The mitochondrial genome sequence analysis of Ophidascaris baylisi from the Burmese python (Python molurus bivittatus).

Ophidascaris species are parasitic roundworms that inhabit the python gut, resulting in severe granulomatous lesions or even death. However, the classification and nomenclature of these roundworms are still controversial. Our study aims to identify a snake roundworm from the Burmese python (Python molurus bivittatus) and analyze the mitochondrial genome. We identified this roundworm as Ophidascaris baylisi based on the morphology and cytochrome c oxidase subunit I (cox1) sequence. Ophidascaris baylisi complete mitochondrial genome was 14,784 bp in length, consisting of two non-coding regions and 36 mitochondrial genes (12 protein-coding genes, 22 tRNA genes, and two rRNA genes). The protein-coding genes used TTG, ATG, ATT, or TTA as start codons and TAG, TAA, or T as stop codons. All tRNA genes showed a TV-loop structure, except trnS1AGN and trnS2UCN revealed a D-loop structure. The mitochondrial large ribosomal subunit 16S (rrnL) and small ribosomal subunit 12S (rrnS) were 956 bp and 700 bp long, respectively. Phylogenetic analysis based on O. baylisi mitochondrial protein-coding genes demonstrated that O. baylisi clustered with the family Ascarididae members and was most closely related to Ophidascaris wangi. These results may enhance the nematode mitochondrial genome database and provide valuable molecular markers for further research on the taxonomy, phylogeny, and genetic relationships of Ophidascaris nematodes.

Successful treatment of Ophionyssus natricis with afoxolaner in two Burmese pythons (Python molurus bivittatus).

The efficacy of afoxolaner was evaluated in two captive Burmese python snakes, which were naturally infested with Ophionyssus natricis mites. The administration of a single oral dose of afoxolaner eliminated live O. natricis mites from both snakes by Day 3. Environmental samples collected from the snakes' terrariums were negative for dead mites by Day 30.

L'efficacité de l'afoxolaner a été évaluée chez deux serpents python birmans captifs, naturellement infestés par des acariens Ophionyssus natricis. L'administration d'une dose orale unique d'afoxolaner a éliminé les acariens O. natricis vivants des deux serpents à jour 3. Les échantillons environnementaux prélevés dans les terrariums des serpents étaient négatifs pour les acariens morts à jour 30.

Se evaluó la eficacia de afoxolaner en dos serpientes pitón birmanas cautivas, que estaban naturalmente infestadas con ácaros Ophionyssus natricis. La administración de una sola dosis oral de afoxolaner eliminó los ácaros O. natricis vivos de ambas serpientes en el día 3. Las muestras ambientales recolectadas de los terrarios de las serpientes dieron negativo para ácaros muertos en el día 30.

A eficácia do afoxolaner foi avaliada em duas cobras python birmanesas de cativeiro, que foram naturalmente infestadas com ácaros Ophionyssus natricis. A administração de uma única dose oral de afoxolaner eliminou ácaros O. natricis vivos de ambas as cobras no dia 3. As amostras ambientais coletadas dos terrários das cobras foram negativas para ácaros mortos no dia 30.

Entamoeba ranarum Infection in a Ball Python (Python regius).

The pathogenic Entamoeba species in snakes is widely regarded to be Entamoeba invadens, which can cause severe amoebiasis with up to 100% mortality. In this case report, we describe a ball python (Python regius) that died after short-term weight loss. Necropsy revealed severe necrotizing colitis with large numbers of intralesional Entamoeba trophozoites. Molecular genetic analysis identified these trophozoites as Entamoeba ranarum, a parasite more usually found in amphibians. Furthermore, the extended history revealed that toads (Rhinella marina) had been housed together with the python. This report illustrates the danger of protozoal cross-infections in exotic animals as well as the importance of molecular genetic tools in Entamoeba diagnosis.

Host-specific phenotypic variation of a parasite co-introduced with invasive Burmese pythons.

