Parasitic Protozoa and Other Vector-Borne Pathogens in Captive Mammals from Brazil

Abstract

In captive environments, mammals are frequently exposed to various parasitic protozoa and other vector-borne pathogens that can impact both animal health and public health. Monitoring these pathogens is essential for animal welfare and zoonotic disease control. This study aimed to investigate the prevalence of parasitic protozoa and other vector-borne pathogens in captive mammals through molecular detection methods at the Belo Horizonte Zoo, Brazil. Between November 2021 and March 2023, whole blood samples were collected from 40 mammals. Molecular analyses identified piroplasms, Leishmania spp., granulocytic/platelet Anaplasma/Ehrlichia spp., monocytic Ehrlichia spp., Bartonella spp. and hemotropic Mycoplasma spp. with a 72.5% positivity rate. Piroplasms were found in 22.5% (two Pantanal cats, two gorillas, one white rhinoceros, one spider monkey, one jaguar, one tufted capuchin and one hippo) and Leishmania spp. in 12.9% (four maned wolves). Granulocytic/platelet Anaplasma/Ehrlichia spp. were found in 12.5% of the samples (one gorilla and four maned wolves), Ehrlichia canis in 2.5% of the animals (one maned wolf), Bartonella spp. in 42.5% (six howler monkeys, two maned wolves, one gorilla, one white rhino, one southern tamandua, one common woolly monkey, one tufted capuchin, one brown brocket deer, one agouti, one cougar and one hippo), hemotropic Mycoplasma spp. in 17.5% (one gorilla, one maned wolf, one white rhino, one howler monkey, two common woolly monkeys and one European fallow deer). Five Artiodactyla members tested negative for A. marginale. Coinfections occurred in 34.5% of the positive samples. Sequencing revealed that Theileria spp. and Cytauxzoon spp. are closely related to Theileria bicornis and Cytauxzoon felis; Ehrlichia canis and Bartonella spp. are closely related to B. clarridgeiae and B. henselae; and hemotropic Mycoplasma spp. are closely related to Candidatus Mycoplasma haemominutum. Our results showed a high occurrence of vector-borne pathogens in captive animals, including zoonotic species, which may pose a risk to animal and human public health.

1. Introduction

Diseases caused by vector-borne pathogens represent a significant challenge to the health and well-being of animals in captivity [1], with direct implications for the management of collections in zoos and conservation centers. These diseases can also affect the human population due to their zoonotic potential. The adverse effects of parasitic infections in zoo animals can vary [2], including the emergence of secondary deficiencies and other infections, reproductive impairment and the risk of mortality in cases of massive and dangerous parasitosis. Additionally, zoos are environments where wild animals have close contact with humans, considerably increasing the risk of transmission of parasitic zoonoses [3]. This poses a threat not only to the health of the animals themselves but also to the health of zoo staff and visitors.

The confined nature of these environments increases the susceptibility of animals to vector-borne infections due to population density, close interactions between species and environmental stress [4]. Additionally, the diversity of host species maintained in zoos provides a conducive environment for the transmission and maintenance of a wide range of vector-borne pathogens. The presence of these pathogens in zoo animals can result in a series of negative health impacts on the host, including anemia, weight loss, tissue damage, immunosuppression and, in severe cases, mortality [5].

Piroplasmids, such as Babesia, Theileria and Cytauxzoon, belong to the phylum Apicomplexa and are transmitted by parasitic ticks to mammals. These protozoa may be asymptomatic in wild animals but cause severe consequences and even death in domestic animals. These infections result in economic losses in livestock, trade restrictions in horses and negative health impacts on dogs and cats, as well as growing concern over cases of human babesiosis [6]. The genus Leishmania, member of the family Trypanosomatidae within the order Kinetoplastida, comprises protozoan pathogens transmitted by sandflies, such as Lutzomyia sp. The Leishmania protozoa infect both humans and a wide range of animals, contributing to the complexity and severity of zoonotic diseases and their impacts on public and veterinary health.

The Anaplasmataceae family, which includes the genera Anaplasma, Ehrlichia, Neorickettsia and Wolbachia, infects a variety of mammalian cells and can be transmitted by a wide range of vectors, such as ticks and trematodes. In Brazil, several species from this family have been detected in a wide range of wild animals, such as deer, wild canids, wild felids, coatis, rodents, peccaries, collared peccaries and opossums [7,8]. Bartonella species, belonging to the family Bartonellaceae, are transmitted by ectoparasites, such as fleas, lice and ticks, and can infect a variety of captive animals, including marine mammals such as dolphins as well as terrestrial mammals and their arthropod ectoparasites. The greater occurrence in some captive cohorts compared to free-ranging animals suggests that captivity may be a risk factor for Bartonella infection [9]. Additionally, hemotropic Mycoplasma species, classified within the class Mollicutes, are known to cause anemia in various mammalian species, transmitted by blood-feeding arthropods [10].

Vector-borne pathogens, including protozoa and bacteria, have been increasingly reported in captive mammals worldwide, with significant implications for both conservation efforts and animal welfare. For example, Protozoan agents have been associated with mortality and the development of clinical signs in captive cervids and canids from Canada and Brazil [11,12]. Bacterial pathogens have already been detected in captive animals around the world, Hemoplasmas have been detected in various captive animals, indicating that these pathogens can infect a wide range of species in captivity, which could potentially lead to health issues if not monitored [9,13,14,15,16].

Thus, the present work aimed to investigate the occurrence of vector-borne pathogens in captive mammals from the Zoo of the Belo Horizonte Zoo-Botanical Foundation, Minas Gerais, Brazil.

In this study, we report the high prevalence of vector-borne pathogens in various mammalian species at the Belo Horizonte Zoo by employing molecular techniques to detect and identify these pathogens. Our findings underscore the complexity of disease management in captive environments and the importance of early detection and integrated control measures to protect both animal and human health.

