**African Lions and Zoonotic Diseases: Implications for Commercial Lion Farms in South Africa**

**Jennah Green <sup>1</sup> , Catherine Jakins <sup>2</sup> , Eyob Asfaw <sup>1</sup> , Nicholas Bruschi <sup>1</sup> , Abbie Parker <sup>1</sup> , Louise de Waal <sup>2</sup> and Neil D'Cruze 1,\***


Received: 21 August 2020; Accepted: 17 September 2020; Published: 18 September 2020

**Simple Summary:** In South Africa, thousands of African lions are bred on farms for commercial purposes, such as tourism, trophy hunting, and traditional medicine. Lions on farms often have direct contact with people, such as farm workers and tourists. Such close contact between wild animals and humans creates opportunities for the spread of zoonotic diseases (diseases that can be passed between animals and people). To help understand the health risks associated with lion farms, our study compiled a list of pathogens (bacteria, viruses, parasites, and fungi) known to affect African lions. We reviewed 148 scientific papers and identified a total of 63 pathogens recorded in both wild and captive lions, most of which were parasites (35, 56%), followed by viruses (17, 27%) and bacteria (11, 17%). This included pathogens that can be passed from lions to other animals and to humans. We also found a total of 83 diseases and clinical symptoms associated with these pathogens. Given that pathogens and their associated infectious diseases can cause harm to both animals and public health, we recommend that the lion farming industry in South Africa takes action to prevent and manage potential disease outbreaks.

**Abstract:** African lions (*Panthera leo*) are bred in captivity on commercial farms across South Africa and often have close contact with farm staff, tourists, and other industry workers. As transmission of zoonotic diseases occurs through close proximity between wildlife and humans, these commercial captive breeding operations pose a potential risk to thousands of captive lions and to public health. An understanding of pathogens known to affect lions is needed to effectively assess the risk of disease emergence and transmission within the industry. Here, we conduct a systematic search of the academic literature, identifying 148 peer-reviewed studies, to summarize the range of pathogens and parasites known to affect African lions. A total of 63 pathogenic organisms were recorded, belonging to 35 genera across 30 taxonomic families. Over half were parasites (35, 56%), followed by viruses (17, 27%) and bacteria (11, 17%). A number of novel pathogens representing unidentified and undescribed species were also reported. Among the pathogenic inventory are species that can be transmitted from lions to other species, including humans. In addition, 83 clinical symptoms and diseases associated with these pathogens were identified. Given the risks posed by infectious diseases, this research highlights the potential public health risks associated with the captive breeding industry. We recommend that relevant authorities take imminent action to help prevent and manage the risks posed by zoonotic pathogens on lion farms.

**Keywords:** zoonotic disease; *Panthera leo*; human health; biosecurity; wildlife farming; wildlife trade; disease transmission

### **1. Introduction**

Zoonotic diseases are infectious diseases caused by pathogenic agents (including bacteria, parasites, fungi, viruses, and prions) that can be transmitted between vertebrate mammals and humans [1]. Outbreaks of zoonotic diseases can have widespread consequences for public health and are thought to cause two billion cases of human illness and over two million human deaths every year [2]. Disease outbreaks from wild animal sources periodically result in hundreds of billions of dollars of economic damage [3]. The most recent global health pandemic, coronavirus COVID-19, which was also thought to originate in a wild animal host [4], is likely to cost the global economy between 5–9 trillion USD [5].

The increasing rate of emerging infectious diseases is thought to be a result of human-induced changes in land use, extraction of natural resources, animal production systems, and the global wildlife trade [6,7]. Wildlife harbor a large and often unknown reservoir of infectious diseases [8] and zoonotic disease transmission to people occurs when wild animals are in close proximity with human activity [6]. Most recent global health pandemics [9], including COVID-19, are thought to have originated in wild animal hosts [4]. A range of solutions could be put in place to prevent future zoonotic epidemics (see Petrovan et al. [10]). However, it has been suggested that efforts to decrease contact between wild animals and humans could prove to be the most practical and cost-effective approach in reducing the global public health threat posed by zoonotic emerging infectious diseases [11].

Commercial use of wildlife, whether legal or illegal, puts humans in direct contact with a range of wild species [12]. Wildlife farms (herein referred to as facilities that breed non-domesticated species for commercial purposes) in particular can create opportunity for pathogen transmission between wild animals and their human caretakers because of regular or prolonged contact for husbandry purposes [13]. Furthermore, conditions often associated with wildlife farms, such as high concentrations of wild animals in the same enclosures, poor hygiene, and stress associated with captive conditions, can reduce resistance to pathogens and increase the risk for transmission of disease [14,15].

A diverse range of wild animal species are farmed around the world for a range of commercial purposes, for example as exotic pets (e.g., snake farms in West Africa [16]), traditional medicine (e.g., bear bile farms in China and South-East Asia [17]), leather (e.g., alligators farms in the USA [18]), or fur (e.g., mink and fox farms in Europe [19]). Cases of infectious disease emergence from pathogen transmission among farmed wildlife have been documented from across the taxonomic spectrum. For example, transmission of zoonotic tape worm *Armillifer armillatus* from snakes to a farm owner was reported in The Gambia [20], and rapid transmission of coronavirus COVID-19 occurred recently between mink and farm workers at a mink farm in the Netherlands [21].

African lions (*Panthera leo*) are bred and kept on commercial farms across South Africa. These lions are bred for a range of purposes that can involve direct contact with people, including interactive tourism experiences (e.g., paying international volunteers working with predators and day tourists involved in cub petting and walking activities), recreational hunting for 'trophies', and bone exports to Asia for use in traditional medicine products [22,23]. For example, lion bone and trophy exports require a number of 'middle-men' who are required to have direct contact with lions and/or handle their derivatives during transport, slaughter, and/or preparation. This relatively high level of direct contact between lions and people (or consumption of their parts and derivatives) provides ample opportunity for zoonotic exchange.

A review of diseases present among lions in the Kruger Park during the 1970s provides an important insight into the variety of infectious diseases that can affect populations in the wild (e.g., trichinosis, filariasis, sarcoptic mange, pentastomiasis, echinococcosis, taeniasis, hepatozoonosis, anthrax, and babesiosis), including some that are considered to be either directly or indirectly transmissible to humans [24]. Likewise, scientific studies have reported the transmission of zoonotic infectious diseases between humans and captive lions. For example, in 2015, a zoo-housed lion cub presented with 'dermatophytosis', a disease caused by infection with pathogenic fungi *Epidermophyton*, *Microsporum*, or *Trichophyton*, was also contracted by a zookeeper caring for the lion as a result of continuous contact with the animal [25].

The number of lions bred on farms in South Africa has grown exponentially in the last two decades to a current captive population of up to 8500 individuals housed across more than 300 facilities [22]. The vast scale of these intensive breeding facilities further increases the number of people in close contact with lions and the opportunities for zoonotic disease transmission. Yet, to the authors' knowledge, despite its value for informing efforts to prevent, monitor, and manage any associated zoonotic disease outbreaks, no attempt has yet been made to compile a list of pathogenic organisms associated with African lions from recent scientific studies. Consequently, this review of the academic literature published in the last ten years provides an initial baseline of pathogenic organisms and discusses the potential animal and public health risks associated with the captive predator industry.

### **2. Materials and Methods**

We conducted a systematic review of the scientific literature using the academic journal database Web of Science (Philadelphia, PA, USA). A total of 13 search terms relating to pathogenic health were searched on the database (Disease, Pathogen, Virus, Viral, Bacteria, Bacterial, Parasite, Parasitic, Fungus, Fungal, Zoonosis, Zoonotic, and Health). Each search term was employed with the Boolean operator 'AND', with the additional term *Panthera leo*. Searches were conducted for the time period 2009–2019, which returned a total of 252 results, comprising 152 individual academic papers. Of the 152 papers returned from the literature search, one could not be sourced due to institutional access and three were excluded because they were not published in English. The remaining 148 papers are included in the analysis.

Each paper was examined by one of six reviewers, who recorded any mention of 'bacteria', 'fungi', 'parasite', 'protozoa', or 'virus' in each article. All disorders, diseases, or conditions were recorded in relation to African and Asiatic lions, with a list of specific named pathogenic organisms compiled. The environment in which the lions were studied was recorded (wild or captive) with specific details on the type of captivity lions were housed in (commercial enterprises, zoos, private ownership, or mixed purposes). In addition, the papers were reviewed for information about disease transmission. The reviewers recorded where it was specified that pathogenic organisms were transmissible between African lions and other animal species, as well as between African lions and humans.

### **3. Results**

A total of 63 different pathogenic organisms, known to affect lions, were reported (Table 1). Over half of the reported pathogenic organisms were parasites (35, 56%), including ticks (order Ixodida) (4, 6%), followed by viruses (17, 27%), and bacteria (11, 17%), with no pathogenic fungi reported. These 63 pathogenic organisms belong to 35 different genera across 30 different taxonomic families. Three novel pathogenic organisms representing unidentified and undescribed species were also reported.

The review also identified a total of 83 clinical symptoms and diseases associated with these pathogenic organisms (Table 2), highlighting the range of detrimental health risks that these pose to their feline hosts. With regards to information on the transmission of infectious disease, 38 (26%) of the scientific papers referred to transmission between lions and other species and three (2%) specifically referred to transmission between lions and humans.


**Table 1.** Pathogenic organisms (categorized into bacteria, parasites, and viruses) specified in the 148 papers in the dataset.


