Molecular detection of trypanosomatids and apicomplexans in owl tissues from the coast of Santa Catarina, BrazilABSTRACT
Owls (Strigiformes) are widely distributed across terrestrial ecosystems and play a key role as top predators, potentially exposing them to diverse infectious agents. This study aimed to investigate the presence of protozoan of veterinary and zoonotic relevance in owl tissues from the southern coast of Santa Catarina, Brazil. A total of 111 tissue samples (lung, liver, spleen, and heart) from seven species-Athene cunicularia, Asio clamator, Strix virgata, Megascops choliba, Tyto furcata, Asio stygius, and Bubo virginianus-were analyzed using conventional and nested PCR targeting Leishmania spp., Trypanosoma spp. and Apicomplexa. DNA extraction was performed using a commercial kit, and all PCR assays included positive and negative controls. Results revealed DNA of Apicomplexa in 1.8% of samples, Trypanosoma spp. in 4.5%, and Leishmania spp. in 2.7%, with a co-infection. These findings indicate that owls are exposed to these pathogens through predation and environmental interactions, highlighting their potential as sentinel species. Despite the low prevalence observed, the study underscores the ecological and epidemiological importance of Strigiformes in the circulation of protozoan parasites. Further investigations using sequencing and serology are recommended to clarify the role of owls in pathogen transmission and support integrated One Health surveillance in overlapping wildlife and human habitats.
Keywords:
strigiformes; molecular detection; protozoan parasites; zoonoses; one health
RESUMO
As corujas (Strigiformes) estão amplamente distribuídas pelos ecossistemas terrestres e desempenham um papel fundamental como predadores de topo, o que as expõe potencialmente a diversos agentes infecciosos. Este estudo teve como objetivo investigar a presença de protozoários de relevância veterinária e zoonótica em tecidos de corujas da região litorânea do sul de Santa Catarina, Brasil. Foram analisadas 111 amostras de tecidos (pulmão, fígado, baço e coração) de sete espécies - Athene cunicularia, Asio clamator, Strix virgata, Megascops choliba, Tyto furcata, Asio stygius e Bubo virginianus - utilizando-se PCR convencional e nested-PCR, com foco em Leishmania spp., Trypanosoma spp. e Apicomplexa. A extração de DNA foi realizada com kit comercial, e todos os ensaios de PCR incluíram controles positivos e negativos. Os resultados revelaram DNA de Apicomplexas em 1,8% das amostras, Trypanosoma spp. em 4,5% e Leishmania spp. em 2,7%, com uma coinfecção Esses achados indicam que as corujas são expostas a esses patógenos por meio da predação e de interações ambientais, destacando seu potencial como espécies sentinelas. Apesar da baixa prevalência observada, o estudo reforça a importância ecológica e epidemiológica das Strigiformes na circulação de protozoários. Investigações adicionais com sequenciamento e sorologia são recomendadas para esclarecer o papel das corujas na transmissão de patógenos e apoiar a vigilância integrada em saúde no conceito One Health em áreas de interface entre fauna silvestre e hábitats humanos.
Palavras-chave:
Strigiformes; detecção molecular; protozoários; zoonoses; one health
INTRODUCTION
Owls (order Strigiformes) are widely distributed across terrestrial ecosystems and play an important role in maintaining the structure and balance of ecological communities by acting as top predators in food webs (Krone et al., 2008; Ponder et al., 2015). Due to their predatory habits and elevated trophic position, owls are continuously exposed to a wide range of infectious agents through the consumption of prey and interaction with contaminated environments. For this reason, these birds may act as ecological indicators or sentinels of pathogen circulation in natural and anthropized ecosystems, reflecting environmental exposure rather than necessarily functioning as reservoirs.
Among the infectious agents of interest, protozoa of the genera Leishmania, Trypanosoma, Sarcocystis, Toxoplasma, and Neospora stand out due to their veterinary and zoonotic relevance and their wide distribution in wildlife (Gradoni, 2018; Rodrigues et al., 2019; Lindsay and Dubey, 2020). The detection of these protozoa in wild birds contributes to understanding host-parasite-environment interactions and supports integrated surveillance approaches within the One Health framework.