2. Materials and Methods

2.1. Ethical Statement

All procedures were conducted in accordance with relevant guidelines and regulations. The samples were collected after the signing of a partnership agreement between the Federal University of Minas Gerais (UFMG) and the Municipal Parks and Zoo-botanical Foundation (FPMZB) in strict compliance with applicable ethical guidelines.

2.2. Study Area

This study was conducted at the Belo Horizonte Municipal Parks and Zoo-Botanical Foundation (FPMZB), a 10.7-million-square-meter complex that houses more than 3500 individuals across 235 species, including 36 mammal species with 117 individuals. Notably, more than 40 species spanning reptiles, birds, fish, amphibians and mammals from all continents are threatened with extinction. Located in Belo Horizonte, Minas Gerais (−19.857889841, −44.0075113912) (Figure 1), the Zoo features a veterinary hospital and a rich history of biodiversity preservation. Established through Decree 16,684 of 31 August 2017, the FPMZB merged the former Municipal Parks Foundations and the Zoo-Botanical Foundation, playing a vital role in conserving local ecosystems and researching mammal species. Its parks and botanical gardens harbor more than a thousand species, representing Brazil’s Cerrado and Atlantic Forest biomes [17].

2.3. Samples

Between November 2021 and March 2023, whole blood samples were collected from 40 captive animals of the class Mammalia at the Zoo. These samples were sent for analysis to the Veterinary Protozoology Laboratory—ProtoVet, as part of the service provided to FPMZB under the code Siex-UFMG 302557—“Diagnosis and control of hemoparasites in domestic and wild animals”. The capture and containment of the animals were conducted by the Zoo’s team of veterinarians following the management schedule of the mammal population. To ensure the representativeness of the sampling, at least one sample of each mammal species present in the zoo was collected.

2.4. DNA Extraction and PCR Amplification

DNA was extracted from 300 µL of whole blood using the “Wizard Genomic DNA Purification Kit” (Promega, Madison, WI, USA) according to the manufacturer’s recommendations. After the extraction, the quality and quantity of the samples were estimated using a NanoDrop (Epoch Microplate Spectrophotometer, Biotek^®, Winooski, Vermont, USA). The extracted DNA was stored at −20 °C until detection via PCR amplification. A fragment of mammalian glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to verify the quality of the extracts [18].

Blood DNA samples from mammals that tested positive for the GAPDH gene were subjected to PCR assays for Piroplasmids, Leishmania, Anaplasma, Ehrlichia, Bartonella and hemotropic Mycoplasma. All agents, target genes, primer sequences, amplicon sizes (bp), thermal cycling conditions and references are shown in

Table 1. Molecular assays used in the present study are categorized by the involved agent, type of PCR, target gene, primer sequences, amplicon size (bp), thermal cycling conditions and references.

Agents	Aim/Primer	Molecular Assay	Primers Sequences	Fragment Size (bp)	Thermal Cycling	Reference
Anaplasma sp. (A. phagocytophilum, A. bovis, A. platys) (16S rRNA gene)	Screening 1ª reaction gE3a gE10R 2ª reaction gE2 gE9f.	nPCR	5′-CACATGCAAGTCGAACGGATTATTC-3′ 5′-TTCCGTTAAGAAGGATCTAATCTCC′-3′	932	94 °C for 5 min 40 cycles: 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, 72 °C for 5 min	[19]
			5′-GGCAGTATTAAAAGCAGCTCCAGG-3′ 5′-AACGGATTATTCTTTATAGCTTGCT-3′	546
Ehrlichia sp. (E. chaffeensis, E. canis) (16S rRNA gene)	Screening 1ª reaction NS16SCH1F NS16SCH1R 2ª reaction NS16SCH2F NS16SCH2R	nPCR	5′-ACGGACAATTGCTTATAGCCTT 5′-ACAACTTTTATGGATTAGCTAAAT	1195	94 °C for 5 min 30 cycles: 92 °C for 1 min, 54 °C for 1 min and 72 °C for 2 min, 72 °C for 8 min	[20]
			5′-GGGCACGTAGGTGGACTAG-3′ 5′-CCTGTTAGGAGGGATACGAC-3′	443
Anaplasma phagocytophilum (msp4 gene)	Characterization 1ª reaction MSP4AP5 MSP4AP3 2ª reaction msp4F msp4R	nPCR	5′-ATGAATTACAGAGAATTGCTTGTAGG-3′ 5′-TTAATTGAAAGCAAATCTTGCTCCTATG-3′	849	94 °C for 5 min 30 cycles: 92 °C for 1 min, 54 °C for 1 min and 72 °C for 2 min, 72 °C for 8 min	[21]
			5′-CTATTGGYGGNGCYAGAGT-3′ 5′-GTTCATCGAAAATTCCGTGGTA-3′	381		[22]
Anaplasma marginale/ A. ovis (msp4 gene)	Characterization 1ª reaction MSP45 MSP43 2ª reaction AnapF AnapR	nPCR	5′-GGGAGCTCCTATGAATTACAGAGAATTGTTTAC-3′ 5′-CCGGATCCTTAGCTGAACAGGAATCTTGC-3′	872	94 °C for 5 min 30 cycles: 92 °C for 1 min, 54 °C for 1 min and 72 °C for 2 min, 72 °C for 8 min	[23]
			5′-CGCCAGCAAACTTTTCCAAA-3′ 5′-ATATGGGGACACAGGCAAAT-3′	294		[14]
Anaplasma platys (16S rRNA gene)	Characterization 1ª reaction 8-F 1448-R 2ª reaction PLATYS-F EHR16S-R	nPCR	5′-AGTTTGATCATGGCTCAG-3′ 5′-CCATGGCGTGACGGGCAGTGT-3′	*	1ª reaction 94 °C for 2 min, 40 cycles: 94 °C for 1 min, 45 °C for 1 min, 72 °C for 40 s, 72 °C for 4 min. 2ª reaction 94 °C for 1 min, 40 cycles: 94 °C for 1 min, 53 °C for 30 s, 72 °C for 30 s, 72 °C for 4 min.	[24]
			5′-GATTTTTGTCGTAGCTTGCTATG-3′ 5′-TAGCACTCATCGTTTACAGC-3′	678
Babesia/Theileira/ Cytauxzoon (18S rRNA gene)	Screening 1ª reaction RIB-19 RIB-20 2ª reaction BabRumF BabRumR	nPCR	5′-CGGGATCCAACCTGGTTGATCCTGC-3′ 5′-CCGAATTCCTTGTTACGACTTCTC-3′	1700	94 °C for 5 min 30 cycles: 92 °C for 1 min, 54 °C for 1 min and 72 °C for 2 min, 72 °C for 8 min	[25]
			5′-ACCTCACCAGGTCCAGACAG-3′ 5′-GTACAAAGGGCAGGGACGTA-3′	430		[26]
Cytauxzoon felis	Characterization CytauxF CytauxR	cPCR	5′-CGAATCGCATTGCTTTATGCTCCAA 5′-TTGATACTCCGGAAAGAG	284	95 °C for 5 min, 40 cycles; 95 °C for 45 s, 59 °C for 45 s and 72 °C for 60 s, 72 °C for 5 min	[27]
Leishmania sp. (ITS1 gene)	Screening LITSR L5.8S	cPCR	5′-CTGGATCATTTTCCGATG-3′ 5′-TGATACCACTTATCGCACTT-3′	300–350	95 °C for 2 min, 37 cycles: 94 °C for 30s, 53 °C for 1 min, 72 °C for 1 min, 72 °C for 6 min.	[28]
Bartonella spp. (16S-23S rRNA intergenic region)	Screening BartF BartR	cPCR	5′-CTCTTTCTTCAGATGATGATCC-3′ 5′-AACCAACTGAGCTACAAGCCCT-3′	145–232	95 °C for 2 min, 45 cycles: 95 °C for 1 min, 60 °C for 1 min, 72 °C for 30 s, 72 °C for 5 min	[29]
Hemotropic Mycoplasma sp. (16S rRNA)	Screening HBTF HBTR	cPCR	5′-ATACGGCCCATATTCCTACG-3′ 5′-TGCTCCACCACTTGTTCA-3′	618	94 °C for 10 min, 40 cycles: 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, 72 °C for 10 min	[30]

