Evaluation of the performance of diagnostic methods of canine parvovirus-2 and canine enteric coronavirus infections under different storage conditions and determination of molecular characterization

Aim: This research was carried out to detect CPV and CCoV infections in dogs in comparison with rapid kit and PCR and to determine the molecular characterization of these infections in Konya region. Besides, it was aimed to determine the sensitivity and specificity rates of the diagnostic tests after fresh or freeze-thawed stool for infection diagnosis. Materials and Methods: Faecal samples were collected from 50 unvaccinated, 0-12 months old dogs with diarrhoea symptoms at the shelter. The samples were analysed for CPV and CCoV by rapid test and PCR test. After freezethawing, the samples were checked again with the same tests. Results: CPV was positively diagnosed by rapid test and PCR in 2 and 29 fresh stool samples, respectively, and CCoV in 14 and 28 samples. CPV positive samples did not change while CCoV was diagnosed as positive in 10 samples and 28 samples by rapid test and PCR, respectively, after the freeze-thaw procedure. Although there were no differences in the diagnosis of CPV, the sensitivity of the rapid test in the diagnosis of CCoV decreased after the freezethaw procedure. In addition, only CPV-2b type was detected in CPV positive samples and both GI and GII subtypes were detected in CCoV positive samples as molecular. In conclusion, it was observed that rapid tests are not sensitive for accurate diagnosis of CPV and CCoV infections. Conclusion: The importance of choosing molecular diagnostic methods and using fresh samples for accurate diagnosis of virological infections can be emphasized

The Serological and Virological Investigation of Canine Adenovirus Infection on the Dogs

Two types of Canine Adenovirus (CAVs), Canine Adenovirus type 1 (CAV-1), the virus which causes infectious canine hepatitis, and Canine Adenovirus type 2 (CAV-2), which causes canine infectious laryngotracheitis, have been found in dogs. In this study, blood samples taken from 111 dogs, which were admitted to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine, with clinical symptoms. Seventy-seven dogs were sampled from Isparta and Burdur dog shelters by random sampling, regardless of the clinical findings. Dogs showed a systemic disease, characterized by fever, diarrhea, vomiting, oculonasal discharge, conjunctivitis, severe moist cough, signs of pulmonary disease and dehydration. Two dogs had corneal opacity and photophobia. In serological studies, 188 serum samples were investigated on the presence of CAV antibodies by ELISA. Total 103 (103/188–54.7%) blood samples were detected to be positive for CAV antibodies by ELISA. However, 85 (85/188–45.2%) blood samples were negative. Blood leukocyte samples from dogs were processed and inoculated onto confluent monolayers of MDCK cells using standard virological techniques. After third passage, cells were examined by direct immunoflourescence test for virus isolation. But positive result was not detected. In conclusion, this study clearly demonstrates the high prevalence of CAV infection in dogs

Serological and virological investigation of bovine viral diarrhea virus infection in cattle with abortion problem

Aim: The aim of this study is to determine the presence of Bovine Viral Diarrhea Virus (BVDV) infection in a cattle herd with abortion problem in Konya. Materials and Methods: Totally 228 blood serum and 228 leukocytes taken from cattle selected according to criteria for infertile and abortion problems were examined for antigens and antibodies to BVDV by Enzyme Linked Immunosorbent Assay. Results: In this research, 41 (17.9%) sera were found seropositive and 4 (1.7%) leukocytes were BVDV antigen positive. Of these 4 BVDV antigen positive cattle, a number of 2 (0.8%) were detected seropositive while 2 (0.8%) were seronegative. The animals being antigen positive and antibody negative were sampled second time after two weeks. The same results were detected for two seronegative cattle. The animals detecting persistent infection status were sent to slaughter. Conclusion: It is recommended that the animals should be checked in terms of BVDV for being negative both antigen and antibody before accepting them to the herds

Comparison of reverse-transcriptase polymerase chain reaction (rt-pcr) and rapid test for the detection of bovine rotavirus and bovine coronavirus in anatolian water

Aim: Coronaviruses and Rotaviruses are important virologic factors for both animal and human health in Turkey and the world. Bovine Rotavirus (BRV) and Bovine Coronavirus (BCoV) in cattle cause significant economic losses. The aim of this study was to determine the presence of BRV and BCoV in Anatolian buffaloes which were on the same farms with cattle. For this purpose, presence of these two viruses were investigated by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and BRV-BCoV Rapid tests and sensitivity and specificity ratios of these two tests were compared. Materials and Methods: In this study, 230 Anatolian buffaloes were clinically evaluated in cattle farms in Afyonkarahisar region. Fecal samples were collected from 27 buffaloes which had clinical signs (weakness, dehydration, vomiting, watery consistency and yellow stool). The fecal samples were evaluated by Rapid Test and RT-PCR for Bovine Rotavirus and Bovine Coronavirus. The analyzes were performed according to the procedure of the commercial RT-PCR and rapid kits. Results: The RT-PCR results were positive as 22.2% (6/27) for BRV and 3.7% (1/27 27/1) for BcoV while Rota-Corona Rapid test results were negative in all samples. When compared with RT-PCR results for both viruses, the rapid test sensitivity and specificity was determined as 0% and 100%, respectively. In addition, positive rates of BRV was statistically important as BCoV rate in analyzed samples (p Conclusion: In conclusion, low sensitivity of rapid test may be due to the change in the amount of virus scattered throughout the course of enteric infections