**Table 2.** Associated diseases and clinical symptoms recorded in the 148 papers in the dataset.

Of the 109 papers that focused on African lions, 45 (41%) were based on data from captive lions, 61 (56%) from wild lions, and three (3%) from a mixture of both. Of the studies focusing on captive lions, only one collected data from a commercial breeding facility in South Africa. The study used samples from three deceased lions to analyze their evolutionary history and was unrelated to pathogens or disease. One further study focused on commercial facilities in South Africa but did not collect first-hand data and instead used literature sources to review the suitability of captive bred lions for reintroduction into the wild. The remainder of the captive data came from lions housed in zoos, wildlife sanctuaries, and reserves (34, 76%), in private ownership (5, 11%), or a combination of both (4, 9%).

### **4. Discussion**

A systematic review of scientific literature confirmed that a range of 63 different pathogenic organisms are known to exist in both captive and wild free ranging lions (Tables 1 and 2). A number of novel pathogenic organisms, in some cases representing unidentified and undescribed species, were reported, including novel *Babesia* species and *Cystoisospora*-resembling oocysts.

There is a paucity of knowledge on disease susceptibility, transmission, epidemiology, and pathology in lions [100]. While the list of known pathogenic organisms will undoubtedly grow, this review provides an important baseline inventory. Given the conditions in which the lion farming industry currently operates, the considerable scale of trade in lions, and their susceptibility to such a wide range of multi-host pathogenic organisms, it is likely that farmed lions could play a central role in the emergence, amplification, and transmission of disease to both people and wild animal populations.

### *4.1. Significance for Lion Health*

Some of the pathogenic organisms reported in this review are of significant health concern for captive and wild lions. For example, *Babesia* parasites, *Mycobacterium bovis* (a bacteria known for causing tuberculosis), canine distemper virus (CDV), canine parvovirus (CP), and feline panleukopenia virus (FPLV) (Table 1) are all highly contagious and cause significant morbidity and mortality in susceptible carnivore species. Infection with these pathogens is associated with a range of clinical symptoms, including but not limited to: emaciation, alopecia, diarrhea, seizures, recurrent twitching, and depression (Table 2) from which infected lions can suffer.

Some of these pathogenic organisms are particularly difficult to manage because infection of susceptible animals does not require direct physical contact. Infected lions shed pathogenic organisms in feces and other bodily secretions, e.g., aerosolized respiratory secretions [105,106] facilitating environmental contamination and rapid spread of disease. Furthermore, some of these organisms have longer incubation periods and can therefore lie undetected in the animals' systems until they reach hazardous levels. For example, tuberculosis onset is slow, in many cases with the majority of infected lions initially appearing healthy [100,107]. In a captive setting, this renders detection and prevention of spread of infections between individuals housed together very difficult.

In addition, some of these pathogenic organisms are likely to present a management challenge on commercial lion farms, as the onset of disease in lions often occurs suddenly after high stress situations, for example after repeated periods of pregnancy and lactation [100]. It has been suggested that intensive farming conditions and poor hygiene may be increasing the incidence of FPLV in captive carnivores, such as lions [108]. Disease transmission is promoted in immunocompromising conditions, and direct human–wildlife contact mixed with limited health and safety standards are all criteria for an emerging zoonosis hotspot [12].

Another challenge for captive facilities is that seemingly innocuous pathogens can cause harm when lions are 'co-infected' with multiple pathogens. For example, severe mortalities have occurred when individual lions were infected with both babesiosis and CDV, resulting in severe diseases like pneumonia and encephalitis, despite appearing healthy when infected with babesiosis alone [81]. This heightens the challenge of identifying infected individuals to manage diseases before transmission can occur.

### *4.2. Significance for Human Health*

In addition to the potential significance for lion health, many of the pathogenic organisms reported in the reviewed scientific literature raise concerns for human health. For example, pathogenic strains of the Enterobacteriaceae *Escherichia coli* [26], the parasitic Sarcocystidae *Toxoplasma gondii* [14,87], and potentially, the parasitic Toxocaridae *Toxascaris leonine* [109] have a possible fecal–oral transmission route from lions to humans. For others, such as the Rickettsiaceae *Anaplasma phagocytophilum* [27], transmission via the bite of an infected arthropod tick is also possible.

Some pathogens possess the capacity to infect human tissue using keratin and therefore only require physical contact with the lion's fur; for example, *Microsporum gypseum*, the cause of dermatomycosis [110]. The adoption of prophylactic measures for sanitary maintenance for these animals and the professionals who maintain contact with them is paramount to reduce possible transmission of infection but is difficult to manage because of the asymptomatic nature of the pathogens in healthy lions [110]. Visitors to lion farms in South Africa have reported that basic hygiene protocols, for example hand sanitizing and stepping points to disinfect shoes between enclosures, are often absent for those intending to interact with the animals [111].

Lions have also been reported as hosts for diseases listed by the World Health Organization (WHO) as 'neglected tropical diseases (NTDs)' [112]. For example, human African trypanosomiasis caused by trypanosomes are multi-host parasites capable of infecting a wide range of wildlife species, including lions [93], that constitute a reservoir of infection for both people and domestic animals. *Echinococcosis*, a parasitic disease caused by tapeworms that reside in the intestines of carnivores, including lions [52], can cause serious morbidity and death in people. The prevalence of *Echinococcosis* is increasing in some African countries due to frequent contact between game animals (reservoir hosts), domestic animal

hosts (such as dogs), and humans who are susceptible to transmission [113]. Neglecting these parasites can have severe socioeconomic consequences [113].

Captive carnivores can be predisposed to infections of *Toxoplasma*, a protozoan parasite with significant zoonotic potential [113]. Lions in particular have been identified as a susceptible host species [113]. Lions infected with *Toxoplasma* can transmit the parasites to people via blood and feces, causing severe pulmonary, cardiac, and brain inflammatory reactions (among others), sometimes with fatal outcomes [113]. Some *Toxoplasma* species have also been reported to cause abortion and fetal death; underestimating the impact of these parasites on humans could lead to a future epidemic where reduction in life expectancy, and increased child and maternal death, are rife [113].

Lions are also vulnerable to bovine tuberculosis (bTB), a disease caused by infection with the bacterial pathogen *M. bovis* [32,47]. Tuberculosis transmission at the wildlife–livestock–human interface is a growing concern worldwide, particularly in sub-Saharan Africa where infection is spreading [114]. Lions initially contracted bTB from infected buffalo carcasses [32], and although no direct spill-over from wildlife to humans (or vice-versa) has yet been documented [114], it is a growing concern, particularly in countries such as South Africa where there are a relatively high number of people living with HIV [115,116] and because HIV is the strongest known risk factor for TB [32]. Transmission of other pathogenic strains of tuberculosis from wild captive animals to humans has already been documented [117].

Epidemics caused by cats are possible (e.g., canine distemper in big cats) but are considered to be relatively rare [118]. While no evidence of lion-to-human transmission of feline coronavirus exists, isolation of pathogens with pandemic potential from any mammalian host is significant as it may provide conditions suitable for the virus to adapt to other mammalian hosts, enabling efficient mammal-to-human, and possibly also human-to-human, transmission, paving the way for a potentially devastating pandemic [119].

For example, it has recently been confirmed that big cats, including lions, can be infected with Sars-CoV-2 [120]. Some experts have publicly stated their belief that it is unlikely Sars-CoV-2 will naturally spread in a wild big cat population [118]. However, given the fact that the lions and tigers that tested positive for Sars-CoV-2 in the Bronx Zoo were likely to be infected by a zoo employee [121,122], there are on-going concerns that this virus could be passed from humans to big cats and vice versa in scenarios that involve captive individuals [118].

### *4.3. Significance for Lion Farming*

The maintenance of wild species in captivity provides an opportunity for unnatural human-wildlife proximity, facilitating interspecies sharing of pathogenic organisms [123]. Lions are kept in captivity in zoos in many cases as part of conservation breeding programs [124], but also, and in far greater numbers, on commercial wildlife farms [125]. While published data detailing the scale of wildlife farming and lion farming in particular in South Africa are scant, the South African Minister of Environment, Forestry and Fisheries stated in July 2019 in response to a Parliamentary question that the captive lion population in South Africa amounted to 7979 lions housed across 366 facilities.

Scientific papers that focus on the welfare conditions on commercial lion farms prevalent across South Africa are currently lacking. However, the living conditions and environments provided are frequently reported as low welfare, involving large numbers of lions, often in poor physical condition and in over-crowded spaces [126,127] (Figure 1). High concentrations of wild animals in the same enclosures can increase the risk for transmission of disease to and from wild animals due to reduced resistance to pathogens from factors associated with captivity, such as poor hygiene, poor diet, or stress [14,128]. Furthermore, cub separation from their mothers and the provision of alternative milk formulas (a practice reported at some lion farms [111]] can lead to nutritional deficiencies [129], which weakens immune systems and leaves animals more susceptible to pathogens [130].

pathogens [130].