* the fragment size is unknown or cannot be determined.

.

For the PCRs, positive DNA controls were obtained from various sources. For tests of Anaplasma phagocytophilum and other granulocytic agents of the Anaplasmataceae family, DNA from IDE8 cell cultures infected with A. phagocytophilum, which was isolated from a German dog and kindly provided by Dr. Erich Zweygarth (Institut für Vergleichende Tropenmedizin und Parasitologie, Ludwig Maximilians Universität München), was used. DNA from Anaplasma marginale was extracted from 300 µL of whole blood collected from a calf experimentally infected with A. marginale (strain UFMG1) [31]. For monocytic ehrlichiosis tests, DNA from a dog infected with Ehrlichia canis (Jaboticabal strain) [32] was used. For the nPCR of piroplasmids, positive controls were obtained from a calf experimentally infected with Babesia bovis (strain BbovMG) and Babesia bigemina (strain BbigMG) [33]. DNA from reference Leishmania strains provided by the World Health Organization (WHO), specifically Leishmania infantum (MCAN/BR/2002/BH400), which was maintained in the cryobank of the Leishmania Biology Laboratory and kindly provided by Prof. Maria Norma Melo (ICB/UFMG/Brazil), was used as a reaction control. Additionally, DNA for hemoplasmas and Bartonella spp. was obtained from the blood samples of naturally infected cats in Belo Horizonte, Minas Gerais, Brazil, confirmed by sequencing [34]. Ultrapure sterile water (Life Technologies^®, Carlsbad, CA, USA) was used as a negative control in all PCR assays.

PCR amplicons were separated by electrophoresis on 1%, 2% and 3% agarose gels (40 min, 100 V), stained with GelRedTM (Biotium, Hayward, CA, USA) and visualized under ultraviolet light.

2.5. Restriction Fragment Length Polymorphism (RFLP) Analysis—PCR for Leishmania spp.

To identify Leishmania spp., the ITS1 amplicon was digested using the restriction enzyme HaeIII (PCR-RFLP). Five microliters of each amplified product were digested with the HaeIII restriction enzyme (Biolabs, Inc., UK) according to the manufacturer’s instructions. The digested products were subjected to 5% polyacrylamide gel electrophoresis to verify the restriction patterns. The restriction patterns obtained were compared with those of WHO reference strains [28].

2.6. Sequencing

Sequencing was performed by ACTGene using an automatic sequencer (ABI 3730xl DNA Analyzer Applied Biosystems ^TM) with POP7 polymers and BigDye v3.1 using the same oligonucleotide primers used in the assays. For sequencing analysis, the raw data in the form of chromatograms generated by the automatic sequencer “Analyzer Applied Biosystems” were aligned, edited and analyzed using the BioEdit program, version 7.0.5.3 [35]. The identity of each sequence was confirmed by comparison with sequences available in GenBank using BLAST software (Basic Local Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/ (accessed on 26 August 2024)) [36]. After comparing the identities, the analyzed sequences were classified according to the degree of similarity with data already deposited in GenBank. All nucleic acid sequences revealed in this study have been deposited in the GenBank database (PP844851, PP948683-PP948688, PP849127, PP849128, PP843077 and PP843078).

