Indirect Assay Research Articles

Indirect Immunofluorescence of Tissue Sections.

Immunofluorescence (IF) on tissue sections allows for the detection of protein species subcellular localization. IF studies further offer the ability to achieve this understanding at the level of single cell granularity. Here, we describe the processes by which tissue is fixed, embedded, sectioned, and subsequently utilized for conducting indirect IF assays. We raise potential opportunities for troubleshooting and optimization at varying stages of the protocol.

Schistosomiasis in the People's Republic of China - down but not out.

Schistosomiasis has been subjected to extensive control efforts in the People's Republic of China (China) which aims to eliminate the disease by 2030. We describe baseline results of a longitudinal cohort study undertaken in the Dongting and Poyang lakes areas of central China designed to determine the prevalence of Schistosoma japonicum in humans, animals (goats and bovines) and Oncomelania snails utilizing molecular diagnostics procedures. Data from the Chinese National Schistosomiasis Control Programme (CNSCP) were compared with the molecular results obtained.Sixteen villages from Hunan and Jiangxi provinces were surveyed; animals were only found in Hunan. The prevalence of schistosomiasis in humans was 1.8% in Jiangxi and 8.0% in Hunan determined by real-time polymerase chain reaction (PCR), while 18.3% of animals were positive by digital droplet PCR. The CNSCP data indicated that all villages harboured S. japonicum-infected individuals, detected serologically by indirect haemagglutination assay (IHA), but very few, if any, of these were subsequently positive by Kato-Katz (KK).Based on the outcome of the IHA and KK results, the CNSCP incorporates targeted human praziquantel chemotherapy but this approach can miss some infections as evidenced by the results reported here. Sensitive molecular diagnostics can play a key role in the elimination of schistosomiasis in China and inform control measures allowing for a more systematic approach to treatment.

Quantitative Detection of Anti-SARS-CoV-2 Antibodies Using Indirect ELISA.

ObjectiveReal-time reverse transcription-polymerase chain reaction is the gold standard for the diagnosis of COVID-19, but it is necessary to utilize other tests to determine the burden of the disease and the spread of the outbreak such as IgG-, IgM-, and IgA-based antibody detection using enzyme-linked immunosorbent assay (ELISA).Materials and MethodsWe developed an indirect ELISA assay to quantitatively measure the amount of COVID-19 IgG, IgM, and IgA antibodies present in patient serum, dried blood, and plasma.ResultsThe population cutoff values for positivity were determined by receiver operating characteristic curves to be 1.23 U/mL, 23.09 U/mL, and 6.36 U/mL for IgG, IgM, and IgA, respectively. After albumin subtraction, the specificity remained >98% and the sensitivity was 95.72%, 83.47%, and 82.60%, respectively, for IgG, IgM, and IgA antibodies to the combined spike subunit 1 receptor binding domain and N proteins in serum. Plasma and dried blood spot specimens were also validated on this assay.ConclusionThis assay may be used for determining the seroprevalence of SARS-CoV-2 in a population exposed to the virus or in vaccinated individuals.

Grafted Organic Selenide Interface for Sensitive Electrocatalytic Detection of Peroxynitrite (PON): Towards Pon Sensing in Brain Tissue

Peroxynitrite is a very reactive and cytotoxic species that plays important pathophysiological roles, particularly in the brain. It is also suspected to be a factor involved in tissue damage that results from long term deep brain neural stimulation. Accurate determination of peroxynitrite concentration is inherently difficult due to its high reactivity and concentration range. Various methods for peroxynitrite determination have been reported including indirect spectroscopic assays and, recently, direct electrochemical methods. Currently, this field is still actively growing in the pursuit of viable electrochemical probes or catalytic materials as interfaces for efficient peroxynitrite detection. A durable and reliable electrochemical sensor would be valuable in order to monitor peroxynitrite generation near sites of electrical neurostimulation.In this work, we prepared and characterized a functional thin film material based on an organic selenide on graphite electrodes and used the interface in sensitive electrochemical determination of peroxynitrite. First, we describe the preparation and grafting of the catalytic material based on the electrodeposition of organic selenides on graphite electrodes. Next, we report on the characterization of the resulting grafted film material on graphite interface using various physicochemical methods including Scanning Electron Microscopy (SEM), Energy Dispersive X-ray spectroscopy (EDX) and X-ray Photoelectron Spectroscopy (XPS). We then tested the performance of resulting aniline selenide catalytic material on modified electrodes as peroxynitrite sensing interfaces using voltammetry and dose-response amperometry. The grafted thin film material showed a significant enhancement in peroxynitrite oxidative current compared to electrodes with aniline only (i.e. materials devoid of selenium) under the same conditions. In the presentation we will show that the enhancement in peroxynitrite signal is the result of an electrocatalytic mechanism where the grafted selenide-based organic material at the oxidized state mediates the electrocatalytic oxidation of peroxynitrite. In terms of selectivity, the electrocatalytic detection of PON on electrodes modified with the selenide compound responds better and selectively toward PON as a target analyte over other potentially interfering analytes such as nitric oxide, nitrite, and nitrate. To the best of our knowledge, this is the first time a selenium-based organic material electrochemically grafted at an electrode surface is used for catalytic detection and quantification of peroxynitrite.

