Deep humoral profiling coupled to interpretable machine learning unveils diagnostic markers and pathophysiology of schistosomiasis.

Schistosomiasis, a highly prevalent parasitic disease, affects more than 200 million people worldwide. Current diagnostics based on parasite egg detection in stool detect infection only at a late stage, and current antibody-based tests cannot distinguish past from current infection. Here, we developed and used a multiplexed antibody profiling platform to obtain a comprehensive repertoire of antihelminth humoral profiles including isotype, subclass, Fc receptor (FcR) binding, and glycosylation profiles of antigen-specific antibodies. Using Essential Regression (ER) and SLIDE, interpretable machine learning methods, we identified latent factors (context-specific groups) that move beyond biomarkers and provide insights into the pathophysiology of different stages of schistosome infection. By comparing profiles of infected and healthy individuals, we identified modules with unique humoral signatures of active disease, including hallmark signatures of parasitic infection such as elevated immunoglobulin G4 (IgG4). However, we also captured previously uncharacterized humoral responses including elevated FcR binding and specific antibody glycoforms in patients with active infection, helping distinguish them from those without active infection but with equivalent antibody titers. This signature was validated in an independent cohort. Our approach also uncovered two distinct endotypes, nonpatent infection and prior infection, in those who were not actively infected. Higher amounts of IgG1 and FcR1/FcR3A binding were also found to be likely protective of the transition from nonpatent to active infection. Overall, we unveiled markers for antibody-based diagnostics and latent factors underlying the pathogenesis of schistosome infection. Our results suggest that selective antigen targeting could be useful in early detection, thus controlling infection severity.

Pre-vaccination Schistosoma mansoni and hookworm infections are associated with altered vaccine immune responses: a longitudinal analysis among adolescents living in helminth-endemic islands of Lake Victoria, Uganda.

Background: Variations in vaccine responses have been observed between populations. A role for helminth infections has been proposed due to their immunomodulatory properties. In a secondary analysis of data from a randomised trial assessing effects of anthelminthic treatment on vaccine responses, we examined associations between helminth infections at baseline prior to vaccine administration, and vaccine responses among adolescents (9-17 years) in Koome Islands, Lake Victoria, Uganda. Methods: Participants received BCG [week 0], yellow fever (YF-17D), oral typhoid (Ty21a), HPV-prime [week 4], and HPV-boost, tetanus/diphtheria [week 28]. Outcomes were BCG-specific interferon-γ ELISpot responses and antibody responses to yellow-fever-, typhoid-, HPV-, tetanus- and diphtheria-specific antigens measured at two time points post vaccination. S. mansoni infection was determined as positive if either the plasma Circulating Anodic Antigen (CAA) assay or stool PCR were positive. Hookworm and Strongyloides were determined by stool PCR. Linear mixed effects regression was used to assess associations. Results: Among 478 adolescents, 70% were Schistosoma mansoni (Sm) infected and 23% hookworm infected at baseline. Sm was associated with lower Salmonella Typhi O:LPS-specific IgG responses (adjusted geometric mean ratio (aGMR) 0.69 (0.57-0.83)), and hookworm with higher diphtheria-specific IgG (aGMR 1.16 (1.02, 1.31)) and lower HPV-16-specific IgG (aGMR 0.70 (0.55, 0.90)) post-vaccination. High Sm intensity was associated with lower BCG-specific interferon-γ and S. Typhi O:LPS-specific IgG. Conclusions: We found inverse associations between Sm and responses to two live vaccines, whereas hookworm was positively associated with diphtheria-specific IgG. These findings support the hypothesis that helminth infections can modulate vaccine responses, while also highlighting potential heterogeneity in the direction of these effects.

Assessment of accuracy of liver fluke diagnostic tests using the gold standard of total worm counts.