Leishmaniases are caused by protozoa of the genus Leishmania, which infect a wide range of vertebrate hosts and are transmitted by phlebotomine sand flies (Bates, 2007; Gradoni, 2018). More than 20 Leishmania species are pathogenic, causing cutaneous, mucocutaneous, or visceral forms of the disease (Bogdan et al., 2024). Although birds are not considered competent reservoirs for Leishmania spp., serological and molecular evidence has demonstrated exposure in avian hosts, particularly in endemic areas. Such findings are generally interpreted as indicators of vector circulation and environmental overlap with mammalian reservoirs rather than active participation in transmission cycles.
Protozoa of the genus Trypanosoma comprises a highly diverse group of hemoparasites with a global distribution, infecting a wide variety of vertebrate hosts, including birds, mammals, and reptiles (Rodrigues et al., 2019). In birds, Trypanosoma infections are commonly detected in raptors and carnivorous species and are often associated with hematophagous vectors. Avian trypanosomes are generally considered to exhibit low pathogenicity, and their detection is frequently interpreted as evidence of ecological interactions among hosts, vectors, and the environment rather than clinical disease (Apanius, 1991; Hamilton et al., 2007).
Protozoa of the phylum Apicomplexa are obligate intracellular parasites with cosmopolitan distribution, particularly prevalent in tropical and subtropical regions. These organisms infect a wide range of warm-blooded animals, including birds and humans (Dubey et al., 2010; Lindsay and Dubey, 2020). Among apicomplexans, the genera Neospora, Sarcocystis, and Toxoplasma are of particular interest due to their epidemiological relevance in wildlife and domestic animals (Moura et al., 2023).
Neospora caninum has a heteroxenous life cycle and is recognized as a major cause of reproductive failure in ruminants (Nayeri et al., 2022a; Dinon et al., 2024). Although its role in wild birds is not fully understood, molecular evidence suggests that avian species may be exposed to Neospora spp. through environmental contamination or ingestion of infected prey, highlighting their potential use as indicators of parasite circulation rather than key hosts in transmission.
Toxoplasma gondii is one of the most widely distributed protozoan parasites worldwide, with felids acting as definitive hosts and a broad range of intermediate hosts, including birds (Dubey et al., 2010; Lindsay and Dubey, 2020). In birds of prey, infection is commonly associated with the ingestion of infected prey species, such as rodents and small mammals. Consequently, the detection of T. gondii in predatory birds has been widely interpreted as an indirect indicator of environmental contamination with oocysts, supporting their role as sentinels rather than reservoirs of infection (Dubey, 2002; Elmore et al., 2010).
Parasites of the genus Sarcocystis form tissue cysts and have life cycles involving carnivorous definitive hosts and herbivorous or omnivorous intermediate hosts. Birds may act as intermediate or definitive hosts for several Sarcocystis species, some of which are pathogenic (Dubey et al., 2015). More than 25 species capable of forming sarcocysts in avian muscle tissue have been described, with Sarcocystis falcatula being among the most frequently reported in the Americas, including infections in Strigiformes (Wünschmann et al., 2009). This genus is therefore particularly relevant when assessing apicomplexan exposure in wild birds.
The predator-prey relationship, combined with the persistence of protozoan stages in the environment, contributes to the maintenance of infection cycles in wildlife (Bogomolni et al., 2008). In this context, owls may reflect the circulation of protozoan parasites present in their prey and surrounding habitats. However, current evidence does not support a major epidemiological role for these birds as reservoirs or amplifiers of infection.
Considering these aspects, the present study aimed to investigate the molecular detection of trypanosomatids and apicomplexans in tissues of owls from the southern coast of Santa Catarina, Brazil. The findings are intended to contribute to the understanding of protozoan exposure in Strigiformes and to evaluate their potential role as environmental sentinels of pathogen circulation in coastal ecosystems.
ETHICAL ASPECTS
Since only deceased animals were used, this work falls under the exemption parameters for submission to an Animal Ethics Committee (CEUA), in accordance with current Brazilian legislation (such as CONCEA Normative Resolution No. 30/2016). Nevertheless, all laboratory procedures followed good biosafety practices and scientific integrity standards.
MATERIALS AND METHODS
A total of 111 biological samples (n=111) from 29 owls (individuals) that were rescued or found dead along the coastal region of the state of Santa Catarina were analyzed. The samples consisted of internal tissues (lung, liver, spleen, and heart) collected in a standardized manner, with approximately 2g of each tissue per individual (the necropsy activities were conducted at Centro Universitário Ritter dos Reis). Due to variation in carcass preservation and availability, not all tissue types were obtained from every individual. Bird identification allowed classification of the samples into seven distinct species: Asio clamator (n = 32), Tyto furcata (n = 22), Strix virgata (n = 17), Asio stygius (n = 16), Megascops choliba (n = 14), Bubo virginianus (n = 6), and Athene cunicularia (n = 3), in addition to one unidentified sample. The total number of samples reflects both the number of individuals and the diversity of tissues obtained for molecular analysis.