Sığırların Sitopatojen Viruslarının Titrasyonunda Mtt Metodunun Kullanımı

Araştırmada, sığırların sitopatojen viruslarının titrasyonunu gerçekleştirmek amacıyla kolorimetrik bir metot geliştirildi. Kolorimetrik MTT metodu, hücre kültürlerinde sadece canlılık aktivitesini devam ettiren hücrelerde bulunan mitokondrial dehidrogenaz enzimi tarafından, sarı renkli MTT maddesinin tetrazolium tuzlarına indirgenerek mavi formazan kristallerine dönüştürülmesi ilkesine dayanmaktadır. Bovine herpesvims type 1 (BHV-1), parainfluenza 3 (PI-3), bovine rota virus (BRV) and bovine adenovirus type 1 (BAV-1) viruslarının titrasyonu, MTT ve DKID50 yöntemleri ile MDBK devamlı hücre kültürleri kullanılarak mikropleytlerde gerçekleştirildi. Virusların titrelerinin saptanması amacıyla kullanılan standart DKID50 ve MTT yöntemleri ile elde edilen sonuçların, birbirleriyle iyi bir korelasyon gösterdiği belirlendi.In the current study, a colorimetric method for titration of cytopathic bovine viruses was developed. MTT colorimetric assay is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells in culture to reduce the tetrazolium salts of the yellow MTT and form a blue formazan crystal. Bovine herpesvims type 1 (BHV-1), parainfluenza 3 (PI-3), bovine rota virus (BRV) and bovine adenoviras type 1 (BAV-1) were titrated using the MTT and TCID50 assays into MDBK continuous cell lines in multi-well microplate. A good correlation was found between the results obtained using the standard TCID50 assay and the MTT assay methods for determining the viral titration endpoint

Seroepidemiology and Comparative Diagnosis of Bovine Herpesvirus Type-1 (BHV-1) in Bulls using Enzyme-Linked Immunosorbent Assay (ELISA), Polymerase Chain Reaction (PCR) and Virus Isolation (VI) I

Araştırmada, Konya ve çevresinden Konya Özel Konet mezbahasına kesim amacı ile getirilen, 100 adet sağlıldı gö rünümlü, yerli ırk ve 1 yaşındaki boğalardan kan, sperma ve nasal swap ?mekleri alındı. Sperma ve nasal swap ?mekleri Fotal Dana Bobrek (FDB) ve Madin Darby Bovine Kidney (MDBK) devamlı hücre kültürlerine inokule edildi. Inkubasyon s?resi sonunda 26 adet sperma ve 2 adet nasal swap omeğinde sitopatik etki (CPE) gözlendi. Izole edilen viruslanın identifikasyonu amacıyla Enzyme Linked Immunosorbent Assay (ELISA/Ag) kullanıldı ve sperma örneklerinden 15 adedi BHV-1 olarak identifiye edilirken, nasal swap ömeklerinin hiçbirisi BHV-1 olarak identifiye edilemedi. Ayrıca sperma ve nasal swap örneklerinde BHV-1'In tespiti amacıyla ELISA ve Polymerase Chain Reaction (PCR) metodundan yararlanıldı. Direkt ELISA le 1 adet sperma ve 1 adet nasal swap ömeği pozitif bulundu. PCR ile ise 23 adet sperma omeği pozitif olarak belirlenirken, nasal swap ömeklerinin hiç birinde pozitif sonuç tespit edilemedi. BHV-1'e karşı oluşan antikorların tespit edilmesi amacıyla ELISA/Ab ve serum mikronötralizasyon (serum mNT) testleri kullanıldı. BHV-1'e karşı oluşan antikortann lespiti amacıyla uygulanan EUSA/Ab ile 23 adet kan serumu pozitif olarak belirlenirken, serum mNT testi ile 14 adet kan serumu pozitif bulundu ve antikor pozitif bu ómeklerin SN50 değerleri ise 1:1.78-1:22.4 arasında tespit edildi.In this study, blood, semen and nasal swap samples were taken from 100 clinically healty, indigenous and one year old bulls brought to Konya Private Konet abbatoir from Konya and its environment with the purpose of slaughter. Semen and nasal swap samples were inoculated into Foetal Bovine Kidney (FBK) and Madin Darby Bovine Kidney (MDBK) cell cultures. At the end of incubation, cytopatogenic effect (CPE) was determined in 26 semen and 2 nasal swap samples. Enzyme Linked Immunosorbent Assay (ELISA/Ag) was used for identification of viruses isolated. Two isolates from nasal swap could not be identified, while 15 out of 26 isolates from semen samples was identified as BHV-1 by ELISA. Following ELISAVAg and Polymerase Chain Reaction (PCR) method was applied for detection of BHV-1 in semen and nasal swap samples. One semen sample and 1 nasal swap sample were found as positive by ELISA/Ag. No positive result could be detected in any of nasal swap samples while 23 semen samples were detected as positive by PCR. Besides, Enzyme linked immunosorbent assay (ELISA) and serum microneutralization test (serum mNT) were used for detection of antibodies aganist to BHV-1. Twenty-three blood sera samples were detected positive by ELISA, while 14 blood sera samples were detected positive by serum mNT. SN50 values of the positive sera samples were detected between 1:1.78 1:22.4