Invasive Burmese pythons (Python bivittatus Kuhl, 1820) have introduced a lung parasite, Raillietiella orientalis, (Hett, 1915) from the python's native range in Southeast Asia to its introduced range in Florida, where parasite spillover from pythons to two families and eight genera of native snakes has occurred. Because these novel host species present a diversity of ecological and morphological traits, and because these parasites attach to their hosts with hooks located on their cephalothorax, we predicted that R. orientalis would exhibit substantial, host-associated phenotypic plasticity in cephalothorax shape. Indeed, geometric morphometric analyses of 39 parasites from five host species revealed significant variation among host taxa in R. orientalis cephalothorax shape. We observed differences associated with host ecology, where parasites from semi-aquatic and aquatic snakes exhibited the greatest morphological similarity. Morphological analyses of R. orientalis recovered from invasive pythons, native pit vipers, and terrestrial snakes each revealed distinct shapes. Our results suggest R. orientalis can exhibit significant differences in morphology based upon host species infected, and this plasticity may facilitate infection with this non-native parasite in a wide array of novel squamate host species.

Armillifer-Infected Snakes Sold at Congolese Bushmeat Markets Represent an Emerging Zoonotic Threat.

African pythons (Pythonidae) and large vipers (Bitis spp.) act as definitive hosts for Armillifer armillatus and Armillifer grandis parasites (Crustacea: Pentastomida) in the Congo Basin. Since the proportion of snakes in bushmeat gradually increases, human pentastomiasis is an emerging zoonotic disease. To substantiate the significance of this threat, we surveyed snakes offered for human consumption at bushmeat markets in the Kole district, Democratic Republic of the Congo, for the presence of adult pentastomids. In Bitis vipers (n = 40), Armillifer spp. infestations exhibited an 87.5% prevalence and 6.0 median intensity. Parasite abundance covaried positively with viper length, but not with body mass. In pythons (n = 13), Armillifer spp. exhibited a 92.3% prevalence and 3.5 median intensity. The positive correlations between parasite abundance and python length or mass were statistically nonsignificant. Ninety-one percent of A. grandis were discovered in vipers and 97% of infected vipers hosted A. grandis, whereas 81% of A. armillatus specimens were found in pythons and 63% of infected pythons hosted A. armillatus. Thus, challenging the widespread notion of strict host specificity, we found 'reversed' infections and even a case of coinfection. In this study, we also gathered information about the snake consumption habits of different tribal cultures in the area. Infective parasite ova likely transmit to humans directly by consumption of uncooked meat, or indirectly through contaminated hands, kitchen tools or washing water.

Borrelia sp. phylogenetically different from Lyme disease- and relapsing fever-related Borrelia spp. in Amblyomma varanense from Python reticulatus.

BACKGROUND: Species of the genus Borrelia are causative agents of Lyme disease and relapsing fever. Lyme disease is the most commonly reported vector-borne disease in the northern hemisphere. However, in some parts of the world Lyme borreliosis and relapsing fever may be caused by novel Borrelia genotypes. Herein, we report the presence of a Borrelia sp. in an Amblyomma varanense collected from Python reticulatus. METHODS: Ticks were collected from snakes, identified to species level and examined by PCR for the presence of Borrelia spp. flaB and 16S rRNA genes. Phylogenetic trees were constructed using the neighbour-joining method. RESULTS: Three A. varanense ticks collected from P. reticulatus were positive for a unique Borrelia sp., which was phylogenetically divergent from both Lyme disease- and relapsing fever-associated Borrelia spp. CONCLUSION: The results of this study suggest for the first time that there is a Borrelia sp. in A. varanense tick in the snake P. reticulatus that might be novel.

Subspectacular nematodiasis caused by a novel Serpentirhabdias species in ball pythons (Python regius).

Subspectacular nematodiasis was diagnosed in three captive-bred juvenile ball pythons (Python regius) from two unrelated facilities within a 6-month period. The snakes were presented with similar lesions, including swelling of facial, periocular and oral tissues. Bilaterally, the subspectacular spaces were distended and filled with an opaque fluid, which contained nematodes and eggs. Histopathology showed nematodes throughout the periocular tissue, subspectacular space and subcutaneous tissue of the head. The nematodes from both facilities were morphologically indistinguishable and most closely resembled Serpentirhabdias species. Morphological characterization and genetic sequencing indicate this is a previously undescribed rhabdiasid nematode.

Comparison of Sanger and next generation sequencing performance for genotyping Cryptosporidium isolates at the 18S rRNA and actin loci.