Scientific papers that focus on the welfare conditions on commercial lion farms prevalent across South Africa are currently lacking. However, the living conditions and environments provided are frequently reported as low welfare, involving large numbers of lions, often in poor physical condition and in over-crowded spaces [126,127] (Figure 1). High concentrations of wild animals in the same enclosures can increase the risk for transmission of disease to and from wild animals due to reduced resistance to pathogens from factors associated with captivity, such as poor hygiene, poor diet, or stress [14,128]. Furthermore, cub separation from their mothers and the provision of

**Figure 1.** Environments provided for lions at commercial captive breeding facilities in South Africa are frequently reported as low welfare, involving large numbers of lions, often in poor physical condition and in over-crowded spaces. (**Top left**) Lioness housed in an enclosure with fecal matter and decaying carcasses. (**Top right**) Lions with little to no fur left as a result of severe and untreated mange. (**Bottom left**) Lion cub born with severe deformities, likely due to inbreeding. (**Bottom right**) Lions housed in overcrowded conditions. Images copyright Blood Lions. A key part of this industry is "ecotourism", where people are provided with the opportunity to **Figure 1.** Environments provided for lions at commercial captive breeding facilities in South Africa are frequently reported as low welfare, involving large numbers of lions, often in poor physical condition and in over-crowded spaces. (**Top left**) Lioness housed in an enclosure with fecal matter and decaying carcasses. (**Top right**) Lions with little to no fur left as a result of severe and untreated mange. (**Bottom left**) Lion cub born with severe deformities, likely due to inbreeding. (**Bottom right**) Lions housed in overcrowded conditions. Images copyright Blood Lions.

come into close and unnatural proximity with lions via cub "petting" and "walking with" interactions or international volunteers paying to hand-rear lion cubs. The process of preparation of carcasses for human consumption also presents a considerable risk for transmission of disease to and from wild animals [131], a risk that is amplified in situations where slaughter and preparation take place at unregulated slaughterhouses, unbound by official hygiene standards [132]. Furthermore, the regulatory body that governs the international export of lion bones (The Convention of International Trade of Endangered Species, 'CITES') dictates quotas based on conservation science [133] and is not specifically aimed at preventing zoonotic disease introduction, despite the major role wildlife trade has as a transmission pathway for pathogenic organisms [12]. A key part of this industry is "ecotourism", where people are provided with the opportunity to come into close and unnatural proximity with lions via cub "petting" and "walking with" interactions or international volunteers paying to hand-rear lion cubs. The process of preparation of carcasses for human consumption also presents a considerable risk for transmission of disease to and from wild animals [131], a risk that is amplified in situations where slaughter and preparation take place at unregulated slaughterhouses, unbound by official hygiene standards [132]. Furthermore, the regulatory body that governs the international export of lion bones (The Convention of International Trade of Endangered Species, 'CITES') dictates quotas based on conservation science [133] and is not specifically aimed at preventing zoonotic disease introduction, despite the major role wildlife trade has as a transmission pathway for pathogenic organisms [12].

It is also important to note that any pathogenic organisms present in the captive lion population may pose a threat to the conservation of wild populations, particularly in scenarios where lion farms are located close to a wild lion habitat and where lion farm staff and visitors are actively engaged in other activities (e.g., conservation-focused field research, hunting, and photo tourism) that bring them into close proximity to free-ranging lions. For example, wild racoon dogs (*Nyctereutes procyonoides*) are thought to have transmitted CDV to a group of zoo-housed tigers in Japan [42] and records of transmission of intestinal nematodes between captive felids and local feral cats have been reported in Brazil [134]. Multi-host pathogenic organisms may pose a particular threat in scenarios where lion farm activity overlaps with areas inhabited by other free-ranging carnivore species (both wild and domesticated).

### *4.4. Mitigating Animal and Public Health Risks*

Remedial measures, such as improved animal welfare standards, veterinary interventions, and biosecurity protocols can partially mitigate the risk of zoonosis at captive lion breeding facilities [135]. However, due to the potential of asymptomatic pathogens affecting lions [110],

biosecurity would require sophisticated disease surveillance, which could prove challenging [136,137]. Even with comprehensive surveillance, identification of emerging pathogens is still a challenge that poses significant animal and public health risks [12]. There is currently no publicly available information detailing the biosecurity protocols and regulatory standards within the lion breeding industry and, from our initial review of the literature, an apparent lack of national norms and standards for the health of the lions housed on commercial farms.

Alternatively, a phased reduction in the scale of, or end to, the commercial captive breeding of lions for non-conservation purposes in South Africa could help to remove the animal and public health risks associated with this industry. However, efforts focused on improving animal husbandry, reducing consumer demand for lions (and their derivatives), increased enforcement effort, and the provision of economic incentives for farm staff would need to be considered to prevent any unintended consequences on lion welfare, conservation, and local livelihoods.

### *4.5. Limitations*

We acknowledge that restricting our search to a ten-year time period and to one academic database will limit the number of relevant articles in our review. In addition, we recognize that additional onsite research is required to determine the incidence and prevalence of particular pathogenic organisms (in both captive and wild lion populations) and to help identify which infectious diseases are more likely to affect them under certain conditions. However, it was not our intention to provide a comprehensive overview of all pathogens affecting African lions or to provide specific statistics on their occurrence. Rather, the aim of our study was to create a baseline inventory of key pathogens and associated diseases and to describe the potential associated health concerns for both lions and people. Although our review may omit some relevant pathogens, we hope to demonstrate that, by only scratching the surface of this field, we identify a previously neglected area of consideration that will stimulate increased attention in future.

### **5. Conclusions**

There are many socio-cultural, political, economic, and conservation factors that create a complex and nuanced debate around the commercial captive lion breeding industry in South Africa [133]. However, all economic, ethical, and environmental considerations aside, the data presented here indicate that the industry poses a potential risk to wild animal and public health. This initial literature review reveals a long and varied list of pathogenic organisms known to affect African lions, some of which can be transmitted to people. Given the range of pathogens identified, the growth of the industry over the last couple of decades, and the increasing number of people who have direct contact with live lions and/or their parts and derivatives, we recommend that a closer examination of the current policies and practices associated with commercial lion farming is required, particularly under a biosecurity lens. Furthermore, to properly safeguard lion and public health, it is paramount that the recommendations of any such examinations should be acted on with clear time-bound objectives relating to both implementation and enforcement.

**Author Contributions:** Conceptualization, N.D., J.G. and L.d.W.; methodology, N.D. and J.G.; formal analysis, J.G.; investigation, N.D., J.G., C.J., L.d.W., E.A., A.P. and N.B.; resources, N.D., J.G., C.J., L.d.W., E.A., A.P. and N.B.; data curation, J.G.; writing—original draft preparation, J.G. and N.D.; writing—review and editing, J.G., N.D., L.d.W., C.J.; visualization, J.G.; supervision, N.D. and L.d.W.; project administration, J.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We would like to thank Gilbert Sape, Edith Kabesiime, Patrick Muinde, and Paul Giess for providing helpful comments and feedback on an earlier version of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Haemogregarines and Criteria for Identification**

**Saleh Al-Quraishy <sup>1</sup> , Fathy Abdel-Ghaffar <sup>2</sup> , Mohamed A. Dkhil 1,3 and Rewaida Abdel-Gaber 1,2,\***


**Simple Summary:** Taxonomic classification of haemogregarines belonging to Apicomplexa can become difficult when the information about the life cycle stages is not available. Using a selfreporting, we record different haemogregarine species infecting various animal categories and exploring the most systematic features for each life cycle stage. The keystone in the classification of any species of haemogregarines is related to the sporogonic cycle more than other stages of schizogony and gamogony. Molecular approaches are excellent tools that enabled the identification of apicomplexan parasites by clarifying their evolutionary relationships.

**Abstract:** Apicomplexa is a phylum that includes all parasitic protozoa sharing unique ultrastructural features. Haemogregarines are sophisticated apicomplexan blood parasites with an obligatory heteroxenous life cycle and haplohomophasic alternation of generations. Haemogregarines are common blood parasites of fish, amphibians, lizards, snakes, turtles, tortoises, crocodilians, birds, and mammals. Haemogregarine ultrastructure has been so far examined only for stages from the vertebrate host. PCR-based assays and the sequencing of the *18S rRNA* gene are helpful methods to further characterize this parasite group. The proper classification for the haemogregarine complex is available with the criteria of generic and unique diagnosis of these parasites.

**Keywords:** haemogregarines; gamogony; sporogony; schizongony; molecular analysis

### **1. Introduction**

Phylum Apicomplexa was described by Levine [1] to include parasitic protozoa sharing unique ultrastructural features known as the "apical complex" (Figure 1). Haemogregarines (Figure 2) are ubiquitous adeleorine apicomplexan protists inhabiting the blood cells of a variety of ectothermic and some endothermic vertebrates [2–4]. They have also an obligatory heteroxenous life cycle (Figure 3), where asexual multiplication occurs in the vertebrate host; while sexual reproduction occurs in the hematophagous invertebrate vector [5]. This family contains four genera, according to Levine [6]: *Haemogregarina* Danilewsky [7], *Karyolysus* Labbé [8], *Hepatozoon* Miller [9], and *Cyrilia* Lainson [10]. Barta [11] conducted a phylogenetic analysis of representative genera in phylum Apicomplexa using biological and morphological features to infer evolutionary relationships in this phylum among the widely recognized groups. The data showed that the biologically diverse Haemogregarinidae family should be divided into at least three families (as suggested by Mohammed and Mansour [12]), were family Haemogregarinidae, containing the genera *Haemogregarina* and *Cyrilia*; family Karyolysidae Wenyon [13], of the genus *Karyolysus*; and family Hepatozoidae Wenyon [13], of the genus *Hepatozoon*, since the four genera currently in the family do not constitute a monophyletic group. The picture is further complicated by evidence from a study by Petit et al. [14] of a new Brazilian toad haemogregarine parasite *Haemolivia stellata*.

**Citation:** Al-Quraishy, S.; Abdel-Ghaffar, F.; Dkhil, M.A.; Abdel-Gaber, R. Haemogregarines and Criteria for Identification. *Animals* **2021**, *11*, 170. https:// doi.org/10.3390/ani11010170

Received: 27 November 2020 Accepted: 7 January 2021 Published: 12 January 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** The general structure for the apical complex for Apicomplexa. **Figure 1.** The general structure for the apical complex for Apicomplexa. **Figure 1.** The general structure for the apical complex for Apicomplexa.

**Figure 2.** Haemogregarines as a part of phylum Apicomplexa. **Figure 2.** Haemogregarines as a part of phylum Apicomplexa.

