Serological Tools Research Articles

Characterization and use in neutralization assays of avian influenza codon-optimized H5 and H7 retroviral pseudotypes

Influenza is a relevant problem for public and animal health, with a significant economic impact. In recent years, outbreaks of avian influenza virus have resulted in devastating losses in the poultry industry worldwide, and although its transmission to humans is very rare, there is always a potential risk for an even more severe outbreak. Currently, vaccination is considered the most effective tool for the control and prevention of influenza infections in both humans and animals. The maintenance of animal welfare and the successful implementation of animal health programs depend on the timely administration of vaccines, which must comply with quality specifications indicated by health authorities; for example, the capability to ensure a minimum antibody titer. The production of viral antigens used in these tests can pose a biosafety risk, and some viral strains can be difficult to grow. Therefore, new biotechnological alternatives are required to overcome these disadvantages. In this study, we produced pseudotypes carrying H5 and H7 hemagglutinins from lowly and highly pathogenic avian influenza viruses. These pseudotypes were used in neutralization assays to detect neutralizing antibodies in avian sera, which were confirmed positive by inhibition of the hemagglutination test. Our results showed that the pseudotype neutralization assay is a viable alternative for the detection of neutralizing antibodies, by demonstrating subtype specificity and requiring reduced biosafety requirements. Therefore, it represents a versatile platform that can facilitate technology transfer protocols between laboratories, and an immediate application in serological tools for quality control of veterinary vaccines against avian influenza.

Tracking Animal Reservoirs of Pathogenic Leptospira: The Right Test for the Right Claim.

Leptospirosis is the most prevalent bacterial zoonosis worldwide and, in this context, has been extensively investigated through the One Health framework. Diagnosis of human leptospirosis includes molecular and serological tools, with the serological Microscopic Agglutination Test (MAT) still being considered as the gold standard. Mammals acting as reservoirs of the pathogen include species or populations that are able to maintain chronic infection and shed the bacteria via their urine into the environment. Animals infected by Leptospira are often identified using the same diagnosis tool as in humans, i.e., serological MAT. However, this tool may lead to misinterpretations as it can signal previous infection but does not provide accurate information regarding the capacity of animals to maintain chronic infection and, hence, participate in the transmission cycle. We employ in this paper previously published data and present original results on introduced and endemic small mammals from Indian Ocean islands to show that MAT should not be used for the identification of Leptospira reservoirs. By contrast, serological data are informative on the level of exposure of animals living in a specific environment. We present a sequential methodology to investigate human leptospirosis in the One Health framework that associates molecular detection in humans and animals, together with MAT of human samples using Leptospira isolates obtained from reservoir animals occurring in the same environment.

Epidemiological and diagnostic aspects of feline leishmaniasis with emphasis on Brazil: a narrative review.

The leishmaniases are a group of diseases caused by the protozoan parasite belonging to the genus Leishmania. In the New World, although dogs are considered the main parasite reservoir, in the last two decades, several studies have confirmed the role of cats (Felis catus) in the epidemiology of the disease and feline leishmaniasis (FeL) is now considered to be an emerging disease. The present review summarizes the current knowledge about FeL, focusing on important immunopathological aspects, epidemiology, and diagnostic methods applied for felines in Brazil. Cats are infected with the same species of Leishmania found in dogs (i.e., Leishmania infantum). Like dogs, skin lesions are the most common in cats with clinical FeL, mainly affecting the cephalic region and less frequently the legs which may be accompanied by generalized signs or visceral involvement. Information on the immune response of cats to Leishmania infection is scarce; however, efficient infection control is seen in most cases. For diagnosis, generally, the same methods as those in dogs are used, mainly serological tools. But there is a lack of studies focusing the performance of these methods for diagnosing FeL. The estimated overall prevalence of FeL in Brazil is 8%, with L. infantum being the most prevalent species. However, infections with Leishmania braziliensis and Leishmania amazonensis have also been reported. In conclusion, although there has been an increase in the publication related to FeL in Brazil in recent years, there is a lack of research relating immune response and diagnosis of these animals. Cats have been shown to be competent hosts for Leishmania parasites, and their role in the epidemiology of the disease cannot be underestimated, especially in areas of Brazil where the disease is historically endemic.