Cryptosporidium is an important enteric pathogen that infects a wide range of humans and animals. Rapid and reliable detection and characterisation methods are essential for understanding the transmission dynamics of the parasite. Sanger sequencing, and high-throughput sequencing (HTS) on an Ion Torrent platform, were compared with each other for their sensitivity and accuracy in detecting and characterising 25 Cryptosporidium-positive human and animal faecal samples. Ion Torrent reads (n = 123,857) were obtained at both 18S rRNA and actin loci for 21 of the 25 samples. Of these, one isolate at the actin locus (Cattle 05) and three at the 18S rRNA locus (HTS 10, HTS 11 and HTS 12), suffered PCR drop-out (i.e. PCR failures) when using fusion-tagged PCR. Sanger sequences were obtained for both loci for 23 of the 25 samples and showed good agreement with Ion Torrent-based genotyping. Two samples both from pythons (SK 02 and SK 05) produced mixed 18S and actin chromatograms by Sanger sequencing but were clearly identified by Ion Torrent sequencing as C. muris. One isolate (SK 03) was typed as C. muris by Sanger sequencing but was identified as a mixed C. muris and C. tyzzeri infection by HTS. 18S rRNA Type B sequences were identified in 4/6 C. parvum isolates when deep sequenced but were undetected in Sanger sequencing. Sanger was cheaper than Ion Torrent when sequencing a small numbers of samples, but when larger numbers of samples are considered (n = 60), the costs were comparative. Fusion-tagged amplicon based approaches are a powerful way of approaching mixtures, the only draw-back being the loss of PCR efficiency on low-template samples when using primers coupled to MID tags and adaptors. Taken together these data show that HTS has excellent potential for revealing the "true" composition of species/types in a Cryptosporidium infection, but that HTS workflows need to be carefully developed to ensure sensitivity, accuracy and contamination are controlled.

Morphological and molecular identification of ticks infesting Boa constrictor (Squamata, Boidae) in Manaus (Central Brazilian Amazon).

The Boa constrictor is one of the world's largest vertebrate carnivores and is often found in urban areas in the city of Manaus, Brazil. The morphological identification of ticks collected from 27 snakes indicated the occurrence of Amblyomma dissimile Koch 1844 on all individuals sampled. In contrast, Amblyomma rotundatum Koch was found on only two snakes. An analysis of the 16S rRNA molecular marker confirmed the morphological identification of these ectoparasites.

Choleoeimeria ghaffari n. sp. (Apicomplexa: Eimeriidae) from the gallbladder of Eryx jayakari Boulenger (Serpentes: Boidae) in Saudi Arabia.

Choleoeimeria ghaffari n. sp. is described from the gallbladder of Eryx jayakari Boulenger in Saudi Arabia. Oöcysts are tetrasporocystic, cylindroidal, 23 × 14 µm, with a smooth bi-layered wall and length/width ratio of 1.5, without micropyle, oöcyst residuum and polar granule. Sporocysts are subspheroidal to ellipsoidal, 8 × 6 µm, with length/width ratio of 1.4, without Stieda, sub-Stieda and para-Stieda bodies but with sporocyst residuum. Sporozoites are banana-shaped measuring 10 × 1.5 µm. The endogenous development was found to occur in the gallbladder epithelium and the extrahepatic bile ducts. Mature meronts are spheroidal, c.10 µm wide, and suspected to produce 12-16 merozoites. Microgamonts are irregular in shape, 13 × 10 µm, whereas macrogamonts are mostly subspheroidal, c.12 µm wide, with a prominent centrally-located nucleus. Based on oöcyst morphology and the site of endogenous development (epithelium of the gallbladder and bile ducts) the new eimeriid coccidian was placed in the genus Choleoeimeria Paperna & Landsberg, 1989.

Molecular identification of Sarcocystis spp. helped to define the origin of green pythons (Morelia viridis) confiscated in Germany.

