Five Years of Partula Snail Pre-Export Health Screening

FIVE YEARS OF PARTULA SNAIL PRE-EXPORT HEALTH
SCREENING
Authors: Flach, Edmund J., Stidworthy, Mark F., Aberdeen, Sam,
Clarke, David, Davidson, Hannah, et al.
Source: Journal of Zoo and Wildlife Medicine, 55(1) : 31-41
Published By: American Association of Zoo Veterinarians
URL: https://doi.org/10.1638/2023-0077
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DOI: 10.1638/2023-0077 Journal of Zoo and Wildlife Medicine 55(1): 31–41, 2024
Copyright 2024 by American Association of Zoo Veterinarians
FIVE YEARS OF PARTULA SNAIL PRE-EXPORT HEALTH

SCREENING
Edmund J. Flach, MA, MSc, VetMB, Dipl Zoo Med (Mammals), DECZM (ZHM), MRCVS,
Mark F. Stidworthy, MA, VetMB, PhD FRC Path, Sam Aberdeen, MSc, David Clarke, FRES,
Hannah Davidson, BSc, MSc (Wild Animal Biology), Helen Donald, BA, MBA, BVetMed, MSc
(Wild Animal Health), MRCVS, Grace Goodey, BSc, RVN, Alysa Hulbert, BA, Shinto John,
MLT, Nic Masters, MA, VetMB, DECZM (ZHM), MRCVS, Shaheed Macgregor, HTec, MSc,
CSci, Paul Pearce-Kelly, Simon Spiro, MA, VetMB, MVetMed, DPhil, DACVP, FRC Path,
MRCVS, and Amanda Guthrie, DVM, DACZM, DECZM (ZHM), MRCVS
Abstract: Between 2015 and 2019, a health screening was carried out annually on captive-bred Partula snails
prior to export for reintroduction as part of an international effort to repopulate areas of French Polynesia, where
the snails were extinct or critically endangered. In total, 129 separate tank populations of 12 different species were
screened at ZSL London Zoo. Wet mounts and smears stained with modified Ziehl–Neelsen (MZN) of 535 fecal
samples were examined, and 45% contained flagellated protozoa, and 35.5% had MZN-positive oocysts, measuring
3–5 lm in diameter. Smaller (2 lm) presumptive spores, MZN-positive bacilli, ciliated protozoa and nematodes
were recorded less frequently. Fecal bacterial culture yielded mixed species, with a clear predominance of Myroides
species (88.9% of samples). The MZN-positive oocysts (3–5 lm) were present in 6.5% of impression smears from
the apices of 432 snails examined postmortem, plus acid-fast bacilli in a few cases, but no 2 lm spores. Mixed bac-
teria were cultured from coelomic swabs, with Myroides species again the most common (63.5%). Histologic exami-
nation was carried out on 292 snails. Autolysis affected almost 90% of those found dead but only 3.4% of
euthanized snails. Histology commonly identified microsporidial sporocysts in the digestive gland and midgut epi-
thelium of all but two species. Intracellular, extracytoplasmic Cryptosporidium-like organisms were also common in
the midgut but were only observed when snails were fixed in 10% formalin (2017–2019), not ethanol. There were no
clear pathologic changes associated with either organism. Pigmented hemocytic nodules were commonly observed,
most frequently in the foot process; these were either age related or evidence of prior chronic inflammatory reaction
and of low clinical significance. With no evidence of poor health and no significant organisms found, a total of
4,978 individuals representing 12 species were exported for reintroduction.
INTRODUCTION many species (estimated to have been 117 originally)