Information regarding age class and sex was not consistently available for all individuals, as the samples were obtained opportunistically from wildlife rescue and monitoring programs. When available, such information was recorded; however, it was not included in the analysis due to incomplete data.
All animals were found dead or died during rehabilitation. Necropsies were performed when possible, and tissue collection was conducted considering macroscopic preservation. However, varying degrees of post-mortem autolysis were observed, which may have influenced DNA quality and detection sensitivity.
The samples used in this study were obtained exclusively from birds found dead or provided by environmental monitoring programs and wildlife rescue institutions, such as the PMP-BS and wildlife screening and rehabilitation centers. No active capture or invasive procedures were performed for the purposes of this study.
Total DNA was extracted from all samples using a commercial kit (Wizard Genomic DNA Purification Kit, Promega, USA), following the manufacturer’s instructions and modified according to the protocol of Bräunig et al. (2016), in which the lysis step was performed at 55°C for 16 hours. Strict measures were adopted to minimize the risk of cross-contamination, particularly due to the high sensitivity of nested PCR assays. DNA extraction, PCR setup, and post-amplification analyses were conducted in physically separated laboratory areas. Aerosol-resistant filter tips were used throughout all procedures.
Negative controls were included in all extraction and amplification steps. Positive controls consisted of previously characterized DNA samples and were handled separately from biological samples to avoid contamination. Samples were stored at −20°C until molecular analyses were performed. The concentration and quality of the extracted DNA from each sample were assessed using a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA), based on the 260/280 nm absorbance ratio to evaluate purity.
PCR amplification targeted the conserved region of the kinetoplast minicircle DNA (kDNA) (between CSB1 and CSB3), using the primers Leishmini-F (5'-GGKAGGGGCGTTCTGC-3') and Leishmini-R (5'-STATWTTACACCAACCCC-3') (KOCHER et al., 2018). The expected product size was 120 base pairs.
The final PCR reaction volume was 10µL, containing 0.5µL of extracted DNA, 1.0µL of 10× PCR buffer (50 mM KCl, 10 mM Tris-HCl; pH 9.0), 0.4µL of MgCl₂ (50mM), 0.2µL of dNTPs (10 mM), 0.6µL of each primer (10), 0.1µL of Platinum Taq DNA Polymerase (Invitrogen, 5 U/µL), and 6.6µL of Milli-Q water.
Amplifications were performed in a T100™ thermocycler (Bio-Rad, USA) under the following conditions: initial denaturation at 95°C for 5 minutes; followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 20 seconds; with a final extension at 72°C for 5 minutes.
A previously characterized Leishmania infantum sample, kindly provided by the Federal University of Pampa (UNIPAMPA), Uruguaiana, Rio Grande do Sul, was used as the positive control. Ultrapure water (Milli-Q) was used as the negative control. The generated amplicons were visualized under UV transillumination after electrophoresis on a 3.0% agarose gel stained with GelRed™ Nucleic Acid Gel Stain (Invitrogen, USA).
DNA samples were subjected to nested PCR targeting a conserved region of the 18S rRNA gene of Apicomplexa, following the protocol described by Silva et al. (2009). In the first amplification round, an external primer pair was used: Tg18s48F (5'-CCATGCATGTCTAAGTATAAGC-3ʹ) and Tg18s359R (5'-GTTACCCGTCACTGCCAC-3ʹ). For the second round, internal primers were employed: Tg18s58F (5'-CTAAGTATAAGCTTTTATACGGC-3ʹ) and Tg18s348R (5'-TGCCACGGTAGTCCAATAC-3ʹ).
Cycling conditions consisted of an initial denaturation at 94 °C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 45 seconds, with a final extension at 72°C for 5 minutes.
The amplified products from the second round, approximately 310 base pairs (bp) in size, were analyzed by electrophoresis on a 2% agarose gel stained with GelRed™ (Biotium, Hayward/CA, USA). As positive controls, previously sequenced and identified samples from the LADOPAR-UFSM sample bank were used for S. neurona, Sarcocystis cruzi, T. gondii, and N. caninum. Ultrapure water was used as the negative control in all reactions.
