Veterinary Research Forum

Abstract The current finding reports the development of iELISA with recombinant AMA-1 (rAMA-1) to identify an infection of Babesia in naturally infected cattle. The 48 kDa protein-encoding recombinant AMA-1 gene was cloned into the pET-28a (+) expression vector and expressed in E. coli. The resulting congregate protein was refined under native settings. Towards the evaluation of the diagnostic potential of AMA-1 as a sero-diagnostic reagent, a panel of sera samples from Babesia infected cattle; uninfected sera as well as Babesia positive samples with other species including B. bigemina, B. divergens, B. major, B. occultans were utilized. Additionally, the efficacy of rAMA-1-based serological assays was compared with commercially available kits using 200 samples taken from cattle suspected of babesiosis. The results demonstrated that the iELISA using rAMA-1 exhibited a diagnostic sensitivity of 88.89%, when compared to commercially available ELISA kit as the reference test. The specificity of this assay was 76.66%. These findings suggest that the iELISA employing rAMA-1 can be utilized on large-scale epidemiological surveys and clinical detection of Babesia infection in cattle.

Abstract Rotavirus is one of the major causes of diarrhea in different animal species and has a bad economic impact due to the losses in neonates and productivity. To investigate the occurrence of this infection in bovine calves, three localities in Khartoum State, Sudan, were selected. A total of 200 fecal samples were collected from diarrheic calves; 100 from Khartoum and 50 from each of Khartoum Bahari and Omdurman provinces. Collected samples were screened for a group A rotavirus antigen using enzyme-linked immunosorbent assay (ELISA). Positive results were seen in 40.00% of samples; the highest prevalence of 42.00% was found in samples from Khartoum province. Five ELISA-positive samples were examined under electron microscope, and characteristic wheel-like appearance of rotavirus was visualized. Polyacrylamide gel electrophoresis was also applied on 15 of the positive samples; eight samples showed different polyacrylamide gel electrophoretic group A rotavirus long profile with different patterns. The results showed that the occurrence of rotavirus infection in cattle in Khartoum State is increasing.

Abstract The Q fever is a zoonotic bacterial infection caused by an obligate intra-cellular bacterium, Coxiella burnetii. Members of the Canidae family (Mammalia), including dogs and foxes, are potential reservoirs of C. burnetii, which has a wide host range from mammals and birds to arthropods (primarily ticks). Infected dogs can transmit the disease to other animals and humans. This study aimed to investigate the presence of C. burnetii in dogs and ticks collected from infested dogs in the Kars, Ardahan, and Iğdir provinces of Türkiye by serological and molecular methods. Three hundred canine serum samples were analyzed for phase I and phase II C. burnetii antibodies using indirect enzyme-linked immunosorbent assay. Whole blood samples (n = 300) from the dogs sampled for sera and 184 ticks randomly collected from these dogs were also analyzed for C. burnetii with touch-down polymerase chain reaction. The ticks were classified according to the taxonomic characteristics. In result, 107 tick DNA samples collected from individual females and pooled males were evaluated. The C. burnetii was detected in 3.73% (of the tick samples. However, C. burnetii was not detected in any of the canine blood samples by polymerase chain reaction. Out of the 300 dogs, 18.33% presented antibodies against C. burnetii in their blood serum. When assessed for location, C. burnetii seropositivity was found to be significantly high especially in the Northeastern Anatolia region (18.33%). Study data highlighted the zoonotic risk of ticks, demonstrating that ticks on dogs can carry C. burnetii.

Abstract Cell-surface proteins of Clostridium chauvoei were purified using a simple method. Bacterial cultures were centrifuged and agitated vigorously in phosphate buffered saline with or without further glycine treatment and ammonium sulfate precipitation. Rabbits were immunized subcutaneously with a blackleg disease vaccine twice with a two-week interval. Immunized sera were collected one week after the second injection. Enzyme-linked immunosorbent assay (ELISA) was performed using the proteins purified by the second method as the coating antigen. Bradford assay results showed a higher protein concentration in the second than the first method. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis showed multiple bands for the cell-surface proteins of C. chauvoei in the first method and a sharp band equivalent to flagellin protein in the second method. The ELISA results indicated that the purified proteins were capable of detecting antibodies against Blackleg disease vaccine. The purified protein would be an alternative antigen for indirect ELISA in order to monitor the immune response in vaccinated farm animals.