Detection of BoHV-1 - Comparative Evaluation of Real Time PCR, Real Time LAMP and Subtyping:

Background: Bovine Herpesvirus 1 (BoHV-1) is an important bovine pathogen found throughout the world. It causes a subclinical, mild, or severe disease. It has different types of clinical manifestations in respiratory, genital, ocular, and encephalomyelitis forms. The aim of this study was to perform comparative diagnosis of BoHV-1 by Real Time PCR and Real Time LAMP and molecular characterization of BoHV-1.  Materials, Methods & Results: In this study, BoHV-1 was investigated in nasal swab samples from cattle with clinical signs of respiratory problems using Real-Time PCR (Polymerase Chain Reaction) methods and the Real-Time Loop-Mediated Isothermal Amplification (LAMP) method, which is a cheap, fast, and reliable technique. BoHV-1 positive samples in both tests were cultured in the Madin-Darby Bovine Kidney (MDBK) cell culture. Conventional PCR was performed for the molecular characterization of the isolated samples. Eight (5.33%) nasal swab samples used in the study were found to be positive by the Real-Time PCR test, while 13 (8.66%) samples were found to be positive by the LAMP test. The results of the McNemar test showed that the difference between Real-Time PCR and Real-Time LAMP tests was not significant (P ˃ 0.05). BoHV-1 was isolated from 3 of 8 samples cultured in cell culture. In the sequence analysis of these three samples after PCR, molecular characterization of the isolates was performed, and they were found to belong to the BoHV-1.1 subtype. These findings indicated that the LAMP method can be used in the rapid diagnosis of BoHV-1 infection, and the BoHV-1.1 subtype should be considered in the fight against infection. Discussion: Many studies have shown that PCR tests are more sensitive than virus isolation (VI). Similarly, in this study, only 3 of the 8 samples found to be positive by Real-Time PCR could be isolated in cell culture. The results of both tests differed in that PCR can detect any viral RNA (infectious whole virus, unpackaged viral genome, even genome strand or subgenomic fragments) whereas VI detects properly packaged and live virus particles. Although no statistical difference was recorded between Real-Time PCR and Real-Time LAMP in this study, a higher rate of positive samples was detected with LAMP, which matched the findings of other reports. These results showed that LAMP is a more resistant to PCR inhibitors and more sensitive test and due to the use of 6 different primers. BoHV-1 continues to cause significant economic losses in the cattle industry around the world. Therefore, a practical, fast, and cost-effective diagnostic tool needs to be used to identify the infection in the affected areas and control it at the earliest. In this context, the LAMP test is a promising method at the field level for the diagnosis and control of BoHV-1 infection and other diseases, as mentioned in other studies. Phylogenetic analysis was performed on 3 swab samples isolated in cell culture, after they were tested by conventional PCR with primers specific to the BoHV-1 gC region, in parallel with other reports. The isolate sequence was aligned using MUSCLE, with the sequences of other isolates obtained by a comparative analysis and multiple sequence alignment was performed in the AliView software. This analysis included the nucleotide sequences of 54 different isolates. All ambiguous locations were removed for each sequence. There were 312 nucleotides in the final dataset. Evolutionary analysis was performed using the Tamura 3-parameter model with the Neighbor-Joining algorithm in MEGA 11; in total, 1,000 iterations (bootstrap) were performed. Similar to the findings of other studies conducted in Turkey, it was determined in this study that the more common BoHV-1 strain in circulation was the BoHV-1.1 strain. Keywords: BoHV-1, molecular characterization, Real-Time LAMP, Real-Time PCR