In many regions of New Zealand liver fluke is endemic, infecting most grazing ruminants, including cattle, sheep, and deer. Restricting the economic losses and welfare costs associated with liver fluke relies on accurately identifying those animals with a production limiting infection. This has proven a difficult goal and although several antemortem quantitative tests are available, including faecal egg counts (FEC), serum ELISA and copro-antigen ELISA, none can be considered a gold standard test of liver fluke infection. The accepted gold standard test for fascioliasis is the total fluke count, which is both laborious and can only be completed at post-mortem. This study aimed to compare the performance of four liver fluke diagnostic tests, against the results of a gold standard total fluke count test. Two groups of cattle were selected, 29 culled mixed age beef cows (MAC) and ten 30-month-old steers. The cattle were blood sampled and faecal sampled prior to slaughter and their whole livers recovered post slaughter at the abattoir. Liveweight was also recorded at slaughter. After collection, each liver was weighed, scored for gross pathology, then serum, faeces and livers were frozen at -20 °C for later analysis. Faecal egg counts and F. hepatica copro-antigen ELISA tests were completed on the faecal samples and total fluke counts were completed on the livers. Fasciola hepatica antibody concentration in serum samples were quantified using a commercial ELISA test. Poisson regression models were built to model the association between each diagnostic test and the total fluke count, and a linear regression model was built to examine the relationship between each diagnostic test and live weight at slaughter. The median fluke count was significantly higher in MAC than steers (p = 0.01), and F. hepatica eggs were present in 100% steers and 66% MAC. There was a significant effect of copro-antigen ELISA value on total fluke count (p < 0.0001), with a coproantigen ELISA value = 20.1 predicting 10 flukes and a value = 44.8 predicting 30 flukes. There was also a significant effect of FEC on total fluke count (p = 0.002) but the R-squared value for this model was lower. There was no association between liver fibrosis score or antibody ELISA test and total fluke count (p = 0.95, p = 0.73, respectively). There was a significant effect of total fluke count (p = 0.03) on liveweight at slaughter, with liveweight falling 20.4 kg for each unit increase in loge (total fluke count). There was no effect of FEC (p = 0.11), antibody ELISA (p = 0.55) or copro-antigen ELISA value (p = 0.16) on liveweight at slaughter. Taken together, these results show that the coproantigen ELISA test is the better test for estimating the true liver fluke burden and that the number of flukes in the liver has a negative effect on cattle live weights at slaughter.

Evaluating the modulation of peripheral immune profile in people living with HIV and (Neuro)cysticercosis.

BACKGROUND: The parasitic infection caused by Taenia solium represents a significant public health concern in developing countries. Larval invasion of body tissues leads to cysticercosis (CC), while central nervous system (CNS) involvement results in neurocysticercosis (NCC). Both conditions exhibit diverse clinical manifestations, and the potential impact of concomitant HIV infection especially prevalent in sub-Saharan Africa on peripheral and CNS immune responses remains poorly understood. This study aimed to identify the potential impact of HIV coinfection in CC and NCC patients. METHODOLOGY: A nested study within a cross-sectional analysis in two Tanzanian regions was performed and 234 participants (110 HIV+ and 124 HIV-) were tested for cysticercosis antibodies, antigens, CD4 counts and serum Th1 and Th2 cytokines via multiplex bead-based immunoassay. 127 cysticercosis seropositive individuals underwent cranial computed tomography (CCT) and clinical symptoms were assessed. Multiple regression analyses were performed to identify factors associated with cytokine modulation due to HIV in CC and NCC patients. RESULTS: Serologically, 18.8% tested positive for cysticercosis antibodies, with no significant difference HIV+ and HIV+. A significantly higher rate of cysticercosis antigen positivity was found in HIV+ individuals (43.6%) compared to HIV- (28.2%) (p = 0.016). CCT scans revealed that overall 10.3% had active brain cysts (NCC+). Our study found no significant changes in the overall cytokine profiles between HIV+ and HIV- participants coinfected CC and NCC, except for IL-5 which was elevated in HIV+ individuals with cysticercosis. Furthermore, HIV infection in general was associated with increased levels of pro-and some anti-inflammatory cytokines e.g. TNF-α, IL-8, and IFN-γ. However, based on the interaction analyses, no cytokine changes were observed due to HIV in CC or NCC patients. CONCLUSIONS: In conclusion, while HIV infection itself significantly modulates levels of key cytokines such as TNF-α, IL-8, and IFN-γ, it does not modulate any cytokine changes due to CC or NCC. This underscores the dominant influence of HIV on the immune system and highlights the importance of effective antiretroviral therapy in managing immune responses in individuals coinfected with HIV and CC/NCC.

