Establishment and application of PDCoV antigen-specific DAS-ELISA detection method.

BACKGROUND: Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus that affects young pigs, causing vomiting, acute diarrhea, dehydration, and even death. There is growing evidence that PDCoV can undergo cross-species as well as zoonotic transmissions. Due to the frequent outbreaks of this deadly virus, early detection is essential for effective prevention and control. Therefore, developing a more convenient and reliable method for PDCoV detection is the need of the hour. RESULTS: This study utilized a high-affinity monoclonal antibody as the capture antibody and a horseradish peroxidase labeled polyclonal antibody as the detection antibody to develop an enzyme-linked immunosorbent assay (DAS-ELSA) for PDCoV detection.Both antibodies target the PDCoV nucleocapsid (N) protein. The findings of this study revealed that DAS-ELISA was highly specific to PDCoV and did not cross-react with other viruses to cause swine diarrhea. The limit of detection of the virus titer using this method was 103 TCID50/mL of PDCoV particles. The results of a parallel analysis of 239 known pig samples revealed a coincidence rate of 97.07% (κ = 0.922) using DAS-ELISA and reverse transcriptase PCR (RT-PCR). The DAS-ELISA was used to measure the one-step growth curve of PDCoV in LLC-PK cells and the tissue distribution of PDCoV in infected piglets. The study found that the DAS-ELISA was comparable in accuracy to the TCID50 method while measuring the one-step growth curve. Furthermore, the tissue distribution measured by DAS-ELISA was also consistent with the qRT-PCR method. CONCLUSION: The developed DAS-ELISA method can be conveniently used for the early clinical detection of PDCoV infection in pigs, and it may also serve as an alternative method for laboratory testing of PDCoV.

Evaluation of chimeric recombinant antigens for the serodiagnosis of Trypanosoma cruzi in dogs: a promising tool for Chagas disease surveillance.

BACKGROUND: Chagas disease (CD), a neglected parasitic disease caused by Trypanosoma cruzi, poses a significant health threat in Latin America and has emerged globally because of human migration. Trypanosoma cruzi infects humans and over 100 other mammalian species, including dogs, which are important sentinels for assessing the risk of human infection. Nonetheless, the serodiagnosis of T. cruzi in dogs is still impaired by the absence of commercial tests. In this study, we investigated the diagnostic accuracy of four chimeric recombinant T. cruzi IBMP antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) for detecting anti-T. cruzi antibodies in dogs, using latent class analysis (LCA). METHODS: We examined 663 canine serum samples, employing indirect ELISA with the chimeric antigens. LCA was utilized to establish a latent variable as a gold standard for T. cruzi infection, revealing distinct response patterns for each antigen. RESULTS: The IBMP (Portuguese acronym for the Molecular Biology Institute of Paraná) antigens achieved area under the ROC curve (AUC) values ranging from 90.9% to 97.3%. The highest sensitivity was attributed to IBMP-8.2 (89.8%), while IBMP-8.1, IBMP-8.3, and IBMP-8.4 achieved 73.5%, 79.6%, and 85.7%, respectively. The highest specificity was observed for IBMP-8.4 (98.6%), followed by IBMP-8.2, IBMP-8.3, and IBMP-8.1 with specificities of 98.3%, 94.4%, and 92.7%, respectively. Predictive values varied according to prevalence, indicating higher effectiveness in endemic settings. CONCLUSIONS: Our findings underscore the remarkable diagnostic performance of IBMP-8.2 and IBMP-8.4 for the serodiagnosis of Trypanosoma cruzi in dogs, representing a promising tool for the diagnosis of CD in dogs. These chimeric recombinant antigens may not only enhance CD surveillance strategies but also hold broader implications for public health, contributing to the global fight against this neglected tropical disease.

Prevalence and risk factors of Toxoplasma gondii infection in dairy cattle from the Western Region of Thailand.

