Hepatozoon caimani in Caiman crocodilus yacare (Crocodylia, Alligatoridae) from North Pantanal, Brazil

Hepatozoon caimani em Caiman crocodilus yacare (Crocodylia, Alligatoridae) do Norte do Pantanal, Brasil

Abstract

Hepatozoon species are the most common intracellular hemoparasite found in reptiles. Hepatozoon caimani, whose vectors are Culex mosquitoes, has been detected in a high prevalence among caimans in Brazil by blood smears examinations. The present work aimed to detect and characterize the Hepatozoon spp. found in 33 caimans (24 free-ranging and 9 captive; 28 males and 5 females) (Caiman crocodilus yacare) sampled at Poconé, North Pantanal, state of Mato Grosso, Brazil, using blood smears examinations and molecular techniques. Hepatozoon spp.-gametocytes were found in 70.8% (17/24) and 88.8% (8/9) of blood smears from free-ranging and captive caimans, respectively. Hepatozoon spp. 18S rRNA DNA was found in 79.2% (19/24) and 88.8% (8/9) of free-ranging and captive caimans, respectively. Comparative analysis of parasitized and non-parasitized erythrocytes showed that all analyzed features were significantly different (P<0.05) for both linear and area dimensions. Phylogenetic analysis based on 18S rRNA sequences grouped the Hepatozoon spp. sequences detected in the present study together with H. caimani, recently detected in caimans in southern Pantanal.

Keywords:
Caimans; Hepatozoon caimani; morphology; morphometry; molecular characterisation; Brazil

Resumo

Espécies do gênero Hepatozoon são os hemoparasitas intracelulares mais comumente encontrados em répteis. Hepatozoon caimani, cujos vetores são mosquitos do gênero Culex sp., têm sido detectados em uma alta prevalência entre jacarés no Brasil, por meio da análise de esfregaços sanguíneos. O presente estudo objetivou detectar e caracterizar parasitas do gênero Hepatozoon spp. em 33 jacarés (24 de vida-livre e 9 de cativeiro; 28 machos e 5 fêmeas) (Caiman crocodilus yacare) amostrados em Poconé, região norte do Pantanal, estado do Mato Grosso, Brasil, por meio da análise de esfregaços sanguíneos e técnicas moleculares. Gametócitos de Hepatozoon spp. foram encontrados em 70,8% (17/24) e em 88,8% (8/9) dos esfregaços sanguíneos de jacarés de via-livre e cativeiro, respectivamente. 18S rRNA DNA de Hepatozoon spp. foi detectado em 79,2% (19/24) e 88,8% (8/9) das amostras de sangue de jacarés de vida-livre e cativeiro, respectivamente. A análise comparativa de eritrócitos parasitados e não parasitados mostrou diferença significativa (P<0,05) em todas as variáveis lineares e de área analisadas. A análise filogenética baseada em sequências de DNA do 18S rRNA agrupou as sequências de Hepatozoon spp. detectadas no presente estudo juntamente com aquelas de H. caimani, recentemente detectadas em jacarés do Pantanal do Mato Grosso do Sul.

Palavras-chave:
Jacarés; Hepatozoon caimani; morfologia; morfometria; caracterização molecular; Brasil

Introduction

Hepatozoon species are apicomplexan protozoans that infect a wide range of vertebrate and invertebrate hosts, and include the widest distributed and most common intracellular hemoparasites found in reptiles (TELFORD, 1984, 2009). Based on morphologic and morphometric characteristics, Hepatozoon caimani have been found described as the only Hepatozoon species parasitizing Caiman latirostris (SMITH, 1996), Caiman crocodilus (LAINSON, 1977; LAINSON et al., 2003) and Caiman yacare (LAINSON et al., 2003; VIANA & MARQUES, 2005; VIANA et al., 2010a) in South America.

