3 to 7 days is needed to determine the SNT titer, which requires labor-intensive or time-consuming procedures. Therefore, a new SNT, based on the luciferase system and using classical swine fever virus, bovine viral diarrhea virus, and border disease virus possessing the 11-amino-acid subunit derived from NanoLuc luciferase was developed and evaluated; this approach enabled the rapid and easy determination of the SNT titer using a luminometer. In the new method, SNT titers can be determined tentatively at 2 days post-infection (dpi) and are comparable to those obtained by conventional SNTs at 3 or 4 dpi. In conclusion, the luciferase-based SNT can replace conventional SNTs as a high-throughput antibody test for pestivirus infections.

Background and Aim: Bluetongue (BT) is a major disease of sheep and goats and is endemic to India. It is known to cause significant economic losses to the sheep industry. The current study aimed to determine the type-specific seroprevalence of BT in sheep population of India during 2018-2019. Materials and Methods: Blood samples (n=405) were collected from 6 months to 1 year old sheep from six districts (Nalgonda, Karimnagar, Khammam, Mahabubnagar, Warangal, and Ranga Reddy) of Telangana state, India. Group- and type-specific seroprevalence (against BT virus [BTV] serotypes BTV-1, 2, 4, 5, 9, 10, 12, 16, 21, 23, and 24) was studied by competitive enzyme-linked immunosorbent assay and serum neutralization test, respectively. Results: Results showed an overall seroprevalence of 14.81% (n=60) with the highest seroprevalence of 50% in Khammam district. Seroprevalence of BTV-1, 2, 4, 5, 9, 10, 12, 16, 21, 23, and 24 was noted as 16.66%, 11.66%, 31.66%, 11.66%, 05%, 6.66%, 16.66%, 8.33%, 13.33%, 6.66%, and 16.66%, respectively. The majority of the sera neutralized more than 1 serotype, indicating superinfection or circulation of multiple serotypes in the sampled flocks. This mixed seroprevalence was observed in 43.33% of the sera with number of BTV serotype-specific antibodies ranging from two to eight in individual animals. Conclusion: Regular monitoring of circulating serotypes, especially in young herds, elucidates pattern of dominating serotypes in a particular area during a season. This knowledge can be applied to design appropriate vaccination strategies by including particular serotypes of virus as part of a multivalent vaccine for a particular period, in a particular area.

Background: Lumpy skin disease (LSD) is one of Ethiopia's most economically significant transboundary livestock illnesses. The disease has a significant economic impact on pastoral household livestock owners, who rely significantly on their cattle as a source of income. Methods: A cross-sectional study was undertaken in selected districts of Afar region from November 2018 to May 2019 primarily intended to estimate the prevalence of lumpy skin disease serologically in local Afar cattle as well as identify potential associated factors. A multistage sampling method was employed to select study districts, peasant association, herd size and study units. A total of 384 sera were processed using serum neutralization test (SNT) method to detect antibodies against lumpy skin disease virus. Relevant data were refined and further analyzed using stata version 14. Results: In the study districts, the overall animal level seroprevalence was found to be 7.6% (N = 29/384; 95% confidence interval: 4.90-10.20) and the overall herd level prevalence was found to be 20.8% (n = 15/72; 95% confidence interval: 11.42-30.18). Only district was shown to be statistically significant (P = 0.004) in terms of LSD occurrence among the relevant factors studied. Cattle in Chifra district were 20.18 times more likely to contract LSD infection than cattle in Dubti district, when Asayita district was used as the reference group. Conclusion: The present study finding confirmed the presence of the disease in the study districts of afar region and coordinated intervention set to be in place. Keywords: afar, cattle, LSD, associated factors, seroprevalence, serum neutralization test

Background and Aim: Canine parvovirus (CPV) is the most important viral cause of enteritis and mortality in pups. Evaluation and monitoring of pre- and post-vaccine immune responses may help to determine the efficacy of the current vaccination schedule being followed in pups in India. This study aimed to evaluate and monitor the pre- and post-vaccine immune responses of CPV vaccinated pups using hemagglutination inhibition (HI) assay. The neutralizing antibody titer levels were also detected using serum neutralization test (SNT). Materials and Methods: The pups were categorized into two groups, the double booster and the single booster groups. In this study, serum samples were subjected to HI and SNT for measuring the CPV antibody titer at frequent intervals for up to 6 months from 27 healthy pups following primary and booster CPV vaccinations. Results: The antibody titers in double booster pups reached their peaks at the 21st day after the second booster vaccination with a geometric mean (GM) of 3.57. The antibody titers in single booster pups reached their peaks at the 21st day after the first booster vaccination with a lower GM of 3.18. Conclusion: The double booster pups maintained a higher immune response throughout the period of the study compared to single booster pups though the difference in titers was not statistically significant. SNT results indicated that the raised antibody titer was also able to yield virus-neutralizing antibodies. No interfering maternally derived antibodies were found in the pups at the age of primary vaccination (45th day) in our study. Therefore, the second booster vaccination may be useful in maintaining the protective titer for a prolonged period.