Abstract Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, an oncogenic deltaretrovirus that has emerged as a potential zoonotic infection. The BLV naturally infects cattle and causes economic losses through a slow persistent infection with various clinical symtoms following preleukosis. The main objective of this study was to determine the seroprevalence of BLV antibodies in cattle and buffaloes in the border provinces of the Eastern Anatolia region, Türkiye, using the agar gel immunodiffusion (AGID) assay and  enzyme-linked immunosorbent assay (ELISA). For this purpose, a total of 1,033 serum samples were collected from 982 cattle and 51 buffaloes from the provinces of Ağrı (n = 178), Iğdır (n = 252), Kars (n = 317), Van (n = 221), and Hakkari (n = 65) during 2021 - 2022. In AGID and ELISA tests, seropositivity for BLV-specific antibodies was not detected in cattle and buffaloes from the mentioned provinces. This study revealed that BLV was not circulating in cattle and buffaloes in the easternmost border provinces of Türkiye during the sampling period and contributed to determine the status of BLV in the mentioned region. Due to the presence of virus in other regions of Türkiye and neighboring countries, Iran and Iraq, it is recommended to control animal movements, continue efforts to combat the transmission of the virus, and maintain control measures.

Abstract Glanders caused by Burkholderia mallei is one of the most dangerous zoonotic diseases in solipeds. Clinical diagnosis of this disease in its early stages in horses, is difficult. This study investigated serological and molecular identification of B. mallei in East Azerbaijan province. In the third and fourth quarters of 2020, throughout 2021, and in the first and second quarters of 2022, the complement fixation test (CFT) was performed on 350 horses. The malleination was used to confirm the positive CFT cases. Blood samples were taken for culture and for preparing serums to perform the enzyme-linked immunosorbent assay (ELISA). Deep eye discharge, nostril, cutaneous ulcers and lymph fluid swabs were cultured, and polymerase chain reaction (PCR) was carried out. Eleven horses were CFT-positive. Based on the malleination on the 11 horses, six were affected by glanders, five were not affected (false positive), and one horse was CFT-negative despite exhibiting clinical signs. It was positive by malleination, ELISA and PCR. A total number of seven positive cases of glanders were diagnosed. The B. mallei could not be isolated, but the Burkholderia cepacia complex was isolated in one case. Except for three cases, the results of the CFT, mallein and ELISA tests were consistent. The amount of confidence interval was 95.00%. It is suggested that ELISA could be used as a complement to CFT in screening and, if positive results are observed in one of the tests, the entire herd must be examined more accurately using the mallein and western blot confirmatory tests.

Abstract A total number of 62 clinically healthy dairy animals of three farms located in Kafr El Sheikh governorate, Egypt, were subjected to field screening surveys of subclinical mastitis (SCM) using California mastitis test (CMT). The obtained results revealed that 38.80% of quarter milk samples were positive to CMT. The most frequently major causative agents isolated from the positive CMT samples were Escherichia coli, Staphylococcus aureus, and environmental streptococcus spp. Acute-phase proteins (APPs), as immunological biomarkers for SCM, including milk serum amyloid A (mSAA) and haptoglobin (Hp) were measured using ELISA. A significant positive correlation was found between the severity of the mammary infection of cow's quarter milk samples represented in somatic cell count (SCC) and each of APPs and pH values. The correlation coefficient (R) between SCC and mSAA, Hp and pH were 0.54, 0.38 and 0.73, respectively. On the other hand, there was a significant negative correlation between casein percentage in milk of SCM cases, and each of APPs, pH and the presence of bacterial pathogens in the milk samples. The obtained results threw light on the inter-relationship between SCC, mSAA, pH value and casein percentage in milk of cows and buffalo suffered from SCM. The percentage of casein in milk is considered a significant accurate tool for diagnosis of SCM and this finding offers the farmers a cheap and fast selection for diagnosis of such disease. These results presented a specific structured view on the efficacy of different diagnostic tools of SCM in dairy herds.