Identification of Schistosoma haematobium and Schistosoma mansoni linear B-cell epitopes with diagnostic potential using in silico immunoinformatic tools and peptide microarray technology.

INTRODUCTION: Immunoinformatic tools can be used to predict schistosome-specific B-cell epitopes with little sequence identity to human proteins and antigens other than the target. This study reports an approach for identifying schistosome peptides mimicking linear B-cell epitopes using in-silico tools and peptide microarray immunoassay validation. METHOD: Firstly, a comprehensive literature search was conducted to obtain published schistosome-specific peptides and recombinant proteins with the best overall diagnostic performances. For novel peptides, linear B-cell epitopes were predicted from target recombinant proteins using ABCpred, Bcepred and BepiPred 2.0 in-silico tools. Together with the published peptides, predicted peptides with the highest probability of being B-cell epitopes and the lowest sequence identity with proteins from human and other pathogens were selected. Antibodies against the peptides were measured in sera, using peptide microarray immunoassays. Area under the ROC curve was calculated to assess the overall diagnostic performances of the peptides. RESULTS: Peptide AA81008-19-30 had excellent and acceptable diagnostic performances for discriminating S. mansoni and S. haematobium positives from healthy controls, with AUC values of 0.8043 and 0.7326 respectively for IgG. Peptides MS3_10186-123-131, MS3_10385-339-354, SmSPI-177-193, SmSPI-379-388, MS3-10186-40-49 and SmS-197-214 had acceptable diagnostic performances for discriminating S. mansoni positives from healthy controls with AUC values ranging from 0.7098 to 0.7763 for IgG. Peptides SmSPI-359-372, Smp126160-438-452 and MS3 10186-25-41 had acceptable diagnostic performances for discriminating S. mansoni positives from S. mansoni negatives with AUC values of 0.7124, 0.7156 and 0.7115 respectively for IgG. Peptide MS3-10186-40-49 had an acceptable diagnostic performance for discriminating S. mansoni positives from healthy controls, with an AUC value of 0.7413 for IgM. CONCLUSION: One peptide with a good diagnostic performance and nine peptides with acceptable diagnostic performances were identified using the immunoinformatic approach and peptide microarray validation. There is need for evaluation of the peptides with true negatives and a good standard positive reference.

Optimizing sheep B-cell epitopes in Echinococcus granulosus recombinant antigen P29 for vaccine development.

Background: Echinococcus granulosus is a widespread zoonotic parasitic disease, significantly impacting human health and livestock development; however, no vaccine is currently available for humans. Our preliminary studies indicate that recombinant antigen P29 (rEg.P29) is a promising candidate for vaccine. Methods: Sheep were immunized with rEg.P29, and venous blood was collected at various time points. Serum was isolated, and the presence of specific antibodies was detected using ELISA. We designed and synthesized a total of 45 B cell monopeptides covering rEg.P29 using the overlap method. ELISA was employed to assess the serum antibodies of the immunized sheep for recognition of these overlapping peptides, leading to the preliminary identification of B cell epitopes. Utilizing these identified epitopes, new single peptides were designed, synthesized, and used to optimize and confirm B-cell epitopes. Results: rEg.P29 effectively induces a sustained antibody response in sheep, particularly characterized by high and stable levels of IgG. Eight B-cell epitopes of were identified, which were mainly distributed in three regions of rEg.P29. Finally, three B cell epitopes were identified and optimized: rEg.P2971-90, rEg.P29151-175, and rEg.P29211-235. These optimized epitopes were well recognized by antibodies in sheep and mice, and the efficacy of these three epitopes significantly increased when they were linked in tandem. Conclusion: Three B-cell epitopes were identified and optimized, and the efficacy of these epitopes was significantly enhanced by tandem connection, which indicated the feasibility of tandem peptide vaccine research. This laid a solid foundation for the development of epitope peptide vaccine for Echinococcus granulosus.

An evaluation of a lateral flow rapid diagnostic test for Strongyloides stercoralis infection.

