Successful Experimental Infant Baboon Model for Childhood Cryptosporidiosis Studies Ngalla E
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Jillani et al. Parasites Vectors (2021) 14:316 https://doi.org/10.1186/s13071-021-04804-4 Parasites & Vectors RESEARCH Open Access Successful experimental infant baboon model for childhood cryptosporidiosis studies Ngalla E. Jillani1* , Atunga Nyachieo1, Daniel C. Chai1 and James Nyabuga Nyariki2 Abstract Background: Cryptosporidiosis causes high morbidity and mortality in children under 2 years of age globally. The lack of an appropriate animal model that mimics the pathogenesis of disease in humans has hampered the develop- ment and testing of potential therapeutic options. This study aimed to develop and validate an infant baboon infec- tion model of cryptosporidiosis. Methods: Eighteen immunocompetent weaned infant baboons aged 12 to 16 months were used. The animals were n 3 controls and three experimental groups of n 5 animals each inoculated with Cryptosporidium parvum oocysts as= follows: group 1: 2 104, group 2: 2 105, group= 3: 2 106 followed by daily fecal sampling for oocyst evaluation. Blood sampling for immunological× assay× was done on the× day of infection and weekly thereafter until the end of the experiment, followed by necropsy and histopathology. Statistical analysis was performed using R, SPSS, and GraphPad Prism software. Analysis of variance (ANOVA) and Bonferroni post hoc tests were used for comparison of the means, with p < 0.05 considered as a signifcant diference. Correlation coefcient and probit analysis were also performed. Results: In all experimental animals but not controls, the onset of oocyst shedding occurred between days 2 and 4, with the highest oocyst shedding occurring between days 6 and 28. Histological analysis revealed parasite establish- ment only in infected animals. Levels of cytokines (TNF-α, IFN-γ, and IL-10) increased signifcantly in experimental groups compared to controls. Conclusion: For developing a reproducible infant baboon model, 2 104 oocysts were an efective minimum quan- tifable experimental infection dose. × Keywords: Cryptosporidiosis, Infant baboon, Cryptosporidium parvum, Children Background with diarrhea in humans of all ages, but more severe in Cryptosporidiosis is an infectious disease caused by children under 2 years worldwide [4, 5]. Tis neglected an apicomplexan oocyst-forming protozoan parasite, zoonotic disease, with a typically underestimated preva- Cryptosporidium spp. [1, 2]. Besides the two main spe- lence of 2–12% in the developing world, has received cies of Cryptosporidium infecting humans, namely C. little research attention, resulting in a limited under- hominis (which infects humans only) and C. parvum standing of its pathogenesis and less efective therapeu- (which infects both humans and bovines), other species tic solutions [3, 6]. Childhood cryptosporidiosis presents such as C. suis, C. meleagridis, C. andersoni, C. felis, C. a huge disease burden in the developing world, causing canis, C. muris, and C. baylei occasionally infect people morbidity and mortality, with possible long-term learn- as well [2, 3]. Tis enteropathogenic disease is associated ing defcits, stunted growth, and impaired physical ftness in afected children [2, 6–8]. *Correspondence: [email protected] Transmission is by the fecal–oral route through direct 1 Institute of Primate Research, Box 24481-00502, Karen Nairobi, Kenya contact with infected humans, animals, contaminated Full list of author information is available at the end of the article drinking water, or agricultural products [9]. Oocysts © The Author(s) 2021. 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. Jillani et al. Parasites Vectors (2021) 14:316 Page 2 of 8 excreted in feces can remain infectious for up to 2 Isolation of Cryptosporidium oocysts months [10]. Tis disease causes opportunistic infections Cryptosporidium parvum oocysts used in this study in both immunocompetent and immunocompromised were isolated by the foatation method and identifed by persons [5]. While the incidence of cryptosporidiosis acid-fast staining (modifed Ziehl–Neelsen method) as varies throughout the year, high levels occur during the described previously [7]. In this method, a lump of the warm rainy months [8]. fecal sample (about 1 g) was put into a container and Despite numerous attempts to control Cryptosporid- 5 ml of distilled water was added, thoroughly mixed, ium infections, there have not been efective animal then sieved into a beaker. Te fltrate was then trans- models that accurately mimic the disease in humans, as ferred into a 15 ml