In total, 901 dairy cow sera and data were collected from 51 farms in Nakhon Pathom, Ratchaburi and Kanchanaburi provinces (Western Region of Thailand). Serum samples were processed via the multispecies ELISA method to detect IgG antibodies against Toxoplasma gondii infection. The results demonstrated that the calculated true prevalence was 1.48% (95% CI, 0.64-2.75%) for the individual-level and 29.41% (95% CI, 18.71-43%) for the farm-level. The univariate risk factor analysis showed that the number of total owned cats, the presence of stray cats, and the frequency of cleaning per day were significant factors (p < 0.2). These three factors were subjected to logistic regression analysis, and the results revealed that the frequency of cleaning farms per day was a potential risk factor for T. gondii-seropositive farms (OR = 2.745, 95% CI, 1.15-8.69, p = 0.02). The frequency of cleaning might increase the T. gondii oocyst distribution within the barn area, thus increasing the possibility of infection. Our findings show that T. gondii continues to circulate in the dairy cow population in the western part of Thailand. The presence of cats on farms was not found to be associated with T. gondii infection, but the high frequency of cleaning the floor was, and contributed to the potential risk of infection.

Title: Prévalence et facteurs de risque de l'infection à Toxoplasma gondii chez les bovins laitiers de la région occidentale de la Thaïlande. Abstract: Au total, 901 sérums de vaches laitières et des données ont été collectés dans 51 fermes des provinces de Nakhon Pathom, Ratchaburi et Kanchanaburi (région occidentale de la Thaïlande). Les échantillons de sérum ont été traités via la méthode ELISA multi-espèces pour détecter les anticorps IgG contre l'infection à Toxoplasma gondii. Les résultats ont démontré que la prévalence réelle calculée était de 1,48 % (IC à 95 %, 0,64­2,75 %) au niveau individuel et de 29,41 % (IC à 95 %, 18,71­43 %) au niveau des exploitations. L'analyse factorielle a montré que le nombre total de chats possédés, la présence de chats errants et la fréquence quotidienne de nettoyage étaient des facteurs significatifs (p < 0,2). Ces trois facteurs ont été soumis à une analyse de régression logistique et les résultats ont révélé que la fréquence quotidienne de nettoyage des exploitations était un facteur de risque potentiel pour les exploitations séropositives à T. gondii (OR = 2,745, IC à 95 % = 1,15­8,69, p = 0,02). La fréquence du nettoyage pourrait favoriser la répartition des oocystes de T. gondii dans les étables, augmentant ainsi le risque d'infection. Nos résultats indiquent que T. gondii continue de circuler dans la population de vaches laitières de l'ouest de la Thaïlande. La présence de chats dans les fermes n'a pas été associée à l'infection à T. gondii, mais la fréquence élevée du nettoyage du sol l'était et contribuait au risque potentiel d'infection.

Seroprevalence and associated risk factors of contagious caprine pleuropneumonia in selected districts of South Wollo Zone Northeast Ethiopia.

Contagious caprine pleuropneumonia (CCPP) is a severe and devastating respiratory disease of goats, which is characterized by severe serofibrinous pleuropneumonia accompanied by high morbidity and mortality. A cross-sectional study was conducted from July 2022 to January 2023 to determine the seroprevalence of CCPP and identify risk factors associated with the occurrence of CCPP in goats in five selected districts of the South Wollo Zone of the Eastern Amhara region. A total of 384 sera samples were collected from goats and examined for antibodies specific to Mycoplasma capricolum subspecies capripneumoniae (Mccp) using Competitive Enzyme-Linked ImmunoSorbent Assay (cELISA) test. Out of the total examined sera, 26 samples were positive for CCPP, giving an overall seroprevalence of 6.7% (95% CI = 6.64-9.77). A seroprevalence of 5.05%, 4.65%, 2.78%, 12.90%, and 10.77% were recorded in Ambasel, Tehuledere, Kalu, Dessie Zuria and Kutaber districts, respectively. However, there was no statistically significant difference among these five districts (p > 0.05). The seroprevalence of CCPP varies significantly between age groups and agroecology (p < 0.05). However, the seroprevalence did not vary with sex, body condition score (BCS), and flock size (p > 0.05). Old-aged goats (OR = 4.10) and goats found in the lowlands (OR = 5.09) were at higher risk of infection with CCPP than young-aged goats and goats found in the highlands, respectively. In conclusion, the present seroprevalence investigation indicated the occurrence of CCPP in those selected study districts of the South Wollo Zone. Therefore, appropriate control measures, including avoiding the mixing of flocks and vaccination should be designed and implemented especially in the lowland areas and older goats to reduce the further spread and magnitude of the disease.