inhabiting different volcanic islands in the Pacific
Snails of the genus Partula belong to the family
ocean.9 However, biologists in the 1970s noticed a
Partulidae within the order Stylommatophora.
catastrophic decline in Partula species numbers,
They were discovered on Captain Cook’s first
and it soon became apparent that a genus-wide
expedition to the Pacific in 1769, and since the early
extinction was in progress.15 The reason for this
19th century have fascinated evolutionary biologists
rapid decline was predation by the Florida rosy
due to the adaptive radiation shown between the
wolfsnail (Euglandina rosea), introduced with gov-
ernment support as a biologic control agent against
the giant African land snail (Achatina fulica), which
From the Wildlife Health and Animal Departments, and the had itself been introduced as a potential food
Institute of Zoology, ZSL London Zoo, Zoological Society of source for humans.1 The wolfsnails preferentially
London, Regents Park, London NW1 4RY, United Kingdom predated the smaller native Partula species, resulting
(Flach [retired], Aberdeen, Clarke, Davidson, Donald, Goodey,
in the extinction (total or in the wild) of 54 species,3
Hulbert, John, Masters, Macgregor, Pearce-Kelly, Spiro, and
most disappearing in just a decade, and leading it
Guthrie); and International Zoo Veterinary Group Pathology,
Station House, Parkwood Street, Keighley, BD21 4NQ, United to be described as the most rapid extinction event
Kingdom (Stidworthy). Present addresses (Flach): Haven, 17 known.9
Grovebury Road, Leighton Buzzard, LU7 4SQ, United Kingdom; In the 1980s, a crisis rescue project was initiated,
(Donald): Chief Scientist’s Directorate, Natural England, Nobel and 25 Partula species were collected and brought
House, 17 Smith Square, London SW1P 3JR, United Kingdom; into captivity in Europe and North America, where
and (Masters): Wildlife Health, Toronto Zoo, 361A Old Finch they successfully survived and bred. Subsequently,
Avenue, Toronto, Ontario, M1B 5K7, Canada. Correspondence the Partula Global Species Management Pro-
should be directed to Dr. Flach (edmund.fl[email protected]). gramme was created in 1986 to coordinate captive
Note: This article contains supplemental material found in breeding and plan future conservation efforts.1
the online version only. Reintroduction was first attempted in 1994 when
31

32 JOURNAL OF ZOO AND WILDLIFE MEDICINE
three species: Partula suturalis vexillum, Partula tae- snails in each varied markedly (range: 12 [all adults]
niata nucleola, and Partula tohiveana were released to 205 [125 newborns, 30 juveniles, and 50 adults
into a protected reserve in Moorea, French Poly- but no subadults]), and in one case (the most popu-
nesia. Unfortunately, the reserve boundary failed lated), some individuals were subsequently moved
to exclude Euglandina, so the remaining Partula to a new tank. The following year, populations of
had to be recaptured. However, the presence of Partula species from four United Kingdom zoos
juvenile snails proved that reintroduction had and one French zoo (similarly varied in numbers
been partially successful.9 of snails per tank) were again quarantined and
Before further reintroductions were attempted, screened at LZ. However, instead of separating
a disease risk analysis (DRA) was performed by those LZ tank populations destined for reintro-
staff of the Zoological Society of London (ZSL) duction and moving them to the quarantine room,
according to IUCN guidelines.6,13,19 Relatively few the whole zoo collection was screened. Two
reports of pathologic investigations of Partula snails additional zoos successfully carried out screen-
have been published,2,5,11 but the DRA reviewed ing in 2016, and others did so in the three subse-
these and other publications related to snail species quent years. Therefore, from 2017 to 2019, LZ
to decide whether translocations might risk transfer- only screened its own tank populations. Because
ring any pathogens they carry and whether sickness the entire LZ collection was tested from 2016 to
and death in these species and native animals could 2019, some tank populations that were maintained
result in failure of the program. Captive breeding for over a year were checked twice or sometimes
in zoos makes it possible to rear large numbers of more often; this is indicated in the results when
individuals for maintenance of the species and the relevant.
return to natural habitat once any factors respon-
sible for decline have been removed or at least Fecal screening
reduced. However, even with good security, there Three fresh fecal samples were obtained from
is a risk of crossover of pathogens from other cap- each tank. Upon receipt, samples were refrigerated
tive species. The DRA identified microsporidia and until examination but were always examined on the
suspected cryptosporidia as potential pathogens day of collection to ensure freshness. All three sam-
but not thought to be critically significant.5,8 How- ples were examined for the presence of parasites
ever, health screening for infectious agents was rec- due to the previous experience of the intermittent
ommended prior to translocation. appearance of various parasites (LZ, unpubl. data).
Following implementation of the DRA recom- In 2018, some tanks (those not destined for export)
mendations, Partula snails were reintroduced to var- were only sampled once, whereas occasionally
ious sites in French Polynesia in 2015 and in each fourth, and even fifth, samples from tanks destined
of the four subsequent years. This study describes for export were collected if large numbers of para-
the results of health screenings carried out at ZSL sites had been detected. In 2015, a bacteriologic cul-
London Zoo (LZ) and formed part of the evidence ture was also attempted from all three samples, but
that enabled the Official Veterinarian (OV) to sign in view of the abundance of mixed bacteria, from
the Animal and Plant Health Agency Export Health 2016, only one sample from each tank was cultured.
Certificate for export to French Polynesia and, ulti-
mately, release to the wild. Postmortem examinations
MATERIALS AND METHODS Dead snails were examined under three circum-
stances: 1) individuals found dead with no gross
Partula populations
signs of autolysis and on a day when they could be
In 2015, individuals of three Partula species submitted for immediate examination; 2) individ-
were sent to LZ from four zoos in the United King- uals found dead, but not fresh, or on a day when
dom and two zoos in the United States and housed immediate examination was not possible; there-
in a biosecure quarantine room, with additional fore, they were fixed in 70% ethanol and then sub-
individuals of two of these species from the zoo’s mitted; and 3) healthy adult individuals randomly
own collection. All were destined for reintroduction selected by keepers for euthanasia (carried out by
that year and were health screened together prior to exposure to an overdose of isoflurane for a mini-
export. Partula snails are typically maintained and mum of 1 h; the standard method used at ZSL and
bred in glass tanks;1 hence, screening was based on found to be rapid and effective) and submitted for
testing each tank population. Imported and resident immediate, fresh examination. The aim was to
tank populations were kept separate. Numbers of examine approximately 10% of the population

