Pathology and infectious agents of unionid mussels: A primer for pathologists in disease surveillance and investigation of mortality events.

Freshwater mussels are one of the most imperiled groups of organisms in the world, and more than 30 species have gone extinct in the last century. While habitat alteration and destruction have contributed to the declines, the role of disease in mortality events is unclear. In an effort to involve veterinary pathologists in disease surveillance and the investigation of freshwater mussel mortality events, we provide information on the conservation status of unionids, sample collection and processing techniques, and unique and confounding anatomical and physiological differences. We review the published accounts of pathology and infectious agents described in freshwater mussels including neoplasms, viruses, bacteria, fungi, fungal-like agents, ciliated protists, Aspidogastrea, Digenea, Nematoda, Acari, Diptera, and Odonata. Of the identified infectious agents, a single viral disease, Hyriopsis cumingii plague disease, that occurs only in cultured mussels is known to cause high mortality. Parasites including ciliates, trematodes, nematodes, mites, and insects may decrease host fitness, but are not known to cause mortality. Many of the published reports identify infectious agents at the light or ultrastructural microscopy level with no lesion or molecular characterization. Although metagenomic analyses provide sequence information for infectious agents, studies often fail to link the agents to tissue changes at the light or ultrastructural level or confirm their role in disease. Pathologists can bridge this gap between identification of infectious agents and confirmation of disease, participate in disease surveillance to ensure successful propagation programs necessary to restore decimated populations, and investigate mussel mortality events to document pathology and identify causality.

Pathobiology and first report of larval nematodes (Ascaridomorpha sp.) infecting freshwater mussels (Villosa nebulosa, Unionidae), including an inventory of nematode infections in freshwater and marine bivalves.

Little information is available on host-parasite relationships between bivalves and larval nematodes. Herein, we describe nematode larvae (likely stage 2) in the infraorder Ascaridomorpha infecting the foot, intestine, and mantle of a freshwater mussel (Alabama rainbow, Villosa nebulosa [Conrad, 1834]) and detail histopathological changes to infected tissues. A total of 43 live mussels from the South Fork of Terrapin Creek, Alabama, were collected between 2010 and 2014, with 14 sectioned for histopathology and 29 dissected. Of the 14 sectioned mussels, 5 appeared to be uninfected, and 7, 1, and 1 had histozoic infections observed in the foot and intestine, intestine only, and mantle edge and foot, respectively. Twenty-three of 29 (79%) of the mussels dissected were infected by live nematodes, and mean nematode abundance was 8.3 (CL = 5.23-13), with 2 mussels infected with >100 nematodes each. Thus, with a total of 32 of the 43 collected mussels observed with nematodes, overall infection prevalence was 74.4% (CL = 0.594-0.855). The 18S rDNA of this nematode was 99% similar to that of several ascaridids (species of Kathlaniidae Lane, 1914 and Quimperiidae Baylis, 1930) that mature in aquatic/semi-aquatic vertebrates; the recovered 18S phylogenetic tree indicated this nematode from V. nebulosa shares a recent common ancestor with Ichthyobronema hamulatum (Ascaridomorpha: Quimperiidae; GenBank Accession Number KY476351). Pathological changes to tissue associated with these infections comprised focal tissue damage, but a cellular response was not evident. The Alabama rainbow possibly represents an intermediate or paratenic host. Given these results, the nematode is likely not pathogenic under normal stream conditions; however, high intensity infections in the foot could inhibit pedal extension and retraction; which would have demonstrable health consequences to a freshwater mussel. Based on our review of the bivalve mollusc parasite literature, a collective biodiversity of 61 nematodes reportedly exhibit some degree of symbiosis (from commensal to parasitic) with 21 bivalves (28 nematode spp. from 17 marine bivalve spp.; 33 nematode spp. from 4 freshwater bivalve spp.); only four records exist of putatively parasitic nematodes from Unionida. The present study represents the first description of a nematode species that invades the tissues of a Unionidae species.

Pathological Changes Associated with Eggs and Larvae of Unionicola sp. (Acari: Unionicolidae) Infecting Strophitus connasaugaensis (Bivalvia: Unionidae) from Alabama Creeks.

We detail gross and histopathological changes associated with infection by the eggs, larvae, and cuticular remnants of Unionicola sp. in the mantle, gill, and visceral mass of 25 Alabama creekmussels, Strophitus connasaugaensis, collected during May 2010 through July 2012 from 2 Alabama streams. A multitude (estimated mean intensity >100) of mite eggs and larvae typically infected mantle, gill, and visceral mass integument. Pathology associated with eggs (prevalence = 0.57) and larvae (prevalence = 0.39) typically consisted of localized distension of the infection site; a host response to these infections was indeterminate. However, larval mites embedded in suprabranchial connective tissues were typically encapsulated (prevalence = 0.89). Mite remnants (prevalence = 0.5) occurred in mantle, gill, visceral mass integument, foot, heart, pericardial gland, intestinal lamina propria, and were typically encapsulated. We speculate that S. connasaugaensis clears some infections but is recolonized by autoinfection or horizontal dispersal of mites in the stream. Noteworthy is that high-intensity infections seemingly do not markedly impact the histological picture of mussel tissues, indicating that mites are relatively benign symbionts that are of little concern to mussels under normal environmental conditions.

Huffmanela cf. carcharhini (Nematoda: Trichosomoididae: Huffmanelinae) from skin of a sandbar shark, Carcharhinus plumbeus, in the Pacific Ocean.