Strongyloides stercoralis in humans often presents as a chronic asymptomatic infection. Diagnosis can be challenging due to the limited sensitivity of faecal-based parasitological techniques. A prototype lateral flow rapid diagnostic test (RDT) for the detection of specific antibodies against Strongyloides stercoralis (SsRapid) was evaluated using 143 samples from the serum bank of the Swiss Tropical and Public Health Institute. Group 1 (n = 30) comprised serum samples from larvae-positive individuals; the RDT's diagnostic sensitivity was 97 % (29/30). Group II comprised serum samples from patients with other parasitic infections (n = 86) and Swiss blood donors (n = 27); the RDT's diagnostic specificity for this group was 90 % (102/113). The RDT showed good diagnostic performance and is a promising point-of-care test for detecting human Strongyloides stercoralis infection.

Characteristics of SARS-CoV-2 and Opisthorchis viverrini coinfections: insights into immune responses and clinical outcomes.

The effects of co-infections with SARS-CoV-2 and parasitic diseases have been little investigated in terms of immune response, disease dynamics, and clinical outcomes. This study aimed to explore the impact of co-infection with Opisthorchis viverrini and SARS-CoV-2 on the immune response concerning clinical symptoms and the severity of pulmonary abnormalities. A cross-sectional study was conducted, including healthy participants as controls, participants with opisthorchiasis, SARS-CoV-2 infection, and a co-infection group with both diseases. Characteristics of SARS-CoV-2 infection were assessed based on clinical parameters and severity of pulmonary abnormalities, whereas opisthorchiasis burden was evaluated by eggs-per-gram (EPG) counts. Immune responses were assessed by measuring levels of interferon-γ (IFN-γ), SARS-CoV-2 anti-spike receptor binding domain (RBD) IgG, and neutralizing antibody against SARS-CoV-2. In the co-infected group, clinical parameters and hospitalization rates were lower than in the SARS-CoV-2 group. Pulmonary abnormalities, such as bronchial fibrosis, were commonly observed in the SARS-CoV-2 group, leading to hospitalization in some cases. Participants with opisthorchiasis had higher IFN-γ levels than healthy individuals. IFN-γ levels were significantly lower in the co-infection group compared with the SARS-CoV-2 group (P = 0.002). There was a significant (P = 0.044) positive correlation between RBD-specific IgG and percent neutralization levels in the SARS-CoV-2 group. Levels of both were somewhat lower (not statistically significant) in the co-infection group. A negative correlation was observed between opisthorchiasis burden (EPG counts) and IFN-γ and RBD-specific IgG levels in the co-infected group. Following vaccination, the increase in IgG levels against the RBD protein was significantly lower in the co-infected group than in the SARS-CoV-2 group. These results suggest that O. viverrini infection suppresses immune responses and may lead to a reduction in severity in cases of SARS-CoV-2 co-infection.

Detection of Anti-Strongyloides Antibodies in the Serum of Liver Transplant Recipients: Need of Screening for This Neglected Helminthiasis.

Immunosuppressed patients, particularly transplant recipients, can develop severe strongyloidiasis. This study aimed to detect anti-Strongyloides IgG antibodies in a panel of sera from liver transplant patients. Two techniques were used: ELISA as the initial screening test and Western blotting as a confirmatory test. ELISA reactivity of 10.9% (32/294) was observed. The 40-30 kDa fraction was recognised in 93.7% (30/32) of the patients, resulting in a positivity rate of 10.2%. These data highlight the importance of serological screening for Strongyloides stercoralis infection in liver transplant recipients.

Evaluation of Somatic Antigens of Adult Toxocara helminthes for Detection of Human Toxocariasis.

We aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) to evaluate the presence of specific IgG against Toxocara canis and Toxocara cati somatic antigens on the serum of patients with toxocariasis. The sensitivity, specificity, positive and negative predictive values for indirect-ELISA were calculated by receiver operating characteristic curve (ROC) analysis and Youden's J using Likelihood ratio. All statistics were analysed and graphs are plotted using GraphPad Prism version 8.4.3 (Graph Pad Software, La Jolla, CA, USA), with 95% confidence interval (CI). The sensitivity, specificity, positive and negative predictive values for T. canis were 100%, 82%, 79% and 100%, respectively. The mentioned variables for T. cati were 97%, 82%, 78% and 98%, respectively. Five immune reactive bands of 38, 40, 72, 100 and 250 kDa were common in both species. Toxocara crude antigens were highly immunogenic in human sera. Immunoreactive bands against T. canis compared to T. cati somatic antigen were about two times more. Unlike Toxocara excretory-secretory antigen, that was homologue in two species, somatic antigens of T. canis and T. cati showed different immunoreactive bands in our western blot.