2.7. Phylogenetic Analysis

Multiple sequence alignments were carried out using the Multiple Alignment using Fast Fourier Transform (MAFTT) algorithm (MAFFT v7.310) [37], with previously published sequences in GenBank. Moreover, representative sample sequences were also downloaded for use as an outgroup in the phylogenetic analysis. Phylogenetic trees were constructed using the Maximum Likelihood (ML) method based on the results of alignment with MEGA11 [38,39,40,41] with suitable models selected with the smallest Bayesian information criterion (BIC) score. The reliability of the tree topology was tested using 1000 bootstrap replicates, as implemented in the program [42]. For piroplasmids, the Kimura 2-parameter method + G evolutionary model (K2 + G (5 categories, parameter = 0.1288)) (430 bp alignment) were used. For Ehrlichia spp., the Kimura 2-parameter model + gamma distribution (K2 + G (5 categories, parameter = 0.3607)) evolutionary model (630 bp) were used. For Bartonella spp., the Jukes–Cantor evolutionary model + gamma distribution + invariable sites (JC + G (5 categories, parameter = 1.0975)) + (I ([+I], 19.19% sites)) (165 bp alignment) were used, and for Mycoplasma spp., the general time reversible model with gamma distribution (GTR +G (5 categories, parameter = 0.3121)) (alignment of 600 bp).

2.8. Data Analysis

The data obtained were collected on an Excel spreadsheet, and descriptive frequency analysis was performed using Microsoft Excel Version 16.71 (Microsoft Corporation, Redmond, WA, USA).

3. Results

In this study, samples were collected from 40 individuals of the class Mammalia. Of these, 57.5% (23/40) were males, and 42.5% (17/40) were females. The animals under study formed a highly heterogeneous group due to the diversity of species found in the zoo, belonging to different orders: 45% (18/40) from the order Primates, 35% (14/40) from the order Carnivora, 12.5% (5/40) from the order Artiodactyla, 2.5% (1/40) from the order Perissodactyla, 2.5% (1/40) from the order Pilosa and 2.5% (1/40) from the order Rodentia. Details regarding the collected animal species can be found in

Table 2. Identification of captive mammals sampled at Zoo of the Belo Horizonte Municipal Parks and Zoo-Botanical Foundation, Minas Gerais, between 2021 and 2023, regarding identification number, collection data, sex and molecular positivity for vector-borne pathogens. Results obtained in the PCR assays for Anaplasma phagocytophilum, A. platys and Anaplasma marginale are not shown since all animals tested negative.

ID	Collection Data	Sex	Species	Granulocytic/Platelet Anaplasma/Ehrlichia sp.	Monocytic Ehrlichia sp.	Bartonella sp.	Hemotropic Mycoplasma sp.	Piroplasmids	Leishmania spp.
AZ01	2021-11-04	M *	Leopardus braccatus	−	−	−	−	Positive	−
AZ02	2021-11-04	M	Leopardus braccatus	−	−	−	−	Positive	−
AZ04	2021-11-22	F **	Gorilla gorilla gorilla	−	−	Positive	Positive	Positive	−
AZ05	2021-11-22	F	Gorilla gorilla gorilla	Positive	−	−	−	Positive	−
AZ06	2021-12-07	F	Puma concolor	−	−	−	−	−	−
AZ07	2021-12-16	F	Chrysocyon brachyurus	−	−	Positive	−	−	Positive
AZ08	2022-02-08	M	Galictis cuja	−	−	−	−	−	−
AZ09	2022-02-08	M	Subulo gouazoubira	−	−	−	−	−	−
AZ11	2022-03-10	F	Chrysocyon brachyurus	Positive	−	−	Positive	−	Positive
AZ12	2022-03-10	F	Chrysocyon brachyurus	Positive	−	−	−	−	Positive
AZ13	2022-03-10	M	Chrysocyon brachyurus	Positive	−	−	−	−	Positive
AZ14	2022-03-16	M	Chrysocyon brachyurus	Positive	Positive	Positive	−	−	−
AZ15	2022-03-16	M	Chrysocyon brachyurus	−	−	−	−	−	−
AZ16	2022-05-16	M	Dama dama	−	−	−	−	−	−
AZ17	2022-05-16	M	Gorilla gorilla gorilla	−	−	−	−	−	−
AZ18	2022-05-27	M	Chrysocyon brachyurus	−	−	−	−	−	−
AZ19	2022-05-16	F	Ceratotherium simum	−	−	Positive	Positive	Positive	−
AZ20	2022-05-16	F	Alouatta sp.	−	−		Positive	−	−
AZ21	2022-05-27	F	Alouatta sp.	−	−		−	−	−
AZ22	2022-07-27	F	Tamandua tetradactyla	−	−	Positive	−	−	−
AZ25	2022-08-25	F	Panthera leo	−	−	−	−	−	−
AZ27	2022-07-01	F	Ateles sp.	−	−	−	−	Positive	−
AZ28	2022-10-25	F	Lagothrix lagotricha	−	−	Positive	Positive	−	−
AZ29	2022-10-25	M	Sapajus apella	−	−	Positive	−	−	−
AZ30	2022-11-15	M	Subulo gouazoubira	−	−	Positive	−	−	−
AZ31	2022-11-21	F	Panthera onca	−	−	−	−	−	−
AZ32	2022-11-24	M	Lagothrix lagotricha	−	−	−	−	−	−
AZ33	2022-11-24	F	Lagothrix lagotricha	−	−	−	Positive	−	−
AZ34	2022-11-24	M	Sapajus apella	−	−	−	−	−	−
AZ35	2022-11-24	F	Lagothrix lagotricha	−	−	−	−	−	−
AZ36	2023-01-24	M	Dama dama	−	−	−	Positive	−	−
AZ37	2023-01-24	M	Dasyprocta sp.	−	−	Positive	−	−	−
AZ38	2023-01-26	M	Alouatta sp.	−	−	Positive	−	−	−
AZ39	2023-01-26	F	Alouatta sp.	−	−	Positive	−	−	−
AZ41	2023-01-26	F	Alouatta sp.	−	−	Positive	−	−	−
AZ42	2023-01-26	M	Alouatta sp.	−	−	Positive	−	−	−
AZ43	2023-01-26	M	Alouatta sp.	−	−	Positive	−	−	−
AZ44	2023-01-25	F	Puma concolor	−	−	Positive	−	−	−
AZ45	2023-02-10	M	Alouatta sp.	−	−	Positive	−	−	−
AZ46	2022-11-15	F	Hippopotamus amphibius	−	−	Positive	−	Positive	−

* Male; ** Female; − Negative.