For the identification of Trypanosoma spp., all PCR reactions were performed in a total volume of 25μL, containing 2 μL of total DNA (100-200 ng), 1.25μL of MgCl₂, 2.5μL of 10× Taq buffer, 1 U of Taq DNA polymerase (0.2μL) (Thermo Fisher Scientific), 0.7μL of dNTPs, 1 μM of each primer, and 16.35μL of ultrapure water. The positive control consisted of Trypanosoma evansi DNA, and ultrapure water was used as the negative control.
The primer sequences used were 18STnF2 (5’-CAACGATGACCCCCCTGAATTGGGGA-3’) and 18STnR3 (5’-TGCTCGACATATATTGCATATAC-3’) according to Geysen et al. (2003), which amplify the 18S gene, a conserved region of 18S rDNA (ribosomal DNA), producing a 780 bp product.
Thermocycling conditions were as follows: initial denaturation at 94°C for 4 minutes; 40 cycles of denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute 30 seconds, and extension at 72°C for 2 minutes; followed by a final extension at 72°C for 5 minutes.
The generated amplicons were detected by electrophoresis on a 2% agarose gel using molecular weight markers (100 bp Ladder, Ludwig Biotec, Brazil) and fluorescent dye (GelRed - Biotium, Hayward, CA, USA) and subsequently visualized under UV light on a transilluminator.
RESULTS AND DISCUSSION
Table 1 presents the positive findings organized by species, tissue analyzed and etiological agent highlighting the potential role of owls as hosts or sentinels of parasitic agents of veterinary and zoonotic relevance.
DNA of Apicomplexa was detected in 1.8% (2/111) of the samples using nested PCR targeting the 18S rRNA gene. Due to the conserved nature of this molecular marker, these findings should be interpreted as evidence of Apicomplexa DNA rather than definitive identification of specific species such as Toxoplasma gondii, Neospora caninum, or Sarcocystis spp. Therefore, the results indicate exposure of these birds to apicomplexan parasites, likely through trophic interactions (Dubey et al., 2010; Lindsay and Dubey, 2020).
In birds of prey, exposure to apicomplexans is commonly associated with the ingestion of infected prey, such as rodents and small mammals, which act as intermediate hosts in the life cycles of several species within this group. Consequently, the detection of Apicomplexa DNA in predatory birds has been widely interpreted as an indirect indicator of environmental contamination and parasite circulation, reinforcing their role as ecological sentinels rather than reservoirs (Dubey, 2002; Elmore et al., 2010).
The identification of Trypanosoma spp. DNA in 4.5% (5/111) of the samples aligns with the wide distribution of these hemoparasites in wild birds. Avian trypanosomiasis occurs on all continents, most frequently reported in raptors and carnivorous birds, but it can also be detected in many other avian species (Apanius, 1991). In owls (Strix aluco) and other birds, Trypanosoma avium is the most commonly found species (Bennett et al., 1994; Duarte et al., 2024).
Avian Trypanosoma species generally have low pathogenicity in their hosts, and their detection highlights potential interactions among birds, hematophagous vectors, and the environment (Hamilton et al., 2007; Fialová et al., 2021). Recent phylogenetic studies show that avian trypanosomes form highly diverse and polyphyletic lineages, often with low host specificity and occurrences of mixed infections (Duarte et al., 2024). The results observed in this study underscore the need for further investigations on the genetic diversity of Trypanosoma spp., particularly given the potential presence of poorly described or unknown species in Neotropical birds.
Findings of Leishmania spp. in 2.7% (3/111) of the samples, although low, are particularly relevant. Literature indicates that the role of birds in the transmission cycle of leishmaniasis remains uncertain and controversial. Previous serological and molecular evidence demonstrates exposure but without confirmation of competence to transmit the parasite to sandfly vectors. Therefore, these results likely indicate contact of owls with areas of vector circulation, possibly due to shared habitats with mammalian reservoirs. Accordingly, the findings should be interpreted as evidence of exposure rather than definitive reservoir status (Abreu-Silva et al., 2018; Valadas et al., 2022; Gomes et al., 2023).
The observation of co-infection, with samples simultaneously positive for Trypanosoma spp. and Apicomplexa DNA, highlights the trophic and ecological interactions of these predators, which are exposed both through ingestion of infected prey and through vector presence in the environment. Co-infections may modulate avian immune responses and interfere with molecular detection of the agents, representing an important consideration in parasitic ecology studies (Elmore et al., 2010; Ferreira et al., 2020).