First report on seroprevalence of Toxoplasma gondii infection in zoo animals in Algeria.

Toxoplasma gondii is one of the world's most widespread polyxenic protozoan parasites that affect all warm-blooded animals, including humans. This survey aims to study, for the first time in Algeria, the seroprevalence of Toxoplasma infection in zoo animals. The study included eight animal species of which 54 serum samples were collected from 30 Australian goats (Capra hircus), four bulls (Bos taurus), one dromedary (Camelus dromedarius), three cuffed sheep (Ammotragus lervia), seven donkeys (Equus asinus), one pony (Equus ferus), four bearded horses (Equus ferus caballus) and four rabbits (Oryctolagus cuniculus). The presence of antibodies to T. gondii was determined using the ID Screen® Toxoplasmosis Indirect Multispecies ELISA kit (IDVet, Grabels, France). A total of 8/54 (14.8%) samples were seropositive, including 5/28 (17.9%) males and 3/26 (11.5%) females. The seroprevalence was 6.7%, 50%, 25% and 75% in Capra hircus, Bos Taurus, Equus ferus caballus, and Oryctolagus cuniculus, respectively. No cases were observed in Camelus dromedarius, Ammotragus lervia, Equus asinus, and Equus ferus. This study indicates, for the first time in Algeria, the seroprevalence of T. gondii in zoo animals.

Frequency of Besnoitia besnoiti and Neospora caninum antibodies in cattle and small ruminants from greater Cairo and Beni Suef governorates, Egypt.

Livestock industry is a pivotal sector for providing food, work places and monetary support for Egyptian people. Besnoitia besnoiti and Neospora caninum are protozoan parasites that are responsible for significant economic losses in ruminants, particularly in cattle. Besnoitia besnoiti can cause fertility problems and a general loss in productivity, while N. caninum is a major cause of abortion and neonatal abnormalities in infected animals. There is little information on the existence of these protozoa in Egypt, thus we conducted this study to reveal the current situation in cattle (n = 264), sheep (n = 151), and goats (n = 25). Serum samples were collected from governorates of Cairo, Giza, and Beni Suef, representing the most densely populated regions in Egypt. Using commercial ELISAs, an overall estimation among all tested animals (n = 440) revealed 7.7%, 13.2%, and 0.9% as seropositive rates for B. besnoiti, N. caninum, and mixed infection, respectively. Animal species (cattle vs sheep vs goat) and age of cattle (less than vs >1 year old) were analyzed as risk factors for infection. Regarding B. besnoiti, the seroprevalence was significantly higher in cattle than in sheep and goats and in adult cattle than calves. For N. caninum infection, no significant differences were recorded, although the seropositive rates were higher in cattle, and in adult cattle. This study provides the first seroprevalence data for B. besnoiti in all surveyed animals in the regions included, and in sheep and goats from Egypt, and supports the current knowledge for the occurrence of N. caninum in Egypt.

Epidemiological factors conditioning sarcoptic mange spreading in wild boar (Sus scrofa).

Sarcoptic mange is a widely distributed disease, with numerous potential hosts among domestic and wild animals. Nowadays it is considered a neglected re-emergent infection in humans. As a difference with domestic pigs, and even with several clinical cases reported in some European countries, it seems that Eurasian wild boars (Sus scrofa) have a low susceptibility to clinical mange. However, because of a case of confirmed transmission from Spanish ibex (Capra pyrenaica) to wild boar in the province of Tarragona, we planned a large-scale ELISA survey in the neighboring Valencian Community (SE Spain). We compared 419 wild boar sera from different management systems (fenced vs. open game estates), different ages (piglets, juveniles, and adults), with different behaviour (gregarious females of all ages and male piglets vs. solitary juveniles and adult males), from areas with different wild boar densities, different wild ruminant densities and different sarcoptic mange epidemiologic situations. The whole prevalence of antibodies against sarcoptic mange in the tested wild boars was 10.5%. No significant differences were found when comparing fenced and free ranging wild boars, males and females, gregarious vs. solitary individuals or among different ages. However, wild boar density was a relevant factor. In areas with a hunting bag of <1 wild boar/km2, considered as a low density of suids, the seroprevalence was 2.94%, but rose to 11.52% in high density districts, constituting a significant difference (p = 0.037). Low wild boar populations would act as a protective factor (OR 0.233; p = 0.049) against coming into contact with the mite. The wild ruminant densities or their sarcoptic mange status did not show any effect on wild boars seroprevalence against this disease. These results reinforce the suggested host-taxon Sarcoptes scabiei specificity and the independence of host-species foci.