**Figure 3.** The life cycle of the apicomplexan parasites. **Figure 3.** The life cycle of the apicomplexan parasites.

It undergoes sporogonic development in its tick host's gut wall and has a complex life cycle that resembles *Karyolysus* species much more than *Hepatozoon*, *Haemogregarina*, and *Cyrilia* species. Haemogregarines can be morphologically classified based on the developmental details of sporogonic phases of the parasite in the vector, which provide the main characters for classification, the morphology of gametocytes in the red blood cells, and an evaluation of the stages of development [15,16]. Although useful, this methodology is not sufficient for a taxonomic diagnosis [17,18] also the classical systematics has been problematic because of the variability to which morphological details are subjected [19]. Therefore, the use of molecular methods from blood or tissue samples [20–22], with appropriate molecular phylogeny study, became an essential adjunct to existing morphological and biological characters for use in the inference of evolutionary history relationships among haemoprotozoan parasites [23–25]. Molecular data has been carried out based using PCR assays targeting the nuclear 18s ribosomal RNA gene, which have been extensively applied to characterize hemoparasites DNA more fully in the absence of complete life cycles [26–32]. It undergoes sporogonic development in its tick host's gut wall and has a complex life cycle that resembles *Karyolysus* species much more than *Hepatozoon*, *Haemogregarina*, and *Cyrilia* species. Haemogregarines can be morphologically classified based on the developmental details of sporogonic phases of the parasite in the vector, which provide the main characters for classification, the morphology of gametocytes in the red blood cells, and an evaluation of the stages of development [15,16]. Although useful, this methodology is not sufficient for a taxonomic diagnosis [17,18] also the classical systematics has been problematic because of the variability to which morphological details are subjected [19]. Therefore, the use of molecular methods from blood or tissue samples [20–22], with appropriate molecular phylogeny study, became an essential adjunct to existing morphological and biological characters for use in the inference of evolutionary history relationships among haemoprotozoan parasites [23–25]. Molecular data has been carried out based using PCR assays targeting the nuclear 18s ribosomal RNA gene, which have been extensively applied to characterize hemoparasites DNA more fully in the absence of complete life cycles [26–32].

In the present critical review of the haemogregarines complex, the proper classification, the criteria of generic and unique diagnosis, and the cosmopolitan distribution of haemogregarines among the vertebrate and invertebrate hosts are examined because of their relevant characteristic and taxonomic revisions. In the present critical review of the haemogregarines complex, the proper classification, the criteria of generic and unique diagnosis, and the cosmopolitan distribution of haemogregarines among the vertebrate and invertebrate hosts are examined because of their relevant characteristic and taxonomic revisions.

#### **2. Materials and Methods 2. Materials and Methods**

This review included all related published scientific articles from January 1901 to December 2020. This article was conducted by searching the electronic databases NCBI, ScienceDirect, Saudi digital library, and GenBank database, to check scientific articles and

M.Sc./Ph.D. Thesis related to the research topic of this review. Studies published in the English language were only included and otherwise are excluded.

Relevant studies were reviewed through numerous steps. In the first step, target published articles were identified by using general related terms related to the morphological features, such as "Haemogregarines" and "Apicomplex". The second step involved screening the resulting articles by using highly specific keywords of the generic features for stages in the life cycle of haemogregarines species, including "Merogony", "Gamogony", "Sporogony", "Infective stages", "Motile stage", "Infection sites", and "sporozoites". The last step of the review focused on selected studies involving the use of molecular analysis for accurate taxonomic identification by using highly specific keywords, including "PCR", "Genetic markers", "Variable regions", "*18S rRNA*", and "Phylogenetic analysis".

The obtained data were presented in tables and figures and were: Table 1 representing the characteristic features for the haemogregarines genera, Tables 2–6 showing haemogregarines species, the vertebrate host, site of the merogonic stage, the invertebrate vectors, site of gamogony and sporogonic stages, geographical locality for hosts, and the authors for publishing data, Table 7 with the primer sets used for the amplification and sequencing for the appropriate gene of *18S rRNA* for haemogregarines, and Table 8 representing all the sequenced and deposited haemogregarines in the GenBank database until now.

### **3. Results and Discussion**

In this review, the different stages of the apicomplexan life cycle were used to identify haemogregarines. However, in most cases, their assignment to one or another genus cannot be considered more than provisional. Accordingly, about 82 haemogregarines in 155 research articles were identified previously. Osimani [33] stated that the differences between the haemogregarines relied more on the host's identity than the parasite's characteristics. Mohammed and Mansour [12] reported that haemogregarines gamonts morphology does not provide generic identification with a reliable key. However, Telford et al. [34], and Herbert et al. [35] stated that the determination of generic haemogregarines should not be based exclusively on the gamonts' form, the type of parasitized host cells, and their effect on the host and site merogony in host cells. While the most characteristic feature for the basic identification via the sporogonic stage.

The reviewed species belonged to the four genera within Hemogregarinidae (Table 1). Following the parsimony analysis in the phylogenetic study of the representative genera in phylum Apicomplexa performed by Siddall and Desser [36] primarily based on ultrastructural observations, it was concluded that the variations between the different haemogregarines genera are mainly reflected by the sporogony features. Besides, Dvoˇráková et al. [37] added that the host specificity, together with the haemogregarine's careful morphological and biological analysis, is a sound criterion for accurate identification. These species are common in different animals as fish (Table 2), amphibians (Table 3), reptiles (Tables 4–7), birds (Table 8), and mammals (Table 9).


**Table 1.** Characters of different groups of haemogregarines used in the parsimony analysis carried out by Barta [19] and Siddall and Desser [36].

**Table 2.** Haemogregarines of fish.


**Table 3.** Haemogregarines of amphibians.




**Table 5.** Haemogregarines of snakes.







**Table 9.** Haemogregarines of mammals.


In the schizogony (merogony) stage, haemogregarines are characterized by their considerable ability to invade and develop within different organs and cell types inside the vertebrate host (Tables 2–9). Bray [127] proposed that haemogregarines with schizonts in the liver should be placed in the genus *Hepatozoon*. In contrast, those species that precede schizogony in other organs should belong to another genus as *Haemogregarina* or *Karyolysus*. However, only in the lung of the river turtle, *Trionyx gangeticus* infected with *Haemogregarina gangetica*, was described by Misra [87]. In addition to the usual location of merogonic development in the liver, lung, and spleen, Ball et al. [71] have found certain merogonic stages in the highly infected snakes' brain and heart. Siddall and Desser [84] described merogonic stages in the lacunar endothelial cells of the circulatory system of the leech and its proboscis, besides the liver, lung, and spleen in the turtle. Yanai et al. [128] also described nodular lesions containing schizonts and merozoites of *Hepatozoon* sp. of the heart's martens, perisplenic, and perirenal adipose tissues, the diaphragm, mesentery, and tongue. Úngari et al. [102] reported that the genus *Haemogregarina* underwent schizogony in the circulating blood cells as in turtles and fish, and the genus *Hepatozoon* underwent schizogony in the liver. Additionally, there are two morphologically different meronts were the micro- and macromeronts. The presence of these two forms of meronts was mentioned to be a fundamental feature of the whole haemogregarine [74,129,130].

Gametocytes are usually the only stages of the parasite detected by scientists. Their morphology, unfortunately, does not provide a reliable clue to the generic differentiation. Together with other relevant data, their morphological characteristics offer a reliable basis for specific identification [35,67]. The haemogregarines gametocytes appeared as sausageshaped and generally lie singly within erythrocytes (Tables 2–9), but sometimes free in extracellular space, which is consistent with Telford et al. [34], Sloboda et al. [79] as the presence of free extracellular gametocytes. They are also observed in the leucocytes of fish (Table 2), birds (Table 8), and mammals (Table 9).

The shape, size, and structure of infected blood-corpuscles often undergo considerable changes. Hypertrophy may result directly from the gametocyte's added intraerythrocytic volume or represent an erythrocyte adaptation to the gametocyte's presence [53,82,131,132]. An entirely different cell response occurred when the gametocytes of *Hemogregarina* sp. invaded erythrocytes of *Rana berlandieri*. The erythrocytes undergo hypertrophy, and the plasmalemma of the infected erythrocyte demonstrated numerous microvilli-like outgrowings. Hussein [133] also described the hypertrophy of *Karyolysus*-infected erythrocytes. Most haemogregarine gametocytes do not invade the host cell's nucleus but instead move it to the opposite side or the other host cell's other pole. This is contrary to the effect of the genus *Karyolysus* on the infected erythrocytes. *Karyolysus* has a karyolytic impact on the host cell's nucleus and is therefore identified *Karyolysus* Reichenow [134].

Little work had been done to identify the actual arthropod vectors of haemogregarines, as the transmission by inoculation of blood was rarely successful. In general, the invertebrate vectors of haemogregarines were the most challenging problem facing this group's research progress [49]. The haemogregarines displayed a wide distribution of vertebrate host infections, and a large number of invertebrate vectors (Tables 2–9). In all haemogregarines, fertilization is of Adelea type; both micro- and macrogamonts lie in syzygy within the same parasitophorous vacuole. Syzygy can stimulate the production of the associated gamonts in haemogregarines, since only the parasites found in pairs were mostly differentiated, which is consistent with Davies and Smit [42]. Regarding the number of microgametes produced by each microgamont, the members of the suborder Adeleidea were characterized by the production of only a few (four or less) microgametes [135]. Simultaneously, the formation of multiple microgametes has been identified in most haemogregarines species [52]. However, there are some suggestions that multiple microgamete formation does not occur in the entire genus *Hepatozoon* [111]. Regarding the number of flagella in microgametes in haemogregarines, contradictions were recorded. While monoflagellated microgametes have been described for haemogregarines species [74], biflagellated microgametes were also recorded for other haemogregarines [52]. On the other hand, Michel [85] reported non-flagellated microgametes in *Hepatozoon mauritanicum*.