The comparison of seroconversion rates among different varicella vaccines administered Turkish children; MAV/06 and vOka

ABSTRACT Varicella is a vaccine-preventable disease, and the incidence of varicella has declined since the introduction of varicella vaccine campaigns. A wild type of varicella zoster virus (VZV) was isolated from a 33-month-old child with varicella in Korea in 1989, a different strain (MAV/06). A live-attenuated varicella vaccine containing strain (MAV/06), Suduvax®, was developed in South Korea in 1994. Turkey introduced the varicella vaccine containing the MAV/06 strain (Varicella Vaccine-GCC, Green Cross, South Korea) in January 2019. Therefore, we aimed to compare the seroconversion rates among MAV/06 vaccine- and vOka-administered children. We prospectively collected blood samples from 98 received vOKA and 98 received MAV/06 children 6 weeks after administration, and seroconversion rates were determined by an indirect fluorescence assay (Anti-VZV IIFT IgG, Euroimmun, Germany). Seroconversion rate was significantly higher in vOka group than MAV/06 group (82.7% vs. 64.3%; p = .004). Of the children vaccinated with vOka strain, 17 children did not develop antibodies, 12 were weakly positive, and the remaining 69 children were strongly positive. Of the children who were administered MAV/06 strain, 35 were negative, 20 were weakly positive, and 43 were strongly positive. In conclusion, this study demonstrated that MAV/06 varicella vaccine had lower seroconversion rates and the strong seropositive cases were less common than vOka-administered children. Larger and prospective studies are needed.

Fusion Expression and Immune Effect of PCV2 Cap Protein Tandem Multiantigen Epitopes with CD154/GM-CSF.

Porcine circovirus associated diseases (PCVAD) is a contagious disease of swine caused by porcine circovirus type 2 (PCV2). The capsid protein (Cap) is the sole structural protein and the main antigen of PCV2. Cap is the principal immunogenic protein and induces humoral and cellular immunity. CD154 and GM-CSF are immune adjuvants that enhance responses to vaccines. However, whether these two cellular molecules could produce an enhanced effect in PCV2 vaccines still needs to be further studied. The results of PCR and restriction enzyme showed that the recombinant lentiviral plasmids pCDH-TB-Cap, pCDH-TB-Cap-CD154 and pCDH-TB-Cap were successfully constructed. Western blot and IFA showed that the three fusion proteins TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF were stably expressed in CHO-K1 cells. Indirect ELISA assay showed that mice immunized with TB-Cap-CD154 and TB-Cap-GM-CSF fusion proteins produced higher PCV2-specific antibodies than mice immunized with the TB-Cap and a commercial vaccine (p < 0.0001). Moreover, lymphocyte proliferation and flow cytometry showed that the cellular immune response of each immune group was significantly enhanced (p < 0.0001). After PCV2 challenge, the results revealed that the viral loads in serum, lung and kidney of all vaccinated groups were significantly lower than the PBS group (p < 0.0001). The transcription levels of IL-2, IFN-gamma, IL-4 and IL-10 cytokines in the TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF groups were significantly higher than those in the PBS and recombinant vaccine groups (p < 0.0001). These results indicated that CD154 and GM-CSF could enhance the ability of TB-Cap protein to induce the body to produce PCV2 specific antibodies and increase the transcription level of cytokines. Thus, CD154 and GM-CSF molecules were a powerful immunoadjuvant for PCV2 subunit vaccines. The novel TB-Cap-CD154 and TB-Cap-GM-CSF subunit vaccine has the potential to be used for the prevention and control of PCVAD.