FLACH ET AL—PARTULA 5-YR HEALTH SCREENING REVIEW 33
of adults in each tank, with at least some snails (microsporidian or protozoal or both). Size (greatest
examined fresh after euthanasia from tanks with diameter) was measured with an eyepiece graticule
sufficient numbers. Exceptions were made if the that had been calibrated from a stage micrometer
number of adults in a tank population was too low slide and was then reported as having diameters of
to risk removing any individuals (generally assessed approximately 2 lm (suspect microsporidial spores)
as less than 10, but dependent on additional factors, or in the range of 3 to 5 lm (presumptive crypto-
such as the number of adults of the same species in sporidial oocysts).
other tanks).
Snails presented fixed in ethanol were not exam- Bacteriologic testing
ined grossly, but a proportion were submitted for
Fecal samples and coelomic swabs were inocu-
histology. All others were weighed and measured
lated onto 5% horse blood agar plates, incubated at
as previously described.16 If the snail did not easily
25°C for 48 h. Single bacterial colonies of predomi-
slide from the shell when traction was applied,
nant types were subcultured onto further plates.
heavy-duty scissors were used to open the shell to
Resulting pure cultures were then identified by
ensure minimal crush and stretching artifacts. The
standard methods: colony morphology; appearance
extracted body was weighed and examined exter- and Gram-staining characteristics; and biochemical
nally. The tip of the apex was removed, and impres- reactions (API commercial test kits and online
sion smears of the cut surface made on a microscope software, bioMérieux UK Limited, Basingstoke,
slide for parasitologic examination (see the follow- Hampshire RG22 6HY, United Kingdom), follow-
ing). The body was then opened with a sterile scalpel ing an initial oxidase test. A 20NE API test identi-
blade, a swab taken for bacteriologic culture, and the fied oxidase-positive organisms, a 20E test detected
internal tissues examined grossly. A small piece of negative bacteria, and a 10S test showed any atypi-
foot tissue was removed and frozen as a source of cal reactions.
DNA, and finally, the remaining carcass, plus the
amputated apex, were fixed for histologic exami- Other evidence
nation. In 2015 and 2016, this was achieved by
immersion in 70% ethanol, but from 2017, 10% buff- Although the majority of the screening was for
ered formalin was used, as this was demonstrated the presence of potential pathogens and abnormal
to yield better quality fixation. Fixed snails were histologic findings, the OV also considered the
bisected longitudinally and processed into forma- keepers’ assessments of the tank populations, in
lin-fixed and paraffin-embedded sections by stan- particular, evidence of successful breeding, normal
dard techniques. Slides were stained with H&E, feeding and activity, and the absence of excessive
modified Ziehl-Neelsen (MZN), Gram–Twort, and mortality (above that expected with normal aging).
periodic acid–Schiff (PAS) using standard labora-
RESULTS
tory protocols,20 plus the Luna stain specifically
for microsporidia,17 and were reported by an ana- Over 5 yr, 12 species of Partula snails, origi-
tomic veterinary pathologist (MFS). nally derived from nine contributing zoos but
managed as a single contributing feeder popula-
Parasitologic testing tion at LZ, were screened (Table 1). These were
Feces underwent direct microscopy of a wet prep- maintained as 129 different tank populations,
aration and microscopy of a dry smear stained with and some of which (at LZ) were screened on
MZN stain. Apex smears were likewise examined more than one occasion.
microscopically after staining with MZN. Wet prep-
Fecal sampling
arations were prepared by mixing a small amount of
feces (a bacteriologic Nichrome wire loopful) in two Of the 535 fecal samples examined (Table 2), 45%
drops of sterile physiologic saline and examined contained flagellated protozoa, 35.5% had MZN
microscopically for the presence of any parasites acid-fast oocysts with a diameter between 3 and
or ova, with particular emphasis on helminths (often, 5 lm, 25.0% had ciliated protozoa, while helminths
but not always, identified further as adult or larval (adults or larvae or both), smaller (2 lm) MZN
nematodes), flagellated protozoa, and ciliated pro- acid-fast-suspect spores, and MZN acid-fast
tozoa. The MZN-stained smears were prepared bacilli were noted less frequently (11.6%, 5.2%, and
and examined microscopically under oil immer- 11.8%, respectively). Interestingly, although hel-
sion for the presence of acid-fast bacilli (possible minths and 3 to 5 lm oocysts were observed each
Mycobacterium species) and spores and oocysts year and flagellated protozoa in four of the 5 yr, the