Fertilization follows, leading to the formation of a zygote that becomes an oocyst. The oocyst is surrounded by a flexible membrane rather than a wall, and it produces sporozoites that may undergo further merogony. Sporogony is elucidated for just a few known haemogregarines species, the vast majority of which is supposed to investigate this aspect of their life-cycle, as reported by Forlano et al. [113]. There is also another potential criterion for distinguishing between *Hepatozoon* and *Haemogregarina* based on the presence or absence of oocysts containing sporocysts in the invertebrate vector, which is consistent with Levine [6]. When the developing mite reaches the nymphal stage, the sporozoites attain their maturity. The sporozoites eventually get the nymph's stomach and pass out with their faeces, which are considered infection sources of the vertebrate host (lizard). The morphological characteristics of the gamonts and meronts found in the blood cells sometimes provide inadequate information for differential diagnoses [37], meaning that assigning species of haemogregarines to one of these genera must be based on the characteristics of its sporogony in the invertebrate vectors [6,64]. However, data on invertebrate vectors and sporogony are missing for the majority of species [23].

Until now, the current taxonomy of haemogregarines is facing a great challenge due to the high variation in gamont morphology, low host specificity, unknown invertebrate hosts in many cases, and fewer details of sporogony. Therefore, molecular approaches are now available to distinguish populations of morphologically identical but genetically different parasites, including DNA and polymerase chain reaction (PCR) based approaches [22,136–141]. Some studies based on PCR-based assays as the reference diagnostic test for epidemiological studies, which given their greater sensitivity, particularly for testing different hosts with intermittent levels of parasitemia via a low infection rate by gamonts, as Otranto et al. [114], Haklová-Koˇcíková et al. [18], Jòzsef et al. [24], Ramos et al. [116], and Mitkova et al. [120]. Notably, all the molecular evidence comes from the complete and partial sequences of the small subunit (SSU) ribosomal DNA (rDNA) 18S gene is a sufficient phylogenetic marker to approximate ordinal level relationships and those within orders [68,98,119,142–145]. Previous molecular studies of Harris et al. [22] and Barta et al. [19] demonstrated that the haemogregarine species are clustered in sister clades with interspecies linked more with the host geographic distribution, rather than host species. There are universal primer sets that were able to molecularly characterize haemogregarines, as mentioned in Table 10. However, many species with sequences deposited in the GenBank database are not identified correctly at the generic level. Table 11 expressed only haemogregarines identified at the species level and others identified at the generic level are excluded.

**Table 10.** Primer sets used in the phylogenetic analysis of haemogregarines by *18S rRNA* gene.



**Table 10.** *Cont.*

**Table 11.** List of sequences for haemogregarines from GenBank database based on the *18S rRNA* gene.



**Table 11.** *Cont.*


**Table 11.** *Cont.*

### **4. Conclusions**

Few haemogregarine characteristics provide a reliable basis for the related parasite to recognized genera. Details of the sporogonic cycle seem to be the only reliable criterion as they are the "Key-stone" in the classification system. Morphological characteristics of the gametocytes do not help in this respect. Features of the schizogonic stages, when these are known, are not much better as criteria of generic value. Molecular phylogenetic studies using the appropriate genetic markers are helpful tools for the accurate taxonomic identification for haemogregarines. Further studies are recommended to include other nuclear and mitochondrial genes to provide more information about the genetic variability among haemogregarines.

**Author Contributions:** Conceptualization, S.A.-Q., F.A.-G. and M.A.D.; methodology, F.A.-G. and R.A.-G.; validation, M.A.D.; formal analysis, R.A.-G. and M.A.D.; investigation, S.A.-Q. and F.A.-G.; resources, R.A.-G. and M.A.D.; data curation, R.A.-G. and M.A.D.; writing—original draft preparation, S.A.-Q., F.A.-G., R.A.-G. and M.A.D.; writing—review and editing, S.A.-Q., F.A.-G., R.A.-G. and M.A.D.; visualization, R.A.-G. and M.A.D.; supervision, S.A.-Q., F.A.-G. and M.A.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Deanship for Research and Innovation, "Ministry of Education" in Saudi Arabia, grant number IFKSURP-131.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data generated or analysed during this study are included in this published article.

**Acknowledgments:** The authors extend their appreciation to the Deanship for Research and Innovation, "Ministry of Education" in Saudi Arabia for funding this research work through the project number IFKSURP-131".

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


### *Review* **Systematic Review of Hepatitis E Virus in Brazil: A One-Health Approach of the Human-Animal-Environment Triad**

**Danny Franciele da Silva Dias Moraes 1,2,3 , João R. Mesquita 3,4,\* , Valéria Dutra <sup>1</sup> and Maria São José Nascimento <sup>5</sup>**


**Simple Summary:** Hepatitis E virus (HEV) is an important causative agent of acute and chronic hepatitis worldwide. Originally identified in epidemics associated with flooding in Asia, it nowadays shows very distinct genetic and epidemiological patterns. While HEV genotypes (HEV-) 1 and 2 are associated with the original outbreaks (waterborne diseases), HEV-3 and HEV-4 present a zoonotic pattern (associated with consumption of meat from infected animals), HEV-5 and 6 have been found only in wild boar in Japan, and HEV-7 and 8 have been detected in camels and dromedary seldom affecting humans. Brazil, with a precarious sanitary structure and being an important world meat producer, was the focus of this study in order to identify patterns of occurrence of HEV. After reviewing scientific studies, it was identified that the only genotype found in Brazil is HEV-3 and the area where there were more reports was the South region of the country. This is the region that produces more pork. These results indicate that HEV-3 is widespread in the country and sanitary surveillance is essential in the national production of pigs, as well as the implementation of monitoring protocols in hospitals.

**Abstract:** Brazil is the fifth largest country in the world with diverse socioeconomic and sanitary conditions, also being the fourth largest pig producer in the world. The aim of the present systematic review was to collect and summarize all HEV published data from Brazil (from 1995 to October 2020) performed in humans, animals, and the environment, in a One Health perspective. A total of 2173 papers were retrieved from five search databases (LILACs, Mendeley, PubMed, Scopus, and Web of Science) resulting in 71 eligible papers after application of exclusion/inclusion criteria. Data shows that HEV genotype 3 (HEV-3) was the only retrieved genotype in humans, animals, and environment in Brazil. The South region showed the highest human seroprevalence and also the highest pig density and industry, suggesting a zoonotic link. HEV-1 and 2 were not detected in Brazil, despite the low sanitary conditions of some regions. From the present review we infer that HEV epidemiology in Brazil is similar to that of industrialized countries (only HEV-3, swine reservoirs, no waterborne transmission, no association with low sanitary conditions). Hence, we alert for the implementation of HEV surveillance systems in swine and for the consideration of HEV in the diagnostic routine of acute and chronic hepatitis in humans.

**Keywords:** Brazil; HEV; zoonotic; One Health

### **1. Introduction**

In the last years, hepatitis E virus (HEV) has captured widespread attention when autochthonous hepatitis E cases started to be reported in industrialized countries [1]. Until

**Citation:** Moraes, D.F.d.S.D.; Mesquita, J.R.; Dutra, V.; Nascimento, M.S.J. Systematic Review of Hepatitis E Virus in Brazil: A One-Health Approach of the Human-Animal-Environment Triad. *Animals* **2021**, *11*, 2290. https://doi.org/10.3390/ ani11082290

Academic Editor: Fabio Ostanello

Received: 16 June 2021 Accepted: 29 July 2021 Published: 3 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

then, hepatitis E was considered a rare disease in these countries and only associated with travelers returning from HEV endemic areas in Africa and Asia [2]. All the autochthonous cases reported in industrialized countries were caused by two HEV genotypes, namely HEV genotypes 3 (HEV-3) and 4 (HEV-4), that showed to have distinct epidemiological and clinical characteristics from the HEV genotype 1 (HEV-1) and HEV genotype 2 (HEV-2) circulating in developing countries. HEV-1 and HEV-2 are restricted to humans, transmitted by orofecal route through contaminated waters (usually linked to the lack of basic sanitation), and associated with large waterborne outbreaks of acute hepatitis in underdeveloped regions [3]. HEV-3 and HEV-4 are zoonotic viruses, common in domestic and wild pigs that infect humans as an accidental host through the consumption of uncooked contaminated pork products, being associated with sporadic human hepatitis cases [2,4]. Clinical features of these genotypes are also unique, with infections mostly asymptomatic in immunocompetent but with the capacity to progress to chronic hepatitis with liver cirrhosis in immunocompromised patients (such as organ transplant recipients and HIV patients), being also associated to diverse extra-hepatic manifestations (neurological and haematological) [2].

HEV is a non-enveloped positive-sense single-stranded RNA virus, belonging to *Hepeviridae* family, genera *Orthohepevirus*, species A, with eight genotypes currently recognized (HEV-1 to HEV-8) [3]. HEV-1 and HEV-4 have been detected in human cases, while HEV-5 and HEV-6 are genotypes strictly found in wild boar, HEV-7 and HEV-8 found in dromedary and Bactrian-camels [3]. There is only one report of HEV-7 in humans [5]. Currently, HEV-3 is subdivided into at least 11 subtypes (3a–3j, 3ra) [6].

Since swine are the main reservoir of HEV-3 as well as the main source of human infection and given that Brazil is the fourth largest pig producer in the world [7], a high HEV-3 circulation in the country is expected. Brazil is divided into 5 regions, namely North, Northeast, Midwest, Southeast and South, 26 states and a Federal District, with a total, of 5570 municipalities [8]. The South region has the highest pig production in the territory, accounting for 66.12% of the national production [7]. Moreover, Brazil is a country with continental dimensions, being the 5th largest country in the world with a population of circa 211 million, having a great extension of rural and urban areas with extremely diverse socioeconomic and sanitary conditions that influence infectious diseases dynamics [9]. There is today an increased awareness to monitor and survey the interfaces of human, animal, and environment in order to manage global health. Hence, the present systematic review aimed to collect and summarize all HEV published data from Brazil (from 1995 to October 2020) performed in humans, swine, other animals, and the environment, from a One Health perspective.