SEROPREVALENCE OF Q FEVER AMONG SHEEP IN MOSUL CITY, IRAQ

The current study aimed to determine the Q fever seroprevalence rate of local sheep in Mosul City in northern Iraq between October 2019 and January 2020. Three hundred and thirty sera were collected and analyzed by a commercial indirect enzyme-linked immune sorbent assays (ELISA) to detect Coxiella burnetii antibodies. A total of 68 animals (seroprevalence: 20.6%) gave positive results. The results showed that the eastern part of Mosul city's prevalence rate was higher than in other areas (28.2%). According to age and sex, the prevalence of adult sheep (21.8%) is higher in females (21.3%) than young sheep (19.4%) and males (19%). Also, we noticed that the infection rate of aborted ewes (26.4%) is higher than that of non-aborted ewes.

Quantum dots‐based fluorescence immunoassay for detection of tiamulin in pork

AbstractTiamulin (TML) is a kind of diterpenoid veterinary antibiotic. If abused, it will cause residues in edible tissues of animals and endanger human health. Thus, a facile, sensitive, and accurate method is essential for TML detection. In this study, TML complete antigen was synthesized by oximation and active ester method, and monoclonal antibodies with high specificity and sensitivity were prepared. ZnCdSe/ZnS quantum dots (QDs) coupled with goat anti‐mouse IgG were used as a fluorescence detection probe to establish indirect competitive fluorescent immunosorbent assay (icFLISA) for the determination of TML in pork. The 50% inhibitory concentration (IC50) of the icFLISA was 0.069 ng/ml, and the limits of detection (LOD) were 0.309 pg/ml. The recovery of the spiked pork samples for the icFLISA ranged from 85.6 to 96.8%, and the coefficient of variation (CV) was less than 9.4%. The results showed that the icFLISA based on ZnCdSe/ZnS QDs was more sensitive than the traditional indirect competitive enzyme‐linked immunosorbent assay for the detection of TML in edible animal tissues.

Neutralization antibody titers against SARS-CoV-2 in an infant born to a mother with COVID-19

Neutralization antibody titers against SARS-CoV-2 in an infant born to a mother with COVID-19

Assessment of Influential Factors for Scours Associated with Cryptosporidium sp., Rotavirus and Coronavirus in Calves from Argentinean Dairy Farms.

Simple SummaryScours is the most common disease in dairy calves, and it is a multifactorial syndrome complex. Cryptosporidium sp., rotavirus group A, and bovine coronavirus are the three main pathogens associated with scours. The objective of this study was to identify potential factors associated with scours and these three pathogens in preweaned dairy calves. The results of this study indicated that scours is a prevalent disease in farms of Salta, Argentina, and that rotavirus and Cryptosporidium sp. infections, along with specific farm management practices, might be important contributing factors that could increase the chance of scours in dairy farms.Scours is the most common disease in dairy calves, and it is a multifactorial syndrome complex. Cryptosporidium sp. (C. sp.), rotavirus group A (RVA), and bovine coronavirus (BCoV) are the three main pathogens associated with scours. The objective of this study was to identify potential factors associated with scours, C. sp., RVA, and BCoV infections in preweaned dairy calves from Lerma Valley in Salta Province, Argentina. A total of 488 preweaned calves from 19 dairy farms located in the Lerma Valley were enrolled in this observational study. One fecal sample was collected from each calf between one week and two months of age for assessment of C. sp., RVA, and BCoV infection status. Cryptosporidium sp. oocysts and RVA and BCoV antigens in fecal samples were assessed using microscopic observation and indirect enzyme-linked immune sorbent assay (iELISA), respectively. A voluntary questionnaire was developed and used to collect data regarding management practices from the participants’ farms. The data were analyzed using multivariable logistic regression models. Scours incidence was 35.4%, and a greater proportion of calves younger than 20 days were affected. Of the fecal samples, 18% and 9.5% tested were positives for C. sp. and RVA, respectively, while BCoV was detected only in two calves. Furthermore, 84.2% and 63.1% of the farms tested positive for Cryptosporidium sp. and RVA, respectively. In addition, the following variables were associated with higher odds of having scours: (1) herd size (>300 milking cows; OR = 1.7), (2) calf age (<20 days of age; OR = 2.2), (3) RVA and C. sp. test (positive test; RVA OR = 2.6; C. sp. OR = 3), calf feeding practices (feeding milk replacer; OR = 1.81), and newborn calf management practices (calf moved from maternity pen <6 h after calving; OR = 1.7). Concerning RVA infection, calves less than 20 days of age (OR = 2.6) had a higher chance of testing positive for RVA, while calves that remained in the calving pen for less than 6 h after calving had a lower chance (OR = 0.3). On the other hand, for C. sp. infection, large farm size (>300 milking cows; OR = 1.2) and young calf age (<20 days of age; OR = 4.4) indicated a higher chance of testing positive for C. sp., while calves belonging to farms that fed frozen colostrum (OR = 0.2) had a lower chance of becoming infected with C. sp. The result of this study indicated that scours is a prevalent disease in farms of the Lerma Valley, Salta, Argentina, and that RVA and C. sp. infections, along with specific farm management practices, might be important contributing factors that could increase the chance of NCS in dairy farms.