.

Among the 40 DNA blood samples collected from mammals, all tested positive by GAPDH gene-based cPCR and were included in subsequent analyses.

Overall, 72.5% (29/40) of the samples tested positive for at least one parasite. Nine animals (9/40–22.5%) tested positive by nPCR for piroplasmids (two Pantanal cats (Leopardus braccatus), two gorillas (Gorilla gorilla gorilla), one white rhinoceros (Ceratotherium simum), one spider monkey (Ateles spp.), one jaguar (Panthera onca), one tufted capuchin (Sapajus apella) and one hippo (Hippopotamus amphibius). Sequencing was performed on all positive samples, but only two were successful. Theileria sp. was detected in a 54-year-old female white rhinoceros, showing 99.55% identity with Theileria bicornis found in a captive white rhinoceros (MF536661) from Australia. Additionally, a female jaguar was found to have a piroplasm with 99.77% identity to Cytauxzoon felis, which was previously identified in an ocelot (Leopardus pardalis) (GU903911) from Brazil. cPCR for Leishmania spp. was performed on 31 animals from the orders Primates and Carnivora, resulting in 12.9% positivity (four maned wolves (Chrysocyon brachyurus)), of which Leishmania infantum was detected in 50% (2/4) by PCR-RFLP.

Phylogenetic analysis using the ML method based on the 18S rRNA gene of piroplasmids (Figure 2) positioned the Theileria spp. sequences detected in white rhinoceroses from this study within the same clade as the T. bicornis sequences identified in white and black rhinoceroses from Australia and South Africa. Moreover, the Cytauxzoon spp. sequence detected in the jaguars in this study was closely related to C. felis sequences found in jaguars, ocelots and domestic cats from Brazil, South Africa and the United States.

In the nPCR analysis for the bacteria of the Anaplasmataceae family, granulocytic/platelet Anaplasma/Ehrlichia spp. were detected in 12.5% (5/40) of the animals, including one gorilla and four maned wolves. However, DNA amplification of A. phagocytophilum and A. platys was unsuccessful in these positive samples. Unfortunately, all samples positive for the 16S rRNA gene by PCR for the Anaplasma/Ehrlichia spp. granulocytes/platelets yielded bands of weak intensity, which precluded sequencing efforts for these fragments. Additionally, 2.5% (1/40) of the animals tested positive for monocytic Ehrlichia spp. (one maned wolf), which was confirmed by sequencing similarity with E. canis. BLASTn analysis revealed 99.31% identity with the sequences of E. canis detected in Canis lupus familiaris (MK507008) sampled in Cuba.

ML analyses based on the 16S rRNA gene of Ehrlichia sp. in this study detected a sequence that can be put into the same clade as previously identified E. canis sequences from domestic dogs in Brazil, Cuba, India and Turkey (Figure 3).

Bartonella spp. were detected in 42.5% (17/40) of the animals (six howler monkeys (Alouatta spp.), two maned wolves, one gorilla, one white rhino, one southern tamandua (Tamandua tetradactyla), one common woolly monkey (Lagothrix lagotricha), one tufted capuchin, one brown brocket deer (Subulo gouazoubira), one agouti (Dasyprocta spp.), one cougar (Puma concolor) and one hippo. Six samples were selected for sequencing due to the good intensity of the bands observed in the electrophoresis gel and the different sizes of the bands according to the primers used. The sequences obtained ranged from 152 bp to 345 bp, with four sequences showing 94.59% to 100% similarity to Bartonella clarridgeiae (the gorilla, tufted capuchin, brown brocket deer and southern tamandua) and two sequences showing 99% similarity to Bartonella henselae (Two howler monkeys).

Phylogenetic analysis using maximum likelihood, based on the internal transcribed spacer 16–23S gene, grouped Bartonella spp. sequences detected in the gorilla, southern tamandua, capuchin monkey and brown brocket deer from this study into the same clade as B. clarridgeiae sequences from cats and fleas. However, Bartonella spp. sequences detected in two howler monkeys from this study clustered with B. henselae previously identified in cats, rodents, elephants, Fea’s muntjacs and humans (Figure 4).

Hemotropics Mycoplasma spp. were detected in 17.5% (7/40) of the animals, including one gorilla, one maned wolf, one white rhino, one howler monkey, two common woolly monkeys and one European fallow deer (Dama dama). Two cases were confirmed by sequencing as hemotropic Mycoplasma sp.: one in the howler monkeys, showing 99.84% identity with “Candidatus Mycoplasma haemominutum” detected in a cat (KR905451) sampled in Italy, and another in a woolly monkey, showing 99.11% identity with a Mycoplasma sp. detected in a howler monkey (MH734376) sampled in Brazil.

Finally, ML analyses of hemotropic Mycoplasma spp. grouped the sequences detected in this study into a large clade, the “Mycoplasma suis group,” alongside sequences previously detected in various hosts from several countries, with 100% clade support (Figure 5). The sequence detected in the howler monkey was closely related to that of Ca. Mycoplasma haemominutum, which had been found in domestic cats from Brazil and Italy. Meanwhile, the sequence detected in a woolly monkey was closely related to hemotropic Mycoplasma spp. found in howler monkeys from Brazil.

Coinfections Detected

Of the 29 positive animals, 10 (34.5%) were coinfected (five maned wolves, two gorillas, one white rhinoceros, one woolly monkey and one hippo). Six animals were coinfected with two pathogens: one gorilla was coinfected with piroplasmids and granulocytic/platelet Anaplasma/Ehrlichia spp., a single maned wolf was coinfected with Leishmania spp. and Bartonella spp., two maned wolves were coinfected with granulocytic/platelet Anaplasma/Ehrlichia spp. and Leishmania spp., a single woolly monkey was coinfected with hemotropic Mycoplasma spp. and Bartonella spp. and one hippopotamus was coinfected with piroplasmids and Bartonella spp. Four animals were coinfected with three pathogens: one gorilla and one white rhino were coinfected with piroplasmids, hemotropic Mycoplasma spp. and Bartonella spp.; one maned wolf was coinfected with granulocytic/platelet Anaplasma/Ehrlichia spp., hemotropic Mycoplasma spp. and Leishmania spp.; and one maned wolf was coinfected with Ehrlichia canis, granulocytic/platelet Anaplasma/Ehrlichia spp. and Bartonella spp. (

Table 3. Coinfections in captive mammals sampled at the Zoo of the Belo Horizonte Municipal Parks and Zoo-Botanical Foundation, Minas Gerais, Brazil between 2021 and 2023.