Epidemiology and diagnostic accuracy of Clostridium perfringens toxins in the intestinal contents of camels, sheep, and cattle: a cross-sectional study in Dakahlia governorate, Egypt.

This study aimed to establish an accurate epidemiological surveillance tool for the detection of different C. perfringens types from 76 diseased and 34 healthy animals in Dakhalia Governorate, Egypt. A total of 110 intestinal content samples were randomly collected from camels, sheep, and cattle. C. perfringens was isolated and biochemically identified by the VITEK2 system. Toxinotyping and genotyping of C. perfringens isolates were specified by a multiscreen ELISA and real-time qPCR (rt-qPCR). The occurrence of C. perfringens was highest among camels (20% in healthy and 25% in diseased) and was lowest in cattle (23.1% and 14.7%). The cpa toxin was detected in all isolates by rt-qPCR and in 7 isolates by ELISA, ext toxin was detected in 7 isolates by rt-qPCR and in 6 isolates by ELISA, and cpb toxin was detected in 2 isolates by both rt-qPCR and ELISA. Four types of C. perfringens were identified by rt-qPCR, type A (65.2%), B (4.3%), C (4.3%), and D (26.1%), and three types by ELISA, type D (17.4%), A (8.7%) and C (4.3%). Our study indicated the prevalence of infection in Dakahlia by C. perfringens type A and D, particularly camels, and recommends adopting an appropriate vaccination strategy among the studied animals.

Monoclonal antibodies for equine IL-1ß enable the quantification of mature IL-1ß in horses.

Interleukin-1ß (IL-1ß) is one of the key mediators of inflammation during innate immune responses. Mature bioactive IL-1ß mediates essential host defense mechanisms but also has a mechanistic role in several autoinflammatory and degenerative diseases. In horses, specific and sensitive assays for IL-1ß are crucial for immunological research on inflammatory processes and diseases. In this article, we describe the development of four monoclonal antibodies (mAbs) against equine IL-1ß. The specificity of the new IL-1ß mAbs was confirmed using a panel of equine recombinant cytokines and chemokines. The mAbs were validated for detection of native mature IL-1ß in a fluorescent bead-based assay and for staining of IL-1ß-producing immune cells by flow cytometry. The bead-based assay for equine IL-1ß had a linear quantification range between 60 pg/ml to 960 ng/ml. Horse peripheral blood mononuclear cells (PBMC) secreted IL-1ß after lipopolysaccharide (LPS) stimulation in time and dose dependent manner as quantified by the new equine IL-1ß bead-based assay. A comparison of two commercial equine IL-1ß ELISA kits with the new IL-1ß fluorescent bead-based assay revealed that the bead-based assay improved the quantification of native equine IL-1ß in LPS stimulated PBMC supernatants by detecting it with high intensity and a broad linear quantification range, while both ELISAs resulted in low signals and poor native IL-1ß recognition. Intracellular staining and flow cytometric analysis confirmed that the main cellular source of IL-1ß in equine PBMC after LPS stimulation were CD14+ monocytes. IL-1ß secretion from PBMC was inhibited by a caspase inhibitor but protein translation within the cells was not, supporting the accumulation of pro-IL-1ß within the cells even when proteolytic cleavage for IL-1ß activation is missing. This confirmed the importance of specific mAbs for analyzing the biologically active, mature IL-1ß in horses.

Newly developed peptide-ELISA successfully detected anti-IgG antibodies against Maedi-Visna virus in sheep.