Evaluation of a SARS-CoV-2 Capture IgM Antibody Assay in Convalescent Sera.

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic with over 152 million cases and 3.19 million deaths reported by early May 2021. Understanding the serological response to SARS-CoV-2 is critical to determining the burden of infection and disease (coronavirus disease 2019 [COVID-19]) and transmission dynamics. We developed a capture IgM assay because it should have better sensitivity and specificity than the commonly used indirect assay. Here, we report the development and performance of a capture IgM enzyme-linked immunosorbent assay (ELISA) and a companion indirect IgG ELISA for the spike (S) and nucleocapsid (N) proteins and the receptor-binding domain (RBD) of S. We found that among the IgM ELISAs, the S ELISA was positive in 76% of 55 serum samples from SARS-CoV-2 PCR-positive patients, the RBD ELISA was positive in 55% of samples, and the N ELISA was positive in 15% of samples. The companion indirect IgG ELISAs were positive for S in 89% of the 55 serum samples, RBD in 78%, and N in 85%. While the specificities for IgM RBD, S, and N ELISAs and IgG S and RBD ELISAs were 97% to 100%, the specificity of the N IgG ELISA was lower (89%). RBD-specific IgM antibodies became undetectable by 3 to 6 months, and S IgM reached low levels at 6 months. The corresponding IgG S, RBD, and N antibodies persisted with some decreases in levels over this time period. These capture IgM ELISAs and the companion indirect IgG ELISAs should enhance serologic studies of SARS-CoV-2 infections.IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has inflicted tremendous loss of lives, overwhelmed health care systems, and disrupted all aspects of life worldwide since its emergence in Wuhan, China, in December 2019. Detecting current and past infection by PCR or serology is important to understanding and controlling SARS-CoV-2. With increasing prevalence of past infection or vaccination, IgG antibodies are less helpful in diagnosing a current infection. IgM antibodies indicate a more recent infection and can supplement PCR diagnosis. We report an alternative method, capture IgM, to detect serum IgM antibodies, which should be more sensitive and specific than most currently used methods. We describe this capture IgM assay and a companion indirect IgG assay for the SARS-CoV-2 spike (S), nucleocapsid (N), and receptor-binding domain (RBD) proteins. These assays can add value to diagnostic and serologic studies of coronavirus disease 2019 (COVID-19).

Development and Application of a Novel Rapid and Throughput Method for Broad-Spectrum Anti-Foodborne Norovirus Antibody Testing.