Diagnosis of Bovine Digital Dermatitis: Exploring the Usefulness of Indirect ELISA.

The precision by which animal diseases are diagnosed affects our ability to make informed decisions with regards to animal health management, from a clinical and economic perspective. Lameness is a major health condition in dairy cattle. The underlying causes of lameness include bovine digital dermatitis (BDD), which is reported as one of the main causes of infectious lameness in dairy cattle. Presently, the gold standard for BDD diagnosis in dairy cattle is visual inspection of lifted hooves—a labour intensive and subjective method. Research has suggested that Treponema spp. are the main pathogens associated with the establishment of BDD. We explored the potential of indirect enzyme-linked immunosorbent assay (ELISA) as a diagnostic serological tool in the identification of cows at different stages of BDD. Additionally, we evaluated the predictive power of this diagnostic tool on the future occurrence of BDD lesions. A total of 232 cows from three farms were used in the study. Serum samples and hoof health data were collected at three time points: ~ 30 days pre-calving, around calving, and approximately 30 days post-calving. The mean absorbance from the ELISA test was compared across different clinical presentations of BDD as assessed by visual inspection of the hooves according to the M-stage classification system. A transition model was developed to estimate the probability of lesion occurrence in time t + 1 based on the spectrophotometer (absorbance) reading in time t. The mean absorbance reading for both IgG1 and IgG2 anti-Treponema antibodies was associated with disease presence—apart from M4.1 lesions, animals with no lesions had a lower mean when compared to animals with lesions regardless of the score. Additionally, the mean absorbance reading of animals with active lesions was higher when compared to animals with no lesions. However, the anti-Treponema antibody assays failed to identify disease presence in a consistent manner. Moreover, indirect ELISA readings were not a predictor of the future occurrence of BDD lesions. In conclusion, although the levels anti-Treponema antibodies were associated with disease presence, the ELISA test failed to detect disease unequivocally and had no predictive value in the future occurrence of BDD lesions.

The potential risk of enzootic Trypanosoma cruzi transmission inside four training and re-training military battalions (BITER) in Colombia

BackgroundColombia’s National Army is one of the largest military institutions in the country based on the number of serving members and its presence throughout the country. There have been reports of cases of acute or chronic cases of Chagas disease among active military personnel. These may be the result of military-associated activities performed in jungles and other endemic areas or the consequence of exposure to Trypanosoma cruzi inside military establishments/facilities located in endemic areas. The aim of the present study was to describe the circulation of T. cruzi inside facilities housing four training and re-training battalions [Battalions of Instruction, Training en Re-training (BITERs)] located in municipalities with historical reports of triatomine bugs and Chagas disease cases. An entomological and faunal survey of domestic and sylvatic environments was conducted inside each of these military facilities.MethodsInfection in working and stray dogs present in each BITER location was determined using serological and molecular tools, and T. cruzi in mammal and triatomine bug samples was determined by PCR assay. The PCR products of the vertebrate 12S rRNA gene were also obtained and subjected to Sanger sequencing to identify blood-feeding sources. Finally, we performed a geospatial analysis to evaluate the coexistence of infected triatomines and mammals with the military personal inside of each BITER installation.ResultsIn total, 86 specimens were collected: 82 Rhodnius pallescens, two Rhodnius prolixus, one Triatoma dimidiata and one Triatoma maculata. The overall T. cruzi infection rate for R. pallescens and R. prolixus was 56.1 and 100% respectively, while T. dimidiata and T. maculata were not infected. Eight feeding sources were found for the infected triatomines, with opossum and humans being the most frequent sources of feeding (85.7%). Infection was most common in the common opossum Didelphis marsupialis, with infection levels of 77.7%. Sylvatic TcI was the most frequent genotype, found in 80% of triatomines and 75% of D. marsupialis. Of the samples collected from dogs (n = 52), five (9.6%; 95% confidence interval: 3.20–21.03) were seropositive based on two independent tests. Four of these dogs were creole and one was a working dog. The spatial analysis revealed a sympatry between infected vectors and mammals with the military population.ConclusionsWe have shown a potential risk of spillover of sylvatic T. cruzi transmission to humans by oral and vectorial transmission in two BITER installations in Colombia. The results indicate that installations where 100,000 active military personnel carry out training activities should be prioritized for epidemiological surveillance of Chagas disease.Graphical abstract

Development of a Dendrimeric Peptide-Based Approach for the Differentiation of Animals Vaccinated with FlagT4G against Classical Swine Fever from Infected Pigs.