Serological diagnosis of strongyloidiasis: An evaluation of three commercial assays.

BACKGROUND: Strongyloidiasis is caused by a neglected nematode, manifesting as chronic intestinal infection with potentially severe manifestations. The disease is an emerging problem in non-endemic countries affecting travelers and migrants. Diagnosis of strongyloidiasis is hampered by the lack of standardization and absence of a gold standard. Since adequate direct methods to detect the motile larvae in stool samples are not widely available, other techniques such as serology have been developed. METHODS: We evaluated three commercial ELISA kits (DRG Instruments, IVD Research, and Bordier Affinity Products) to detect IgG antibodies against Strongyloides stercoralis assays utilizing serum samples from travelers with microscopically confirmed strongyloidiasis (n = 50) and other imported helminthic infections (n = 159) as well as healthy controls (n = 50). RESULTS: The DRG, IVD, and Bordier assays showed sensitivities of 58.0%, 64.0%, and 56.0%, respectively. Specificity values were 96.0%, 96.0%, and 92.0% in healthy controls, and 67.3%, 62.9%, and 76.7% in cases with other helminth infections, respectively. Cross-reactions were mostly observed in cases with other nematodes (37.5%, 42.5%, and 20.0%, respectively), but also in trematode (33.3%, 38.1%, and 19.0%, respectively) and in cestode infections (25.0%, 30.0%, and 32.5%, respectively). CONCLUSION: The study demonstrates the diagnostic limitations of serological assays to detect or exclude cases of strongyloidiasis in returning travelers, who frequently present with recent or acute infections.

Production and evaluation of a new set of recombinant antigens for the serological diagnosis of human cysticercosis.

Human cysticercosis caused by Taenia soliun (T. soliun) is endemic in certain areas of Latin America, Asia and Sub-Saharan Africa. Neurocysticercosis (NCC) is mainly diagnosed by neuroimaging, which, in most cases, is unavailable in endemic areas. Due to their high sensitivity and specificity, serological tests such as enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) based on the glycosylated fraction of the cyst CS50 are widely used for the detection of the anti-cysticercus IgG antibodies despite their significant cost and the need of cysticercus material. Given their cost-effectivess and simplicity, immunoassays based on recombinant proteins could provide new alternatives for human cysticercosis diagnosis: such tests would be aimed at screening those people living in remote areas who need further examination. To date, however, no test using recombinant antigens is commercially available. Herein, five recombinant proteins (R14, R18, R93.1, R914.1, and R915.2) were produced, three of which (R93.1, R914.1, and R915.2) were newly identified from the cyst fluid. Evaluation of the diagnostic performance of these recombinant antigens by ELISA was done using sera from 200 epileptic and non-epileptic individuals in comparison with the WB-CS50 as the reference serological method. Recombinant proteins-based ELISA showed a level of diagnostic performance that is inferior than the reference serological method, but similar to that of the native antigen ELISA for human cysticercosis (commonly used for screening). Further optimization of expression conditions is still needed in order to improve proteins solubility and enhance diagnostic performance for human cysticercosis detection. However, this preliminary evaluation of the recombinant antigens has shown their potential valuable use for screening cysticercosis in patients with epilepsy attending dispensaries in remote areas. Future studies should be conducted to evaluate our recombinant antigens in a large group of patients with different stages of NCC, and in correlation with imaging findings.

A silent threat behind bars: Serosurvey and associated risk factors for strongyloidiasis in female inmates and correctional officers.