Background In 2008, a program to eradicate bovine virus diarrhea (BVD) in cattle in Switzerland was initiated. After targeted elimination of persistently infected animals that represent the main virus reservoir, the absence of BVD is surveilled serologically since 2012. In view of steadily decreasing pestivirus seroprevalence in the cattle population, the susceptibility for (re-) infection by border disease (BD) virus mainly from small ruminants increases. Due to serological cross-reactivity of pestiviruses, serological surveillance of BVD by ELISA does not distinguish between BVD and BD virus as source of infection. Results In this work the cross-serum neutralisation test (SNT) procedure was adapted to the epidemiological situation in Switzerland by the use of three pestiviruses, i.e., strains representing the subgenotype BVDV-1a, BVDV-1h and BDSwiss-a, for adequate differentiation between BVDV and BDV. Thereby the BDV-seroprevalence in seropositive cattle in Switzerland was determined for the first time. Out of 1,555 seropositive blood samples taken from cattle in the frame of the surveillance program, a total of 104 samples (6.7%) reacted with significantly higher titers against BDV than BVDV. These samples originated from 65 farms and encompassed 15 different cantons with the highest BDV-seroprevalence found in Central Switzerland. On the base of epidemiological information collected by questionnaire in case- and control farms, common housing of cattle and sheep was identified as the most significant risk factor for BDV infection in cattle by logistic regression. Conclusion This indicates that pestiviruses from sheep should be considered as a source of infection of domestic cattle and might well impede serological BVD surveillance.

Neutralising antibodies to bluetongue virus (BTV) in convalescent cattle have been described as persistent. Controlled laboratory studies, however, rarely last longer than a couple of weeks and long-term field data are lacking. This study followed twelve cattle that had been naturally infected with bluetongue virus serotype 8 (BTV-8) in Germany in 2006. Using ELISAs and a serum neutralisation test, we found a strong humoral immune response four to six years after the last exposure to BTV-8; based on data from long-term vaccine studies, it is highly likely that this coincides with immunity to reinfection with the same serotype.

Aim: The present work deals with different methods for foot and mouth disease virus (FMDV) inactivation for serotypes O/pan Asia, A/Iran05, and SAT-2/2012 by heat, gamma radiation, and ultraviolet (UV) in comparison with the traditional methods and their effects on the antigenicity of viruses for production of inactivated vaccines. Materials and Methods: FMDV types O/pan Asia, A/Iran05, and SAT-2/2012 were propagated in baby hamster kidney 21 (BHK21) and titrated then divided into five parts; the first part inactivated with heat, the second part inactivated with gamma radiation, the third part inactivated with UV light, the fourth part inactivated with binary ethylamine, and the last part inactivated with combination of binary ethylamine and formaldehyde (BEI+FA). Evaluate the method of inactivation via inoculation in BHK21, inoculation in suckling baby mice and complement fixation test then formulate vaccine using different methods of inactivation then applying the quality control tests to evaluate each formulated vaccine. Results: The effect of heat, gamma radiation, and UV on the ability of replication of FMDV \"O/pan Asia, A/Iran05, and SAT-2/2012\" was determined through BHK cell line passage. Each of the 9 virus aliquots titer 108 TCID50 (3 for each strain) were exposed to 37, 57, and 77°C for 15, 30, and 45 min. Similarly, another 15 aliquots (5 for each strain) contain 1 mm depth of the exposed samples in petri-dish was exposed to UV light (252.7 nm wavelength: One foot distance) for 15, 30, 45, 60, and 65 min. Different doses of gamma radiation (10, 20, 25, 30, 35, 40, 45, 50, 55, and 60 KGy) were applied in a dose rate 0.551 Gy/s for each strain and repeated 6 times for each dose. FMDV (O/pan Asia, A/Iran05, and SAT-2/2012) were inactivated when exposed to heat ≥57°C for 15 min. The UV inactivation of FMDV (O/pan Asia and SAT-2) was obtained within 60 min and 65 min for type A/Iran05. The ideal dose for inactivation of FMDV (O/pan Asia, A/Iran05, and SAT-2/2012) with gamma radiation were 55-60 and 45 kGy, respectively. Inactivation of FMDV with binary was 20, 24 and 16 hr for O/pan Asia, A/Iran05, and SAT-2/2012, respectively while inactivation by (BEI+FA) was determined after 18, 19 and 11 hr for O/pan-Asia, A/Iran 05, and SAT-2/2012, respectively. The antigenicity of control virus before inactivation was 1/32, it was not changed after inactivation in case of gamma radiation and (BEI+FA) and slightly decrease to 1/16 in case of binary and declined to 1/2, 1/4 in case of heat and UV inactivation, respectively. The immune response induced by inactivated FMD vaccines by gamma radiation and (BEI+FA) lasted to 9 months post-vaccination, while the binary only still up to 8 months post-vaccination but heat and UV-inactivated vaccines were not effective. Conclusion: Gamma radiation could be considered a good new inactivator inducing the same results of inactivated vaccine by binary with formaldehyde (BEI+FA).