Table 1. Partula species health screened from 2015 to 2019.
Tank populations screened:

first time (screened before)c
Species Original range IUCN classificationa Zoosb 2015 2016 2017 2018 2019
Partula affinis Tahiti Critically endangered 4 10 — — — —

Partula garrettiid Raiatea Extinct in the wild 3 — 3 3 (1) 4 (2) 4 (2)
Partula hebe bella Raiatea Extinct in the wild 2 — 5 5 8 (4) 5 (3)
Partula hyalina Multiisland, endemic Vulnerable 2 3 1 1 (1) 1 (1) —
to Tahiti
Partula mirabilis Moorea Extinct in the wild 1 — 1 — 4 (1) 2 (1)
Partula mooreana Moorea Extinct in the wild 4 — 12 8 (5) 3 (3) —
Partula navigatoriad Raiatea Extinct in the wild 3 — 6 3 (2) 4 (3) 5 (4)
Partula nodosa Tahiti Extinct in the wild 2 4 — — — —
Partula rosea Huahine Extinct in the wild 1 — — — — 2
Partula suturalis vexillum Moorea Extinct in the wild 2 — 7 3 (2) 3 (3) 2 (2)
Partula taeniata nucleola Moorea Subspecies extinct in the wild 3 — 4 4 (2) 5 (3) 5 (2)
Partula tohiveana Moorea Extinct in the wild 3 — 13 13 (7) 10 (5) 9 (3)
a
Reference 3.
b
Number of zoos contributing snails for screening.
c
Relating to tank populations at Zoological Society of London London Zoo screened more than once.
d
Species that were previously misidentified: P. garrettii as Partula tristis; P. navigatoria as Partula dentifera.
other organisms were absent in two or three of the each year but in low numbers. They were most
years. Also, flagellated protozoa were extremely commonly detected in Partula mooreana (12.0% of
abundant in 2017 and 2019, reasonably so in 2018, samples), followed by Partula navigatoria (11.6%),
and very rare in 2015. The results for the different Partula hebe bella (10.5%), and Partula hyalina
Partula species were also very variable (Table 3). (10.0%), with low numbers in Partula suturalis vexil-
Mixed bacteria were grown from all 199 fecal lum, Partula taeniata nucleola, and Partula tohiveana,
samples cultured. Myroides species were present, and none in the other species. Acid-fast bacilli
and often predominant, in almost 90% of these were only observed in these smears in 2017 (four
(Table 4). The second most common identification cases) and 2019 (one case). Bacterial culture of the
was Aeromonas hydrophila/caviae (species indistin- coelomic swabs invariably yielded a mixture of
guishable using current methods), but these bacte- species, with Myroides species again being predom-
ria were only present in 15.6% of samples and inant (63.5% of all cultures), especially in snails
found in only 3 yr. Another 15 bacterial types that had died (122 of 151 [80.8%] compared with 69
were identified to genus or species level but were of 150 [46.0%] euthanized snails). However, in 2015,
only found infrequently. no Myroides species were identified. Aeromonas
hydrophila/caviae were the second most common
Postmortem examinations but only cultured from 4.6% of swabs. Twenty-six
other bacterial genera and species were identified
Over 5 yr, 479 snails were examined postmortem,
but none consistently.
with the majority having died, but with an increas-
ing percentage euthanized so that they could be
Histology findings
examined fresh (Table 5). No 2 lm diameter spores
were observed in the MZN-stained apex impression In total, 292 snail carcasses (61.0% of postmor-
smears, whereas 3 to 5 lm oocysts were detected in tem examinations) were examined histologically
Table 2. Fecal parasite screening 2015–2019 by year.
Modified Ziehl–Neelsen acid positive
Helminths Flagellates Ciliates 2 mm 3 to 5 mm