Culex (Meloconion) mosquitoes are the H. caimani natural vectors, where sporogonic phase takes place (LAINSON et al., 2003; VIANA et al., 2010b). Despite of that, the possibility of the infected-invertebrate hosts being eaten by caimans is minimal (LAINSON et al., 2003; PAPERNA & LAINSON, 2003). Caiman yacare become infected for the first time as juveniles, when its diet changes from ingestion of invertebrates to predation of anurans and fishes (VIANA et al., 2010a). In South America, predation of insectivorous vertebrates appears to be the main transmission route of H. caimani (VIANA et al., 2012). Hepatozoon caimani-cystozoites have been found in amphibian tissues of the following species: Leptodactylus fuscus, Leptodactylus chaquensis, Leptodactylus podicipinis, Scinax nasicus and Rana catesbeiana (LAINSON et al., 2003; VIANA et al., 2012), suggesting that anurans are paratenic hosts for H. caimani, although frogs are not part of caiman's diet. The characid fish Metynnis sp., when fed with C. quinquefasciatus previously engorged on naturally H. caimani-infected caiman showed cysts harbouring cystozoites identical to those of H. caimani, suggesting its role as paratenic hosts for this haemoprotozoa (PEREIRA et al., 2014). Although some attempts were unsuccessful in proving the experimental transmission of Hepatozoon to crocodilians (PESSÔA et al., 1972; LAINSON et al., 2003), structures similar to Hepatozoon oocystis have been recently described in these invertebrates collected from Caiman yacare’s oral cavity (SOARES et al., 2017a).

Although H. caimani has been detected in a high prevalence in C. crocodilus (76.7%) in the Amazon region (LAINSON, 1977) and in C. yacare in western (71.4%) and southeastern (76-79.5%) Pantanal (VIANA & MARQUES, 2005; VIANA et al., 2010b; SOARES et al., 2017a) in Brazil, by blood smear examinations, molecular characterization studies are scarce. In fact, only recently, a molecular confirmation has been performed in a population of Caiman yacare sampled in Miranda, state of Mato Grosso do Sul, central-western Brazil (SOARES et al., 2017a) and in Cayman crocodilus in Amazon (SOARES et al., 2017b). The present work aims to detect and characterize the Hepatozoon spp. found in wild and captive Caiman yacare sampled at Poconé, North Pantanal, Mato Grosso state (MT), Brazil, using blood smear examinations and molecular techniques.

Material and Methods

Blood samples of C. yacare were collected from wild and captivity animals from August to October 2010, for a total of 2 collections. In the wild, the samples were taken at the area called Corixo Verde (16°25′,08,3″S, 56°37′ 39,2″W) at Piuval Farm in Poconé-MT (Figure 1). Thirty-three caimans (24 free-ranging and 9 captive; 28 males and 5 females) were captured during the day and night by hand or by using nooses attached to long poles. Afterwards they were tied, weighed and also marked (just free-ranging caimans) in their tail ridges with a numbered plastic ring and their snout-vent lengths were measured. Sex was determined by exposure of genitalia.

Figure 1
Capture site. Map of Mato Grosso state, central-western Brazil, showing the locality where caimans were sampled in the present study.

Blood samples from tails from caimans were used for DNA extraction. DNA samples were extracted using QIAamp DNeasy Blood & Tissue Kit (QIAGEN®, Valencia, CA, USA), following manufacturer’s instructions. Initially, the detection of the presence of Hepatozoon spp. DNA was made using 0.3 µM of primers targeting part of 18S rRNA gene, namely HEMO1 (5’-TAT TGG TTT TAA GAA CTA ATT TTA TGA TTG- 3’) and HEMO2 (5’-CTT CTC CTT CCT TTA AGT GAT AAG GTT CAC3-’) (Síntese Biotecnologia®, Belo Horizonte, MG, BR), previously described by Perkins & Keller (2001). PCR amplifications were performed at 94 °C for 3 min followed by 35 repetitive cycles of 94 °C for 30 sec, 48 °C for 30 sec, and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min (HARRIS et al., 2011). Each 5 µL sample of extracted DNA was used as a template in 25 µL reaction mixtures containing 1X PCR buffer (Life Technologies®, Carlsbad, CA, USA), 2.5 mM MgCl₂, 0.2 mM deoxynucleotide triphosphate (dNTPs) mixture (Life Technologies®, Carlsbad, CA, USA), 1.5 U Taq DNA Polymerase (Life Technologies®, Carlsbad, CA, USA). DNA samples were also used in another PCR targeting a region of 18S rRNA gene, using primers HepF300 (5'- GTT TCT GAC CTA TCA GCT TTC GAC -3') and HepR900 (5’- CAA ATC TAA GAA TTT CAC CTC TGA C -3'), described by Ujvari et al. (2004). The amplification conditions were performed with 25 µL PCR reactions, containing 5 µL DNA template, 1X PCR buffer (Life Technologies®, Carlsbad, CA, USA), 0.2 mM of each dNTPs (Life Technologies®, Carlsbad, CA, USA), 1.5 mM MgCl₂, 0.6 µM of each primer (HepF300 and HepR900) (Síntese Biotecnologia®, Belo Horizonte, MG, BR), 1U Taq DNA Polymerase (Life Technologies®, Carlsbad, CA, USA). The cycling conditions were conducted following O'Dwyer et al. (2013) protocol, at 94 °C for 3 min followed by 35 repetitive cycles of 94 °C for 45 sec, 56 °C for 1 min, and 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. Hepatozoon sp. DNA positive control was obtained from a naturally infected wild canid (ANDRÉ et al., 2010). Ultra-pure sterile water (Life Technologies®, Carlsbad, CA, USA) was used as negative control. In order to prevent PCR contamination, DNA extraction, reaction setup, PCR amplification and electrophoresis were performed in separated rooms. The reaction products were purified using Silica Bead DNA Gel Extraction Kit (Thermo Fisher Scientific®, Waltham, MA, USA).