Maedi Visna Virus (MVV) is a retrovirus that can infect sheep. There is still no effective therapy or vaccine against this virus and timely diagnosis is important to combat the complications of the disease. In this study, we aimed to develop an ELISA using peptides derived from gag protein as antigen. For this purpose, B cell epitopes of gag protein were predicted and a docking analysis with the B cell receptor was performed to select peptides to be used in ELISA. After three soluble epitopes with the highest antigenicity were produced as peptides, the immunogenicity of each peptide was determined by ELISA using sheep serum samples categorized as MVV positive (n=24) and negative (n=13). Subsequently, in house ELISA using above mentioned immunogenic peptides as antigen was used to investigate MVV seroprevalence in sheep (n=88). According to the results, among three peptides, two of them strongly reacted with MVV positive serum samples and the mean absorbance values detected among positive and negative serum samples were statistically significant, indicating that these peptides were immunogenic (P=0.016 and P=0.038). The third peptide also reacted with positive serum samples but the mean absorbance value was not statistically significant and this peptide was considered non-immunogenic (P=0.175). The immunogenic two peptides showed the same high sensitivity and specificity values of 91.60 and 92.80 according to the commercial kit. Moreover, MVV seroprevalence detected by peptide-ELISAs using CKQGSKE and CRPQGKAGHKG peptides as antigen was 3.40 % and 4.5 %, respectively. As a result, it was shown that these peptides can be successfully used for serological diagnosis of MVV.

Seroprevalence of Toxoplasma gondii and Neospora spp. in horse population of Tehran, Iran.

Neospora spp. and Toxoplasma gondii are two closely related protozoan parasites that are widely distributed throughout the world. Horses can act as intermediate hosts for both parasites and can acquire disease. Blood samples were taken from 487 clinically healthy horses from 17 different mechanized stables in Tehran, the capital of Iran, during September and November of 2022. IFAT and ELISA were employed to detect antibodies directed against Neospora spp. and T. gondii. The anti-N. caninum antibodies were detected in 52 of the horses (10.67%) based on IFAT and in 86 of the 487 horses (17.65%) based on the ELISA test. Also, antibodies against T. gondii were detected in 41 horses (8.42%) based on IFAT and in 63 of 487 horses (12.94%) based on the ELISA test. Also, in 6 of the horses (1.23%) based on IFAT and in 13 of the 487 horses (2.67%) based on the ELISA test, double positivity suggested co-infection with both parasites. Gender, age groups, and the presence of dogs for neosporosis, and age groups and the presence of cats for toxoplasmosis, could be considered factors having an influence on the seroprevalences (P < 0.05). The results proved the importance of the urgent implementation of stringent regulatory measures to prevent and control the spread of these parasites.

Evaluation of commercial ELISA kits' diagnostic specificity for FAST diseases in wild animals.

Wild animals, sharing pathogens with domestic animals, play a crucial role in the epidemiology of infectious diseases. Sampling from wild animals poses significant challenges, yet it is vital for inclusion in disease surveillance and monitoring programmes. Often, mass surveillance involves serological screenings using enzyme-linked immunosorbent assay (ELISA) tests, typically validated only for domestic animals. This study assessed the diagnostic specificity of commercially available ELISA tests on 342 wild ruminant serum samples and 100 from wild boars. We evaluated three tests for foot-and-mouth disease: two for Peste des petits ruminants, two for Rift Valley fever and one for Capripox virus. Diagnostic specificity was calculated using the formula True Negative/(False Positive + True Negative). Cohen's kappa coefficient measured agreement between tests. Results showed high specificity and agreement across all tests. Specificity for foot-and-mouth disease (FMD) ranged from 93.89% for Prionics to 100% for IDEXX, with IDvet showing 99.6%. The highest agreement was between FMD IDvet and IDEXX at 97.1%. Rift Valley fever (RVF) tests, Ingezim and IDvet, achieved specificities of 100% and 98.83%, respectively. The optimal specificity was attained by retesting single reactors and inactivating the complement.Contribution: Commercially available ELISA kits are specific for foot-and-mouth disease and similar transboundary animal diseases and can be used for highly specific wild animal testing.

PRESENCE OF ANTI-NEOSPORA CANINUM ANTIBODIES IN TEMAZATE DEER (MAZAMA TEMAMA) AND WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) FROM THE CENTRAL REGION OF VERACRUZ, MEXICO.

