Le et al. Parasites Vectors (2020) 13:109 https://doi.org/10.1186/s13071-020-3980-z Parasites & Vectors SHORT REPORT Open Access Chronic whipworm infection exacerbates Schistosoma mansoni egg-induced hepatopathology in non-human primates Loc Le1,2†, Sabiha Khatoon1,2†, Paola Jiménez1,2, Christopher Peterson2, Rebecca Kernen2, Weidong Zhang1,2, Adebayo J. Molehin1,2, Samra Lazarus1,2, Justin Sudduth1,2, Jordan May1,2,3, Souvik Karmakar4, Juan U. Rojo5, Gul Ahmad6, Workineh Torben7, David Carey8, Roman F. Wolf8,9, James F. Papin8 and Afzal A. Siddiqui1,2* Abstract Background: Schistosomiasis continues to infict signifcant morbidity and mortality in the tropical and subtropical regions of the world. The disease endemicity overlaps with the transmission of other parasitic diseases. Despite the ubiquity of polyparasitism in tropical regions, particularly in rural communities, little is known about the impact of multiple helminth infections on disease progression. In this pilot study, we describe the infuence of chronic Trichuris trichiura infection on Schistosoma mansoni egg-induced hepatopathology in infected baboons. Methods: Baboons with or without underlying whipworm infection were challenged with S. mansoni cercariae to establish schistosomiasis. Adult S. mansoni worms were recovered by perfusion and enumerated, hepatic granulomas were quantifed via light microscopy, and transcriptional profling of tissues were completed using RNA sequencing technologies. Results: Co-infection with both S. mansoni and T. trichiura resulted in higher female schistosome worm burden and signifcantly larger liver granuloma sizes. Systems biology analyses of peripheral blood mononuclear cells (PBMC) revealed pathways associated with increased liver damage in co-infected baboons. Conclusions: Underlying chronic whipworm infection intensifed schistosome egg-induced liver pathology in infected baboons. RNA-Seq analysis provided insight into pathways associated with increased liver damage, corrobo- rating histological fndings. Keywords: Schistosomiasis, Trichuriasis, Helminths, Co-infection, NTDs, Hepatopathology Background poverty despite current eforts to control and interrupt Neglected tropical diseases (NTDs) are a group of dis- transmission using mass drug administration. Currently, eases that disproportionately afect communities of 252 million people are infected with schistosomiasis, poverty. Te lack of adequate infrastructure and pub- an estimate that continues to rise as diagnostic meth- lic sanitation in these communities contribute to main- odologies become more refned [1]. Chronic intestinal taining the vicious cycle of chronic disease burden and schistosome infection can result in hepatosplenomegaly and eventually death due to internal bleeding. It is not *Correspondence: [email protected] uncommon for the same populations to be infected or †Loc Le and Sabiha Khatoon contributed equally to this work be at risk for infection with soil-transmitted helminths 1 Center for Tropical Medicine and Infectious Diseases, Texas Tech (STH) [2]. Over 465 million people currently live with University Health Sciences Center, Lubbock, TX, USA Full list of author information is available at the end of the article whipworm infection [1]. Chronic whipworm, or Trichuris © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Le et al. Parasites Vectors (2020) 13:109 Page 2 of 7 trichiura, infection can result in abdominal pain, anemia, colonies commonly harbor T. trichiura and, in those and wasting, particularly in children. Anemia and Tri- cases, deworming with anthelmintics is routine. In this churis Dysentery Syndrome contribute to protein energy study, baboons selected for the co-infection group did malnutrition which leads to an immunocompromised not undergo deworming and specifc-pathogen-free state that can exacerbate subsequent helminth infections baboons were utilized for the single infection group. We [3, 4]. As major sources of morbidity and disability rather subsequently exposed all baboons to 1000 S. mansoni than mortality, the disability-adjusted life years attrib- cercariae percutaneously as previously described [7]. uted to schistosomiasis and trichuriasis are 3.31 million/ Eight weeks after infection with S. mansoni, necropsies