### **2. Materials and Methods**

Exhaustive searches were carried out in the electronic databases: Latin American and Caribbean Health Sciences Literature (LILACs), Mendeley, PubMed, Scopus, and Web of Science. Two independent investigators (DFSDM and JRM) searched the databases, and included all studies published until October 2020. The study followed the protocol of the Preferred Reporting of Systematic Reviews and Meta-Analysis (PRISMA) [10], and the studies included should necessarily be published, indexed, and peer reviewed. No filters or other forms of search restrictions were used to achieve the greatest possible reach.

The literary search was made in the databases already mentioned above using the keywords (HEV OR Hepatitis E Virus) AND (Brazil). After reading the title and the abstract, papers that did not address Brazil as a scope or part of the scope, papers that did not study HEV, duplicate studies, review articles and experimental studies were excluded from this systematic review. Papers that did not make clear the information in the title and abstract were read in full and only those that contained the target content were included.

For the purpose of constructing this systematic review, all studies found in the databases that aimed at the parsing HEV in Brazil on their study scope were included, regardless of language, studied population or sample size. All authors independently

screened the databases, and relevant information was extracted. Differences in opinions about whether to include an article were solved by consensus between all the authors.

### **3. Results**

A total of 2173 papers were retrieved from the 5 databases used for the search (Figure 1). After removal of duplicated papers (*n* = 542), exclusion criteria were applied to eliminate non-related papers, namely papers classified as "non-Brazilian" (*n* = 24), "non-HEV" (*n* = 1519), as well as review articles and in vivo animal experimental studies.

Application of inclusion and exclusion criteria generated a total of 71 eligible papers. They were all included in the study after being assessed by full-reading. The distribution of published papers by regions of Brazil and type of study can be observed in Figure 2. HEV studies in humans, swine and animal products, animals other than swine, and environment are summarized in Tables 1–4, respectively.


**Table 1.** HEV in humans, Brazil.





52










61


(*morcilla*) *recom*Well® HEV IgM/ *recom*Well® HEV IgG (Mikrogen, Diagnostik, Munich, Germany); 2 *recom*Line® HEV IgG/IgM (Mikrogen, Diagnostik, Munich, Germany); 3 PrioCHECK™ AB HEV antibody ELISA kit (Thermo Fisher, Zurich, Switzerland); 4 HEV antibody (Abbiotec™, California, USA); 5 in-house: a two HEV recombinant proteins, a mosaic protein (MP-II) and a protein containing region 452–617 aa of the ORF2 of the HEV Burma strain used as coating antigens; b in-house indirect ELISA containing recombinant HEV-ORF2p antigen.

South

Rio Grande

1

do Sul 2015 Pork products

Pâtés, blood sausage

RNA (Nested

RT-PCR) 18/50 (36%) 3 - [76]

Rio Grande

do Sul 2015–2016 Edible products

of animal origin

RNA (Nested RT-PCR)


Bovine, swine, chicken and capybara raw meats, processed meats (mortadella, sausage, salami, ham, pâté)

Bovine 0/57 (0%), swine 0/30 (0%), chicken 0/29 (0%), capybara 0/1 (0%), mortadella 0/8 (0%), sausage 0/12 (0%), salami 0/14 (0%), ham 0/4 (0%), pâté 0/4 (0%)






**Figure 2.** Distribution (number) of HEV studies according to the regions of Brazil and the origin (human, swine and animal products, animals other than swine, and environmental).

### *3.1. HEV in Humans*

HEV studies performed in humans in Brazil (Table 1) were focused on a variety of population groups and most were serological surveys.

Studies performed in populations from regions with lower sanitation and hygiene conditions in the North region found an anti-HEV IgG seroprevalence of 0.3% in afro descendants [14]. Studies done in poor communities in the Midwest region found an anti-HEV IgG seroprevalence of 3.3% and 10.66% in adults [28,30] and 4.5% in children [27]. In the Southeast region, a seroprevalence of 2.4% was found also in poor communities [34].

Seroprevalence studies focusing on rural settlements (Table 1) found anti-HEV IgG seroprevalences of 12.9% in the North [11], 3.4% [25], 3.9% [23], and 8.4% [26] in the Midwest, and 2.1% [35] and 20.7% [41] in the Southeast. Three of these studies performed in rural settlements were also focused on current and/or recent infections. The study of the Midwest region found 0% of anti-HEV IgM and HEV RNA [25] and the study of the North found 0.3% of anti-HEV IgM [14].

Several investigations were conducted in HIV patients from Brazil and found anti-HEV IgG seroprevalence of 4.1% [19] in the North, 0% [32], 6.7% [53] and 10.7% [42] in the Southeast. Anti-HEV IgM and HEV RNA in HIV patients was searched only in the Southeast region and found anti-HEV IgM in 0% [32], 0.83% [53], and 1.4% [42], while HEV RNA was detected in 2.23% [53] and 3.6% [32].

HEV studies in Brazil have also focused on transplant recipients (Table 1). Among those with kidney transplants, anti-HEV IgG seroprevalence was found to be of 2.5% [21] in the Midwest, and 3.1% [45] and 15% [43] in the Southeast. HEV RNA was found in 3.1% [45] and 10% [43] of kidney transplant recipients. Only two studies investigated HEV infection in liver transplant recipients, namely a case report in a pediatric patient [40] and a study in the Southeast region that found a seroprevalence of anti-HEV IgG and IgM of 8.1% and 2.6%, respectively [39].

Several investigations in Brazil were conducted in healthy blood donors and pregnant women (Table 1). Anti-HEV IgG seroprevalence in blood donors was found to be 0.45% [13] in the North, 2% [18] in the Northeast, 4% [26] in the Midwest, 4% [47], 4.3% [35] and 9.8% [37] in the Southeast, and 2.3% [50], 7.1% [53], 10% [54], 26% [49], and 40.25% [52] in the South. Of these studies, three also investigated current and/or recent infections by detecting anti-HEV IgM/HEV RNA, having found 0.33% and 0% [54], and 0.35% and 0.35% [53] respectiely, in the South. In the study of Southeast, anti-HEV IgM/RNA was 2.4% and 0%, but only IgG positive samples were tested [37].

Seroprevalence studies were also conducted in populations with occupational, exposure risk to HEV infection. In hospital employees anti-HEV IgG seroprevalences of 4.34% [48] and 5.9% [47] were found, while in recyclable waste pickers [24] and pig handlers [11] seroprevalences were 5.1% and 6.3%, respectively.

Molecular characterization of the HEV strains detected in humans in Brazil showed that all belonged to HEV-3 [32,33,40,45,53]. Further characterization of some of the strains identified subtypes 3b [33,40] and 3i [45].

### *3.2. HEV in Swine and in Animal Products*

All studies performed in swine (Table 2) found evidence of HEV infection, either by using the detection of anti-HEV IgG and/or HEV RNA. Seroprevalence studies in younger pigs (<10 months) found an anti-HEV IgG prevalence of 8.6% in North region of Brazil [57] and 69.7% in the Midwest region [60]. The detection of HEV RNA in stools in this age group was 1.7% in the Northeast region [58] 7.94% in the North region [57] and 87.5% in Southwest [67].

In pigs from family-scale the anti-HEV IgG prevalence was 0% [61] and 67% [60] in the Midwest region, and 77.6% in the South region [71]. Regarding the detection of HEV RNA in stools of pigs from family-scale farms, 8% [61] and 24% [62] were found positive in the Midwest region, and 20% [68] in the South region.

Seroprevalence studies on slaughtered pigs showed anti-HEV IgG in 81.2% in Midwest [64] and 81.3% in the Northeast [59]. The detection of HEV RNA in bile from slaughtered pigs showed to be positive in 9.6% [66] and 15.2% [65] in Southeast and 0.84% in South [69].

The molecular characterization of the HEV found in pigs showed several subtypes (Table 2), namely 3b [62,66,68–72] 3c [57,65,71], 3d [61], 3f [57,58,62], 3h [61,71], and 3i [61,65].

Concerning wild boar, only two HEV seroprevalence studies were performed, both in the South region, having found a seroprevalence of 14.29% in Rio Grande do Sul state [73] while in Santa Catarina state, 1.55% [73] and 13.1% [74] seroprevalences were observed.

Regarding the HEV contamination of meat and meat products derived from swine and other animals (Table 2), HEV RNA was detected in 36% of the pig pâtés and blood sausages (morcilla) derived from pork [76]. In another study, no HEV was detected either in pig processed meats such as mortadella, sausage, salami, ham, and pate, or in the raw meat of bovine, swine, chicken, and capybara [75].

### *3.3. HEV in Animals Other Than Swine*

None of the studies performed in free-living monkeys has found evidence of HEV infection, either by using the detection of anti-HEV IgG [11] or HEV RNA [77] (Table 3). Anti-HEV IgG was detected in cows (1.42%), dogs (6.97%), chickens (20%), and wild rodents (50%), but not in sheep and goats [11]. Two new viruses were detected in wild rodents, *Calomys* HEV (CaHEV) and *Necromys* HEV (NeHEV), and a new orthohepevirus species was proposed [78] (Table 3).

### *3.4. HEV in Environment*

The detection of HEV RNA in waters (bathing/recreation waters, pig farm draining waters, settlement influenced waters), bivalve molluscs, and sediments was nega-

tive [55,76,79] (Table 4). In the two studies performed on pig slurry lagoons, HEV RNA was detected in 50% [66] and 100% [72] of the samples.

