Occurrence of Clostridium perfringens in Wild Mammals in the Amazon Biome

1. Introduction

Clostridium perfringens is a gram-positive, spore-forming bacillus that is classified into seven toxinotypes (A – G) according to the production of six major toxins: alpha (cpa), beta (cpb), epsilon (etx), iota toxin (itx), enterotoxin (cpe) and necrotic enteritis type B (netB) [1,2,3]. C. perfringens is found mainly as a commensal of the gastrointestinal tract of humans and animals and is ubiquitous in the environment [3].

Despite being a common commensal, C. perfringens can cause a number of diseases in animals and humans, including gas gangrene and various enteric conditions [1,4]. In humans, it is the second most common cause of foodborne illness in the United States and the sixth most common in Brazil [5]. Some studies have demonstrated the presence of C. perfringens as a commensal in some species of wild animal in addition to confirming the participation of this agent as a cause of enteritis and enterotoxaemia [6,7,8,9,10]. However, the description of this bacterium and its toxinotypes in wild animals of the Amazon Biome, either as a commensal or as a pathogen, is lacking.

It remains largely unknown which species are colonized by this agent, and among those that have C. perfringens as a commensal species, it is not known which toxinotypes are present or which toxins are potentially produced by those strains. Thus, the objective of the present study was to evaluate the isolation frequency and types of C. perfringens in wild animals in the Amazon biome.

2. Materials and Methods

2.1. Data Collection

This study was conducted in accordance with the Ethical Principles of Animal Research adopted by the National Council for the Control of Animal Experimentation (CONCEA) and was approved by the Committee on Ethics in the Use of Animals of the Federal University of Pará (CEUA/UFPA) under protocol n° 8888280618. The study was also authorized by the Chico Mendes Institute for Biodiversity Conservation (ICMBio) under n° 67300-1.

A non-probabilistic sampling method (for convenience) was used [11] due to all the particular characteristics of the species of the animals treated and hospitalized during the period from August 2017 to October 2022. The collection procedures were monitored by veterinarian residents to monitor animal welfare, including the physical, mental and behavioural states of each species [12].

Stool samples were collected during feeding, which occurred twice a day (morning and afternoon), to reduce contact and handling stress. Pieces of PVC film paper were placed under the animals’ enclosure, with subsequent collection of fresh, individual faeces produced without much soiling of the substrate. The collection of samples also occurred during medication applications, physical examinations and weighing [13]. Cloacal and anal swabs were collected under the same conditions, and proper physical and chemical protocols for each species were applied to reduces the risk of accidents and stress [14].

The stool samples were placed in microtubes (2 ml), and the swab samples were transferred to a saline solution, after which the samples were labelled with the animal identification, species and collection date. After labelling, the samples were stored in a refrigerator at -20 °C [15].

2.2. Processing

All samples were sent to the Laboratory of Bacteriosis and Research of the Federal University of Minas Gerais (UFMG) for research and typing of C. perfringens [15]. For the isolation of C. perfringens, between 0.08 and 0.12 g of faeces were immersed in 1 ml of brain heart infusion broth (BHI, Difco Laboratories, USA) for enrichment and incubated in an anaerobic chamber (Thermo Scientific, BR) for 24 hours at 37 °C. Cloacal and anal swabs were inoculated directly into tubes with BHI and subjected to the same culture conditions. After incubation, 10 μL aliquots of each sample were plated on Shahadi-Ferguson perfringens agar (SFP, Oxoid, UK) and incubated again in an anaerobic chamber at 37 °C for 48 hours [15].

For each plate, up to three characteristic colonies containing sulfite-reducing microorganisms were suspended in 400 μL of ultrapure water (Milli-Q®) in Eppendorf tubes (2 ml) and subjected to thermal DNA extraction at 98 °C for 20 min in a thermoblock (HDV, BR) [15,16]. Subsequently, the microtubes were centrifuged at 3000 x g for 10 min, and the resulting supernatant was used as template DNA in a multiplex PCR to identify the genes encoding the alpha, beta, epsilon and iota toxins and enterotoxin [16]. For all the PCRs, the amplifications were performed in a thermocycler (Thermal Cycler Px2, Thermo Electron Corporation, USA), and the bands were visualized with ultraviolet light on a 2% agarose gel stained with ethidium bromide (Sigma‒Aldrich Corporation, USA) [15,16].

2.3. Statistical Analyses

The data were summarized using frequency tables. To measure the association between the categorical variables (presence of diarrhoea, host, sample type [swabs or faeces]) and isolation of C. perfringens, a univariate analysis was performed using Fisher’s exact test with a significance level of P ≤ 0.05. The odds ratios with 95% confidence intervals were also calculated. All analyses were performed using R Software 4.0.9 (R Development Core Team, NZ).