Purified amplified DNA fragments were submitted for sequence confirmation in an automatic sequencer (ABI Prism 310 DNA Analyser – Applied Biosystem®, Foster City, CA, EUA) (SANGER et al., 1977) in house and used for subsequent phylogenetic analysis. Phylogenetic reconstructions were based on DNA sequence alignment of positive samples. Samples showing positive results for both PCR protocols had their sequences concatenated (HepF300/HepR900 and HEMO1/HEMO2), using the Fragment Merger software version 1 (BELL & KRAMVIS, 2013). Comparisons with sequences deposited in GenBank (BENSON et al., 2002) were done using the nucleotides basic local alignment search tool (BLASTn) (ALTSCHUL et al., 1990). The sequences were aligned with sequences published in GenBank using MAFFT software, version 7 (KATOH & STANDLEY, 2013, 2016; YAMADA et al., 2016). Adelina sp. (Genbank access no. AF494059), Eimeria sp. (AF311644), Haemogregarina spp. (KX691418, HQ224959, KM887507, KM887508), Isospora sp. (U97523), Sarcocystis sp. (U97524) and Theileria sp. (FJ213586) were used as outgroups. Aligned sequences were edited by BioEdit Sequence Alignment Editor version 7.0.5.3 (HALL, 1999). Phylogenetic inference was based on Maximum Likelihood (ML) method. The Maximum-likelihood (ML) analysis was inferred with software IQ-TREE (NGUYEN et al., 2015) using W-IQ-TREE (TRIFINOPOULOS et al., 2016) (which includes an estimation of bootstrap node support), using 1000 bootstrapping replicates. The best model of evolution was selected using the software IQ-TREE (NGUYEN et al., 2015) by W-IQ-TREE (TRIFINOPOULOS et al., 2016), under the Akaike Information Criterion (AIC) (POSADA & BUCKLEY, 2004). The trees were examined in Treegraph 2.0.56-381 beta (STOVER & MULLER, 2010).

Additionally, an analysis of nucleotide polymorphisms of the 18S rRNA sequences obtained in the present study was performed. The sequences were aligned using MAFFT software, version 7 (KATOH & STANDLEY, 2013, 2016; YAMADA et al., 2016). The number of haplotypes, haplotype diversity (Hd) and nucleotide diversity (Pi) were determined using the program DnaSP 5, version 5.10.01 (LIBRADO & ROZAS, 2009).

Furthermore, the blood samples were used in blood smears for microscopic examination. Slides were air-dried, fixed with methanol and stained with Giemsa. Morphologic and morphometric features of gamonts, as well as the changes caused by the presence of parasites in erythrocytes were analyzed using the CellSens Imaging software (Olympus) in the Immunoparasitology Laboratory, Department of Veterinary Pathology, Universidade Estadual Paulista (UNESP Jaboticabal). Mean morphometric variables (nuclear and erythrocytes length, width, and area) of parasitized and non-parasitized cells were compared using non-paired t-test with Welch’s Correction; F test was used for comparing variance values. The results were analyzed using GraphPrism 7.03.