### **4. Discussion**

The HEV studies in humans in Brazil started in the early 90s. The majority of these initial investigations were conducted in rural areas, possibly motivated by the HEV-1 and HEV-2 data from endemic regions in developing countries with similar poor sanitary conditions. The first HEV reports in Brazil focused on communities with low levels of sanitation, such as gold miners [29] and poor communities [28,30] from the Amazon area of the Midwest region, and from the Southeast region [34]. In these reports, the fecally contaminated water was pointed as a potential route of HEV transmission and the seroprevalences within these communities ranged from 0.45% in children to 10.66% in adults [27,28].

After the recognition of HEV-3 as being responsible for autochthonous hepatitis E in industrialized countries [81,82], HEV studies in Brazil started to focus on cases of acute non-A-C viral hepatitis in order to clarify the potential role of HEV in these undiagnosed cases [17,28,35], efforts that still motivate publications nowadays [15,36]. In general, markers of current and/or recent HEV infection (anti-IgM HEV and HEV RNA) have been detected but at a low prevalence, indicating that HEV was not the causal agent of the majority of these acute hepatitis cases.

Based on the knowledge that HEV-3 infection may progress to a chronic hepatitis in immunocompromised patients [3], some HEV studies in Brazil have focused on organ transplant recipients [39] and HIV patients [42]. In kidney transplants, HEV seroprevalence varied from infrequent (2.5%) [21] to frequent (15%) [43]. In liver transplant recipients the prevalence of anti-HEV antibodies showed to be higher than immunocompetent populations in Brazil, suggesting HEV infection as a possible cause of liver injury [39]. Concerning HIV patients, studies showed similar HEV seroprevalences when compared with blood donors indicating that HIV patients are not at risk for HEV infection [19,53].

Hepatitis E caused by HEV-1 and HEV-2 has been associated with morbidity and mortality in pregnant women [3]. Possibly motivated by this, some HEV seroprevalence studies have been performed in pregnant women in Brazil, however no risk for HEV seropositivity has been shown in this particular group when compared with the general population [13,35,49].

Several studies have evaluated the HEV seroprevalence in the general population of Brazil, with the majority using blood donors as the sampled group. A great range of HEV seroprevalence was observed, with the lowest detected in the North (0.45%) [13] and Northeast regions (2%) [18]. Mid-range levels of HEV seroprevalence were observed in the Midwest (4%) and Southeast (4%, 9.8%) regions [26,37,47]. In the South region, the five seroprevalence studies showed values of 2.3% [50], 7.1% [53], 10% [54], 26% [49], and 40.25% [52]. The high seroprevalence detected in the South has been justified for being the region in Brazil with the highest density of pig farms and the largest consumption of pig meat and related products [52]. In fact, pig breeding has been suggested to influence human HEV seroprevalence in other countries [83,84]. Epidemiologic surveys performed in rural population of Brazil, namely in the North [11] and in the Southeast regions, have found higher seroprevalences in these populations (12.9% and 20.7%, respectively) when compared to those previously reported on blood donors from the same regions [11,41]. This difference has been attributed to the lower sanitary conditions of the rural populations. Overall, the range of seroprevalences observed in Brazil has to be interpreted with caution since some studies were performed several decades apart and using different immunoassays. It is widely known that the different anti-HEV IgG immunoassays and their performance characteristics strongly influence HEV seroprevalence data [85].

Despite the strong evidence of widespread HEV circulation in Brazil, the recent report of the official governmental databases presented no notification of hepatitis E among the notified 216,379 hepatitis cases [86]. This draws attention to an underdiagnosis and/or underreporting of hepatitis E in Brazil. The underdiagnosing of hepatitis E cases has been reported elsewhere and is partly attributed to the fact that HEV testing has not been traditionally included in hepatitis differential diagnostic algorithms [87].

Many HEV studies in Brazil have focused on swine, which is understandable given the fact that this country is the 4th largest pig producer in the world, with more than 2 million breeders and producing 3975 thousand tons/year of pork meat, with the South region representing 66.12% of the national production [88]. Circulation of HEV in pigs of Brazil was observed either in large or family-scale herds, and in all age groups, based on HEV RNA presence in stools/biological fluids/organs (0.8–88.9%) or anti-HEV IgG seroprevalence (0–77.6%) [61,62,68,72]. Evidence for HEV infection in slaughtered pigs was also shown by the high seroprevalence (>80%) detected [59,64]. The circulation of HEV was also demonstrated in wild boars of Brazil with seroprevalences ranging from 1.55% to 14.29% [73,74]. HEV was inclusively found in pig pâtés and blood sausages derived from pork [76]. Overall, HEV is highly disseminated in the swine population throughout Brazil and might present a risk to animal handlers and pork consumers, mainly if pork meat and meat products are eaten raw or undercooked. The presence of HEV in pigs and derived pig products has been widely reported in other countries [84,88–90].

In the past years there has been an interest in studying HEV infection in non-human primates, inclusively *Macaca fascicularis* were used on experimental in vivo studies performed in Brazil to evaluate HEV pathogenesis [91–93]. HEV seroprevalences have been reported in farmed *Rhesus* monkeys in China (70.8%) [94] and in captive non-human primates in Italy (4.2%) [95] but the only seroprevalence study performed in Brazil in wild non-human primates did not detect any (0%) anti-HEV antibodies [11]. Furthermore, no HEV RNA was detected in the stools and livers of Golden-headed lion tamarins of Brazil [77].

Serological studies in Brazil also focused on other animals, having reported the presence of antibodies anti-HEV in cows, dogs, chicken, and wild rodents, but not in sheep and goats [11]. Antibodies against HEV have also been detected in dogs in the United Kingdom [96], in chicken, cows, wild rodents, sheep, and goats in China [97–100], chickens in Korea [101], sheep in Italy [102], but the zoonotic importance of these animals concerning HEV remain to be clarified. Noteworthy, two novel HEV strains were discovered in wild rodents from Brazil (*Calomys tener* and *Necromys asiurus*) [78].

Concerning the HEV studies that focused on the environment in Brazil, only water samples under the influence of swine farm effluents, namely slurry lagoons, were found positive for HEV [66,72]. Samples from the southern region of Brazil, with a high density of swine production, detected HEV in up to 100% of the samples analyzed [72]. This same region coincides with the highest rates of human seropositivity for HEV and is also the region with the highest concentration of pig production in the country. This fact, analyzed from the One Health perspective, highlights the zoonotic character of this virus. Swineinfluenced waters contaminated by HEV have been frequently detected and reported in other countries [103,104]. In the studies of Brazil, HEV was not detected in bivalve molluscs, recreation waters, or even in waters that drained effluents from pig farms or waters of poor quality, very close to human settlements [76,79,80]. However, studies in other countries have reported HEV in bivalve molluscs [105–107], seawater [108], and wastewater [109,110]. These discrepancies of detection of HEV in environment samples could be in part due to the low concentration of HEV and complexity of the matrices, two well-known limiting factors of the detection of enteric viruses in environmental samples.

Concerning the molecular characterization of HEV strains detected in Brazil, studies showed that all HEVs found in Brazil were classified as HEV-3 (6 studies in humans, 15 in swine and animal products, and 2 on environmental samples). HEV-3 is known to have a zoonotic (swine) origin and the subtypes 3b and 3i were detected in humans [33,40,45] and pigs [61,62,65,66,68–72], while the subtypes 3c [57,65,71], subtype 3d [61], subtype 3f [57,58,62] and subtypes 3h [61,71] have been only detected in pigs. As molecular studies have been performed using several molecular assays and primer choices, different regions

of HEV have been targeted and characterized. This clearly hampers the robust classification of HEV subtypes and, consequently, a solid comparison between subtypes, hence caution must be taken when analyzing this data. In fact, attention should be paid to several factors that could bias the interpretation of results here presented. A clear focus has been given to human samples with little attention to animal or environmental matrices, most likely due to the initial understanding of this disease, not known to be zoonotic at that time. Additionally, not only a higher number of studies have also focused on the South where the highest density of pig farms is present but also a vast diversity of sample sizes has been used throughout the studies, making it difficult to robustly compare results. Further studies spatially dispersed are for these reasons recommended.

The present systematic review is not the first that targets HEV in Brazil. The two published so far have centered only on human infection [111,112] while here we present for the first time a perspective focusing on the One Health triad, having included HEV studies on humans, animals, and environment. A One Health approach makes it possible to look at issues such as zoonotic diseases, food safety, and food security, as well as environmental contamination and other aspects. In this perspective this review evidenced that the scientific community has approached the topic of HEV on every aspect of environment, human, and animal systems individually, however when compiled, this translates into data that broadens the scope to One Health.

### **5. Conclusions**

Overall, this systematic review shows that HEV-3 was the only retrieved genotype in humans, animals, and environment in Brazil. The South region showed the highest HEV seroprevalence in humans, which curiously is also the region with the highest pig density, swine industry, and pig HEV circulation, suggesting a zoonotic link. HEV- 1 and HEV-2 were not detected in any of the studies performed in Brazil, even in those focusing on low sanitary condition communities. This allowed us to infer that HEV epidemiology in Brazil is similar to that of industrialized countries (only HEV-3 circulation, swine reservoirs, no waterborne transmission, no association with low sanitary conditions). Hence, we alert for the implementation of HEV surveillance systems in swine and for the inclusion of HEV in the diagnostic routine of acute and chronic hepatitis in humans. More sequence data are needed on HEV strains circulating in humans, animals, and the environment to further evidence the zoonotic origin of HEV infection in Brazil.