3. Results

Sixty-five stool samples and cloacal or anal swabs were obtained from 16 species and three genera of mammals in good health or with diarrhoea (Table 1). Among the species included in the study, five were the most common: Nasua nasua (South American coati), with 14 samples (21.4%); Bradypus variegatus (common ground sloth), with eight samples (12.3%); Didelphis sp. (opossum), with seven samples (10.8%); and Choloepus didactylus (sloth), Saimiri collinsi (squirrel monkey), and Sapajus apella (Guyana capuchin), each with five samples (7.7%). In the present study, 59 (90.8%) of the samples were from healthy individuals, and six (9.2%) were from individuals with diarrhoea. Regarding the type of sample, there was a balance between stool samples (32/65=49.2%) and anal and cloacal swabs (33/65=50.8%). The number of animals sampled in situ (19, 29.2%) was lower than the number of animals sampled ex situ (46, 70.8%).

C. perfringens was isolated from 40 (61.5%) samples. All the isolates were positive only for the gene encoding the alpha toxin and were classified as type A (Table 1). Approximately half of the healthy animals (36/59 =55.4%) were positive for C. perfringens type A, while four of the six animals with diarrhoea (4/6 =66.7%) were positive for this toxinotype, with no difference between groups (p=1.0). The animals that were positive for C. perfringens Type A and that presented diarrhoea were of the genus Didelphis sp. (opossum) and of the species Sapajus apella (Guyana capuchin), Puma yagouaroundi (buckwheat) and Nasua nasua (South American coati).

The Isolation of C. perfringens was greater in the stool samples than in the swab samples (p = 0.0105): a stool sample was approximately four times more likely to be positive than a swab sample (OR = 4.28; CI: 1.47-12.6). There was no difference in the frequency of isolation between ex situ and in situ animals (p=0.16), with 31 (47.7%) and nine (13.8%) positive animals, respectively.

Samples from Alouatta caraya (black howler monkey, n=1), Saguinus ursulus (marmoset, n=1), Mazama americana (Mateiro deer, n=1), Mazama nemorivaga (brown deer, n=1) and Dasypus novemcinctus (nine-banded armadillo, n=1) were negative for C. perfringens in the present study.

4. Discussion

Most previous studies that investigated the prevalence of C. perfringens in wild animals have focused on a single animal species or on groups belonging to the same family and with similar feeding habits [9,15,17,18,19]. This appears to be the first study covering this diversity of forest mammals within Brazil, allowing a better understanding of the occurrence of C. perfringens in the Amazon biome.

The finding that more than 60% of the samples were positive for C. perfringens type A corroborates the findings of previous studies of other species of wild animals, showing that type A is the most prevalent commensal [9,15,17,18,19,20]. Some species included in the present study were negative for this anaerobic microorganism. This result may be related to the low sampling rate of some animals, especially for those with only one sample. A previous study also proposed the hypothesis of non-colonization or low colonization of some species [21].

In addition, the present study revealed that swabs had a lower rate of C. perfringens isolation than faeces and excreta, which may also have influenced C. perfringens detection in some species. Interestingly, a study conducted with toucans in Brazil revealed no difference between the isolation rate of C. perfringens from faeces and that from swabs [21]. Furthermore, it is worth noting that most of the stool samples obtained in the present study were collected ex situ, representing more than two-thirds of the samples obtained. This pattern could have affected the rate of isolation, since studies have suggested that C. perfringens is apparently more likely to be observed in ex situ samples than in in situ samples [18].

Despite the importance of C. perfringens as an enteropathogen in domestic animals and humans, the actual role of type A in diarrhoeal diseases remains uncertain in several wild species due to the absence of a marker that allows for the differentiation of C. perfringens when it is present as an enteropathogen or as a commensal [15,17,18]. Nevertheless, there are reports suggesting that type A is the cause of lethal haemorrhagic enteritis in several species, such as Panthera tigris altaica (Siberian tiger), Panthera leo (lion) [10], Vulpes vulpes (red fox) [22], Loxodonta africana (elephant) [23], Selenarctos thibetanus (Asian black bear) [24] and Papio hamadryas (hamadryas baboon) [25]. Previous studies have suggested that factors other than the toxinotype may be associated with the occurrence of enteric disease caused by C. perfringens type A in animals and humans, such as stress, dysregulation of the intestinal microbiota, and parasitic and viral infections [5,18,22,24,25,26,27,28,29,30].

The results of the present study corroborate previous findings suggesting that C. perfringens type A is a commensal in N. nasua (coati) [15], C. thous (crab-eating fox) [17], P. yagouaroundi (black-tailed cat), and L. pardalis (ocelot) [19]. On the other hand, this seems to be the first report of C. perfringens in some species, such as B. variegatus (common ground sloth), C. didactylus (two-toed sloth), T. tetradactyla (anteater), Didelphis sp. (opossum), S. apella (Guyana capuchin), S. collinsi (squirrel monkey), S. niger and P. flavus (Jupará).

5. Conclusions

The findings of the present study suggest that C. perfringens is a commensal bacterium of several species of wild mammals in the Amazon biome. Similar to the vast majority of domestic animals, type A seems to be the most common type among the animals sampled.