Results

Hepatozoon sp.-gametocytes (Figure 2) were found in 70.8% (17/24) and 88.8% (8/9) of blood smears from free-ranging and captive caimans, respectively. DNA of Hepatozoon spp. was found in 83.3% (20/24) and 88.8% (8/9) of free-ranging and captive caimans, respectively. Thirteen (54.2%) free-ranging caimans showed Hepatozoon sp.-gametocytes in blood smear examinations and were also positive to 18S rRNA PCR for Hepatozoon sp.; four (16.6%) showed positive results only in microscopic examinations. Seven (77.7%) captive caimans showed Hepatozoon sp.-gametocytes in blood smear examinations and were also positive to 18S rRNA PCR for Hepatozoon sp.; one (11.1%) showed positive results only in microscopic examinations.

Figure 2
Hepatozoon caimani gamonts found in Cayman yacare sampled in Poconé, state of Mato Grosso, central-western Brazil. 1000x.

The average morphometric measures of H. caimani gametocytes were: parasite whole cell (area = 53.2 µm² ± 14.6; lenght =12.9 µm ± 1.6; width = 4.81 µm ±1.1); nucleus (area = 13.1 µm² ± 4.71; lenght = 5.67 µm ± 1,5; width= 2.73 µm ± 0.88). Morphometric measures of non-parasitized and parasitized erythrocytes were shown in Table 1. Mean morphometric variables (nuclear and erythrocytes length, width, and area) showed statistical differences between parasitized and non-parasitized cells (P<0.05), except for length parasite cell.

Table 1
Comparative analysis between non-parasitized and H. caimani-parasitized erythrocytes found in Caiman crocodilus yacare blood smears.

	Cell			Nucleus
	Area (µm2)	Lenght (µm)	Width (µm)	Area (µm2)	Lenght (µm)	Width (µm)
N	130.9 (24.9)a	25.5 (10.4)a	8.66 (1.1)a	15.8 (4.4)a	5.8 (0.8)a	3.4 (0.6)a
PA	162.6 (32.6)b	19.3 (2.3)a	10.5 (1.6)b	17.5 (6.8)b	6.62 (1.3)b	3.3 (0.7)b

• Same letters in columns = no statistic differences (P≥0,05); different letters in columns = statistic differences (P≤0,05); (Standard deviation); N = non-parasitized erythrocytes; PA = parasitized erythrocytes.

The two 18S rRNA Hepatozoon sequences obtained from C. yacare showed 98-99% identity (98-99% of coverage) with Hepatozoon spp. from lizards from North Africa, previously deposited in GenBank (HQ734787, HQ734789, HQ734807) by BLAST analysis. Additionally, the Hepatozoon sequences obtained showed 99% identity (97-98% of coverage) with H. dormerguei sequence obtained from chameleon and snake from Madagascar (KM234646 and KM234649) and 99% identity (54-57% of coverage) with H. caimani sequence amplified from C. yacare from Brazil (KU495924 and KU495925).

The phylogenetic tree of Hepatozoon spp. 18S rRNA sequences clustered in a monophyletic group. The phylogenetic tree was basically in two branches: one of them composed by Hepatozoon sequences amplified in the present study and sequences of H. caimani, recently detected in caimans in southern Pantanal, and Hepatozoon spp. from other reptile taxa (snakes, lizards and tortoise) and amphibians retrieved from Genbank. The other branch grouped Hepatozoon sequences amplified from mammals and ticks previously deposited in Genbank. Adelina sp., Haemogregarina spp., Theileria sp., Isospora sp., and Sarcocystis sp. were used as outgroups (Figure 3).

Figure 3
Phylogenetic tree based on an alignment of 1200bp fragment of Hepatozoon spp. 18SrRNA sequences from Caiman yacare blood samples, using Maximum Likelihood (ML) method and TVM+G+I evolutionary model. Bootstrap values for ML (≥50) are given above relevant nodes. The sequences indicated in bold represent those from this study and original sample codes were named.

Nucleotide polymorphisms and DNA divergence between sequences obtained in the present study were also analyzed. The analysis of nucleotide polymorphisms of 18S rRNA sequences showed two haplotypes (haplotype diversity (Hd): 1.000; variance of haplotype diversity: 0.25000; Standard Deviation (SD): =0.500) and nucleotide diversity (Pi) of 0.02113 (SD= 0.01057).

Discussion

The occurrence of Hepatozoon sp. found in our study based on blood smears examinations (70.8%) among free-ranging C. yacare was lower than that found in C. crocodilus (76.7%) in the Amazon region (LAINSON, 1977) and in C. yacare in western (71.4%) and southeastern (76-79.5%) Pantanal (VIANA & MARQUES, 2005; VIANA et al., 2010a; SOARES et al., 2017a) in Brazil.