**Author Contributions:** D.F.d.S.D.M.: Conceptualization, data curation and investigation (search of articles in electronic databases and their respective cataloguing), formal analysis, methodology, and writing original draft preparation; J.R.M.: Conceptualization, data curation and investigation (search of articles in electronic databases and their respective cataloguing), review of the writing and substance of this article, supervision, and validation; V.D.: Conceptualization, methodology, revised this paper, supervision, and validation; M.S.J.N.: Conceptualization, data curation and investigation, wrote full text and revised the article improving the technical quality of the manuscript, supervision, and validation. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

### **References**


**Abdelaziz Ghanemi 1,2 , Mayumi Yoshioka <sup>2</sup> and Jonny St-Amand 1,2,\***


**Simple Summary:** High-fat (HF) diet induces both immune-mediated damage and trefoil factor family member 2 (*Tff2*) expression. As TFF2 has tissue repair and protection properties, this suggests that HF diet-induced *Tff2* production and the resulting TFF2 mucosal protective effects would be a mechanism to counteract the HF diet-induced tissue damage. On the other hand, the induction of *Tff2* by HF diet could indicate that TFF2 is a food intake regulator (appetite control) since *Tff2* is also expressed in the brain. This highlights the importance of exploring TFF2-related pathways in the context of obesity management towards potential therapies.

**Abstract:** Physiological homeostasis requires a balance between the immunological functions and the resulting damage/side effects of the immunological reactions including those related to high-fat (HF) diet. Within this context, whereas HF diet, through diverse mechanisms (such as inflammation), leads to immune-mediated damage, trefoil factor family member 2 (*Tff2*) represents a HF diet-induced gene. On the other hand, TFF2 both promotes tissue repair and reduces inflammation. These properties are towards counteracting the immune-mediated damage resulting from the HF diet. These observations suggest that the HF diet-induction of *Tff2* could be a regulatory pathway aiming to counteract the immune-mediated damage resulting from the HF diet. Interestingly, since *Tff2* expression increases with HF diet and with *Tff2* also expressed in the brain, we also hypothesize that TFF2 could be a HF diet-induced food intake-control signal that reduces appetite. This hypothesis fits with counteracting the immune damage since reducing the food intake will reduce the HF intake and therefore, reduces the HF diet-induced tissue damage. Such food intake signaling would be an indirect mechanism by which TFF2 promotes tissue repair as well as a pathway worth exploring for potential obesity management pharmacotherapies.

**Keywords:** trefoil factor family member 2 (TFF2); high-fat diet; immunity; damage; mice

Animal physiological homeostasis requires a balance between the immunological functions and the damage/side effects of those immunological reactions. Knowing that immunological reactions can be triggered by diverse factors, the homeostasis supposes that parallel or secondary pathways are activated or stimulated with these immunological reactions to repair the damage. The immune system is a complex network of cells and circulating fluids that is modulated by the nervous system [1], endocrine system [2], infections [3], and even diet. Indeed, different types of diets, such as high-sucrose and highfat (HF) diets, have been shown to impact immune functions [4,5], among other factors and genes [6,7]. HF diets characterize our modern life, and are associated with diverse diseases and health problems, such as obesity, dyslipidemia, diabetes, fatty liver disease and cardiovascular diseases [7–10]. However, such HF diet-induced immune modulations, which could be implicated in the HF diet-induced risks and diseases, are yet to be fully

**Citation:** Ghanemi, A.; Yoshioka, M.; St-Amand, J. High-Fat Diet-Induced Trefoil Factor Family Member 2 (TFF2) to Counteract the Immune-Mediated Damage in Mice. *Animals* **2021**, *11*, 258. https:// doi.org/10.3390/ani11020258

Received: 22 December 2020 Accepted: 19 January 2021 Published: 21 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

understood. Within this context, the molecules and signals that are either upregulated or downregulated with HF diets could be the mechanistic answer, as per the examples we provide below from studies on mice.

For instance, trefoil factor family member 2 (TFF2), known as spasmolytic peptide [11], is well involved in mucosal repair, protection and proliferation, as it represents an important stabilizer of the gastric mucus, with roles in tissue remodeling [12]. Herein, we go beyond its mucosal protective role to explore the hypothesis linking this diet-induced molecule, TFF2, to the diet-induced immunomodulation. Indeed, whereas *Tff2* has been reported as a gene that is specifically induced by HF diets in mice [13,14], its knockout protected mice from HF diet-induced obesity [15] through a metabolic phenotype that contributes to more energy expenditure and reduced energy storage [16]. The importance of the studies that identified *Tff2* as a gene specifically induced by HF diets is that the control groups were, unlike in other studies, fasted mice [13,14]. Based on the HF induction of TFF2, we notice a correlation between the HF diet-induced immunological changes and the TFF2-related immunological effects and benefits (as illustrated below). This correlation suggests that TFF2 would be involved in mediating the protective effects against such HF diet damage.

On one side, a HF diet has important immunological impacts. For instance, a HF diet increases TNFα and IL1β in young mice's hippocampus [17], and leads to chronic systemic inflammation [18]. Moreover, a chronic HF diet is also associated with obesity [19,20], which also affects the immunity [21] and might explain some of the impacts obesity has on regeneration impairment through diverse processes, including inflammation [22], which is important in the context of TFF20 s roles in tissues repair.

On the other hand, TFF2, beyond its well-known roles in injured mucosa healing [23–25], has a noticeable role in the immune response [25,26], as suggested by its expression in immune organs [27] and its expression during inflammations [12]. Indeed, *Helicobacter* infection upregulated it in gastric tissues, macrophages and lymphocytes [11], whereas *Helicobacter pylori* eradication decreased TFF2 level in patients' sera [28]. Furthermore, TFF2 deficiency leads to a deregulation of macrophages' and lymphocytes' proliferative responses [11], and an accelerated gastritis progression [29] during *Helicobacter* infection. This correlates with both the ulceration role of *Helicobacter pylori* [30] and the tissue repair/protections roles of TFF2 in animal selected tissues [12].

TFF2 expression during such immunological changes seems to be an attempt to limit the negative impacts of these immune reactions, such as inflammation [12], due to the HF diet. For instance, TFF2 could both limit the recruitment of leukocytes and the monocyte production of nitric oxide [25], and decrease macrophage responsiveness [27], which would contribute to promoting the tissue repair environment. Therefore, this TFF2 induced downregulation of selected immunological responses would be a step required to accomplish the healing and protecting effects TFF2 governs.

These illustrative examples present TFF2 as a mediator of the HF diet-triggered mechanisms attempting to correct the HF diet's negative impacts, mediated through the immune system. Interestingly, unlike glucose, which causes insulin as a hormone to be secreted immediately following meal ingestion [31], there is no equivalent hormone for lipid ingestion. TFF2 could be that missing signal within animal endocrinology, since in the studies in which *Tff2* was shown to be unregulated at 3 h following a low-fat meal ingestion, it was upregulated with a HF meal [13,14]. The acute character of this expression indicates an immediate effect of the HF diet on Tff2 expression. Therefore, TFF2 could be a short-term lipid-specific signal that controls lipid intake by limiting lipid ingestion through a TFF2-dependant feedback acting on food intake centers. This is supported by the differential *Tff2* expression in the hypothalamus of fasted, and low-fat and HF dietfed, mice (lipid ratio-dependent expression) [15]. This hypothesis is further supported by the increase in the drive to consume a HF meal, as well as the appetite enhancement as a consequence of TFF2 deficiency [15]. This would suggest that TFF2 counteracts HF diet-induced damage indirectly through reducing the HF intake. The other remarkable link is that TFF2 is mostly expressed in the digestive system [32,33], which represents

the site whereat the animal's neuroendocrine receptors first interact with the ingested food, including HF meals; this further suggests the acute responsiveness of the HF diet's induction of TFF2 in the mouse intestine. Always within the digestive system, the HF diet impacts the local microbiome [34,35], which could be another key link between the diet and the immunological changes, especially with the known interactions between the immune system and the microbiome [36–38], the microbiota richness reduction [39], and dysbiosis, in all of which the HF diet has been implicated [40]. In addition, since several effects of a HF diet are mediated by microbiota [18] with probiotics that upregulate TFF2 [41], these microbiota-mediated effects of the HF diet could be through TFF2 expression changes.

These elements highlight TFF2 expression (HF diet-induced) as a feedback aiming to counteract the immune-mediated HF diet-induced damage. However, the correcting potential and efficacy of TFF2 would depend on the severity and the chronic or acute character of such a HF diet. This explains why during obesity (such as in HF diet-induced obesity in animal models), those TFF2-correcting mechanisms are less efficient due to the strong immune-mediated damage that overcomes the TFF2-counteracting ability. Further explorations of diets' impacts on TFF2 expression, such as high-salt diets [42], within an immunological context would expand this emerging field linking the type of diet to the immunological changes via identifying the linking factors. Importantly, combining these metabolic and immunological properties of TFF2 would allow us to further understand how mice immunologically react to a HF diet, and elucidate more diet-induced effects on immunology, infections and inflammation. Importantly, extrapolating these concepts from mice to humans and building clinical trials based on animal experiments could lead to developing novel TFF2-based therapies for diseases and conditions, such as inflammation, and, most importantly, a potential control for lipid intake (appetite control) towards a better obesity management strategy, which requires urgent solutions due obesity's epidemiological profile and its impacts on health and the economy [43–46].

**Author Contributions:** A.G. drafted the manuscript; A.G., M.Y. and J.S.-A. critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Abdelaziz Ghanemi received a merit scholarship for foreign students from the Ministry of Education and Higher Education of Quebec, Canada. The Fonds de recherche du Québec—Nature et technologies (FRQNT) is responsible for managing the program (Bourses d'excellence pour étudiants étrangers du Ministère de l'Éducation et de l'Enseignement supérieur du Québec, Le Fonds de recherche du Québec—Nature et technologies (FRQNT) est responsable de la gestion du programme). The graphical abstract was created using images from: http://smart. servier.com. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


*Letter*