Classical swine fever virus (CSFV) causes a viral disease of high epidemiological and economical significance that affects domestic and wild swine. Control of the disease in endemic countries is based on live-attenuated vaccines (LAVs) that induce an early protective immune response against highly virulent CSFV strains. The main disadvantage of these currently available LAVs is the lack of serological techniques to differentiate between vaccinated and infected animals (DIVA concept). Here, we describe the development of the FlagDIVA test, a serological diagnostic tool allowing for the differentiation between animals vaccinated with the FlagT4G candidate and those infected with CSFV field strains. The FlagDIVA test is a direct ELISA based on a dendrimeric peptide construct displaying a conserved epitope of CSFV structural protein E2. Although FlagDIVA detected anti-CSFV anti-bodies in infected animals, it did not recognize the antibody response of FlagT4G-vaccinated animals. Therefore, the FlagDIVA test constitutes a valuable accessory DIVA tool in implementing vaccination with the FlagT4G candidate.

Identification of Immunogenic Linear B-Cell Epitopes in C. burnetii Outer Membrane Proteins Using Immunoinformatics Approaches Reveals Potential Targets of Persistent Infections.

Coxiella burnetii is a global, highly infectious intracellular bacterium, able to infect a wide range of hosts and to persist for months in the environment. It is the etiological agent of Q fever—a zoonosis of global priority. Currently, there are no national surveillance data on C. burnetii’s seroprevalence for any South American country, reinforcing the necessity of developing novel and inexpensive serological tools to monitor the prevalence of infections among humans and animals—especially cattle, goats, and sheep. In this study, we used immunoinformatics and computational biology tools to predict specific linear B-cell epitopes in three C. burnetii outer membrane proteins: OMP-H (CBU_0612), Com-1 (CBU_1910), and OMP-P1 (CBU_0311). Furthermore, predicted epitopes were tested by ELISA, as synthetic peptides, against samples of patients reactive to C. burnetii in indirect immunofluorescence assay, in order to evaluate their natural immunogenicity. In this way, two linear B-cell epitopes were identified in each studied protein (OMP-H(51–59), OMP-H(91–106), Com-1(57–76), Com-1(191–206), OMP-P1(197–209), and OMP-P1(215–227)); all of them were confirmed as naturally immunogenic by the presence of specific antibodies in 77% of studied patients against at least one of the identified epitopes. Remarkably, a higher frequency of endocarditis cases was observed among patients who presented an intense humoral response to OMP-H and Com-1 epitopes. These data confirm that immunoinformatics applied to the identification of specific B-cell epitopes can be an effective strategy to improve and accelerate the development of surveillance tools against neglected diseases.

Performance of SPINK1 and SPINK1-based diagnostic model in detection of hepatocellular carcinoma.