Foodbone norovirus (NoV) is the leading cause of acute gastroenteritis worldwide. Candidate vaccines are being developed, however, no licensed vaccines are currently available for managing NoV infections. Screening for stimulated antibodies with broad-spectrum binding activities can be performed for the development of NoV polyvalent vaccines. In this study, we aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) for testing the broad spectrum of anti-NoV antibodies. Capsid P proteins from 28 representative NoV strains (GI.1–GI.9 and GII.1–GII.22 except GII.11, GII.18, and GII.19) were selected, prepared, and used as coating antigens on one microplate. Combined with incubation and the horseradish peroxidase chromogenic reaction, the entire process for testing the spectrum of unknown antibodies required 2 h for completion. The intra-assay and inter-assay coefficients of variation were less than 10%. The new method was successfully performed with monoclonal antibodies and polyclonal antibodies induced by multiple antigens. In conclusion, the indirect ELISA assay developed in this study had a good performance of reliability, convenience, and high-throughput screening for broad-spectrum antibodies.

Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1.

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149–811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.

Identification of novel B-cell epitopes on the capsid protein of type 1 porcine astrovirus, using monoclonal antibodies

Porcine astrovirus (PAstV) is prevalent in pigs worldwide and could cause clinical symptoms such as diarrhea and encephalitis. The capsid protein (Cap) of PAstV plays a determinant role for virus immunological characteristics. In this study, the major antigenic regions of PAstV1 Cap were expressed through prokaryotic expression systems and immunized to BALB/c mice. Finally, two anti-Cap monoclonal antibodies (named mAb F4-4 and D3F10) were screened by indirect immune-fluorescence assay (IFA). A series of truncated GST-fused or artificially synthesized peptides were used to detect their reactivity with the mAbs and PAstV positive serum. Two novel B cell epitopes (120-GNNTFG-125, 485-RISDPTWFSA-494) were identified by using these two mAbs. Moreover, sequence alignment result showed that epitope 120-GNNTFG-125 was highly conserved in type 1 PAstV capsid protein. Cross-reactivity analysis further confirmed the genotype-specificity of mAb F4-4. The results of this study demonstrated to be the first description of monoclonal antibody preparation and B-cell epitope mapping on PAstV capsid protein, which may provide new information on the biological significance of PAstV capsid protein and lay a foundation for the development of PAstV immunological tests and genotype diagnostic methods.

PREVALENCE OF MEASLES IGG ANTIBODIES AMONG HEALTHCARE WORKERS IN BULGARIA

Background: Healthcare workers are at greater risk of acquiring measles than the general population. Transmission occurs from infected patients to staff and from infected staff to patients and colleagues. In inpatient and outpatient settings, susceptible patients suffering from other conditions, especially the elderly and severely ill patients in intensive care units are at a high risk of severe disease or death if infected with measles. The most effective preventive measure against measles is vaccination with two doses of a measles-containing vaccine. Aim: To perform a serological assessment of the presence of measles IgG antibodies among healthcare workers. Materials and Methods: The present study involves serological testing performed on samples from persons working in hospitals and Regional Health Inspectorates in the country. Screening started in connection with a reported measles outbreak in Bulgaria in 2019, and continued into early 2020. An indirect ELISA assay for the detection of specific measles IgG antibodies in serum samples was used. Results: The tested 152 healthcare workers were from 5 regions of the country, i.e. Sofia-capital (n=87), Burgas (n=6), Blagoevgrad (n=5), Dobrich (n=8) and Pazardzhik (n=46). Anti-measles IgG antibodies were detected in 85.5% of the participants (130/152). Negative results, indicating a possible lack of protective immunity against measles, were obtained primarily in samples from persons younger than 40 years of age. Conclusion: Screening to identify healthcare workers who lack presumptive evidence of protective immunity against measles should be reinforced, especially among younger healthcare workers, in order to minimize the risk of measles both among healthcare workers and among patients.

8-acetoxy-trisulfopyrene as the first activatable fluorogenic probe for add-and-read assessment of Organic anion-transporting polypeptides, OATP1B1, OATP1B3, and OATP2B1.