Year Samples examined positive (%) positive (%) positive (%) spores (%) oocysts (%) Bacilli (%)
2015 54 2 (3.7) 3 (5.6) 0 (0.0) 0 (0.0) 7 (13.0) 1 (1.9)

2016 168 23 (13.7) 0 (0.0) 0 (0.0) 0 (0.0) 64 (38.1) 0 (0.0)
2017 120 17 (14.2) 106 (88.3) 46 (38.3) 0 (0.0) 21 (17.5) 62 (51.7)
2018 93 11 (11.8) 42 (45.2) 51 (54.8) 13 (14.0) 63 (67.7) 0 (0.0)
2019 100 9 (9.0) 90 (90.0) 37 (37.0) 15 (15.0) 35 (35.0) 0 (0.0)
Total 535 62 (11.6) 241 (45.0) 134 (25.0) 28 (5.2) 190 (35.5) 63 (11.8)

Table 3. Fecal parasite screening 2015–2019 by species.
Modified Ziehl–Neelsen
acid positive
Tank populations
First time Samples Helminths Flagellates Ciliates 2 mm 3 to 5 mm
Species (retested)a examined positive positive positive spores oocysts Bacilli
Partula affinis 10 (0) 30 1 1 0 0 2 0

Partula garrettii 14 (5) 33 3 15 4 2 7 4
Partula hebe bella 23 (7) 57 1 25 10 2 19 8
Partula hyalina 6 (2) 18 2 5 2 0 4 1
Partula mirabilis 7 (2) 16 0 10 8 1 8 0
Partula mooreana 23 (8) 71 10 30 18 2 27 15
Partula navigatoria 18 (9) 48 4 23 13 2 25 6
Partula nodosa 4 (0) 15 0 1 0 0 5 1
Partula rosea 2 (0) 6 0 4 1 1 1 0
Partula suturalis vexillum 15 (7) 49 6 34 25 1 17 6
Partula taeniata nucleola 18 (7) 54 6 35 25 6 23 7
Partula tohiveana 45 (15) 136 32 76 45 11 51 15
a
Relating to tank populations at Zoological Society of London London Zoo screened more than once.
(Supplemental Table 1). Moderate to severe autol- from 70% ethanol to 10% buffered formalin. They
ysis was a regular feature of Partula histologic sec- were then reported each year and in eight of the
tions (86 cases; 29.5%) and was much more 10 species examined. The percentage of cases posi-
common in snails that had died (79 of 88; 89.8%) tive for microsporidial sporocysts also increased in
than those euthanized (7 of 204; 3.4%). Hence, it the years following this change (Table 6), although
also declined over 5 yr, as the proportion of eutha- this was not consistent across species (Table 7).
nized snails examined increased (2015, 23 of 49; The abundance of microsporidial sporocysts in
2016, 44 of 114; 2017, 14 of 53; 2018, 2 of 43; and examined sections was most commonly described
2019, 3 of 33). In cases that were relatively unaf- as occasional or rare (68.6%), followed by moderate
fected by autolysis, the tissues routinely identified numbers (29.1%) and large numbers (2.3%), with
were radula, foot process, kidney, ganglion, ovotes- the majority present in the digestive gland acinar
tis, reproductive ducts, digestive gland, salivary epithelial cells (digestive cells; 71.0%). Others were
gland, shell gland, lung and pulmonary cavity, heart observed in mucosal epithelial cells of the intestine
and gastrointestinal tract, but in addition, the albu- (39.5%). Sometimes, they were present in both sites.
men gland, pedal mucus gland, and eyestalk were The Cryptosporidium-like organisms were most com-
occasionally seen.7,22 monly present in large numbers (65.2%), followed
Microsporidial sporocysts were detected in all by occasional (19.6%) and moderate numbers
5 yr (Table 6 and Fig. 1) and in all except two spe- (15.2%), typically affecting focally extensive or
cies (Partula nodosa and Partula garrettii; Table 7). In extensive populations of epithelial cells of the
contrast, apical, extracytoplasmic, intracellular digestive gland duct (73.9%) or midgut intestine
protozoal stages consistent with developing Crypto- (65.2%), and commonly in both. Occasionally, free
sporidium oocysts were only detected in 2017 and presumptive oocysts were reported in the digestive
subsequent years, following the change of fixative gland duct lumen.
Table 4. Fecal bacterial screening from 2015 to 2019.
Samples Myroides sp. Aeromonas hydrophila/