ObjectivesTo identify the SPINK1 or SPINK1‐based model as a more reliable biomarker for the diagnosis of hepatocellular carcinoma (HCC).MethodsSerum samples and related laboratory parameters were collected from 540 subjects (119 healthy donors, 113 patients with chronic hepatitis B, 122 patients with liver cirrhosis, and 186 patients with HCC). SPINK1 was determined by ELISA assay. Differences in each variable were compared by one‐way ANOVA or Kruskal‐Wallis test. ROC (receiver operating characteristic) curve analysis was conducted to compare the diagnostic efficiency of alpha‐fetoprotein (AFP), SPINK1, and a SPINK1‐based combine model constructed by binary Logistic regression.ResultsIn detecting HCC using the other three groups as control, ROC curve analysis revealed that SPINK1 alone reached AUC of 0.899 (0.866–0.933), with the sensitivity of 0.812 of and specificity of 0.953. The combined model increased the AUC to 0.945 (0.926–0.964) with the sensitivity and specificity of 0.860 and 0.910, respectively. For AFP, significantly lower AUC (p < 0.0001) was shown, which was 0.695 (0.645–0.745) with the sensitivity and specificity of 0.634 and 0.718, respectively. In discriminating HCC from liver disease control, AUC of SPINK1 was 0.863(0.826–0.894), the sensitivity and specificity were 0.823 and 0.906, respectively. For combined model, the AUC, sensitivity, and specificity were 0.915 (0.884–0.940), 0.863, and 0.916, respectively. For detecting early‐stage HCC, SPINK1 and combined model achieved the sensitivity of 0.788 and 0.818, respectively, much higher than AFP of 0.485 (p < 0.05); however, the difference between SPINK1 and combined model was not statistically significant (p = 1).ConclusionWe provided solid evidence for SPINK1 as a robust serological tool for HCC diagnosis.

Evaluation of Two Serological Assays for Diagnosing Zika Virus Infection.

Zika virus (ZIKV) emerged and spread rapidly in South American countries during 2015. Efforts to diagnose ZIKV infection using serological tools were challenging in dengue-endemic areas because of antigenic similarities between both viruses. Here, we assessed the performance of an in-house developed IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the plaque reduction neutralization test (PRNT) to diagnose ZIKV infection. Acute and convalescent paired serum samples from 51 patients who presented with clinical symptoms suggestive of an arbovirus illness in dengue-endemic areas of Honduras, Venezuela, Colombia and Peru were used in the assessment. Samples were tested for ZIKV, dengue and chikungunya virus using a variety of laboratory techniques. The results for the ZIKV-RNA screening and seroconversion detected by the microneutralization test were used to construct a composite reference standard. The overall sensitivity and specificity for the MAC-ELISA were 93.5% and 100.0%, respectively. Contrastingly, the overall sensitivity and specificity for the PRNT were 96.8% and 95.0%, respectively. Restricting the analysis according to IgM or neutralizing antibodies against dengue, the performances of both serological assays were adequate. The findings of this study reveal that the MAC-ELISA and PRNT would provide initial reliable laboratory diagnostic assays for ZIKV infection in dengue-endemic areas.

Socioeconomic status is related to pubertal development in a German cohort

INTRODUCTION Current health literature suggests that there has been a decline in the age of pubertal onset and that pubertal onset/duration of puberty may, besides weight status, be influenced by socioeconomic context. OBJECTIVE The goal of this study was to determine whether pubertal onset/duration and puberty-triggering hormones luteinizing hormone (LH) and follicle-stimulating hormone (FSH) vary according to socioeconomic status (SES). Moreover, we aimed to propose cutoff values of serum LH and FSH for predicting gonadarche in boys. METHODS 2,657 apparently healthy children and adolescents between 5.5 and 18 years from the area of Leipzig were recruited from the LIFE Child study. Age at pubertal onset/end of puberty was given in 738/573 children, respectively. Anthropometric parameters of puberty, blood measurements of LH and FSH, and questionnaires assessing SES were evaluated. RESULTS Lower SES was associated with earlier thelarche and longer duration of puberty in overweight/obese girls, whereas age of menarche was not affected. In boys with low SES, a trend versus earlier puberty onset can be seen. Lower SES was significantly associated with boys' age at mutation. No significant differences in boys' and girls' serum levels of LH and FSH during puberty according to SES were observed. Serum LH levels of 0.56 IU/L and serum FSH levels of 1.74 IU/L showed the best prediction of gonadarche in boys. CONCLUSION Puberty onset/duration and boys' age at mutation is affected by SES. The proposed cutoff levels for serum LH and FSH could provide a serological tool to determine gonadarche in boys.

Serological Detection of SARS-CoV-2 Antibodies in Naturally-Infected Mink and Other Experimentally-Infected Animals.

