Severe Amoebic Placentitis in a Horse Caused by an Acanthamoeba hatchetti Isolate Identified Using Next-Generation Sequencing

Abstract

A case of amoebic placentitis in a mare from eastern Australia was diagnosed postpartum by histopathological examination of the placenta. The identity of the etiological agent was confirmed as Acanthamoeba hatchetti by use of diversity profiling based on a next-generation sequencing approach.

CASE REPORT

A normal underweight term foal was born to an 11-year-old multiparous Thoroughbred mare in the Hunter region of New South Wales, Australia. A thick mucopurulent tan discharge covered the body of the placenta encompassing the pregnant horn (Fig. 1A). Routine culture of the discharge grew mixed organisms comprised of Escherichia coli, Streptococcus agalactiae, Bacillus cereus, and an unidentifiable Gram-negative bacillus (identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry) (1). The mare had been present on the same farm for 6 years, with no history of reproductive problems, placentitis, or abortion with her previous six pregnancies.

FIG 1.

Horse placenta with Acanthamoeba hatchetti. (A) Mucopurulent orange-brown discharge in the placenta containing numerous A. hatchetti organisms within the allantochorion (hematoxylin and eosin stain [H&E]); (B and C) detailed view of the cyst of A. hatchetti; (D) H&E stain of a cyst; (E) Grocott's methenamine silver stain of a cyst; (F and G) Calcofluor White-labeled A. hatchetti cysts (green) under fluorescence microscopy (fluorescein isothiocyanate [FITC] filter set). Scale bars, 10 µm.

Histologically, the allantochorion of the pregnant horn was extensively effaced by a severe chronic active placentitis (Fig. 1B). The chorion was primarily involved, with limited extension through the extra embryonic coelom into the allantois. There was extensive chorionic villous loss and shortening, with variable trophoblastic epithelial hyperplasia and squamous metaplasia. A thick adherent layer of amorphous eosinophilic material with extensive multifocal mineralization and foci of necrotic inflammatory cells, primarily neutrophils, covered the surface. The residual villi and adjacent chorionic stroma were expanded by large numbers of plasma cells, macrophages, some multinucleated giant cells, and neutrophils with multifocal active villous necrosis (Fig. 1B). Acute villous necrosis and suppurative inflammation were evident in less affected areas, with large numbers of 3- to 15-μm-diameter, round to irregular amoeboid organisms (presumed to be trophozoites) with a characteristic large dark endosome. These organisms were located primarily within trophoblastic cells of the chorionic villi (Fig. 1C). Large numbers of degenerate encysted stages up to 24 μm in diameter were present in the intervillous spaces and superficial necrotic debris (Fig. 1D and E).

Diagnostic material was submitted to the Veterinary Pathology Diagnostic Services (University of Sydney) for characterization and identification. Initially, Calcofluor White (Fluka, Sigma-Aldrich) was used on unstained histological sections (Fig. 1F and G) to confirm the presence of cellulose and chitin in pathogen cell walls, including cysts of Acanthamoeba spp., microsporidia, or yeasts (2, 3). Calcofluor White staining confirmed the structures to be the same as those identified in the stained histological section (Fig. 1C, D, and E). The morphology and size of the Calcofluor White-stained structures were not consistent with the known morphologies and sizes of microsporidia (1- to 3-μm cysts) or of yeasts (budding).