Abstract The aim of the present study was to determine seroprevalence of Toxoplasma gondii infectionin stray cats and correlation with oocyst shedding and IFN-γ concentration. From April to August 2016, one hundred fifty-nine stray cats were captured from various localities in Mashhad area. The blood and fecal samples were collected from each cat. The serum samples were examined to detect antibodies against T. gondii infection by ELISA assay and the fecal samples were microscopically examined for T. gondii oocyst detection. The concentration changes of IFN-γ in serum samples of seropositive and seronegative cats were measured using ELISA kit. The results showed that59.12% (94/159) of cats had antibodies against T. gondii infection. The seroprevalence of T. gondii infection in the adult cats above three years olds was higher than other groups. Regarding gender, month and region factors, the difference of seroprevalence of T. gondii infection was not significant. In this study, the Toxoplasma/Hammondia like oocyst (THLO) were detected in 2.56% (4/156) in fecal samples of one seropositive and three seronegative cats. Results also showed that the mean value for IFN-γ concentration in the seropositive cats was significantly higher than that of the seronegative cats. Based on the results, the high percentages of stray cats were infected with T. gondii in this area. The IFN-γ concentration of seropositive cats was higher than that of the seronegative cats.

Abstract Neospora caninum is an obligate intracellular parasite causing abortion and reproductive failure in ruminants. Here, the seroprevalence of Neospora DNA and anti-Neospora antibodies and the correlation between the DNA and the antibody using polymerase chain reaction (PCR) and a new developed whole cell-based enzyme-linked immunosorbent assay (ELISA) in water buffalo (Bubalus bubalis) were investigated. To determine the level of anti-Neospora antibody, 83 serum samples were collected from buffaloes in the northwest of Iran. Plates were coated with 2 × 10⁶ whole Neospora tachyzoites and the anti-Neospora antibody level was determined by calculating the ratio of sample/positive control (S/P) optical densities (ODs) in the ELISA. All samples with the ration of 0.50 or above were accounted as positive. To confirm the presence of Neospora DNA, the serum samples were directly subjected to PCR and nested PCR for detection of Neospora NC5 gene without the DNA isolation process. A total number of 83 buffalo serum samples were examined for the presence of anti-N. caninum immunoglobulin G and Neospora DNA. All samples with the S/P ratio of 0.50 or above (16 samples, 19.27%) were also positive for Neospora DNA. All samples with OD less than 0.50 (34 samples, 40.96%) were negative for Neospora DNA. However, 33 samples with the S/P ratio of bellow 0.50 (39.75%) showed a significant level of antibody. A 100% correlation was observed between high levels of the anti-Neospora antibody and Neospora DNA in the serum of water buffalo, and the whole N. caninum tachyzoites have the potency to be used as antigens for detection of the parasite in ELISA.

Abstract Monoclonal antibodies (MAbs) are invaluable molecules which have several advantages over polyclonal immunoglobulins (Igs) including consistency and higher specificity and hence can be used in biological researches, diagnosis and treatment of diseases. The present study was conducted to produce monoclonal antibody against chicken IgG.TheIgG molecules were purified from chicken serum and used as antigens to immunize several mice. Thereafter, a well-immunized mouse was chosen and used for fusion process. After production of hybridoma cells, several rounds of cloning were carried out and produced MAbs were examined by various immunological assays including enzyme-linked immunosorbent assay (ELISA) and western and dot blotting. Assessment of grown hybridomas indicated that only one clone (5B8) has produced desired MAb against chicken IgG. Meanwhile, using an indirect ELISA, it was shown that this MAb successfully recognizes chicken IgG molecules attached to influenza virus nucleoprotein. Evaluation of cross reactivity of MAb 5B8 with several avian serum samples revealed that this molecule specifically identifies chicken antibody molecules. However, it also recognized turkey antibodies with less affinity. In addition to research applications like isolation and purification of chicken and turkey IgG molecules, such a MAb can be applied to design and development of various immunoassays (e.g. ELISA) in these avian species.

