Emily A. Wright1, Madison B. Reddock2, Emma K. Roberts2,3, Yoseph W. Legesse4,5, Gad Perry6, Robert D. Bradley1,2
Published June 27, 2024Read the peer review reports
Abstract Transmissible spongiform encephalopathies (TSEs) are a fatal neurogenerative disease that include Creutzfeldt–Jakob disease in humans, scrapie in sheep and goats, bovine spongiform encephalopathy (BSE), and several others as well as the recently described camel prion disease (CPD). CPD originally was documented in 3.1% of camels examined during an antemortem slaughterhouse inspection in the Ouargla region of Algeria. Of three individuals confirmed for CPD, two were sequenced for the exon 3 of the prion protein gene (PRNP) and were identical to sequences previously reported for Camelus dromedarius. Given that other TSEs, such as BSE, are known to be capable of cross–species transmission and that there is household consumption of meat and milk from Camelus, regulations to ensure camel and human health should be a One Health priority in exporting countries. Although the interspecies transmissibility of CPD currently is unknown, genotypic characterization of Camelus PRNP may be used for predictability of predisposition and potential susceptibility to CPD. Herein, eight breeds of dromedary camels from a previous genetic (mitochondrial DNA and microsatellites) and morphological study were genotyped for PRNP and compared to genotypes from CPD–positive Algerian camels. Sequence data from PRNP indicated that Ethiopian camels possessed 100% sequence identity to CPD–positive camels from Algeria. In addition, the camel PRNP genotype is unique compared to other members of the Orders Cetartiodactyla and Perissodactyla and provides an in–depth phylogenetic analysis of families within Cetartiodactyla and Perissodactyla that was used to infer the evolutionary history of the PRNP gene.
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Introduction Spongiform encephalopathies are a fatal neurogenerative disease (Prusiner, 1982; Prusiner, 1998) that include Creutzfeldt–Jakob disease and Kuru in humans, scrapie in domestic sheep and goats, chronic wasting disease (CWD) in cervids, bovine spongiform encephalopathy (BSE), transmissible mink encephalopathy, feline spongiform encephalopathy, among others (Abdalla & Sharif, 2022; Aguzzi & Polymenidou, 2004; Collinge & Clarke, 2007; Davenport et al., 2015; Greenlee & Greenlee, 2015). Spongiform encephalopathies can be contracted through a variety of means: (1) consumption of infected flesh or contact with bodily fluids (transmissible, Collins, Lawson & Masters, 2004; Haywood, 1997; Weissmann, 1999), (2) genetic transfer of a mutated prion gene from one or both parents to offspring (familial, Nitrini et al., 1997; Riek et al., 1998), or (3) spontaneous production of an alternative prion protein (sporadic, Brown et al., 2006; Casalone et al., 2004). Additionally, dietary intake may influence transmission of prion diseases through consumption of infected animal products (meat, milk, etc.) or through infectious prions on or within plants and other biotic and abiotic material in the environment (Bartelt-Hunt, Bartz & Yuan, 2023; Gough & Maddison, 2010; Inzalaco et al., 2023; Johnson et al., 2011; Konold et al., 2008; Kuznetsova et al., 2023; Lacroux et al., 2008; Prusiner, 1997).
Evidence, obtained from the genotypic characterization of the exon 3 region of the prion protein gene (PRNP), has been relevant in determining the distribution of populations susceptible to TSE infection and in managing the spread of prion diseases (Arifin et al., 2023; Buchholz et al., 2021; Fernandez-Borges, Erana & Castilla, 2018; Goldmann, 2008; Jewell et al., 2005; Mead et al., 2009; Otero et al., 2021; Perucchini et al., 2008). The most common isoform, PrPc, is inherited and is present during embryogenesis (Westergard, Christensen & Harris, 2007). However, mutated, protease–resistant isoforms (PrPSc) cause abnormal folding of the prion protein, aggregations of amyloid plaques (Horwich & Weissman, 1997), and ultimately the fatal presentation of a prion disease. Although the function of PrP remains unknown, the protein is involved with the circadian rhythm, homeostasis of metal ions, mitochondria, and myelin, intercellular signaling, and neuroprotection (reviewed in Kovač & Šerbec, 2022).
Some mammalian species have amino acid substitutions that may confer low susceptibility in wild populations. For example, there is evidence of strong salt bridges that link the β2- α2 loop of the prion protein to suggest that water buffalo (Bubalus bubalis) has low susceptibility to TSEs similar to members of Canidae, Equidae, Leporidae, Mustelidae, and Suidae (Zhang, Wang & Chatterjee, 2016). However, most members of Suborder Ruminantia are thought to be highly susceptible to prion diseases; codon positions A136V, R154H, and R171Q/K as well as Q95H, S96G, and S225F are known to be important in the susceptibility of domestic sheep and North American deer, respectively (Belt et al., 1995; Goldmann, 2008; Jewell et al., 2005). Given the recent increase in CWD cases in the US and other prion diseases in Old-World ruminants, this is a critical area for determining species that might be increasingly at risk for prion exposure.