centrifuge tube and topped up with tools for testing newly developed or repurposed mol- distilled water to 15 ml, then centrifuged at 1500 rpm ecules for potential drugs that could efectively treat this for 10 min. Te supernatant was discarded, and 5 ml of condition [2, 7]. To better understand the disease, various sucrose solution of specifc gravity 1.27 was added and animal models, mainly murine and porcine models, have vortexed for 1 min. More sucrose solution was added to been utilized [11]. Te fndings from these lower animal bring it to 15 ml, until a convex meniscus was formed. models, however, cannot be extrapolated to humans, It was then covered with a coverslip and centrifuged since the disease pattern does not mirror that in humans at 1500 rpm for 10 min. Te coverslip was then care- due to the huge phylogenetic gap. Non-human primates, fully removed and washed by sprinkling PBS on it into which are phylogenetically closer to humans, are there- a glass beaker. Te 15 ml tube was again topped up fore attractive as models for understanding this disease with sucrose solution until it formed a meniscus, cov- [7]. Indeed, previous studies in pigtailed macaques dem- ered with a coverslip, centrifuged for another 10 min onstrated that these non-human primates were good at 1500 rpm, and oocysts were recovered as described models of the disease [7]. Similarly, in another study earlier. Tis process was repeated three times to ensure involving a cross-sectional survey in captive baboons the maximum recovery of oocysts. Te resultant solu- [12], C. hominis infection was detected mostly in infant tion with recovered oocysts from the beaker was then baboons, confrming that baboon infants are susceptible put into a 15 ml tube, topped up with PBS, and cen- to Cryptosporidium hominis infection and could be use- trifuged at 1500 rpm for 10 min. Te supernatant was ful in the development of a reproducible experimental discarded and the resultant pellets were suspended in infection model of childhood cryptosporidiosis. How- 1.5 ml PBS. One microliter of this solution was exam- ever, there is no study showing dose-controlled Crypto- ined under a microscope to determine the number of sporidium infection in baboon infants. Tus, the main oocysts in a 1 μl volume. Tis volume estimation for objective of this study was to validate and develop a oocyst numbers was then used to constitute appropri- reproducible infant baboon infection model of childhood ate inoculation doses. cryptosporidiosis through a quantifable Cryptosporid- ium dosage using Cryptosporidium parvum. Inoculation of animals Methods All selected experimental animals (n = 18) were starved Selection of animals in the morning of inoculation, and the procedures were carried out under general anesthesia. Tis involved the Weaned immunocompetent infant baboons (n = 18) of both sexes (10 males and 8 females) aged between 12 and administration of ketamine HCl at 10 mg/kg body weight 16 months and weighing between 3.0 and 7.1 kg were mixed with xylazine 5%, injected intramuscularly. During recruited into the study. Te sample size (n) was calcu- this time, blood samples, temperature, and body weight 2 measurements were taken. Te animals were randomized lated using the power analysis equation n = 1 + 2C(s/d) . Te animals were screened and treated for enteric worms to three experimental groups using sealed opaque enve- and diarrhea-causing bacteria, namely Shigella spp. and lopes. Quantifed parasite doses were administered to the Salmonella spp., before inoculation with doses of Crypto- animals on the table according to the groups. Te doses sporidium parvum oocysts. Tey were then housed in comprised three concentrations of the parasite: group 1: 2 104 (n 5), group 2: 2 105 (n 5), and group cages that meet international standards in size and space × 6 = × = based on the NIH Guide for the Care and Use of Labora- 3: 2 × 10 (n = 5). Group 4 comprised control animals tory Animals, eighth edition. Animals were fed commer- (n = 3), which were given normal saline. Parasite dose cial chow made by Unga Group Limited, based on our administration was done through a thin tube pushed into formulation, while water was given ad libitum. Tey were the orogastric route and the parasite’s oocysts delivered given fruits three times a week and provided with enrich- through a syringe connected to the tube and washed ment devices, which were changed weekly. several times into the gastrointestinal tract. Te animals Jillani et al. Parasites Vectors (2021) 14:316 Page 3 of 8 were then returned to their cages and closely monitored Sequencing until they recovered from the efects of anesthesia.