Organic anion-transporting polypeptides, OATP1B1, OATP1B3, and OATP2B1 are multispecific membrane proteins mediating the hepatocellular uptake of structurally diverse endo- and exogenous compounds, including various kinds of drugs. Co-administration of OATP1B/2B1 substrates may lead to altered pharmacokinetics or even toxicity. Therefore, the study of the interaction with these OATPs is essential in drug development and is recommended by international regulatory agencies, the FDA, EMA, and PMDA. In general, radiolabeled indicators are used to measure drug interactions of OATPs, and, lately, fluorescent probes are also gaining wider application in OATP tests. However, all of the currently available methods (either radioactive or fluorescence-based) comprise multiple steps, including the removal of the indicator in the end of the experiment. Hence, they are not ideally suited for high-throughput screening. In the current study, in order to find an indicator allowing real-time assessment of hepatic OATP function, we searched for an activatable fluorogenic OATP substrate. Here, we show that 8-acetoxypyrene-1,3,6-trisulfonate (Ace), a fluorogenic derivative of the hepatic OATP substrate pyranine (8-hydroxypyrene-1,3,6-trisulfonate) enters the cells via OATP1B1/3 or OATP2B1 function. In living cells, Ace is then converted into highly fluorescent pyranine, allowing "no-wash" measurement of OATP function and drug interactions. Furthermore, we demonstrate that Ace can be used in an indirect assay termed as competitive counterflow suitable to distinguish between transported substrates and inhibitors of OATP1B1. The fluorescence-based methods described here are unique and open the way toward high-throughput screening of interactions between new molecular entities and OATPs.

Sero Status and Presumed Risk Factors Assessment for Bovine Herpesvirus-1 in North Western, Ethiopia

A cross sectional study was conducted from November 2020 to June 2021 on a total of 442 randomly selected indigenous zebus (bos indicus) of extensively and semi-intensively managed cattle’s. These cattle’s were sampled from East Gojam (n=137), west Gojam (n=160) and Awi zone (n=145) north western part of Ethiopia. Indirect Enzyme-Linked Immune-Sorbent Assay (I-ELISA) was used to detect antibodies specific to Bovine Herpes Virus-1 (BoHV-1). The relationship of the categories under the variables was analyzed using a chisquare descriptive statistic. Logistic regression analyses were also used to assess potential predictive factors associated with the outcome variable. An overall animal level sero-prevalence of BoHV-1 antibodies were 77.6% (95% CI: 73.5-81.3%) for the present study. In a random-effects of an adjusted logistic regression model, geographically the sero-prevalence of BoHV-1 exposure was higher for cattle’s from East Gojam (Odds ratio [OR] =0.2; p=0.002) than in Awi zone (OR=1.4; p=0.377) and West Gojam. The likelihood of disease occurrence for introduced cattle’s were relatively higher (OR=0.4; p= 0.001) than homebred. Age of cattle’s and parity status of heifers/cows were significantly associated with BoHV-1 infection (p&lt;0.001). The risk of infection was also positively correlated with the occurrence of respiratory problem (OR=2.2; P=0.048). Thus, this result signifies that BoHV-1 is widely circulating among the indigenous zebus in the study areas. Given that there was no BoHV-1 vaccination delivery in the study area and in the country as a whole, control and prevention measures using marker vaccines were highly recommended.

European Guidelines (S3) on diagnosis and management of mucous membrane pemphigoid, initiated by the European Academy of Dermatology and Venereology - Part II.

This guideline has been initiated by the task force Autoimmune Blistering Diseases of the European Academy of Dermatology and Venereology, including physicians from all relevant disciplines and patient organizations. It is a S3 consensus‐based guideline that systematically reviewed the literature on mucous membrane pemphigoid (MMP) in the MEDLINE and EMBASE databases until June 2019, with no limitations on language. While the first part of this guideline addressed methodology, as well as epidemiology, terminology, aetiology, clinical presentation and outcome measures in MMP, the second part presents the diagnostics and management of MMP. MMP should be suspected in cases with predominant mucosal lesions. Direct immunofluorescence microscopy to detect tissue‐bound IgG, IgA and/or complement C3, combined with serological testing for circulating autoantibodies are recommended. In most patients, serum autoantibodies are present only in low levels and in variable proportions, depending on the clinical sites involved. Circulating autoantibodies are determined by indirect IF assays using tissue substrates, or ELISA using different recombinant forms of the target antigens or immunoblotting using different substrates. The major target antigen in MMP is type XVII collagen (BP180), although in 10–25% of patients laminin 332 is recognized. In 25–30% of MMP patients with anti‐laminin 332 reactivity, malignancies have been associated. As first‐line treatment of mild/moderate MMP, dapsone, methotrexate or tetracyclines and/or topical corticosteroids are recommended. For severe MMP, dapsone and oral or intravenous cyclophosphamide and/or oral corticosteroids are recommended as first‐line regimens. Additional recommendations are given, tailored to treatment of single‐site MMP such as oral, ocular, laryngeal, oesophageal and genital MMP, as well as the diagnosis of ocular MMP. Treatment recommendations are limited by the complete lack of high‐quality randomized controlled trials.