To identify the potential intracellular and free eukaryotes present in the diagnostic specimen, we used a community profiling-based amplification, 454 sequencing approach of conserved eukaryotic single-subunit (SSU) ribosomal DNA (rDNA) (4). Genomic DNA (gDNA) was isolated from approximately 25 mg of frozen (−20°C) allantochorion using the Isolate II genomic DNA kit (Bioline, Australia). The gDNA was submitted to the diversity profiling service at the Australian Genomic Research Facility, Australia. The SSU rDNA assay applied was based on primer pair Euk1A (5′-CTGGTTGATCCTGCCAG-3′) and Euk516 (5′-CCAGACTTGCCCTCC-3′), amplifying approximately 500 to 550 bp of the 5′ end of the eukaryotic SSU rDNA. The barcoded PCR amplicon was pooled and sequenced on the 454 GS-FLX platform. Eight thousand six hundred thirty-four reads were obtained, and the number of reads was subsequently reduced to 2,294 high-quality reads with an average read length of 501 bp. We removed homopolymers (>8) and sequences of <150 bp in length and kept only sequence reads with a cutoff quality score of 20 across 80% of each sequence read. The high-quality reads were clustered using CD-HIT (cd-hit.org) into pools of sequence clusters with >97% identity. Eighteen clusters (98.8% of which were duplicates) and six singleton sequences were identified and used as queries in BLASTN within CLC Main Workbench 6.9 (CLC bin; Qiagen, Denmark). A single cluster represented by two sequences and two singletons was most closely related to Acanthamoeba hatchetti (GenBank accession number AF019068). A remaining singleton was most closely related to a fungus, Cystofilobasidium infirmominiatum (DQ645524), suspected of being a contaminant with no direct relevance to the case. Horse SSU rDNA matched 17 of the clusters and 5 of the singletons. Querying of all reads (including the low-quality reads) using BLASTN with A. hatchetti (AF019068) confirmed the presence of 4 hits in the high-quality pool of reads as well as an additional 5 sequence reads in the previously discarded poor-quality reads. A phylogenetic tree revealed monophyly of the horse Acanthamoeba sequences with A. hatchetti, which together formed a sister group to Acanthamoeba stevensoni (Fig. 2A). Pairwise nucleotide comparison revealed over 98.5% identity with available SSU rDNA sequences of A. hatchetti strains BH2 and 4RE (Fig. 2B). Strain BH-2 (ATCC 30730) is the type species of A. hatchetti and forms a distinct SSU rDNA clade, T11, together with A. stevensoni (5). BH2 was isolated from brackish sediment from Brewerton Channel in Baltimore Harbor, MD (6). 4RE was derived from a contact lens storage case in Austria (7). Further querying (BLASTN) revealed high (>98%) similarity with a phylotypes from fecally contaminated water from Equatorial Guinea (KF433820) and a sink plughole in Japan (AB859622).

FIG 2.

Evolutionary relationships of Acanthamoeba hatchetti from a horse with placentitis based on SSU rDNA. (A) The tree was inferred using the minimum-evolution (ME) method. The bootstrap supports in percentages are shown next to the branches (1,000 replicates for ME and 100 replicates for maximum likelihood [ML]). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method, and the ME tree was searched using the close-neighbor-interchange (CNI) algorithm in MEGA6 (www.megasoftware.net). All ambiguous positions were removed for each sequence pair. There were 589 positions spanning a partial A. hatchetti SSU rDNA. (B) Pairwise comparison of nucleotide distances (uncorrected) for published A. hatchetti SSU rDNA sequences and the sequences obtained using 454 sequencing, cluster 16 (represented by IJMZ0LK01BAKMG and IJMZ0LK01AO988 reads), and two singletons (IJMZ0LK01ATWF7, IJMZ0LK01AKNTK). A. stevensoni is the closest sister species to A. hatchetti.

Initial attempts at direct pathogen PCR identification with the Acanthamoeba species-specific SSU rDNA primers ACAN18SF0 (5′-TCCTGCCAGTAGTCATATGC-3′) and ACAN18SR0 (5′-CTTCTCCTTCCTCTAAATGGT-3′) (8) were unsuccessful. Subsequently, successful amplification was achieved with primers targeting a partial SSU rDNA fragment, JDP1 (5′-GGCCCAGATCGTTTACCGTGAA-3′) and JDP2 (5′-TCTCACAAGCTGCTAGGGAGTCA-3′) (9). PCR used MyTaq Red Mix (Bioline, Australia) and the following cycling conditions: 95°C for 15 s, 57°C for 15 s, and 72°C for 15 s for 35 cycles in a Veriti PCR cycler (Life Sciences, Australia). A PCR product band of the expected size was bidirectionally sequenced using amplification primers at Macrogen Ltd. (Seoul, South Korea) and analyzed in CLC Main Workbench. The obtained sequence was 100% and 99.5% identical to the A. hatchetti 4RE and BH2 strains, respectively.