The detection sensitivity was slightly higher in PCR (83.3%) when compared to blood smear examinations (70.8%) of free-ranging caimans, corroborating previous studies involving molecular detection of Hepatozoon spp. in reptiles (UJVARI et al., 2004; HARRIS et al., 2011).

In the present study, morphometric alterations in parasitized cells by Hepatozoon spp. gamonts were markedly verified in all sampled yacares. The induction of changes in reptile parasitized cells by Hepatozoon spp. have already been reported in Caiman crocodilus yacare (VIANA & MARQUES, 2005; SOARES et al., 2017a) and in several species of snakes sampled in Brazil (MOÇO et al., 2002; O’DWYER et al., 2004; MOÇO et al., 2012). These changes in parasitize cells is probably mainly due to alterations in cellular erythrocyte cellular membrane’s permeability (VIANA & MARQUES, 2005).

Herein, the average size of Hepatozoon sp. gametocytes observed (12.9 × 4.81 µM) was closely related to previously described Hepatozoon caimani gamonts in Caiman crocodilus yacare (LAINSON et al., 2003; VIANA & MARQUES, 2005; SOARES et al., 2017a) and Caiman crocodilus crocodilus (LAINSON et al., 2003). Morphometric and morphologic features of sporogonic stages in the invertebrate vector, Culex (Meloconion), and meronts in vertebrate hosts will allow a better characterization of Hepatozoon parasitizing caimans in Brazil. Recently, meronts structures were described in the wall of vessels from liver and kidney ducts of H. caimani-naturally infected C. yacare in southern Pantanal (SOARES et al., 2017a).

The variety of morphological and morphometric forms of gamonts verified in yacare’ erythrocytes emphasizes the need for molecular confirmation of the involved Hepatozoon species. According to Moço et al. (2012), morphologically similar parasites showing different morphometric features may or may not represent different species. Furthermore, Hepatozoon spp. blood forms could show difference in their morphology depending on the host species involved (TELFORD, 2009). Besides, immature and mature gamonts, showing different morphological and morphometric features, could lead to a misdiagnosis of different species of Hepatozoon spp. (SMITH, 1996). Herein, we used a combination of morphometrical and phylogenetic assessment to confirm the occurrence of H. caimani in sampled caimans.

It seems that Hepatozoon species show limited host specificity, switching easily between different host species (MAIA et al., 2011). Regarding this high spectrum of host specificity found among Hepatozoon species, the host ecology appears to play a more important role in the complex relationship Hepatozoon – different host species than the phylogenetic relatedness among the hosts properly (SLOBODA et al., 2007). For instance, similar Hepatozoon spp. isolates appears to infect different genera of lizards, as previously reported (MAIA et al., 2011). On the other hand, some Hepatozoon species found in snakes have shown high specificity being restricted to determined hosts (TELFORD et al., 2001). Because of that, identification of new Hepatozoon species based solely on blood smears examinations should be avoided (MAIA et al., 2011). The identification of Hepatozoon species in different caiman species in Brazil should be done based on blood smears examinations and molecular phylogeny. Also, future studies should assess the phylogenetic relationships among Hepatozoon sp. isolates in different alligator species, Culex mosquitoes (vectors), anurans (paratenic hosts) and leeches in Brazil. Regarding the molecular characterization based on rDNA coding regions, such as that used in the present study (18S rRNA), although highly conserved and used frequently in molecular characterization of Hepatozoon spp. in several hosts (ANDRÉ et al., 2010; BORTOLI et al., 2011; MOÇO et al., 2012; O'DWYER et al., 2013; SOARES et al., 2017a; SOUSA et al., 2017), this target sequence may not be suitable for phylogenetic inferences of parasites which share some of intraspecific divergences (MOÇO et al., 2012), preventing attempts to differentiate those species. The search for new targets genes which allow a better phylogenetic differentiation of this group of parasites are much needed as an attempt to elucidate the diversity of Hepatozoon species that parasitize reptiles.

In summary, the present work showed through morphological, morphometric and molecular approaches, the occurrence of H. caimani in free-ranging and captive Cayman yacare in northern Pantanal.

Acknowledgements

The authors would like to thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support, Process nº 2015/14896-1.

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Publication Dates

• Publication in this collection
  24 Aug 2017
• Date of issue
  Jul-Sep 2017

History

• Received
  14 June 2017
• Accepted
  29 June 2017