According to Köhler-Rollefson (1991), Dromedary camels (Camelus dromedarius) have been extinct in the wild for approximately 2,000 years and have been under considerable exploitation by humans. The population structure of and subsequent underlying genetic and evolutionary forces on Dromedary camels most likely has been human mediated for millennia (Köhler, 1981). In Ethiopia, populations of Dromedary camels (Camelus dromedarius) are mostly restricted to the Ethiopian regional states of Afar, Oromia, and Somali (Abebe, 2001). Although Dromedary camels are, in some instances, free-ranging, these populations and those under captive operations are actively maintained and used for pastoralism, including the production of milk and the sales for pack animals or slaughter (Habte et al., 2021; Kena, 2022; Mirkena et al., 2018).
In 2018, a novel camel prion disease (designated by Babelhadj et al. (2018) as CPD, termed CPrD by Khalafalla (2021)) in Dromedary camels was detected in the Ouargla abattoir (slaughterhouse) in Algeria, using traditional histological, immunohistochemical, and western blot techniques (Babelhadj et al., 2018). DNA sequences obtained from the PRNP gene were examined and then used to generate a genotype of CPD positive individuals; however, the authors made no inference from those data as unfortunately no CPD-negative individuals were sequenced for the PRNP gene (Babelhadj et al., 2018). Based on this initial study, Babelhadj et al. (2018) and Watson et al. (2021) suggested several hypotheses (e.g., CPD naturally developed and was not related to scrapie or BSE, prion-contaminated waste dumps as a source of food in the Ouargla region, etc.) to explain the occurrence of CPD in Algeria; however, no patterns for transmission pathways were identified (Orge et al., 2021).
With the confirmed case of prion disease in Dromedary camels in Algeria (Babelhadj et al., 2018; Khalafalla, 2021) and a second case reported in Tunisia (World Organization of Animal Health, 2019), there was a developing need for prion research and surveillance in Ethiopia and other regions in Africa, the Middle East, and the United Kingdom (Breedlove, 2020; Faye, 2019; Gallardo & Delgado, 2021; Horigan et al., 2020; World Organization of Animal Health, 2019; Teferedegn, Tesfaye & Ün, 2019). Given the increased level of local camel consumption in northern Africa, exportation of meat and milk on a world–wide scale, and lack of regulations in animal husbandry (Teferedegn, Tesfaye & Ün, 2019), it is crucial to develop methods for genotypic characterization of the PRNP gene in camels.
Previous genetic studies of dromedary camels in Algeria, Egypt, and Ethiopia (Cherifi et al., 2017; Legesse et al., 2018) reported a lack of morphological, genetic variation, and population structure indicating homogeneity in the nuclear genome of C. dromedarius. In addition, low variability of camel PRNP sequences has been reported compared to other sequences representative of dromedary camels (Abdel-Aziem et al., 2019; Babelhadj et al., 2018; Kaluz, Kaluzova & Flint, 1997; Tahmoorespur & Jelokhani Niaraki, 2014; Xu et al., 2012; Zoubeyda et al., 2020). Given the broad distribution of camel breeds across northern Africa and the apparent lack of genetic variation among breeds, it is hypothesized that Ethiopian dromedary camels will have similar PRNP genotypes to other dromedary camels. Therefore, the goal of this study is to determine the genotypic characterization of the PRNP gene in camels to ascertain the significance of predicting potential susceptibility or resistance to CPD.
Materials & Methods
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Conclusions Although more studies are needed to determine if there is a difference in the genotypic profile of PRNP among CPD–positive and CPD–negative individuals, this preliminary study demonstrates the genetic similarity in the PRNP gene between Algerian and Ethiopian camels. The lack of nucleotide and corresponding amino acid differentiation, as well as a lack of genetic diversity between Ethiopian and Algerian dromedaries leads us to conclude that dromedaries from both of these regions have equivalent susceptibility to developing PRNP mutations, infection, and transmission rates. Considering Camelus products, such as milk and meat, are distributed widely in Africa and Europe (World Organization of Animal Health, 2019), CPD transmission may mirror the BSE outbreak, which was the causal agent of variant CJD (Mead et al., 2009), if health and safety precautions are ignored. Camelid antibodies have been used in trials for the treatment of neurodegenerative diseases and others (David, Jones & Tayebi, 2014; Jones et al., 2010; Tayebi et al., 2010) and possess unique characteristics that allow these antibodies to cross the blood–brain barrier (Hamers-Casterman et al., 1993; Steeland, Vandenbroucke & Libert, 2016). Further, knock–out and inoculation trials with mice using PRNP of Camelus need to be conducted to examine the susceptibility of Camelus to BSE, CWD, and other prion diseases and the zoonotic potential for transmission from camels to other artiodactylids as well as humans (Watson et al., 2021).
Considering the novelty of CPD, surveillance studies should be implemented in regions where abattoirs are common. Collaborations among international universities, federal agencies, and agricultural workers are essential to these research areas. Further investigations in Ethiopia are in stages of development to: (i) identify potential routes of CPD transmission and document standard practices involved in camel husbandry, butchery, and sale, (ii) incorporate a human dimensions aspect, with current CPD awareness among abattoir meat inspectors, (iii) assess the prevalence of CPD, and (iv) generate policy recommendations for CPD. The implementation of a CPD surveillance program will contribute to the overall One Health of humans, camels, and the environment.
Supplemental Information Supplemental Information Tables 1–3 list all individuals and their associated data that were used in this study.
DOI: 10.7717/peerj.17552/supp-1
Friday, May 12, 2023
Terry S. Singeltary Sr.