Strongyloidiasis has been a neglected parasitic infection caused by Strongyloides genus parasites. Despite assessment of S. stercoralis exposure in different vulnerable populations, seroprevalence in inmates worldwide remains to be fully established. Due to poor sanitation and lack of personal hygienic practices, incarcerated individuals have been considered prone to spread infectious illnesses. Accordingly, the present study has assessed exposure and associated risk factors for strongyloidiasis in women inmates and correctional officers at the Women's State Penitentiary of Parana, part of the third largest incarceration complex in Brazil at the time. Blood samplings were performed in 2020 and 2021from a total of 503 women inmates and 92 correctional officers. Participants voluntarily responded to an epidemiological questionnaire to assess associated risk factors to strongyloidiasis. Serological analysis was performed by ELISA for anti-S. stercoralis IgG detection. Statistical analysis was performed using R software, adopting a 5% level of significance. The data were submitted to univariate analysis by chi-square or Fisher´s Exact test for assessing the association among seropositivity and the variables. The variables with p-value < 0.2 in the univariate analysis were considered fit to be included in the logistic regression. In overall, 356/503 (70.8%; 95% CI: 66.7-74.6) inmates were seropositive for anti-S. stercoralis antibodies, with no statistically associated risk factor to seropositivity. A total of 57/92 (62.0%; 95% CI: 51.8-71.2) correctional officers were seropositive, and logistic regression revealed that individuals older than 50 years were more likely seropositive. In conclusion, the high endemicity observed herein has indicated a history of previous exposure to S. stercoralis and warned for a systematic strongyloidiasis screening for inmates, to prevent long term morbidity and disseminated infection during incarceration.

Design and expression of a chimeric recombinant antigen (SsIR-Ss1a) for the serodiagnosis of human strongyloidiasis: Evaluation of performance, sensitivity, and specificity.

BACKGROUND: The sensitivity of parasitological and molecular methods is unsatisfactory for the diagnosis of strongyloidiasis, and serological techniques are remaining as the most effective diagnostic approach. The present study aimed to design and produce a chimeric recombinant antigen from Strongyloides stercoralis immunoreactive antigen (SsIR) and Ss1a antigens, using immune-informatics approaches, and evaluated its diagnostic performance in an ELISA system for the diagnosis of human strongyloidiasis. METHODOLOGY/PRINCIPAL FINDINGS: The coding sequences for SsIR and Ss1a were selected from GenBank and were gene-optimized. Using bioinformatics analysis, the regions with the highest antigenicity that did not overlap with other parasite antigens were selected. The chimeric recombinant antigen SsIR- Ss1a, was constructed. The solubility and physicochemical properties of the designed construct were analyzed and its tertiary structures were built and evaluated. The construct was expressed into the pET-23a (+) expression vector and the optimized DNA sequences of SsIR-Ss1a (873 bp) were cloned into competent E. coli DH5α cells. Diagnostic performances of the produced recombinant antigen, along with a commercial kit were evaluated in an indirect ELISA system, using a panel of sera from strongyloidiasis patients and controls. The physicochemical and bioinformatics evaluations revealed that the designed chimeric construct is soluble, has a molecular with of 35 KDa, and is antigenic. Western blotting confirmed the immunoreactivity of the produced chimeric recombinant antigen with the sera of strongyloidiasis patients. The sensitivity and specificity of the indirect ELISA system, using the produced SsIR-Ss1a chimeric antigen, were found to be 93.94% (95% CI, 0.803 to 0.989) and 97.22% (95% CI, 0.921 to 0.992) respectively. CONCLUSIONS/SIGNIFICANCE: The preliminary findings of this study suggest that the produced SsIR-Ss1a chimeric antigen shows promise in the diagnosis of human strongyloidiasis. However, these results are based on a limited panel of samples, and further research with a larger sample size is necessary to confirm its accuracy. The construct has potential as an antigen in the ELISA system for the serological diagnosis of this neglected parasitic infection, but additional validation is required.

Efficacy of a Haemonchus contortus vaccine under field conditions in young alpacas.

