Rabies in chozna ‘Potus flavus’: a warning of a potential threat to public and animal health

Abstract

We reported an outbreak of wild rabies in Potus flavus (choznas), a carnivorous mammal associated with insectivorous bats in Kosñipata, Cusco, Peru, from February 2021 to October 2022. Eight human exposures were reported, with aggressive attacks resulting in injuries. The outbreak prompted a public health response and raised concerns about zoonotic diseases due to habitat fragmentation and urbanization. The study involved antigenic and genetic characterization of the virus in the affected animals, revealing similarities with variants in non-hematophagous bats. This outbreak marked the first time rabies had been reported in choznas in the region, highlighting the need for early intervention programs and wildlife protection. Pre-exposure rabies vaccinations were administered to residents, and community surveillance was established to control the outbreak. The study emphasizes the importance of continuous surveillance and education to prevent rabies transmission from choznas to humans and other animals, as their role as potential secondary transmitters of the virus warrants further investigation.

Keywords:

• mammals
• outbreak
• rabies

Introduction

Rabies, caused by a virus of the Lyssavirus genus, produces an acute, progressive and fatal encephalomyelitis in humans exposed to saliva or tissues of rabid animals (Hemachudha et al. 2013). Throughout its evolutionary history, the rabies viruses (RV) have established species-specific transmission cycles as a consequence of interspecies transmission, mainly through host changes, overcoming species, geographic and ecological barriers (Davis et al. 2015). In addition to known transmission cycles, there is also evidence of the circulation of the rabies virus in other wild animals through the detection of neutralizing antibodies (Almeida et al. 2001; Jorge et al. 2010).

In Peru, reports of rabies outbreaks in non-hematophagous bats in the high jungle of Peru date as back as the late 1960s (De Yosti et al. 1971). The circulation of the rabies virus in Potus flavus (chozna) was first reported in 2002 in Madre de Dios, and additional isolated cases were reported in 2012 and 2015 (Condori-Condori et al. 2013; Vargas-Linares et al. 2014). This small species of wild carnivore (42–58 cm height and 2–5 Kg weight) inhabits tropical areas in central-southern Peru and are likely to be susceptible to habitat fragmentation, deforestation, and climate change. The continuous growth of cities has favored the deforestation of natural areas, as well as the capture of animals likes this for adoption as pets by the rural population, which can favor the transmission of zoonotic diseases. It is very likely that due to the COVID-19 pandemic, measures in the control and prevention of wild rabies have been weakened in the past few years (Raynor et al. 2021). Understanding these threats is crucial for wildlife protection, as well as control of wild rabies. Therefore, it is necessary to review the epidemiological description of outbreaks, identification of species and conservation areas for chozna, which will favor early and timely intervention programs in the transmission of wild rabies and eventually anticipate the presentation of human rabies transmitted by these animals, in addition to expanded studies to detect and characterize new reservoirs and hosts of rabies.

We present an epidemiological description of an outbreak of wild rabies in choznas occurring for the first time in the district of Kosñipata, Paucartambo, Cusco, Peru.

Materials and methods

The present investigation describes nine cases of rabies in chozna that occurred in a period of 20 months (February 2021 – October 2022), in a range of approximately 10 km affecting six different rural populations in the Kosñipata district, Cusco, Peru: Pillcopata (Túpac Amaru, Sabaluyoc, Villa San Francisco, Santa Rosa de Huacaria) and Patria (Mistiana, Sonia) (Figure 1). The district of Kosñipata, is located at 527 meters above sea level, has a population of 5,609 inhabitants, whose main economic activity is agriculture, mainly coca plant cultivation. The cases occurred in buffer zones, which are areas adjacent to the limits of protected natural areas that due to their location require special treatment to guarantee compliance with the purposes of habitat, flora and fauna conservation.

Figure 1.  Temporal (A) and geographic (B) distribution of Potus flavus cases with rabies in Pillcopata, Cusco, 2021–2022.

Antigenic characterization was performed using eight monoclonal antibodies made against the nucleoprotein and donated by the United States Centers for Disease Control and Prevention which identified variant 4, subsequently genetic characterization was performed. For this, total RNA was extracted from a portion of the infected brain using the TRI- reagent kit (Sigma, https://www.thermofisher.com). Next, the complementary DNA (cDNA) was synthesized using the SuperScript ® IV Reverse Transcriptase kit (https://www.thermofisher.com) and used as a template for the sequencing of the complete nucleoprotein gene (71-1423). Two pairs of oligonucleotides Lys 001, 921B and 550 F, 304 (López-Ingunza 2004; Velasco-Villa et al. 2006; Mantari Torpoco et al. 2019) were used for the PCR. Subsequently, the amplified products were purified with the QIAquick PCR Purification Kit (QIAGEN, https://www.qiagen.com/) and sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, https://www.thermofisher.com) and the same pairs of primers (Supplementary Table 1).

This analysis was evaluated by the Continental University Ethics Committee (letter N°0261-2022-VI-UC), the data was collected by the researchers as part of the routine epidemiologic surveillance.

Results

There were reports of human being attacked by choznas exhibiting the aggressive behavior characteristic of furious rabies in six of the nine animal cases (66.6%). People reported having been bitten in arms, hands or legs, inside of their houses or in the road of the town (Table 1). Additionally, in two instances the choznas attackeed hens or dogs, not causing any bites in humans. Eight of the nine choznas (88.8%) were captured and killed by the inhabitants of the area after defending themselves from animal attacks. Generally, after the incidents, villagers went to the local health center for care and treatment, receiving anti-rabies serum and rabies vaccine.

Table 1. Characteristics of the Potos flavus cases in the Kosñipata district, Paucartambo province, Cusco region, Peru, 2021–2022.