This study documents the presence of anti-Neospora caninum antibodies and their association with certain risk factors in 2 deer species from the central region of Veracruz State, Mexico. A total of 90 blood samples, 20 from temazate deer (Mazama temama) and 70 from white-tailed deer (Odocoileus virginianus), were taken from 3 farms, and serum samples were subjected to ELISA indirect test to detect N. caninum antibodies; the association between the serological status and the possible risk factors was then estimated. The overall presence of anti-N. caninum antibodies was 57.7% (52/90; 95% CI 46.9-67.9), with positive animals identified on all farms; in white-tailed deer it was 57% and in temazate deer 60%. Prevalence was higher in females than males. Adult animals had a higher prevalence than young ones. The risk analysis identified the age in the adult animal category (odds ratio 5.8) as being associated with the presence of anti-N. caninum antibodies. These results provide evidence of the significant contamination of oocysts in the environment and allow us to estimate the contribution of deer to the sylvatic cycle.

Studies on the association of malondialdehyde as a biomarker for oxidative stress and degree of malignancy in dogs with mammary adenocarcinomas.

BACKGROUND: Mammary adenocarcinomas are one of the most common tumour diseases in bitches. The relationship between oxidative stress and the degree of malignancy of the tumour has not been sufficiently researched in veterinary medicine. OBJECTIVES: The main objective was to investigate the potential role of MDA as a practice-relevant biomarker for the assessment of systemic oxidative stress and to determine whether this parameter can indicate the malignancy grade of a mammary adenocarcinoma. METHODS: In the present pilot study, MDA plasma concentrations were analysed in 55 bitches with (n = 28) and without (n027) malignant adenocarcinomas of the mammary gland using two different measurement methods and the relationship to tumour size was investigated. RESULTS: The mean MDA concentration measured by enzyme-linked immunosorbent assay (ELISA) was 289 ng/mL (range 365-634 ng/mL) in dogs with grade 1 adenocarcinoma (n = 13), 288.5 ng/mL (range 85-752 ng/mL) in dogs with grade 2 adenocarcinoma (n = 10), 332 ng/mL (range 239-947 ng/mL) in dogs with grade 3 (n = 5) adenocarcinoma and 293 ng/mL (range 175-549 ng/mL) in dogs without a mammary tumour (n = 27). When MDA was measured by HPLC, the average MDA concentration in the study group (n = 11) was 0.24 µmol/L (range 0.16-0.37) and that of the control group (n = 15) was 0.27 µmol/L (range 0.16-1.62). Thus, there were no significant differences between the study group with malignant adenocarcinomas and the control group in both examination methods (p > 0.05). Furthermore, there was no correlation between the MDA concentrations and the approximate volume of the mammary tumour. CONCLUSION: The results highlight the challenges of providing a prognosis for the malignancy of a mammary adenocarcinoma based on MDA concentrations in plasma using ELISA or HPLC. As a result, histopathological examination remains the gold standard for diagnosing and differentiating adenocarcinomas of the mammary gland.

Development and validation of an ELISA to measure regenerating island-derived protein 3E in canine blood.

BACKGROUND: Regenerating island-derived proteins (REG) are upregulated in people with sepsis, pancreatitis, and gastrointestinal diseases. One member of the REG family, namely REG3E, was recently identified in pancreatic tissue and plasma of dogs, with high expression in pancreatitis and sepsis. OBJECTIVES: We aimed to develop and validate an ELISA to measure REG3E concentrations in canine blood. METHODS: An indirect sandwich ELISA was developed using recombinant canine REG3E protein and polyclonal anti-canine REG3E antibodies raised in guinea pigs and rabbits. Antibody specificity was assessed using western blot and mass spectrometric analysis of protein purified from canine plasma. Assay validation included evaluation of dilutional linearity, parallelism, spiking recovery, repeatability and reproducibility, stability, interferences, and comparison of serum and heparinized plasma. RESULTS: Antibodies bound specifically to REG3E with no evidence of cross-reactivity with other proteins. The limit of detection of the ELISA was 15 ng/mL, and the lower limit of quantification was 30 ng/mL. The assay demonstrated good to excellent linearity, dilutional and mixing parallelism, and recovery, with mean observed-to-expected ratios of 104%, 107%, 102%, and 92%, respectively, and no evidence of a hook effect. Coefficients of variation were ≤8.5% for repeatability and ≤14.3% for reproducibility at three different levels. Measurements of REG3E in plasma were not significantly influenced by different storage conditions, freeze-thawing cycles, hemolysis, lipemia, or icterus. There was no significant difference between REG3E concentrations in heparinized plasma and serum samples. CONCLUSIONS: The canine REG3E ELISA has acceptable precision, accuracy, linearity, and reproducibility for the measurement of REG3E in canine plasma and serum.