GNAI2 Promotes Proliferation and Decreases Apoptosis in Rabbit Melanocytes.

GNAI2 (G protein subunit alpha i2) is a signaling modulator or transducer, involved in several transmembrane signaling systems, that plays a vital role in the melanogenesis signaling pathway. However, whether GNAI2 regulates cell proliferation and apoptosis in rabbit melanocytes is not known. We found that GNAI2 was differentially expressed in rabbits with different coat colors using qRT-PCR and Wes assays. Furthermore, it was observed that the rabbits with black skin had the highest GNAI2 levels, and those with white skin had the lowest expression. The coding sequence of GNAI2 was successfully cloned and inserted into pcDNA3.1 and pcDNA3.1-Myc vectors. It was observed that the GNAI2 protein was mainly localized in the cytoplasm using the indirect immunofluorescence staining assay. Overexpression of GNAI2 significantly increased melanin content, promoted melanocyte proliferation, and inhibited melanocyte apoptosis. On the contrary, the knockdown of GNAI2 using siRNA had the opposite effect. In addition, GNAI2 significantly increased the mRNA expression levels of the melanin-related genes TYR, GPNMB, PMEL, and DCT in rabbit melanocytes. The results suggested that GNAI2 regulated melanocyte development by promoting melanocyte proliferation and inhibiting apoptosis.

Serological prevalence and risk factors of Toxoplasma gondii in Zoo Mammals in Chile

Toxoplasma gondii is a zoonotic cosmopolitan protozoan that causes a high mortality rate among zoo mammals such as New World primates, meerkats, marsupials and Pallas’ cat. It has been recently reported in Chile, mainly among wild populations, but also as the cause of death of a kangaroo and a mara. However, there has not been a T. gondii report at a Zoo population level in Chile in the last 35 years. The aim of the study was to estimate the seroprevalence and risk factors associated with T. gondii infection in mammals housed in a zoo located in the Metropolitan Region of Chile between 2011 and 2018. In this study, we analyzed 350 samples, from 324 animals, belonging to 57 species of carnivores, non-human primates, macropodids, ungulates and rodents to detect the presence of anti-T. gondii antibodies. Additionally, 20 animals were longitudinally sampled to evaluate intra-zoo infection. Using a commercial indirect Enzyme-Linked Immuno Sorbent Assay (ELISA) test, we detected T. gondii antibodies in 72 (22.2 %) samples. The overall seroprevalence estimates were 48.4 % in felines, 22.9 % in non-feline carnivores, 21.1 % in ungulates and 15.0 % in non-human primates. There were no positive samples from rodents or marsupials. Of animals sampled longitudinally, only a culpeo fox (Lycalopex cualpaeus) became seropositive along the study indicating exposition inside the facility. T. gondii seroprevalence differed significantly in taxonomic groups (p = 0.003), felines are statistically different from non-feline carnivores (NFC) (p = 0.040), ungulate (p = 0.027) and non-human primates (NHP) (p = 0.009). Annual prevalence comparison was performed showing no statistical difference (p = 0.941). A multivariable logistic regression was performed to ascertain the effect of taxonomic groups, proximity to water sources, diet, sex and type of housing on seropositivity. Only taxonomic group was statistically significant, indicating that NFC (OR = 0.35; 95 % CI = 0.15 – 0.83; p = 0.017), ungulates (OR = 0.30; 95 % CI = 0.13 – 0.69; p = 0.005), and NHP (OR = 0.25; 95 % CI = 0.09 – 0.72; p = 0.010) have lower risk of positivity to T. gondii compared to felines. Additionally, a black-faced spider monkey (Ateles chamek) and a siamang (Symphalangus syndactylus) were seropositive, being the first description of T. gondii infection in these species worldwide. As seen in previous studies, the widespread presence and exposure of T. gondii in zoo mammals was confirmed, and there may be contact with the agent and transmission within the zoo, which was confirmed by one animal became seropositive over the time. This fact could be a health problem for animals susceptible to fatal toxoplasmosis.