ID	Species	Granulocytic/Platelet Anaplasma/Ehrlichia sp.	Monocytic Ehrlichia sp.	Bartonella sp.	Hemotropic Mycoplasma sp.	Piroplasmids	Leishmania spp.	Total Number of Pathogens Coinfected
AZ04	Gorilla gorilla gorilla	−	−	+	+	+	−	3
AZ05	Gorilla gorilla gorilla	+	−	−	−	+	−	2
AZ07	Chrysocyon brachyurus	−	−	+	−	−	+	2
AZ11	Chrysocyon brachyurus	+	−	−	+	−	+	3
AZ12	Chrysocyon brachyurus	+	−	−	−	−	+	2
AZ13	Chrysocyon brachyurus	+	−	−	−	−	+	2
AZ14	Chrysocyon brachyurus	+	+	+	−	−	−	3
AZ19	Ceratotherium simum	−	−	+	+	+	−	3
AZ28	Lagothrix lagotricha	−	−	+	+	−	−	2
AZ46	Hippopotamus amphibius	−	−	+	−	+	−	2

+ Positive, − Negative.

).

4. Discussion

Vector-borne diseases involving various infectious agents, hosts and vectors are highly important to human and animal health. Disease surveillance and control in zoological environments are crucial for preventing outbreaks and protecting both the species on display and nearby human populations [43]. The study results demonstrated a high prevalence of vector-borne pathogens in wild mammals at the Belo Horizonte Zoo-Botanical Foundation, with 72.5% of the tested samples showing positivity for at least one vector-borne pathogen.

It is worth noting that Belo Horizonte is considered an endemic region for most of the agents studied, which corroborates the high occurrence described here. Since the first report of E. canis in the city, the pathogen has spread throughout the territory and is being found in various regions of Brazil [44]. Additionally, A. platys is endemic in Brazil, with the prevalence varying according to the geographic region, target population and diagnostic methods used [45]. A study conducted in Minas Gerais with Belo Horizonte as the coverage area reported a prevalence of 4.1% and 17.2% of A. platys in dry and rainy periods, respectively, both in rural areas, while a lower prevalence was observed in urban areas (5%). Regional and climatic variations directly affect vector population density, providing specific conditions that influence the dissemination of the agent [33].

Canine visceral leishmaniasis (CVL) is a significant public health issue in Belo Horizonte, Brazil, with domestic dogs being the main reservoir host for the causative parasite Leishmania infantum [46,47]. Studies have shown that a large portion, estimated at 40–60%, of seropositive dogs are asymptomatic carriers. These asymptomatic dogs can still harbor the parasite, especially on their skin, and contribute to the maintenance of the disease transmission cycle [48]. Furthermore, research in Belo Horizonte has identified a high percentage of infected dogs that were asymptomatic and PCR positive but seronegative, posing challenges for health authorities in implementing effective control measures [49]. The prevalence of CVL in the city’s neighborhoods can vary widely, from 0 to 166.7 cases per 1000 dogs [47]. Spatial and temporal analyses have been used to identify priority areas for targeted surveillance and control efforts [46]. Human visceral leishmaniasis (HVL) cases in Belo Horizonte have also been found to be associated with canine seroprevalence and the human-to-dog ratio, underscoring the importance of addressing the canine reservoir in reducing disease transmission to humans [49].

Despite strict regulations prohibiting the entry of domestic animals into the zoo, stray dogs and cats often infiltrate the area, potentially introducing ectoparasites and pathogens to captive animals [50]. This uncontrolled access increases the risk of transmission of vector-borne diseases, underscoring the importance of stringent biosecurity measures and continuous, comprehensive monitoring of both zoo and stray animals for the health of the captive population and potential public health risks.

For piroplasmids, 22.5% (9/40) of the tested animals were positive, with confirmed detections of Theileria sp. related to T. bicornis and Cytauxzoon sp. related to C. felis, indicating a diversity of piroplasma species infecting different hosts. The infection rates in our study align with the literature, which reports values ranging from 5.2% to 96.7% for piroplasms, including Babesia spp., Theileria spp. and Cytauxzoon spp., in wild mammals [51]. For instance, ref. [51] reported a 5.5% prevalence of piroplasms in free-ranging mammals of the Superorder Xenarthra from four Brazilian states, whereas another study in Brazil reported a prevalence of 26.3% (40/152) in wild mammals [52,53]. Ref. [53] reported a 96.7% prevalence in free-ranging jaguars [54]. These differences are possibly due to variations in host species, geographic location and environmental factors [55].

Theileria bicornis has been reported in black and white rhinoceroses in Africa, with implications for their health and conservation. T. bicornis infections can be fatal in rhinoceroses, particularly when combined with other stressors such as injury, pregnancy or translocation [56], while C. felis is known to infect felids such as jaguars in Brazil [57]. The available evidence strongly suggests that jaguars can act as reservoirs for C. felis, harboring and potentially transmitting the parasite to other susceptible hosts. Moreover, C. felis infections appear to be capable of causing fatal disease in jaguars, similar to the severe outcomes observed in domestic cats [58,59]. Despite its predominance in wild felids, cases of infection in domestic felids are increasing in Brazil [59].