Canine visceral leishmaniasis: Seroprevalence and georeferencing in the state of Santa Catarina, Brazil.

Canine visceral leishmaniasis (CVL) has long been considered an endemic disease in the northern and northeastern regions of Brazil, while the southern region remains non-endemic. However, in recent years, several cases of CVL have been reported in southern states. The objective of this work was to determine the seroprevalence of CVL in dogs in the state of Santa Catarina, Brazil, through immunochromatographic tests (DPP®) and ELISA (Enzyme-Linked Immunosorbent Assay) and its correlation with environmental characteristics through georeferencing. Blood samples from dogs (n = 1227) were collected in six mesoregions of the state and evaluated by the rapid test (DPP®). Positive samples were sent to Lacen (Central Public Health Laboratory) in Santa Catarina to be tested using ELISA. Information obtained from the epidemiological questionnaire was subjected to statistical analysis (Chi-square and Student's t-test; P < 0.05) to verify the correlation between serology and the analyzed variables. The locations (GPS) of the samples were used for georeferencing and creating heatmaps (Kernel Method). Four animals that died from CVL were necropsied and organ samples were collected for molecular analysis (PCR), immunohistochemistry, and histopathology (HE). Of the 1227 dogs analyzed, 22 (1.8%) were reactive in the DPP® and of these, 7 (0.6%) were also positive in the ELISA. A correlation (P < 0.01) was observed between positive serology and region, environment, access to the street, and clinical signs. The positive cases were concentrated in the eastern region of the state, in low-altitude areas with average rainfall and higher average temperatures, and in more populated areas close to forest fragments. PCR, HE, and immunohistochemistry, along with serology, have proven to be efficient for characterizing positive cases.

High-affinity monoclonal antibodies against the porcine epidemic diarrhea virus S1 protein.

The porcine epidemic diarrhea virus (PEDV) infection inflicted substantial economic losses upon the global pig-breeding industry. This pathogen can infect all pigs and poses a particularly high fatality risk for suckling piglets. The S1 subunit of spike protein is a crucial target protein for inducing the particularly neutralizing antibodies that can intercept the virus-host interaction and neutralize virus infectivity. In the present study, the HEK293F eukaryotic expression system was successfully utilized to express and produce recombinant S1 protein. Through quantitative analysis, five monoclonal antibodies (mAbs) specifically targeting the recombinant S1 protein of PEDV were developed and subsequently evaluated using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and flow cytometry assay (FCA). The results indicate that all five mAbs belong to the IgG1 isotype, and their half-maximal effective concentration (EC50) values measured at 84.77, 7.42, 0.89, 14.64, and 7.86 pM. All these five mAbs can be utilized in ELISA, FCA, and IFA for the detection of PEDV infection. MAb 5-F9 exhibits the highest sensitivity to detect as low as 0.3125 ng/mL of recombinant PEDV-S1 protein in ELISA, while only 0.096 ng/mL of mAb 5-F9 is required to detect PEDV in FCA. The results from antigen epitope analysis indicated that mAb 8-G2 is the sole antibody capable of recognizing linear epitopes. In conclusion, this study has yielded a highly immunogenic S1 protein and five high-affinity mAbs specifically targeting the S1 protein. These findings have significant implications for early detection of PEDV infection and provide a solid foundation for further investigation into studying virus-host interactions.

Improved serodiagnosis of Trypanosoma vivax infections in cattle reveals high infection rates in the livestock regions of Argentina.