Eggs of Huffmanela cf. carcharhini from the skin of an aquarium-held, juvenile sandbar shark, Carcharhinus plumbeus , from the Pacific Ocean were studied using light and scanning electron microscopy. Grossly, eggs imparted a scribble-like skin marking approximately 130 × 60 mm on the right side of the shark's snout adjacent to its eye and nostril. Fresh (unfixed) eggs were elliptical, 75-95 µm long (x¯  =  85 µm, SD  =  ±4.5; n  =  75), 48-63 µm wide (53 ± 3.4; 75), 8-10 µm in shell thickness (9 ± 1.3; 27), 45-68 µm in vitelline mass length (52 ± 6.9; 8); had a smooth shell surface and nonprotruding polar plugs 8-13 µm wide (10 ± 1.5; 73); lacked thin filaments, superficial envelope, and shell spines; sank in 35 ppt artificial seawater; and did not spontaneously hatch after 12 hr in 35 ppt artificial seawater. Formalin-fixed eggs measured 193 days postfixation were 75-95 µm long (84 ± 3.9; 150), 45-60 µm wide (50 ± 2.2; 150), 5-10 µm in shell thickness (8 ± 1.2; 87), 45-60 µm in vitelline mass length (51 ± 3.0; 92), and 30-40 µm in vitelline mass width (33 ± 2.0; 84), and had nonprotruding polar plugs that were 10-15 µm long (11 ± 1.4; 93) and 8-10 µm wide (9 ± 1.1; 108). Forcibly hatched first-stage larvae (unfixed) were filiform, 188-273 µm long (212 ± 25.5; 13), 8-13 µm wide (10 ± 1.2; 13), and had fine transverse striations. Eggs infected the epidermis only. Histology revealed intra-epithelial inflammation with eosinophilic granulocytes and hyperplasia, plus dermal lymphofollicular hyperplasia associated with the infection. The eggs of H. cf. carcharhini likely undergo considerable ex utero development before being sloughed (unhatched) from the host, along with epidermal cells.

Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge.

The adhesion dynamics of Flavobacterium columnare to fish tissues were evaluated in vivo by immersion challenge followed by bacterial plate count and confirmatory observations of gill-adhered bacterial cells using scanning electron microscopy. Adhesion of F. columnare genomovar I (ARS-1) and II (BGFS-27) strains to skin and gill of channel catfish Ictalurus punctactus and gill of zebrafish Danio rerio was compared. At 0.5 h post-challenge, both strains adhered to gill of channel catfish at comparable levels (10(6) colony forming units [CFU] g(-1)), but significant differences in adhesion were found later in the time course. Channel catfish was able to effectively reduce ARS-1 cells on gill, whereas BGFS-27 persisted in gill beyond the first 24 h post-challenge. No significant difference was found between both strains when adhered to skin, but adhered cell numbers were lower (10(3) CFU g(-1)) than those found in gill and were not detectable at 6 h post-challenge. Adhesion of BGFS-27 cells to gill of zebrafish also occurred at high numbers (> 10(6) CFU g(-1)), while only < 10(2) CFU g(-1) of ARS-1 cells were detected in this fish. The results of the present study show that particular strains of F. columnare exhibit different levels of specificity to their fish hosts and that adhesion to fish tissues is not sufficient to cause columnaris disease.

Distribution of Kroeyerina elongata (Kroyeriidae: Siphonostomatoida, Copepoda) in the olfactory sacs of the blue shark, Prionace glauca.

The infection pattern of Kroeyerina elongata (Kroyeriidae, Copepoda) in the olfactory sacs of the blue shark, Prionace glauca, was investigated using 4,722 copepods from 54 olfactory sacs. Copepod prevalence and mean intensity of infection per olfactory sac were 94.0 and 91.1%, respectively, and the most intensely infected olfactory sac and shark hosted 218 and 409 copepods, respectively. There were significant linear relationships between the number of female and total copepods per left olfactory sac and shark fork length as well as between the numbers of female, male, and total copepods per shark and mean olfactory sac width and cumulative olfactory sac width. Female copepods typically outnumbered males within olfactory sacs (mean intensity  =  65.7 and 26.3, respectively), and no statistical differences were detected between the numbers of copepods inhabiting the left and right olfactory sacs. Copepods were not evenly distributed within olfactory sacs. Typically, female copepods occupied olfactory chambers located centrally along the length of the olfactory sac, while males infected lateral olfactory chambers nearest the naris. The orientation of most copepods (84.6%) suggested positive rheotaxis relative to the path of water through the olfactory sac. Within olfactory chambers, most mature females (68.2%) infected the first third of the peripheral excurrent channel and the adjacent fringe of olfactory lamellae, while most males (91.7%) infected the olfactory lamellae, and the 4 larval females collected were attached within the lamellar field and grasped by males. Based on the observed infection patterns and the pattern of water flow throughout the olfactory sac, a hypothesis regarding the life cycle of K. elongata is advanced wherein infective copepodids are swept into the olfactory sac from the surrounding sea and initially colonize the olfactory lamellae. Copepodids feed and mature among the olfactory lamellae, and adult males search for mates and copulate with young females among the olfactory lamellae. Inseminated females move to the peripheral excurrent channels to mature and produce ovisacs. Hatching ovisacs release free-swimming nauplii into the excurrent water flow to be swept into the milieu, where they can molt into infective copepodids that may infect new hosts.