Sarcocystis spp. represent apicomplexan parasites. They usually have a heteroxenous life cycle. Around 200 species have been described, affecting a wide range of animals worldwide, including reptiles. In recent years, large numbers of reptiles have been imported into Europe as pets and, as a consequence, animal welfare and species protection issues emerged. A sample of pooled feces from four confiscated green pythons (Morelia viridis) containing Sarcocystis spp. sporocysts was investigated. These snakes were imported for the pet trade and declared as being captive-bred. Full length 18S rRNA genes were amplified, cloned into plasmids and sequenced. Two different Sarcocystis spp. sequences were identified and registered as Sarcocystis sp. from M. viridis in GenBank. Both showed a 95-97% sequence identity with the 18S rRNA gene of Sarcocystis singaporensis. Phylogenetic analysis positioned these sequences together with other Sarcocystis spp. from snakes and rodents as definitive and intermediate hosts (IH), respectively. Sequence data and also the results of clinical and parasitological examinations suggest that the snakes were definitive hosts for Sarcocystis spp. that circulate in wild IH. Thus, it seems unlikely that the infected snakes had been legally bred. Our research shows that information on the infection of snakes with Sarcocystis spp. may be used to assess compliance with regulations on the trade with wildlife species.

Myiasis by Megaselia scalaris (Diptera: Phoridae) in a python affected by pulmonitis.

Myiases are caused by the presence of maggots in vertebrate tissues and organs. Myiases have been studied widely in humans, farm animals, and pets, whereas reports of myiasis in reptiles are scarce. We describe a case of myiasis caused by the Megaselia scalaris (Loew) in an Indian python (Python molurus bivittatus, Kuhl) (Ophida: Boidae). The python, 15 yr old, born and reared in a terrarium in the mainland of Venice (Italy), was affected by diffuse, purulent pneumonia caused by Burkholderia cepacia. The severe infestation of maggots found in the lungs during an autopsy indicated at a myiasis.

Determinação de hemogregarina em Boa constrictor constrictor mantidos em cativeiro / Determination of hemogregarine in Boa constrictor constrictor kept in captivity

O presente estudo teve como objetivo determinar a presença de hemogregarina em boídeos mantidos em cativeiro no Estado do Pará, bem como, relacionar a hemoparasitose com pre-disposição sexual, alterações clínicas e hematológicas e a presença de ectoparasitos. Esta pesquisa teve autorização do Sistema de Autorização e Informação em Biodiversidade do Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis para ser realizado. Utilizaram-se 19 serpentes da família Boidae mantidas em cativeiro, pertencentes ao "Museu Paraense Emilio Goeldi" (Belém/PA) e "Sítio Xerimbabo" (Santo Antônio do Tauá/PA). A pesquisa de hemogregarina foi realizada em esfregaços sanguíneos examinados no aumento de 400x, enquanto que a parasitemia foi determinada contando- se 550 hemácias em aumento de 1000x. Do total de animais estudados (n=19), nove encontraram-se parasitados (47,36%), não havendo correlação entre presença de hemogregarina, pré-disposição sexual, alterações clínicas e hematológicas nas serpentes hospedeiras. A correlação da hemoparasitose foi detectada apenas quanto à presença de ectoparasitas nas serpentes, no entanto, estudos adicionais são necessários para verificar a prevalência de hemogregarinas em animais mantidos em cativeiro no Estado do Pará, visto que, existe grande lacuna de dados na literatura veterinária especializada no que diz respeito à fauna da região amazônica.

We aimed to determine hemogregarines presence in snakes of the Boidae family kept in captivity in Pará (PA), Brazil, and to relate it with sex, clinical and hematological and ectoparasitism. This study had authorization from Sisbio/IBAMA to be done. Nineteen Boa constrictor snakes were used, belonging to the "Museu Paraense Emílio Goeldi" (Belém/ PA) and "Xerimbabo Farm" (Santo Antônio do Tauá/PA). Blood smears were examined with 400x magnification, while the parasitemia percentage was determined by counting 550 red blood cells with 1000x magnification. From the snakes studied (n=19), nine were parasitized (47.36%) and there was no correlation between hemogregarines presence, sex, clinical and hematological changes. Hemoparasitosis correlation was detected only with the ectoparasites presence; however further studies are needed to determine the real hemogregarines prevalence in snakes kept in captivity in Pará, since there is a huge gap of data in the veterinary specialized literature about the fauna of the Amazon region.

Acariasis on pet Burmese python, Python molurus bivittatus in Malaysia.