Bovine trypanosomosis, caused by Trypanosoma vivax, currently affects cattle and has a significant economic impact in sub-Saharan Africa and South America. The development of new diagnostic antigens is essential to improve and refine existing methods. Our study evaluated the efficacy of two recombinant antigens in detecting specific antibodies in cattle. These antigens are derivatives of an invariant surface glycoprotein (ISG) from T. vivax. A fraction of a previously described antigen (TvY486_0045500), designated TvISGAf, from an African strain was evaluated, and a new ISG antigen from an American isolate, TvISGAm, was identified. The two antigens were expressed as fusion proteins in Escherichia coli: TvISGAf was fused to the MBP-His-tag, and TvISGAm was obtained as a His-tag fused protein. An ELISA evaluation was conducted using these antigens on 149 positive and 63 negative bovine samples. The diagnostic performance was enhanced by the use of a combination of both antigens (referred to as TvISG-based ELISA), achieving a sensitivity of 89.6% and specificity of 93.8%. Following the validation of the TvISG-based ELISA, the seroprevalence of T. vivax infection in 892 field samples from cattle in the central region of Argentina was determined. The mean seroprevalence of T. vivax was 53%, with variation ranging from 21% to 69% among the six departments studied. These results support the use of the TvISG ELISA as a valuable serological tool for the detection and monitoring of T. vivax infection in cattle. Furthermore, we report for the first time the seroprevalence of T. vivax in Argentina, which highlights the widespread endemic nature of the disease in the region. In order to effectively manage the increasing spread of T. vivax in the vast livestock production areas of South America, it is essential to implement consistent surveillance programs and to adopt preventive strategies.

Seroprevalence of brucellosis and associated risk factors in camels and its herders in selected districts of Somali Pastoral Region, Eastern Ethiopia.

Brucellosis poses a major public and animal health problem in many parts of the world, particularly in pastoral settings, however, seroepidemological studies are scarce. A cross-sectional study was conducted from December 2021 to April 2022 to estimate the prevalence of brucellosis and to identify the associated risk factors for camels and occupational individuals from three purposively selected districts of the Somali pastoral region in Eastern Ethiopia. Serum samples were serially diluted using the Rose Bengal Plate Test (RBPT) as a screening test and a competitive Enzyme-Linked Immunosorbent Assay (cELISA) test as a confirmatory test. From a total of 450 camels and 250 human serum samples tested, the overall seroprevalence was confirmed to be 2.9 % (95 % CI, 1.5-4.9) in camels and 2.0 % (95 % CI, 0.2-3.7) in humans. In camels, abortion and retained fetal membrane (RFM) were significant risk factors for Brucella seropositivity (p<0.05). However, in humans, RFM disposal differed significantly (p<0.05). The fact that brucellosis is found in both camels and humans highlights the importance of implementing a coordinated One Health approach to control and eliminate the disease. This would ensure improved public health and increased livestock productivity.

Serosurvey and associated risk factors for Chlamydia abortus infection in Dromedary camels in Egypt.

Chlamydia abortus (C. abortus) is a gram-negative, obligate intracellular bacterium that causes major public health problems in human and reproductive problems in animals. The information about the epidemiology of this pathogen among camels in Egypt is very rare. This study aimed to evaluate the existence of antibodies against C. abortus in camels and assess the related risk factors for infection. A total of 410 blood samples were collected from camels from three Egyptian governorates and examined using commercial ELISA kit. The overall seroprevalence rate was 6.6% and the higher C. abortus seropositivity rate was found in Giza governorate. Location, sex and infestation by ectoparasites did not influence on the seroprevalence of the disease. In addition, age, herd size, contact with small ruminants and history of abortion were identified as risk factors for C. abortus infection according to the univariate analysis. Based on multivariate analysis, age group of 4-8 years, small herd size, contact of camels with sheep and goats, and history of abortion were found to be significant risk factors for chlamydiosis transmission in camels. These factors had odds ratios of 4.23, 3.51, 2.84, and 2.5, respectively. These results suggest that camels have a role in the epidemiology of C. abortus infection. This promotes awareness and severe public health concern about infectious camel illnesses, allowing for additional diagnostic advancements and effective management techniques to be developed.