The recent emergence of SARS-CoV-2 in humans from a yet unidentified animal reservoir and the capacity of the virus to naturally infect pets, farmed animals and potentially wild animals has highlighted the need for serological surveillance tools. In this study, the luciferase immunoprecipitation systems (LIPS), employing the spike (S) and nucleocapsid proteins (N) of SARS-CoV-2, was used to examine the suitability of the assay for antibody detection in different animal species. Sera from SARS-CoV-2 naturally-infected mink (n = 77), SARS-CoV-2 experimentally-infected ferrets, fruit bats and hamsters and a rabbit vaccinated with a purified spike protein were examined for antibodies using the SARS-CoV-2 N and/or S proteins. From comparison with the known neutralization status of the serum samples, statistical analyses including calculation of the Spearman rank-order-correlation coefficient and Cohen’s kappa agreement were used to interpret the antibody results and diagnostic performance. The LIPS immunoassay robustly detected the presence of viral antibodies in naturally infected SARS-CoV-2 mink, experimentally infected ferrets, fruit bats and hamsters as well as in an immunized rabbit. For the SARS-CoV-2-LIPS-S assay, there was a good level of discrimination between the positive and negative samples for each of the five species tested with 100% agreement with the virus neutralization results. In contrast, the SARS-CoV-2-LIPS-N assay did not consistently differentiate between SARS-CoV-2 positive and negative sera. This study demonstrates the suitability of the SARS-CoV-2-LIPS-S assay for the sero-surveillance of SARS-CoV-2 infection in a range of animal species.

Effect of pig serum storage conditions on detection of anti-ASFV antibodies by ELISA

One of the measures used to control and prevent African swine fever spread in the Russian Federation involves testing pig and boar sera using inter alia serological tools based on enzyme-linked immunosorbent assay (ELISA) for anti-viral antibody detection. However, there is no unified regulatory document specifying storage conditions for sera used in the ELISA for anti-ASFV antibody detection. There are also lack of published data on the maximum admissible shelf life of the pig sera, and the effect of storage conditions on the serological status of the pig sera as for ASF is understudied. The paper demonstrates results of the experiment aimed at the determination of the effect of storage temperatures and shelf life on the serological status of ASFV seropositive and seronegative pig sera when tested by INgezim PPA Compac (Ingenasa, Spain) ELISA as well as on the possibility of false results. During the experiment and analysis of its results, the new data were obtained, and they indicated from none to non-significant effect of the simulated storage conditions on the serological status of sera used for ASFV detection, while hemolyzed sera demonstrated more significant changes proportional to hemolysis degree and storage duration. Although the results of detection of antibodies against the agents of some diseases cannot be used in case of other pathogens, this study has a substantial applied significance as it allows to specify the dependence of the valid results of ASF serodiagnosis on the storage conditions of the samples.

The Potential of O’nyong-nyong Virus Strain SG650 Murine Monoclonal Antibodies for Detection of O’nyong-nyong and Chikungunya Viruses

O’nyong-nyong virus (ONNV) and Chikungunya virus (CHIKV) are antigenically related alphaviruses responsible for febrile illnesses common to the tropics and associated with relatively high morbidity and mortality. Murine monoclonal antibodies (mAbs) targeting alphaviruses like Chikungunya have been developed and used to make commercially available kits. However, few studies have been conducted to develop antibodies specific to ONNV and no commercial kits are available for use in endemic regions where outbreak potential is high. We demonstrate the potential of in-house generated monoclonal antibodies against ONNV to detect both ONNV and CHIKV. The objective of this study was to generate mAbs using hybridoma technology, characterize the developed mAbs, determine their specificity against selected alphaviruses and check their diagnostic potential using an indirect IgG enzyme-linked immunosorbent assay (ELISA) and focus neutralization assay (FRNT50). BALB/c mice were immunized with ONNV purified proteins from ONNV infectious culture fluid. After four rounds of booster injections, the mice were sacrificed, spleen cells harvested and fused with parental myeloma cells then cultured in selective media and the successful hybrid clones with antibody-producing ability purified to yield the desired mAbs. Five monoclonal antibodies targeting the ONNV E1 protein of isotypes IgG2a/kappa, IgG2b/kappa and IgM/kappa (P1B12, P1E9, P1G11, P1B4 and P1G6) demonstrated a potential to detect both ONNV and CHIKV isolates by indirect IgG ELISA but no potential for neutralization of the viruses by FRNT50. This study demonstrates the potential efficacy of in-house serological tools as an alternative in the absence of commercial assays in screening and diagnosis of ONN and CHIK viruses which are often co-circulating. It is our recommendation that this work may be pursued further to design and optimize ELISA assays, using the developed mAbs, for the detection of both ONN and CHIK viruses in the research laboratory set-up