Abstract Bovine ephemeral fever is an acute and arthropod-borne viral disease of cattle and water buffalo which occurs seasonally in most of the world tropical and subtropical regions. The epizootic feature of the disease has been reported in Iran with serious economic consequences. The surface glycoprotein G of bovine ephemeral fever virus (BEFV) is composed of 4 antigenic sites (G1-G4) and plays the main role for eliciting neutralizing antibodies and protective immunity. The G1 – epitope is a linear antigenic site and conserved among BEFV strains. In order to develop an ELISA test based on G1-epitope as coating antigen, this study was carried out to express the recombinant G1-epitope of BEFV in prokaryotic system. Using PCR and specific primers, a length of 88 amino acid of the G glycoprotein of BEFV including G1- epitope was amplified and cloned into the expression vector pGEX-4T-1, with the GST moiety. The recombinant plasmid (pGEX-4T-1-G1) was then transformed into Escherichia coli BL₂₁ and expression of fusion protein was induced by 0.10 mM IPTG. The maximum expression of the fusion protein was obtained at 16 hr post induction as verified by SDS-PAGE electrophoresis, and it was also confirmed that this protein bearing G1- epitope is sufficiently biologically active to bind to anti-BEFV serum in western blot experiment.

Abstract Maedi-Visna (MV) virus and caprine arthritis encephalitis (CAE) virus known as small ruminant lentiviruses (SRLVs) cause chronic diseases in susceptible animals. The main reservoirs of these viral agents are sheep and goat. In sheep, MV virus causes a disease as the same name of the virus. This is the first seroprevalence survey of SRLVs in sheep population of Khorasan-e-Razavi province in Iran. Two hundred and twenty sheep from 30 flocks in 12 regions of the province were selected by random cluster sampling method. Serum samples were analyzed for the presence of antibodies against MV/CAE viruses. The seroprevalence in sheep was 34.5% (95.0% CI: 28.3 to 40.7%). Totally, the seroprevalence was in the range of 6.7 to 72.2 %. In 26 flocks of sheep (89.6%; 95.0%CI: 74.4 to 98.8%), at least one seropositive case was detected. The relationship between seropositivity and age, sex, flock size and breeds of sheep were statistically analyzed. In logistic regression model, only age was correlated with SRLV seroprevalence (p < 0.05). This study showed relatively high seroprevalence against SRLVs in sheep population in this area of the country. Due to difficulty in clinical diagnosis, chronic course of the disease, the absence of effective vaccine and treatment and huge economic loss, more epidemiological studies with regards to prevention and control of the disease are necessary.

Abstract Bluetongue (BT) is a viral disease of ruminants transmitted by Culicoides biting midges and has the ability to spread rapidly over large distances. The disease occurs almost worldwide between latitudes approximately 35˚ S and 50˚ N. Among the numerous diseases of ruminants, BT has gained considerable importance in recent years as one of the best examples of the effects of climate change on disease spread. Sheep are major livestock species in Iran, but studies of BT have not gained the priority compared to other diseases. Thus, the objective of this study was to describe the distribution and seroprevalence of bluetongue virus (BTV) infections in sheep in Kohgiluyeh and Boyer-Ahmad province of Iran, and to identify factors associated with the exposure of these sheep to BTV infection. Sera from 262 apparently healthy sheep were collected during the year 2011. The collected sera of the animals were screened with competitive enzyme like immunosorbent assay (c-ELISA). Two hundred and three (77.48%) out of 262 sera tested were positive to BTV antibodies. Statistically significant differences were found in the seroprevalence BT, between sex and age of sheep (p < 0.001). No statistically significant differences were observed in BTV seroprevalence among different seasons, nor among recently aborted and normally delivered.

Abstract A cross-sectional study with a random cluster sampling design was carried out to estimate the seroprevalence of bovine herpesvirus type 1 (BHV-1) in non-vaccinated dairy herds in Hamedan province, west of Iran. Simple random sampling was used for selection of cattle in each herd. Informative data about each herd and selected animals were recorded by the farm manager in a provided questionnaire. Blood samples were collected from 492 animals in 41 industrial herds. A commercial indirect ELISA test was used to determine the seropositivity against BHV-1. The individual and herd seroprevalence for BHV-1 were 58.74% and 82.93%, respectively. The intra-herd prevalences were ranged from 16.70% to 100%. Geographical characteristics of Hamedan province may explain the high sero-prevalence rates found in this study compared to those of others obtained from different parts of the country. The proportion of seropositive cows were increased with age (p <0.05). Animals from large and moderate sized herds had higher odds of seropositivity than those of small size herds. These findings could be related to the presence of a considerable number of BHV-1 carriers in this region. The high herd and animal prevalence found in the present study suggested necessity of implementing an intensive control program for reducing BHV-1 infection rates.