Anthelmintic resistance to Haemonchus contortus creates increasing management challenges with small ruminants and camelids. The commercial vaccine, Barbervax®, contains H11 and H-gal-GP antigens, derived from gut mucosal membrane enzymes of H. contortus involved in digesting blood. Antibody neutralization of these antigens causes failure of H. contortus to digest blood, resulting in parasite death. H11 and H-gal-GP are considered "hidden" antigens, meaning the host immune system does not encounter these proteins under natural infection. Therefore, repeat immunization is required to maintain protective humoral responses. One previous study evaluated the safety of Barbervax® in camelids but the efficacy could not be assessed due to lack of successful infection in the controls. The objective of the current study was to evaluate clinical parameters of anemia, fecal egg counts (FECs), and humoral immune responses of healthy alpacas after immunizing with Barbervax® compared to non-vaccinated controls, all under natural environmental exposure on parasite-laden pastures. A crossover-like study was performed where twenty alpacas (298 ± 66 days of age) were assigned to be initially vaccinated with Barbervax® (n=10) or receive no treatment (n=10). Three doses of Barbervax® were administered at three-week intervals. Feces and blood were collected on Day -10, 0, 21, 43, 64, 85, 106, and 135 to evaluate FECs, packed cell volume (PCV), and antibody titers. Each group was kept on separate adjacent pastures. Tracer sheep (n=2 per study group) were introduced on Day 43 for a three-week period to ensure parasite acquisition. For the crossover-like component on Day 85, the initial non-vaccinated group was administered Barbervax® with dosing repeated on Day 106 and 135. Results indicated all initially vaccinated alpacas produced antibody titers to vaccine antigen that corresponded to lower mean FECs compared to the initially non-vaccinated group. A reduced mean FEC in the vaccinate group was observed 21 days after peak antibody titers. Similarly, when pooled vaccinate antibody titers were noted to wane on Day 106, an increase in FEC was observed at the following time point (Day 135). Conclusions from our study support the use of Barbervax® to reduce H. contortus burdens in alpacas. Furthermore, a less than 30-day lag time between antibody titer and resultant effect in FECs was observed. Additional studies assessing the ability of Barbervax® to reduce H. contortus burdens during subsequent grazing seasons would provide even greater information regarding the use of Barbervax® within alpaca herds to modulate H. contortus infections, refugia, and anthelmintic use.

Construction and mouse antibody response evaluation of juvenile stage-specific chimeric protein from Fasciola gigantica.

Fasciolosis, caused by the liver fluke Fasciola gigantica, is a major parasitic disease that affects livestock and therefore causes significant economic losses in tropical countries. Although anthelminthic drugs can kill the parasite, drug-resistant liver fluke populations are increasing. In this study, a recombinant F. gigantica chimeric protein (rFgCHI) consisting of cathepsin L1H (FgCL1H), cathepsin B3 (FgCB3), and Saposin-like protein 1 (FgSAP1) was designed and expressed in Escherichia coli (BL21). The molecular weight of rFgCHI was 61 kDa. To study the antibody response, male BALB/c mice were immunized via the subcutaneous injection of rFgCHI combined with Quil A. Immunization with rFgCHI showed the induction of IgG1 and IgG2a with a higher IgG1 isotype level, indicating the potential of mixed Th1/Th2 immune responses, with Th2 predominating. However, the results showed high levels of IgG against the single proteins, except for rFgSAP1. Through Western blotting, mouse anti-rFgCHI polyclonal antibodies could be detected to the native proteins obtained from the parasite at all stages. Immunolocalization also revealed that the anti-rFgCHI antibodies could detect targeted antigens in the cecal epithelium of the parasite. These results demonstrated that rFgCHI is immunogenic to the mouse immune system and may potentially be a protein candidate for the development of a fasciolosis vaccine.

Evaluation of Seroprevalence and Risk Factors of Heartworm Infection for Dogs in Rio de Janeiro with Access to Veterinary Care.

Heartworm infection is a chronic disease with clinical signs and effects ranging from an asymptomatic condition to severe disease and death. The prevalence of heartworm disease in the state of Rio de Janeiro has been reported to be high (21.3%). The present study was conducted to evaluate the seroprevalence and risk factors of heartworm infection for the canine population with access to veterinary services in different areas of the state of Rio de Janeiro, Brazil. A total of 1787 canine blood samples were obtained from 135 practices across 8 different areas of Rio de Janeiro state (Rio de Janeiro municipality, São Gonçalo municipality, Niterói municipality, Baixada Fluminense, and the northern, southern, eastern, and mountainous areas) and tested for the presence of Dirofilaria immitis antigens and antibodies against several tick-borne disease pathogens using a commercial immunochromatography technique (Vetscan® Flex 4 Rapid Test; Zoetis; NJ USA). Pet owners reported living conditions, physical characteristics, demographics, and clinical signs for evaluation of risk factors for heartworm infection. Only two evaluated risk factors were shown to enhance the risk for D. immitis infection, including having a short hair coat vs. having a medium or long hair coat (OR 2.62) or positive for antibodies to tick-borne disease parasites (OR 3.83). Clinical signs reported for dogs with heartworm disease were typical for that condition. The overall prevalence of heartworm disease in the state was 8.2%, ranging from 2.4% in the mountainous region to 29.4% in the eastern area. It could not be determined if veterinarians were not diligent about dispensing heartworm preventatives or if poor levels of compliance by dog owners were responsible for higher infection rates in some areas of the state.