The APAC Score: A Novel and Highly Performant Serological Tool for Early Diagnosis of Hepatocellular Carcinoma in Patients with Liver Cirrhosis

(1) Background: Surveillance of at-risk patients for hepatocellular carcinoma (HCC) is highly necessary, as curative treatment options are only feasible in early disease stages. However, to date, screening of patients with liver cirrhosis for HCC mostly relies on suboptimal ultrasound-mediated evaluation and α-fetoprotein (AFP) measurement. Therefore, we sought to develop a novel and blood-based scoring tool for the identification of early-stage HCC. (2) Methods: Serum samples from 267 patients with liver cirrhosis, including 122 patients with HCC and 145 without, were collected. Expression levels of soluble platelet-derived growth factor receptor beta (sPDGFRβ) and routine clinical parameters were evaluated, and then utilized in logistic regression analysis. (3) Results: We developed a novel serological scoring tool, the APAC score, consisting of the parameters age, sPDGFRβ, AFP, and creatinine, which identified patients with HCC in a cirrhotic population with an AUC of 0.9503, which was significantly better than the GALAD score (AUC: 0.9000, p = 0.0031). Moreover, the diagnostic accuracy of the APAC score was independent of disease etiology, including alcohol (AUC: 0.9317), viral infection (AUC: 0.9561), and NAFLD (AUC: 0.9545). For the detection of patients with (very) early (BCLC 0/A) HCC stage or within Milan criteria, the APAC score achieved an AUC of 0.9317 (sensitivity: 85.2%, specificity: 89.2%) and 0.9488 (sensitivity: 91.1%, specificity 85.3%), respectively. (4) Conclusions: The APAC score is a novel and highly accurate serological tool for the identification of HCC, especially for early stages. It is superior to the currently proposed blood-based algorithms, and has the potential to improve surveillance of the at-risk population.

Development of Nanobodies Targeting Peste des Petits Ruminants Virus: The Prospect in Disease Diagnosis and Therapy.

Simple SummaryPeste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR) of sheep and goats, that threatens food security, small ruminant production, and the conservation of wild small ruminants. Current efforts are directed towards the global control and eradication of PPRV, an initiative of the World Organisation for Animal Health and Food and the Agriculture Organisation of the United Nations. A plethora of diagnostic tools for PPR were primarily developed for livestock. New innovative diagnostic tools are needed to detect PPRV in atypical hosts (e.g., Camelidae, Suidae, and Bovinae), in wildlife ecosystems, and in complex field situations. Recent studies confirmed that single-domain antigen binding fragments (nanobodies) derived from heavy-chain-only camelid antibodies have proven to be a powerful tool in diagnostics and therapeutics due to their unique properties, such as small size and strong antigen-binding affinity. Therefore, the main objective of this study was to generate PPRV-reactive nanobodies in order to set a pace for the development of diagnostic and possibly therapeutic nanobodies in the future. Initially, a strategy was developed whereby an alpaca was immunized with PPRV in order to raise an affinity-matured immune response, from which an immune nanobody library was constructed. Following phage display, nine nanobodies that specifically recognise PPRV were identified on enzyme-linked immunosorbent assay. This study has generated PPRV-reactive nanobodies and have significant implications in the development of cost-effective diagnostic tools in context with the planned eradication of PPR in the world.Peste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR) of sheep and goats, that threatens food security, small ruminant production, and the conservation of wild small ruminants in many developing countries, especially in Africa. Robust serological and molecular diagnostic tools are available to detect PPRV infection, but they were mainly developed for domestic sheep and goats. The presence of a wide host range for PPRV does present serological diagnostic challenges. New innovative diagnostic tools are needed to detect PPRV in atypical hosts (e.g., Camelidae, Suidae, and Bovinae), in wildlife ecosystems and in complex field situations. Interestingly, single-domain antigen binding fragments (nanobodies) derived from heavy-chain-only camelid antibodies have emerged as a new hope in the development of accurate, rapid, and cost-effective diagnostic tools in veterinary and biomedical fields that are suitable for low-income countries. The main objective of this study was to construct an immune nanobody library to retrieve PPRV-reactive nanobodies that enable the development of diagnostic and therapeutic nanobodies in the future. Here, a strategy was developed whereby an alpaca (Vicugna pacos) was immunized with a live attenuated vaccine strain (PPRV/N/75/1) to raise an affinity-matured immune response in the heavy-chain-only antibody classes. The nanobody gene repertoire was engineered in pMECS-GG phagemid, whereby a ccdB gene (encoding a lethal protein) was substituted by the nanobody gene. An immune nanobody library with approximately sixty-four million independent transformants was constructed, of which 100% contained an insert with the proper size of nanobody gene. Following phage display and biopanning, nine nanobodies that specifically recognise completely inactivated PPRV were identified on enzyme-linked immunosorbent assay. They showed superb potency in rapidly identifying PPRV, which is likely to open a new perspective in the diagnosis and possible treatment of PPR infection.