year and 0.64 million/year, respectively [1]. were performed. Briefy, the hepatic portal system and There are insufficient studies on the interactions mesenteric vasculature were perfused to recover adult of trichuriasis and schistosomiasis, specifically on schistosome worms. Blood was collected for PBMC iso- the influence of co-infection on Schistosoma man- lation by density gradient centrifugation using HISTO- soni egg-induced hepatopathology. Others have found PAQUE-1077 (Sigma-Aldrich, St. Louis, Missouri, USA) that concomitant infections of Schistosoma japoni- and stored in freezing media (10% DMSO in fetal bovine cum and Trichuris significantly increased the odds of sera and RPMI) until later use for RNA-purifcation. Liver anemia in children [4] and that mice with established samples were collected for tissue egg burden assessment chronic Trichuris muris infection and challenged with and granuloma quantifcation via histology. For determi- S. mansoni developed significantly higher S. mansoni nation of schistosome egg burden in tissue, liver samples worm burden and egg and granuloma burden in the were digested overnight in 4% potassium hydroxide at liver [5]. To prepare for deployment of interventions 37 °C without CO2. Tis suspension was then washed and such as vaccines against schistosomiasis [6], it is criti- resuspended with a solution of 1.2% (w/v) NaCl and eggs cal to understand the interactions between potential were enumerated under light microscopy to determine confounding factors that will be present in endemic the number of eggs per gram of tissue [7]. Excised spleen areas, such as polyparasitism. This present study samples and mesenteric lymph nodes were mashed aimed to evaluate whether concurrent infection with through nylon cell strainers and stored in freezing media T. trichiura could influence S. mansoni egg-induced until RNA purifcation for RNA-sequencing. hepatopathology using the non-human primate model (baboons). Baboons develop a human-like acute schis- Histology tosomiasis syndrome after exposure to the cercariae Sections of liver were fxed in 10% neutral-bufered for- and chronic hepatic/intestinal clinical manifestations. malin, dehydrated in ascending grades of alcohol, and Herein, we describe the first transcription profiling embedded in parafn. Parafn blocks were cut into using next-generation sequencing for liver damage in 5-micron sections and processed for staining with hema- baboons infected with schistosomiasis and trichuriasis, toxylin and eosin. Stained slides were observed via light indicating several pathways by which co-infection may microscopy at 100× magnifcation for granuloma quan- exacerbate hepatopathology. tifcation as previously described [8]. Te diameter of each granuloma was measured via a straight line bisect- Methods ing the central egg and the area of each granuloma was Animals calculated assuming an area of a circle (Additional fle 1: Male and female olive baboons (Papio anubis) aged 2 to Figure S1). 4 years-old were obtained from the University of Okla- homa Health Sciences Center (OUHSC) and were housed RNA purifcation in facilities accredited by the AAALAC. Total RNA was isolated from PBMCs, splenocytes, and Schistosoma mansoni-infected Biomphalaria glabrata mesenteric lymph nodes of each animal using GenElute™ (Puerto Rican strain) snails were obtained from the mammalian total RNA miniprep kit (Millipore Sigma, Schistosomiasis Resource Center, Biomedical Research St. Louis, MO, USA) as previously described [7]. Total Institute (Rockville, MD, USA). RNA concentrations were measured using the Qubit® 3.0 Fluorometer and RNA HS assay kit (Termo Fisher Sci- Parasite challenge and necropsy entifc, Waltham, MA, USA). RNA quality was assessed We selected baboons that had prior exposure to T. trichi- using Agilent 2200 TapStation (Agilent, Santa Clara, CA). ura for the co-infection group (n = 10) and baboons that had no prior exposure to T. trichiura for the single infec- RNA sequencing and pathway analysis tion group (n = 10) as identifed by microscopic iden- Total RNA from PBMCs, spleen, and mesenteric lymph tifcation of T. trichiura eggs in stool samples. Baboon node cells were used to prepare the libraries as previously Le et al. Parasites Vectors