In this study, we detected sequences of Theileria spp. in a 54-year-old female white rhinoceros originating from Africa, housed at the Belo Horizonte Zoo and donated from Germany. To date, there has not been a description of T. bicornis in this animal species in Brazil, suggesting that the animal arrived at the zoo with a pre-existing infection, although there are recent reports of a piroplasm with 91.8 to 93.6% identity with T. bicornis in bats [60]. Phylogenetic analysis grouped the sequences detected in our study in the same clade as sequences of T. bicornis identified in white and black rhinoceroses from Australia and South Africa [56,61]. The phylogenetic similarity between these sequences may indicate a common origin of these pathogens or transmission via vectors sharing a common habitat across these regions [62,63].

Furthermore, Cytauxzoon spp. sequences detected in jaguars were closely related to the sequences of C. felis found in jaguars, domestic cats and other wild felids from Brazil, South Africa and the United States. This finding supports the global distribution of this protozoan and indicates its ability to infect a variety of feline species across different geographic regions [54,58]. Although the pathogenicity of Cytauxzoon spp. genotypes found in Brazil is unknown, free-ranging jaguars likely play a crucial role in the survival of C. felis in the wild [54]. Therefore, continuous surveillance and monitoring of the spread of C. felis are important, especially in environments where multiple feline species coexist, such as in zoos [58].

The presence of Leishmania spp. in maned wolves is a significant finding, highlighting the zoonotic nature of this parasite [64]. Leishmaniasis, particularly visceral leishmaniasis, is a serious disease in both humans and animals. The presence of Leishmania spp. in maned wolves is consistent with findings in other wild canids, underscoring the epidemiology of this disease and the need for severe vector control techniques and continuous health monitoring to prevent outbreaks [65]. A study by Silveira et al. (2016) [63] reported a case of Rangelia vitalii in a free-ranging maned wolf, which also tested positive for Leishmania sp., among other pathogens [64]. Similarly, a study by Mol et al. (2015) [64] demonstrated the transmissibility of L. infantum from maned wolves to the invertebrate vector Lutzomyia longipalpis, further emphasizing the role of wildlife in the epidemiology of leishmaniasis [65].

The detection of granulocytic/platelet Anaplasma/Ehrlichia spp. in 12.5% of the studied animals is consistent with previous reports of infections in wild and captive animals. Studies conducted by Calchi et al. (2020) and Pereira et al. (2016) [7,66] reported similar prevalences in wild animals in Brazil (Xenarthra mammals) and Portugal (cervids and wild boars), with prevalence rates of 27.57% and 24.82%, respectively. The unsuccessful amplification of DNA from A. phagocytophilum and A. platys might indicate the presence of Anaplasma species or genetic variants not detectable by the primers used, limited PCR sensitivity or a low parasitic load in the samples [67]. Previous studies have documented similar challenges in the molecular detection of Anaplasma species, where primer specificity and parasitic load significantly influence the success of amplification [68]. Although the DNA of A. marginale in members of the Artiodactyla order was not amplified in this study, this pathogen has already been detected in South American deer from Brazil [14].

The identification of E. canis in the maned wolf supports the hypothesis that stray dogs may act as carriers of arthropod-borne pathogens to captive wild animals. Serological and molecular studies have detected the antibodies and DNA of E. canis in free-ranging maned wolves in Brazil, indicating serious health risks due to the severe nature of ehrlichiosis [69]. Despite being collected from a maned wolf, a wild carnivore native to South America, the results showed a close phylogenetic relationship with strains found in domestic dogs (C. familiaris) from Brazil, Cuba, India and Turkey, underscoring their global distribution and the importance of disease surveillance and control. From a taxonomic perspective, the close phylogenetic relationship suggested that the sample may belong to the same species or a species closely related to E. canis [70].

This study detected Bartonella spp. in 42.5% (17/40) of the animals, including a variety of species. This finding aligns with previous research indicating a significant incidence of Bartonella in various mammalian species, both in wild and captive habitats [71,72]. The prevalence in this study is consistent with the findings of Rao et al. (2021) [73], who reported significant proportions of Bartonella (38.61%) among small mammals in the Qaidam Basin, western China. A study in Italian Nature Reserve Parks detected Bartonella infection in 97 red foxes, eight European badgers, six Eurasian wolves, six European hedgehogs, three beech martens and two deer. The prevalence was 9.84%, with zoonotic species detected in wolves (83.3%), hedgehogs (33.33%) and foxes (4.12%) [73]. Bartonella rochalimae was the most common species found in foxes and wolves. Zoonotic species were significantly more frequent in Eurasian wolves (p < 0.0001), indicating that they may be reservoirs for infection in humans and domestic animals [74]. In Brazil, several studies have investigated the occurrence of Bartonella in wild and domestic animals, revealing a wide diversity of genotypes and potential zoonotic risks [72,75].

ITS phylogenetic analysis grouped B. clarridgeiae sequences from cats and fleas with our sequences from gorillas, southern tamanduas, capuchin monkeys and brocket deer. Conversely, B. henselae sequences from cats, deer, rodents and humans clustered with our sequences from howler monkeys. These findings expand the known host range for B. clarridgeiae and B. henselae, suggesting potential underestimation of their prevalence in captive animals [29]. Despite the small sequences used for constructing the phylogeny, it was possible to distinguish between the species. The ITS gene, a noncoding region of ribosomal DNA, varies between Bartonella species, enabling their molecular differentiation [76].

Our study revealed a 17.5% prevalence of hemotropic Mycoplasma spp. among tested animals infected with multiple species. These bacteria have been detected in numerous nonhuman primates and captive mammals globally [77,78]. In Brazil, several investigations have focused on hemoplasma infections across diverse animal populations. For instance, captive Allouata in São Paulo’s zoos exhibited a 26.47% positivity rate for hemoplasmas [79], while captive wild carnivores showed high prevalence rates, such as 45.5% in wild felids and 83.3% in wild canids [80]. Moreover, studies involving non-human primates, both captive and free-ranging, reported an infection rate of 25%, with notable prevalence among black howler monkeys (64.3%) and black-horned capuchins (4.2%) [77]. In the southern Pantanal region, Mycoplasma spp. infections were detected in various wild mammals, including Nasua nasua, Cerdocyon thous and Leopardus pardalis, indicating exposure to multiple hemoplasma species [10].