Immunogenic Peptides from Pap31 and SCS-α of Bartonella bacilliformis: One Step Closer to a Rapid Diagnostic Tool for Carrion's Disease.

Bartonella bacilliformis is the causal agent of Carrion’s disease, an overlooked illness endemic in the Andean Mountains with Peru being the most affected country. The diagnostic of this illness is a challenge due to the limited resources and the common symptomatology with other infectious diseases. The goal of this study was to identify immunogenic peptides from Pap31 and succinyl-CoA synthetase α (SCS-α) of B. bacilliformis that might be suitable for developing a serologic tool. The immunodominant character of Pap31 and SCS-α was determined by Western blotting and in-silico analysis. Subsequently, 35 peptides were selected for epitope mapping and their immunoreactivity was tested by enzyme-linked immunosorbent assay (ELISA). A total of 30 sera were tested including pre-exposed people with high IgM levels for Pap31/SCS-α (23 sera), patients (2 sera) as well as 5 sera with no reactivity to Pap31/SCS-α. The results indicate that Pap31-8 (187QAIGSAILKGTKDTGT202) and SCS-α-12 (59IFASVAEGKEKTGANA74) are the most immunogenic peptides, with Pap31-8 showing potential to discriminate between B. bacilliformis and the remaining Bartonella spp., and SCS-α-12 differentiating Bartonella spp. from other microorganisms.

Brucellosis in Iranian buffalo: prevalence and diagnostic methods

The water buffalo (Bubalus bubalis) in Iran represent an important source of meat and milk products with high biological value. Given the importance of water buffalo in Iran and the prevalence of brucellosis as one of the most important zoonotic diseases in this ruminant species, this study summarized available data on history, epidemiology, diagnosis, and control of brucellosis in water buffalo from previous studies that have been carried out in Iran. According to the documented data, there are three main groups of Iranian buffalo, including the Khuzestan ecotype (Khuzestan province); the Azary ecotype (Western/ Eastern Azarbaijan and Ardabil provinces); and the North ecotype (Gylan and Mazandaran provinces). Preliminary studies conducted on Iranian buffaloes either by serological or molecular tools reported that buffaloes' infection occurred due to natural exposure to a wild strain of Brucella abortus and Brucella melitensis. Previous studies dealing with brucellosis in Iranian buffalo are next to none. This review notifies the importance of reliable and detailed epidemiological investigations of Iranian buffaloes through continuous monitoring systems of the health status of buffalo populations. Continuous test and slaughter strategy, vaccination, and re-planning of veterinary activities are required to mitigate buffalo's role in disseminating and maintaining brucellosis in Iran.