Among the methodological limitations of this study are the restriction of samples to specific organs, the lack of confirmation of amplicons by sequencing, and the potential for degraded DNA in necropsy tissues. Additionally, the use of nested PCR, while increasing sensitivity, may elevate the risk of false-positive results due to cross-contamination. Therefore, complementary analyses such as sequencing, culture isolation, and serology are recommended to confirm the identity of detected parasites (Marques et al., 2021; Gomes et al., 2023).
From an epidemiological perspective, the detection of protozoa of veterinary and zoonotic relevance in owls reinforces their utility as sentinel hosts for environmental monitoring, especially in peri-urban areas where wild and anthropogenic habitats overlap. These results support the need for integrated health surveillance approaches aligned with the One Health concept, considering the interface among wildlife, domestic animals, vectors, and humans (Abreu-Silva et al., 2018; Valadas et al., 2022).
CONCLUSION
In this study, tissues from seven owl species (Athene cunicularia, Asio clamator, Strix virgata, Megascops choliba, Tyto furcata, Asio stygius, and Bubo virginianus) were analyzed using conventional and nested PCR assays to investigate the presence of protozoan parasites of veterinary and zoonotic relevance. Molecular detection revealed the presence of Leishmania spp., Trypanosoma spp., and Apicomplexa DNA in a subset of the analyzed samples. These findings indicate that owls from the coastal region of southern Santa Catarina are exposed to protozoan parasites circulating in their environment, likely through trophic interactions and contact with vector-infested habitats. However, given the use of conserved molecular targets, the absence of sequencing confirmation, and the limitations related to sampling design and tissue availability, the results should be interpreted as evidence of exposure rather than confirmation of active infection, transmission competence, or reservoir status. From an ecological and epidemiological perspective, the detection of these protozoa supports the use of owls as potential environmental sentinels, reflecting the circulation of parasitic agents in coastal ecosystems where wildlife, domestic animals, vectors, and human activities overlap. Further studies incorporating standardized sampling, parasite sequencing, serological assays, and vector investigations are necessary to clarify the dynamics of protozoan circulation and to better define the role of Strigiformes within integrated One Health surveillance frameworks.
REFERENCES
- ABREU-SILVA, A.L.; GOMES, L.I.; LOPES, E.G. et al Leishmania (Leishmania) infantum in wild mammals in endemic areas of Maranhão, Brazil. Parasitol. Int, v.67, p.48-52, 2018.
- APANIUS, V. Avian trypanosomes as models of hemoflagellate evolution. Parasitol. Today, v.7, p.87-90, 1991.
- BATES, P.A. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. Int. J. Parasitol, v.37, p.1097-1106, 2007.
- BENNETT, G.F., WILSON, P.; HENDERSON, B.; HARTLEY, W.J. Trypanosomes of some Fennoscandian birds. Mem. Inst. Oswaldo Cruz, v.89, p.531-537, 1994.
- BOGDAN, C.; KRAUSE, P.; XU, X. et al The immunomicrotope of Leishmania control and persistence. Trends Parasitol, v.40, p.788-804, 2024.
- BOGOMOLNI, A.; KRAUSE, P.; XU, X. et al Mortality in marine mammals: infections and disease risks. Ecohealth, v.5, p.278-289, 2008.
- BRÄUNIG, P.; HARTL, R.; KRAUSE, P. et al DNA extraction methods and multiple sampling to improve molecular diagnosis of Sarcocystis spp. in cattle hearts. Parasitol. Res, v.115, p.3913-3921, 2016.
- DINON, A.; LOPES, E.G.; MENEZES, R.C. et al The role of genetic variability of the host on the resistance to Neospora caninum infection in cattle. Anim. Genet, v.55, p.304-318, 2024.
- DUARTE, R.G.; LOPES, E.G.; MENEZES, R.C. et al Molecular diversity and polyparasitism of avian trypanosomes in the Brazilian Atlantic Rainforest. Acad. Bras. Ciênc, v.96, p.e20230629, 2024.
- DUBEY, J.P. A review of toxoplasmosis in wild birds. Vet. Parasitol, v.106, p.121-153, 2002.
- DUBEY, J.P.; SHEN, S.K.; FRENKEL, J.K. Sarcocystosis of Animals and Humans Boca Raton: CRC Press, 2015. 502p.
- DUBEY, J.P.; SHEN, S.K.; FRENKEL, J.K. Toxoplasmosis of Animals and Humans 2.ed. Boca Raton: CRC Press, 2010. 313p.