OBJECTIVE: To identify the acari present on pet Burmese pythons in Malaysia and to determine whether there is any potential public health risk related to handling of the snakes. METHODS: Two sub-adult Burmese pythons kept as pets for a period of about 6 to 7 months by different owners, were brought to an exotic animal practice for treatment. On a complete medical examination, some ticks and mites (acari) were detected beneath the dorsal and ventral scales along body length of the snakes. Ticks were directly identified and mites were mounted prior to identification. RESULTS: A total of 12 ticks represented by 3 males, 2 females and 7 nymphal stages of Rhipicephalus sanguineus (R. sanguineus) were extracted from the first python while the other one was with 25 female Ophionyssus natricis (O. natricis) mesostigmatid mites. Only adult female mites were found. These mites are common ectoparasites of Burmese pythons. CONCLUSIONS: Both the acarine species found on the Burmese pythons are known vectors of pathogens. This is the first record that R. sanguineus has been reported from a pet Burmese python in Malaysia.

Novel Hepatozoon in vertebrates from the southern United States.

Novel Hepatozoon spp. sequences collected from previously unrecognized vertebrate hosts in North America were compared with documented Hepatozoon 18S rRNA sequences in an effort to examine phylogenetic relationships between the different Hepatozoon organisms found cycling in nature. An approximately 500-base pair fragment of 18S rDNA common to Hepatozoon spp. and some other apicomplexans was amplified and sequenced from the tissues or blood of 16 vertebrate host species from the southern United States, including 1 opossum (Didelphis virginiana), 2 bobcats (Lynx rufus), 1 domestic cat (Felis catus), 3 coyotes (Canis latrans), 1 gray fox (Urocyon cinereoargenteus), 4 raccoons (Procyon lotor), 1 pet boa constrictor (Boa constrictor imperator), 1 swamp rabbit (Sylvilagus aquaticus), 1 cottontail rabbit (Sylvilagus floridanus), 4 woodrats (Neotoma fuscipes and Neotoma micropus), 3 white-footed mice (Peromyscus leucopus), 8 cotton rats (Sigmodon hispidus), 1 cotton mouse (Peromyscus gossypinus), 1 eastern grey squirrel (Sciurus carolinensis), and 1 woodchuck (Marmota monax). Phylogenetic analyses and comparison with sequences in the existing database revealed distinct groups of Hepatozoon spp., with clusters formed by sequences obtained from scavengers and carnivores (opossum, raccoons, canids, and felids) and those obtained from rodents. Surprisingly, Hepatozoon spp. sequences from wild rabbits were most closely related to sequences obtained from carnivores (97.2% identical), and the sequence from the boa constrictor was most closely related to the rodent cluster (97.4% identical). These data are consistent with recent work identifying prey-predator transmission cycles in Hepatozoon spp. and suggest this pattern may be more common than previously recognized.

First reports of ectoparasites collected from wild-caught exotic reptiles in Florida.

We collected ectoparasites from 27 of 51 wild-caught, free-ranging exotic reptiles examined in Florida from 2003 to 2008. Sampled animals represented eight species, five of which yielded ectoparasites. Reported new parasite distribution records for the United States include the following: the first collection of the African tick Amblyomma latum (Koch) from a wild-caught animal [ball python, Python regius (Shaw)] in the United States; the first collection of the lizard scale mite Hirstiella stamii (Jack) from any wild-caught animal [green iguana, Iguana iguana (L.)]; and the first collection of the lizard scale mite Geckobia hemidactyli (Lawrence) in the continental United States from a wild-caught tropical house gecko, Hemidactylus mabouia (Moreau de Jonnès). We also report the first collections of the Neotropical ticks Amblyomma rotundatum (Koch) and Amblyomma dissimile (Koch) from wild-caught Burmese pythons, Python molurus bivittatus (Kuhl); the first collections of A. dissimile from a wild-caught African savannah monitor, Varanus exanthematicus (Bosc); and from wild-caught green iguanas in the United States; and the first collections of the native chiggers Eutrombicula splendens (Ewing) and Eutrombicula cinnabaris (Ewing) from wild-caught Burmese pythons. These reports may only suggest the diversity of reptile ectoparasites introduced and established in Florida and the new host-parasite relationships that have developed among exotic and native ectoparasites and established exotic reptiles.

A new species of Caryospora Léger, 1904 (Apicomplexa: Eimeriidae) from the endangered Round Island boa Casarea dussumieri (Schlegel) (Serpentes: Bolyeridae) of Round Island, Mauritius: an endangered parasite?