Year cultured positive (%) caviae positive (%) Other bacterial species identified
2015 51 47 (92.2) 15 (29.4) Acinetobacter sp., Bacillus sp., Brevundimonas vesicularis,

Pantoea sp., Sphingobacterium multivorum, Weeksella virosa
and Empedobacter brevis
2016 53 42 (79.2) 11 (20.8) Acinetobacter sp., Bacillus sp., Burkholderia cepacia,
Citrobacter freundii, Enterobacter cloacae, Leclercia
adecarboxylata, Pseudomonas putida
2017 40 33 (82.5) 5 (12.5) Bacillus sp., Proteus mirabilis, Serratia odorifera
2018 22 22 (100.0) 0 (0.0) Weeksella virosa and Empedobacter brevis
2019 33 33 (100.0) 0 (0.0) Chryseobacterium indologenes, Serratia odorifera, Streptococcus
mutans
Total 199 177 (88.9) 31 (15.6)

Pseudostratification or presumptive hyperplasia
Enterobacter species (Entb), Enterobacter cloacae (Entbc), Enterococcus

species (Entc), Escherichia coli (Ec), Escherichia vulneris (Ev), Klebsiella
Acinetobacter species (Ac), Bacillus species (Ba), Citrobacter freundii (Cf),
Corynebacterium species, Ec, Entc, Pseudomonas fluorescens, Sphingobac-
Cf, Chryseobacterium indologenes, Entc, Ev, Klepsiella p.ozaenae, Mi, Pa,

(La), Micrococcus species (Mi), Ochrobactrum anthropi (Oa), Pantoea
of the epithelium of the digestive gland duct or mid-
oxytoca (Ko), Klebsiella p.pneumoniae (Kp), Leclercia adecarboxylata
Ac, Aeromonas species, Ba, Cf, Citrobacter koseri/analonaticus (Ck),

Mannheimia haemolytica/Ibersteinia trehalosi, Proteus mirabilis (Pm),
gut intestine was often associated with the Crypto-
sporidium-like organisms (36 of 46 positive cases;
78.3%) but was never present in their absence. The
clinical significance of this change is uncertain in
otherwise apparently healthy snails randomly
Entb, Entc, Ko, Kp, Pa, Sphingomonas species

selected for sampling. No other pathologic reaction,
such as inflammation or erosion, was observed.
Weeksella virosa/Empedobacter brevis (Wv)
Nematodes and gram-positive and gram-negative

Other
bacteria of a range of morphologies were commonly

visible in the lumen of the intestinal tract and fre-
quently were present in body tissues of snails that
had died and were showing evidence of autolysis.
terium paucimobilis, Wv
Occasionally, nematodes were observed in the tis-

Streptococcus mutans
Bacterial culture
sues of fresh snails (Fig. 2), but in only one case

species (Pa), Pm
was a histologic reaction present: a Partula tohi-

veana that was euthanized in 2017 had larval nema-
todes in the foot process surrounded by dense
macrophage-like hemocytes.
The main histologic change noted was infiltration
of tissues with macrophage-like hemocytes (Fig. 2).
4 (14.8)
3 (10.3)
A. hydroa
14 (4.7)
2 (1.4)
3 (5.7)
2 (4.5)
These often contained yellow and brown pigment,

(%)
but they were also nonpigmented, or there was a

Table 5. Postmortem examination parasitology and bacteriology results, 2015–19 by year.
mix of both. Distribution was described as scattered

Myroides sp.
191 (63.5)
113 (76.4)
31 (58.5)
15 (51.7)
32 (72.7)
or multifocal, and in some cases, focal accumula-

0 (0.0)
(%)
tions were considered equivalent to granulomas.