Case	Epidemiological week	Location	Circumstances of the accident	Distance between consecutive cases	People attacked	Age of people attacked (years)	Antigenic variant
1	8 − 2021	Tupac Amaru Sector, Pillcopata	A person finds a small animal on a road when he treated to adopt it like a pet, he was attacked by a furious animal.	---------	1	12	4
2	15-2021	Sabaluyoc Sector, Pillcopata	Furious animal attacked a person who treated to get the animal out of his house.	Between case 1 and 2: 4 108 m	1	32	4
3	26-2021	Tupac Amaru Sector, Pillcopata	The dogs of a person were attacked by a furious animal, where the owner of the dogs was biting.	Between case 2 and 3: 3 561 m	1	38	4
4	28-2021	Pillcopata	A dying animal was finding in Pillcopata city by the people of the town	Between case 3 and 4 2 931 m	0	-----	4
5	49-2021	Villa San Francisco	Furious animal entered in a house and attacked the overnight room.	Between case 4 and 5 6 243 m	2	25/1	4
6	09 − 2022	Mistiana Patria	Furious animal entered and attacked the chicken coop of a house biting the owner of the house who treated to get the animal out.	Between case 5 and 6: 10 536 m	1	52	4
7	32-2022	Sonia Sector, Patria	Furious animal attacked and biting in the road where people were walking	Between case 6 and 7: 4 174 m	2	15/17	4
8	34-2022	Santa Rosa de Huacaria (native community), Pillcopata	Two children kill the chozna because it was chasing their chickens. They hadn’t bitten.	Between case 7 and 8: 9 478 m	0	-----	4
9	43-2022	Patria	Furious animal attack to dogs of the town with one dog injured	Between case 8 and 9: 6 920 m	0	-----	4

The extraction of the encephalic samples was carried out in all (9/9) cases at the Pillcopata Health Center, Kosñipata. In most cases, the initial diagnosis using direct immunofluorescence was made by the Reference Health Laboratory of Cusco and confirmed by the Viral Zoonoses Laboratory of the Peruvian National Institute of Health.

The edited sequences allowed us to build a molecular phylogeny tree where we observed an identity of 99.9% across the four rabies sequences from choznas from Cusco (Figure 2). Likewise, the similarities of the rabies virus sequences in choznas with variants circulating in non-hematophagous bats were evidenced. The closest sequences were identified in Eptesicus sp and Myotis yumanensis bats with an identity of 88.8% and 88.6% respectively. Comparison of the sequences from this outbreak to those from a previous outbreak in Madre de Dios revealed the presence of a second RV clade circulating in populations of chozna in Cusco. Phylogenetic reconstruction showed that they share a common ancestor, but it is independent lineage from the isolates reported in Madre de Dios (Identity: 88.4%); probably related to distinctive spatio-temporal dynamics throughout their geographic ranges. Similar findings were reported in outbreaks that occurred in Mexico, with variants of the rabies virus circulating in coatis and in an unidentified reservoir in Oaxaca, whose occurrence would be related to the circulation of variants in insectivorous bats of the Molossidae family (Aréchiga-Ceballos et al. 2010). However, the reservoir for the lineages found in choznas have not yet been identified in this study or in other studies from the Americas. Similar data were also reported in a Potus flavus case in Brazil; suggesting the associated evolutionary origin in non-hematophagous bats (Dell’Armelina Rocha et al. 2020).

Figure 2. Phylogenetic tree of the RV isolated in Potos flavus and Reference sequences associated with bat transmission in Peru and america, including three sequences of choznas isolated in the department of Madre de Dios in the years 2007, 2013 and 2015. The maximum likelihood evolutionary tree was obtained using the IQ-TREE v1.6.12 program (model GTR + G + I, 1000 bootstraps).

Discussion and conclusions

This is the first report of an outbreak of rabies in choznas in Kosñipata, Paucartambo province, Cusco, Peru. Human exposures occurred due to the aggression of choznas, both in the open field, and inside homes. Nine cases of rabies in choznas led to eight people being injured during animal attacks or while attempting to defend and/or capture the animals. All humans exposed received treatment with anti-rabies serum and rabies vaccine. The genetic analysis found that the cases of rabies in choznas were more closely associated with insectivorous bats, with a still unknown reservoir, not identified in this study or in other studies (Salmón-Mulanovich et al. 2009; Kuzmin et al. 2012; Meske et al. 2021).

As part of outbreak control measures, an epidemiological alert was issued to confirm wild rabies in these mammals with a high risk of human rabies during the COVID-19 health emergency scenario in the area affected. Likewise, residents of the Tupac Amaru and Sabaluyoc sectors, received pre-exposure rabies vaccination (Ministerio de 2017) with 66.67% coverage for the first dose, 58.3% for the second dose and 36% for the third dose. The remaining 33.3% of people received post-exposure vaccination. Community surveillance of wild rabies was implemented through meetings with local committees such as the free-school-breakfast (Vaso de Leche) program committee, teachers and parents, and radio broadcasts by local stations, in addition to meetings with local authorities to strengthen community actions in the short and long term. Subsequently, research and surveillance actions for rabies in animals were complemented through the National Agricultural Health Service (SENASA) and the National Forest and Wildlife Service (SERFOR).

Finally, it is necessary to recognize the risk presented by the choznas as probable secondary transmitters of rabies for humans and animals, making it necessary to carry out an active epidemiological search for more cases to assess whether it is a new reservoir. Likewise, it is important to educate the members of the small communities where the choznas are kept as pets, due to a possible risk of bites and consequent transmission of rabies to humans.

Acknowledgments

The authors thank all the participants for their ongoing contributions and commitment to this study; all the physicians and biologist which work in Pillcopata Health Center.

Additional information

Funding

This work was not supported by any monetary contributions from public or private institutions.