A new species of Caryospora Léger, 1904 (Apicomplexa: Eimeriidae), C. durelli n. sp., is described from the endangered Round Island boa Casarea dussumieri (Schlegel) (Serpentes: Bolyeridae) from Round Island, Mauritius. Six of 11 hosts were infected. Oöcysts are spherical to subspherical, 19.2 × 18.2 (17.5-21 × 16-21) µm, n = 20, and have a shape index (mean length/mean width) of 1.05 (1.02-1.09). The bi-layered wall is composed of an outer layer of c.0.6 µm thick and an inner layer of c.0.4 µm thick. A micropyle, oöcyst residuum and polar granule are absent. Sporocysts are ellipsoidal, 14.7 × 11.0 (13-16 × 9.5-11.5) µm, n = 20, and have a shape index of 1.33. Both Stieda and substieda bodies are present. The sporocyst residuum measures c.12 × 4.5 µm, is surrounded by sporozoites and composed of numerous granules. Refractile bodies are present but not clearly visible. This is the first coccidian parasite reported from the family Bolyeridae and the first species of Caryospora durrelli [corrected] reported from the Mascarenes. Conservation issues concerning parasites of endangered host species are discussed.

Trypanosoma cf. varani in an imported ball python (Python reginus) from Ghana.

Peripheral blood from a ball python (Python reginus) imported from Ghana was cultured in Barbour-Stoenner-Kelly (BSK) medium for Borrelia spp. isolation, resulting in the prominent appearance of free, and clusters of, trypanosomes in a variety of morphological forms. The molecular phylogenetic characterization of these cultured trypanosomes, using the small subunit rDNA, indicated that this python was infected with a species closely related to Trypanosoma varani Wenyon, 1908, originally described in the Nile monitor lizard (Varanus niloticus) from Sudan. Furthermore, nucleotide sequences of glycosomal glyceraldehyde-3-phosphate dehydrogenase gene of both isolates showed few differences. Giemsa-stained blood smears, prepared from the infected python 8 mo after the initial observation of trypanosomes in hemoculture, contained trypomastigotes with a broad body and a short, free flagellum; these most closely resembled the original description of T. varani, or T. voltariae Macfie, 1919 recorded in a black-necked spitting cobra (Naja nigricollis) from Ghana. It is highly possible that lizards and snakes could naturally share an identical trypanosome species. Alternatively, lizards and snakes in the same region might have closely related, but distinct, Trypanosoma species as a result of sympatric speciation. From multiple viewpoints, including molecular phylogenetic analyses, reappraisal of trypanosome species from a wide range of reptiles in Africa is needed to clarify the relationship of recorded species, or to unmask unrecorded species.

Dracunculus brasiliensis sp. n. (Nematoda: Dracunculidae) from the anaconda, Eunectes murinus (Ophidia: Boidae).

Dracunculus brasiliensis sp. n. (Dracunculidae), is described based on a single female specimen found in the body cavity of the anaconda, Eunectes murinus (L.) (Ophidia: Boidae), from the Mexiana Island, Amazon River delta, Brazil and one female previously recorded from the subcutaneous tissue of this host species imported from South America into Europe (ZOO in the Czech Republic). The new species is characterised mainly by markedly large, anteriorly protruding dorsal and ventral double papillae of the internal circle and small lateral papillae of the same circle, a widely rounded caudal end, the excretory pore situated just posterior to the nerve ring, a distinctly transversely striated cuticle and by the length (396-429 mum) of larvae from uterus. This is the first species of Dracunculus described from reptiles in South America.

Rodents as intermediate hosts of Hepatozoon ayorgbor (Apicomplexa: Adeleina: Hepatozoidae) from the African ball python, Python regius?

Two experimental trials were performed to elucidate the role of rodents in the life cycle of Hepatozoon species using snakes as intermediate hosts. In one trial, two ball pythons, Python regius Shaw, 1802 were force fed livers of laboratory mice previously inoculated with sporocysts of Hepatozoon ayorgbor Sloboda, Kamler, Bulantová, Votýpka et Modrý, 2007. Transmission was successful in these experimentally infected snakes as evidenced by the appearance of intraerythrocytic gamonts, which persisted until the end of trial, 12 months after inoculation. Developmental stages of haemogregarines were not observed in histological sections from mice. In another experimental trial, a presence of haemogregarine DNA in mice inoculated with H. ayorgbor was demonstrated by PCR in the liver, lungs and spleen.