- ELMORE, S.A.; SALLIS, E.S.V.; KRAUSE, P. et al Toxoplasma gondii: epidemiology, feline clinical aspects, and prevention. Trends Parasitol, v.26, p.190-196, 2010.
- FERREIRA, R.C.; LOPES, E.G.; MENEZES, R.C. et al Diversity of avian trypanosomes in Brazil: insights from molecular and morphological analyses. Int. J. Parasitol. Parasites Wildl, v.11, p.23-32, 2020.
- FIALOVÁ, M.; KRAUSE, P.; WANG, Z.; KAPOOR, S. Complete life cycle of Trypanosoma thomasbancrofti, an avian trypanosome transmitted by culicine mosquitoes. Microorganisms, v.9, p.2101, 2021.
- GEYSEN, D.; DELESPAUX, V.; GEERTS, S. PCR-RFLP using SSU-rDNA amplification as an easy method for species-specific diagnosis of Trypanosoma species in cattle. Vet. Parasitol, v.110, p.171-180, 2003.
- GOMES, L.A.; LOPES, E.G.; MENEZES, R.C. et al Molecular detection of Leishmania DNA in wild birds from endemic areas: evaluating their role in the epidemiology of leishmaniasis. J. Infect. Dev. Ctries, v.17, p.278-285, 2023.
- GRADONI, L. A brief introduction to leishmaniasis epidemiology. In: BRUSCHI, F.; GRADONI, L. (Eds). The Leishmaniases Cham: Springer, 2018. p.1-12.
- HAMILTON, P.B.; LOPES, E.G.; MENEZES, R.C. et al Patterns of co-evolution between trypanosomes and their hosts deduced from ribosomal RNA and protein-coding gene phylogenies. Mol. Phylogenet. Evol, v.44, p.15-25, 2007.
- KOCHER, A.; KRAUSE, P.; WANG, Z.; KAPOOR, S. High-throughput sequencing of kDNA amplicons. Parasitology, v.145, p.585-594, 2018.
- KRONE, O.; KRAUSE, P.; WANG, Z.; KAPOOR, S. Haemosporidian blood parasites in European birds of prey and owls. J. Parasitol, v.74, p.709-715, 2008.
- LINDSAY, D.S.; DUBEY, J.P. Neosporosis, toxoplasmosis, and sarcocystosis in ruminants. Vet. Clin. North Am. Food Anim. Pract, v.36, p.205-222, 2020.
- MARQUES, M.V.R.; LOPES, E.G.; MENEZES, R.C. et al Seroprevalence and risk factors of Toxoplasma gondii in birds of prey in Brazil. Pesqui. Agropec. Bras, v.56, p.e02362, 2021.
- MOURA, G.H.F.; LOPES, E.G.; MENEZES, R.C. et al Occurrence of Apicomplexa protozoa in wild birds in Northeast region of Brazil. Rev. Bras. Parasitol, v.32, p.e014722, 2023.
- NAYERI, T.; LOPES, E.G.; MENEZES, R.C. et al Neospora caninum infection in sheep and goats. Front. Vet. Sci, v.9, p.1-10, 2022a.
- PONDER, J.B.; MILLER, R.E.; FOWLER, M.E. Strigiformes. In: MILLER, R.E.; FOWLER, M.E. (Eds.). Fowler’s Zoo and Wild Animal Medicine St Louis: Elsevier, 2015. p.189-198.
- RODRIGUES, M.S.; LOPES, E.G.; MENEZES, R.C. et al Diversity of Trypanosoma spp. in wild mammals. Int. J. Parasitol. Parasites Wildl, v.8, p.171-181, 2019.
- SILVA, R.C.; SU, S.; LANGONI, H. First identification of Sarcocystis tenella (Railliet, 1886) Moulé, 1886 (Protozoa: Apicomplexa) by PCR in naturally infected sheep from Brazil. Vet. Parasitol, v.165, p.332-336, 2009.
- VALADAS, S.; LOPES, E.G.; MENEZES, R.C. et al Diversity of Sarcocystis spp. shed by opossums in Brazil and molecular identification of species infecting livestock. Int. J. Parasitol. Parasites Wildl, v.19, p.1-10, 2022.
- WÜNSCHMANN, A.; LOPES, E.G.; MENEZES, R.C. et al Sarcocystis falcatula-associated encephalitis in a free-ranging great horned owl (Bubo virginianus). J. Vet. Diagn. Invest, v.21, p.283-287, 2009.