The identity A. hatchetti was further supported by cyst morphology. Calcofluor White visualization of the placenta revealed myriad 14.0 (12.4 to 15.7)-μm-diameter cysts morphologically compatible with those for the BH-2 A. hatchetti isolate, originally reported to form cysts that measured 13.1 (11.5 to 16.1) μm in diameter (6).

Acanthamoeba spp. are ubiquitous free-living amoebae that have received a bad reputation as the cause of a sight-threatening keratitis in contact lens wearers and a rare but fatal granulomatous encephalitis (10). In humans, Acanthamoeba spp. enter the central nervous system (CNS) through either the respiratory track or skin injury, followed by amoebic invasion of the blood vessels or the nasal passage (10, 11). Infections are generally diagnosed by visualization of typical cysts, culture, and PCR (10). Free-living amoebae have rarely been documented to cause encephalitis in animals, including horses; this is most likely due to underreporting (12). Animals as sentinels for human Acanthamoeba species exposure are scarcely explored (13). In domestic animals, only encephalitis is recognized as an Acanthamoeba-associated disease.

We report a unique placentitis in a mare with a documented presence of A. hatchetti associated with histopathological lesions of focal chronic active inflammation of the chorioallantois. To our knowledge, this is the first opportunistic Acanthamoeba infection of the reproductive tract of any species and confirms the experimental potential of A. hatchetti to cause significant disease (6). A. hatchetti is a common free-living amoeba that has not previously been documented to cause significant pathology under natural conditions (14). However, experimental intranasal inoculation of laboratory mice with A. hatchetti BH-2 results in death due to extensive inflammation and necrosis of the brain (6). This case report of amoebic equine placentitis is the first documented case of clinical disease caused by A. hatchetti.

Several opportunities exist for A. hatchetti to enter into the horse reproductive system. A vascular route of transmission (the route for CNS infection) is supported by the literature if respiratory tract or skin injury is present (10, 11). However, the mare was clinically healthy prior to and throughout the pregnancy. Recently, setae (hairs) of the Processionary caterpillar (Ochrogaster lunifer) were documented to migrate from the gastrointestinal tracts of experimentally exposed mares into the uterus and fetal membranes, ultimately causing abortion (15). Mechanical transport of A. hatchetti by the setae into the uterus may be possible due to the hollow interior of the setae or unknown adherence factors related to biofilm or any seta-associated bacteria. Alternatively, A. hatchetti may have localized in the uterus after vascular invasion or via cervical incompetence with a bacterial focal placentitis. This lesion can occur as sequelae to caterpillar exoskeleton ingestion in early gestation, and any bacteria may have served as a food source for A. hatchetti in the role of opportunistic pathogen (16). Acanthamoeba cysts themselves carry a myriad of pathogens within their cysts, including the infamous Legionella (10, 11). Therefore, if it is evidenced that Acanthamoeba cysts were carried into the uterus on caterpillar hairs, this would represent one of the most remarkable cases of pathogen transmission.

The diagnosis of Acanthamoeba spp. is challenging because of the cryptic morphology of their trophozoites, which resemble macrophages. This case demonstrates the advantages of the use of diversity profiling approaches for the identification of eukaryotic pathogens, including emerging pathogens for which routine diagnostic approaches are not available.

Nucleotide sequence accession numbers.

Nucleotide sequence data from this study are available in the GenBank database under accession number KJ801938 and the SRA database under accession number SRP041013.