Evaluation of copromicroscopy, multiplex-qPCR and antibody serology for monitoring of human ascariasis in endemic settings.

BACKGROUND: The standard diagnosis of Ascaris lumbricoides and other soil-transmitted helminth (STH) infections relies on the detection of worm eggs by copromicroscopy. However, this method is dependent on worm patency and shows only limited accuracy in low-intensity infection settings. We aimed to decipher the diagnostic accuracy of different antibodies using various Ascaris antigens in reference to copromicroscopy and quantitative PCR (qPCR), four months after national STH preventative chemotherapy among school children in western Kenya. METHODOLOGY: STH infection status of 390 school children was evaluated via copromicroscopy (Kato-Katz and mini-FLOTAC) and qPCR. In parallel, Ascaris-specific antibody profiles against larval and adult worm lysates, and adult worm excretory-secretory (ES) products were determined by enzyme-linked immunosorbent assay. Antibody cross-reactivity was evaluated using the closely related zoonotic roundworm species Toxocara cati and Toxocara canis. The diagnostic accuracy of each antibody was evaluated using receiver operating curve analysis and the correspondent area under the curve (AUC). PRINCIPAL FINDINGS: Ascaris was the predominant helminth infection with an overall prevalence of 14.9% (58/390). The sensitivity of mini-FLOTAC and Kato-Katz for Ascaris diagnosis reached only 53.5% and 63.8%, respectively compared to qPCR. Although being more sensitive, qPCR values correlated with microscopic egg counts (R = -0.71, P<0.001), in contrast to antibody levels. Strikingly, IgG antibodies recognizing the ES products of adult Ascaris worms reliably diagnosed active Ascaris infection as determined by qPCR and microscopy, with IgG1 displaying the highest accuracy (AUC = 0.83, 95% CI: 0.75-0.91). CONCLUSION: IgG1 antibody responses against adult Ascaris-ES products hold a promising potential for complementing the standard fecal and molecular techniques employed for monitoring Ascaris infections. This is of particular importance in the context of deworming programs as the antibody diagnostic accuracy was independent of egg counts.

Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens.

Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein's specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system.

Atypical Toxocara canis-Induced Hepatic Visceral Larva Migrans: Diagnostic Challenges and Literature Review.

Toxocariasis, a zoonotic infection transmitted by Toxocara canis (from dogs) and Toxocara cati (from cats) larvae, poses rare but severe risks to humans. We present a case of hepatic visceral larva migrans (VLM) caused by Toxocara canis in a 21-year-old male with a history of close contact with a pet dog. Initial symptoms and imaging findings mimicked a pyogenic liver abscess. The initial laboratory investigations revealed neutrophilia and elevated levels of IgE. Despite broad-spectrum antibiotics, persistent fever prompted further investigation. Subsequent serological testing for Toxocara antibodies and histopathological analysis of liver tissue demonstrating eosinophil infiltrates and Charcot-Leyden crystals led to a confirmed diagnosis of a liver abscess caused by Toxocara canis. Serological testing for Toxocara antibodies and histopathological analysis of liver tissue confirmed a Toxocara canis-induced liver abscess. Albendazole treatment yielded significant clinical improvement. This case highlights the necessity of considering toxocariasis in liver abscess differentials, particularly in high-seroprevalence regions like Vietnam. Relying solely on serological tests may be insufficient, emphasizing the need for corroborative evidence, including invasive procedures like liver biopsy, for accurate hepatic toxocariasis diagnosis.