Poor performance of the utility of the serological diagnosis of pulmonary tuberculosis in Nigeria

Introduction Pulmonary tuberculosis (PTB) is the most common form of tuberculosis. In Nigeria, PTB is diagnosed by sputum smear microscopy. The problems associated with the diagnosis of PTB using sputum smear for acid-fast bacilli and culture has provided an urgent need to assess the utility of serological tools as an adjunctive diagnostic tool in presumptive PTB cases. This study is aimed at testing the diagnostic utility of a specific immunoglobulin G (IgG) serological assay in the diagnosis of PTB. Patients and methods A utility study was conducted on 184 participants of which 92 were smear-positive PTB cases and 92 were healthy controls at the TB and Leprosy Centre and Ahmadu Bello University Teaching Hospital, Zaria. Data were collected on age and sex, and both PTB patients and controls were screened for anti-TB IgG antibodies by enzyme-linked immunosorbent assay technique. Results The mean age of the participants was 35, and the male-female ratio was 1 : 4. Twenty-one (5.7%) of the PTB patients tested positive for IgG antibodies, and 11 (1.3%) of the controls were positive. The sensitivity of the IgG antibody assay was 18.4%, with the specificity being 92.3%, while the predictive values were 70.8 and 53.1% for positive and negative results, respectively. Conclusion The IgG antibody assay is poorly sensitive; serology may not have a role in the diagnosis of PTB in resource-limited settings, but the much higher specificity suggests that it may be useful as an adjunctive diagnostic tool, or for monitoring of patients on treatment for PTB.

Investigation of Brucellosis Using Molecular and Serological Tools in Selected Sites of Maekel Region, Eritrea

Brucellosis is a zoonotic disease primarily affecting animals and accidentally exposed humans. In Eritrea, brucellosis endemicity is proven by several sero-prevalence studies in domestic animals and occupationally exposed humans. However, there is a gap of information on the commonly occurring brucella species and cause of human/animal brucellosis. The objective of this study is to identify brucella species in sheep and goats which possibly pose risk to human brucellosis. Out of a total 71 serum and 71 vaginal swab samples collected from sheep and goats, eight serum samples by Rose Bengal plate test (RBPT) and 4 vaginal swabs by polymerase chain reaction (PCR) were found positive for brucellosis. Combinatorial PCR detected Br. melitensis from the four PCR positive samples. Questionnaire interviews collected from 68 confirmed brucellosis patients showed that habits of drinking unpasteurized yoghurt and raw milk and handling of aborted materials were common. This investigation study suggests, Br. melitensis could be the major cause of brucellosis in humans and animals in Maekel region Eritrea. Therefore, a large-scale epidemiological study is recommended to confirm the true extent of the problem in animals and humans.

Role of S100β Glial Protein as a Serological Marker for Analysis of Acute Ischemic Stroke

Background: Stroke is a major cause of neurological disability, which can be often predicted with serological markers. Glial-derived S100β protein is a potential biomarker for cerebral ischemia and may be helpful in predicting the severity, outcome, and recovery of stroke. Aim: This study aimed to study the role of S100β glial protein as a serological marker in predicting the severity of acute ischemic stroke (AIS), outcome, and functional recovery after 1 month. Methods: A hospital-based prospective case control study included 43 consecutive patients, &gt;18 years old, who were admitted with acute middle cerebral artery (MCA) territory infarcts within 72 h of onset of neurological deficits. Control group comprised of 43 age-matched asymptomatic volunteers. Independent t-test and chi square test were used to compare the means and evaluate the association between protein level and various parameters. P ≤ .05 was statistically significant. Results: S100β protein level in AIS patients was significantly higher compared to controls ( P &lt; .05). Elevated serum S100β protein level was found to be associated with larger infarct volumes, higher National Institute Health Stroke Scale scores, and higher modified Rankin Scale scores at admission ( P &lt; .05). Patients with higher S100β protein levels at admission had poor recovery at 1 month compared to patients having normal S100β protein levels. Conclusion: S100β protein levels at admission after an acute MCA territory infarct may be used as a reliable serological tool in predicting the severity, outcome, and functional recovery in stroke.