Moreover, our study identified hemotropic Mycoplasma spp. in D. dama, consistent with previous findings in cervids by André et al. (2020) [80], who reported these bacteria in 40% of free-ranging deer in Brazil, broadening our understanding of hemoplasma distribution in deer populations [81]. These results underscore the importance of further research to elucidate the ecology of these pathogens in captive populations, aligning with the One Health concept, given reports of infection in immunocompromised humans.

Phylogenetic analysis revealed that the sequences identified in this study for hemotropic Mycoplasma species are phylogenetically closely related to those of the M. suis group. Previous research has demonstrated that hemotropic Mycoplasma species form specific evolutionary groups based on their hosts. The placement of the sequences detected in non-human primates within the “M. suis group” clade is consistent with this pattern, suggesting a common ancestor between the species that infect NHPs and other animals, reflecting possible specific adaptations to their hosts [82]. Although “Ca. M. haemominutum” was detected by 16S rRNA cPCR in the howler monkeys, presumably, this cPCR assay revealed a sequence of the novel Mycoplasma sp. because several regions of homology may be found with “Ca. M. haemominutum” sequences [77]. The 16S rRNA gene can show 99% homology with sequences of other pathogenic Mycoplasma species from different hosts, suggesting a close relationship between the species. However, when other genes are used for molecular characterization, little identity is observed, indicating that they are different species. Therefore, it is necessary to characterize isolates using multiple genes to investigate genetic diversity and accurately determine the species [4,83].

The tree showed that the sequence detected in a captative woolly monkey in the Belo Horizonte Zoo, Minas Gerais, clustered within a clade with hemotropic Mycoplasma spp. detected in howler monkeys from southern Brazil. This suggests a possible relationship between the pathogens in different species of Neotropical primates and indicates that these hemoplasmas occur in close geographic locations [77,79]. Furthermore, phylogenetic trees have shown an evident separation of the hemoplasma detected in the woolly monkey from “Ca. M. kahanei” was found in squirrel monkeys in Brazil, and hemotropic Mycoplasma spp. were detected in S. apella in Brazil, indicating the presence of different species, an event that has been previously reported [77,79].

Our study revealed a high prevalence (34.7%) of coinfections by vector-borne pathogens in mammals at the Belo Horizonte Zoo, Brazil. Perles et al. (2023) [4] reported that only a small percentage (8.1%) of the studied animals tested negative for the evaluated agents, with bacterial coinfections being the most common (18.3%), particularly involving Mycoplasma spp. and Bartonella spp. Comparing it with our findings, we see those coinfections with bacterial pathogens, especially Mycoplasma spp. and Bartonella spp., are prevalent in both studies. Although vector-borne infections often remain subclinical in wild animals, they may act as opportunistic pathogens in immunocompromised animals under stressful conditions or in the presence of coinfections [4].

For example, hemotropic Mycoplasma species can cause severe anemia in cats with concurrent FeLV infection or immunosuppression [4,84]. Coinfections can complicate diagnosis and treatment and worsen prognosis in animals. In a study on cats, 22 out of 624 (4%) tested positive for more than one vector-borne pathogen, with nine cats infected with Hepatozoon spp. having antibodies against Leishmania spp., Rickettsia spp. and Ehrlichia spp. [84]. Additionally, the authors of [63] reported a fatal case of parasite coinfection in a threatened maned wolf from Minas Gerais, Brazil [64].

The detection of possible reservoirs of zoonotic pathogens in wildlife is crucial to public health because it allows us to anticipate and prevent the spread of diseases between animals and humans, in line with the concept of One Health. In a study conducted in Switzerland, antibodies against SARS-COV-2 were found in red foxes, Eurasian lynxes and wild cats, indicating that these animals were exposed to the virus, but without signs of active infection [85]. Similarly, our research at the Belo Horizonte Zoo found a significant incidence of zoonotic infections such as Bartonella spp., Mycoplasma spp. and Leishmania spp. in captive mammals, reinforcing the potential risk of disease transmission between animals and humans. These results emphasize the importance of continually monitoring the circulation of zoonotic pathogens in different animal environments to preserve public health and prevent future pandemics.

Our study demonstrated that wild and exotic animals kept in zoos are also at risk of infection with several species of vector-borne pathogens. Our findings showed the high prevalence of these agents that infect and coinfect mammals at the Belo Horizonte Zoo in Brazil, reinforcing the urgent need to improve control measures, including vector control strategies and routine parasitological examinations, to protect animal health and prevent the spread of diseases, while advancing our understanding of parasite–host dynamics in animals, benefiting both public health and wildlife conservation.

Limitations of the present study should be considered. A notable limitation of the present study is that the phylogeny was based on a small fragment (165 to 630 bp) of a conserved gene, which precluded additional phylogenetic inferences to confirm species identification, potentially limiting the resolution of our analyses. Unfortunately, it was not possible to sequence more samples or amplify other gene targets to evaluate the genetic diversity between the vector-borne pathogens. Additionally, short-read sequencing platforms targeting regions of 16S rRNA or 18S rRNA may not achieve complete taxonomic resolutions consistently obtained through whole-genome sequencing [84]. Future studies should prioritize larger sample sizes and more detailed genetic analyses, including the use of other genes, to improve our understanding of phylogenetic relationships and species differentiation [86].

Although this study identified several pathogens in captive mammals in Brazil, it was not possible to evaluate all pathogens of interest, such as Toxoplasma gondii. Detection of T. gondii requires specific methods that were not implemented in this study. We recognize the importance of T. gondii in wildlife parasitology and public health studies due to its zoonotic potential and significant impact on domestic and wild animals [87,88]. Future research should include the evaluation of this and other relevant pathogens to provide a more comprehensive understanding of the epidemiology of parasitic diseases in captive environments.