Fibropapillomatosis and Chelonid Alphaherpesvirus 5 Infection in Kemp's Ridley Sea Turtles (Lepidochelys kempii)

Abstract

Fibropapillomatosis (FP), a debilitating, infectious neoplastic disease, is rarely reported in endangered Kemp's ridley sea turtles (Lepidochelys kempii). With this study, we describe FP and the associated chelonid alphaherpesvirus 5 (ChHV5) in Kemp's ridley turtles encountered in the United States during 2006-2020. Analysis of 22 case reports of Kemp's ridley turtles with FP revealed that while the disease was mild in most cases, 54.5% were adult turtles, a reproductively valuable age class whose survival is a priority for population recovery. Of 51 blood samples from tumor-free turtles and 12 tumor samples from turtles with FP, 7.8% and 91.7%, respectively, tested positive for ChHV5 DNA via quantitative polymerase chain reaction (qPCR). Viral genome shotgun sequencing and phylogenetic analysis of six tumor samples show that ChHV5 sequences in Kemp's ridley turtles encountered in the Gulf of Mexico and northwestern Atlantic cluster with ChHV5 sequences identified in green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtles from Hawaii, the southwestern Atlantic Ocean, and the Caribbean. Results suggest an interspecific, spatiotemporal spread of FP among Kemp's ridley turtles in regions where the disease is enzootic. Although FP is currently uncommon in this species, it remains a health concern due to its uncertain pathogenesis and potential relationship with habitat degradation.

Keywords: ChHV5; disease ecology; enzootic; green turtle; marine turtle; molecular diagnostics; transmission; tumor; virus; whole genome sequencing.

Figure 1

Locations of 22 cases of Kemp’s ridley sea turtles (Lepidochelys kempii) observed with fibropapillomatosis in the coastal United States during 2006–2020.

Figure 2

Gross morphology of fibropapillomatosis tumors in afflicted Kemp’s ridley sea turtles (Lepidochelys kempii). Masses can develop anywhere on a turtle’s body, and tumor morphology varies from flat plaques, to verrucous, arborizing masses (a–e), to smooth polypoid masses (f–h).

Figure 3

Photomicrograph of a fibropapilloma in a Kemp’s ridley sea turtle (Lepidochelys kempii). Histopathological features are diagnostic for this disease and include well-differentiated neoplastic epidermal and stromal components. Short epidermal pegs (*) extend into the underlying dense collagenous stroma, which includes loosely arranged streams and individually dispersed fibroblasts and supportive vessels. A crust of degenerate heterophils with accumulations of bacteria (arrow) is formed on the surface at the top of the image. Hematoxylin and eosin. Scale bar = 200 µm.

Figure 4

Mitochondrial DNA analysis of tissue samples collected from a Kemp’s ridley sea turtle (Lepidochelys kempii) that stranded in Massachusetts, USA (Lk15), alongside other published Kemp’s ridley mitochondrial D-loop control regions [44,45]. Lk15 (shaded red) grouped within the haplotype 1 sub-group. Haplotype defining sequences are shaded green. Kemp’s ridley turtles along the east coast USA are 80% likely to be haplotypes 1 and 2 (predominantly haplotype 1), and 79% of haplotypes 1 and 2 originate from rookeries along the Texas coast. Combined with ChHV5 phylogenetic data (Section 3.4, Figures 6 and 7), these results suggest that Lk15 likely originated from Texas (based on currently available data) and harbors a Florida variant of ChHV5.

Figure 5

Coverage of chelonid alphaherpesvirus 5 (ChHV5) hypothetical protein-32 (HP32) gene in each of the Kemp’s ridley sea turtle (Lepidochelys kempii) DNA-seq samples in transcripts per kilobase million (TPM). HP32-aligning reads in these samples ranged from 3 to 31,209 reads.

Figure 6

Chelonid alphaherpesvirus 5 (ChHV5) viral reads across sample types based on results of whole-genome sequencing (DNAseq). Comparing tumor ChHV5 loads (reads per ten million) shows significantly lower viral loads in samples from Kemp’s ridley sea turtles (Lepidochelys kempii) versus those typically observed in green sea turtles (Chelonia mydas; green turtle ChHV5 data adapted from Farrell et al. [14]). Abbreviations: ChHV5, chelonid alphaherpesvirus 5; Cm, C. mydas; FP, fibropapillomatosis; Lk, L. kempii; WGS, whole genome sequencing.

Figure 7

Phylogenetic analysis of partial chelonid alphaherpesvirus 5 (ChHV5) UL30 consensus genes obtained from two Kemp’s ridley sea turtle (Lepidochelys kempii) tumor samples along with the reference UL30 gene (GenBank accession: HQ878327.2) and other available ChHV5 UL30 genes obtained from the NCBI GenBank database, including Florida variants. Generated sequences denote turtles’ geographic locations. Generated sequences match length and position of known variants (483 bp) for direct phylogenetic comparison. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All samples, where appropriate, have accession numbers. The two Kemp’s ridley ChHV5 gene sequences analyzed in the current study are shaded in red. Abbreviations: Cc, Caretta caretta; Cm, Chelonia mydas; Lk, L. kempii; Lo, Lepidochelys olivacea; At, Atlantic; Pa, Pacific; GG, Gulf of Guinea; PR, Puerto Rico; BR, Brazil; HA, Hawaii; AUS, Australia; CAR, Caribbean; FL, Florida; CA, California; MEX, Mexico.

Figure 8

Phylogenetic analysis of chelonid alphaherpesvirus 5 (ChHV5) hypothetical protein-32 (HP32) consensus gene obtained from six Kemp’s ridley sea turtle (Lepidochelys kempii) tumor samples, along with the reference HP32 gene from the NCBI GenBank database (GenBank accession: HQ878327.2) sequenced from a green turtle (Chelonia mydas) in Hawaii. Generated sequences denote the geographic location of each turtle. Sequences match the length and position of the reference gene (1685 bp) for direct phylogenetic comparison. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Samples generated as part of the current study are shaded red. Abbreviations: Cm, C. mydas; Lk, L. kempii; HA, Hawaii.