The common sites affected were the foot process
and visceral interstitial connective tissue, with the
most frequently referenced organs including the
Tissue swabs
cultured
digestive gland, intestine, and pulmonary cavity,

27
148
53
29
44
301
and occasionally, the kidney and ovotestis. In a

few cases (one in 2017 and three in 2019), PAS-
positive debris was observed within these hemo-
cytes, with no evidence of any etiologic agent, sug-
oocysts (%)
6 (10.9)
5 (11.1)
3–5 mm
28 (6.5)
1 (3.2)
8 (4.1)
8 (8.3)
gesting that this may be the result of age-related

“wear and tear” pigments in mature adults. In
some cases, particularly when discrete granuloma-
MZN acid-positivity
like lesions were found, such as within viscera or in

2 mm spores
the foot process, lesions were suspected to be the

0 (0.0)
0 (0.0)
0 (0.0)
0 (0.0)
0 (0.0)
0 (0.0)
(%)
result of prior local inflammatory responses. How-

ever, the panel of special stains for potential etio-
logic agents was reviewed routinely, with negative
Apex samples
results in all cases.

examined
31
205
96
55
45
432
The histologic appearance of the digestive gland

varied with life stage and digestive activity, as
Aeromonas hydrophila/caviae.
described for Helix.7 Snails whose connective

tissues contained numerous large, clear, and vacuo-
Dead Euthanized
examinations
lated fat cells (which contain glycogen and lipid

Postmortem
26
65
38
42
33
204
droplets) and had abundant gastrointestinal ingesta

typically had narrow digestive gland acinar lumina,
plump digestive cells with rounded brush-bordered
31
147
72
13
12
275
apical surface, and abundant mixed cytoplas-

mic granularity (endolysosomes, heterolyso-
Total
Year
2015
2016
2017
2018
2019
somes, and residual bodies). In contrast, snails

a

Table 6. Microsporidial and Cryptosporidium-like infections reported histologically by year.
No. of No. of histology No. of microsporidia No. of Cryptosporidium-like

Yeara species examinations positive (%) positive (%)
2015 3 49 9 (18.4) 0 (0.0)

2016 9 114 33 (28.9) 0 (0.0)
2017 8 53 17 (32.1) 20 (37.7)
2018 9 43 18 (41.9) 10 (23.3)
2019 8 33 12 (36.4) 16 (48.5)
Total 2015–2016 11 163 42 (25.8) 0 (0.0)
Total 2017–2019 10 129 47 (36.4) 46 (35.7)
a
Partula snails fixed in 70% ethanol in 2015 and 2016 but fixed in 10% buffered formalin 2017–2019.
without conspicuous vacuolated fat cell popula- between the presence or absence of microsporidia or
tions, absent or scant mucoid intestinal content, Cryptosporidium-like organisms and these changes.
or increased numbers of yellow- and brown-pig-
mented connective tissue hemocytes frequently Comparisons between parasitologic findings from
had dilated digestive gland acini, small flattened different tests
digestive cells containing brown granules and vac- At postmortem examination, microsporidial spo-
uoles, and more conspicuous excretory cells. The rocysts were only detected histologically; no
latter appearance was interpreted as digestive gland microsporidial spores were observed in MZN-
inactivity or atrophy. There was no correlation stained apex smears. However, Cryptosporidium-
Figure 1. Microsporidiosis and cryptosporidiosis in Partula snails. (a) Digestive gland, Partula taeniata nucle-
ola, captive-bred specimen. H&E, original magnification 6003. Arrows, intracytoplasmic microsporidian
spores. Inset: Luna stain, 1,000 3 oil immersion. (b) Intestine, Partula suturalis vexillum, captive-bred speci-
men. H&E, original magnification 6003. Arrows, intracytoplasmic microsporidian spores. (c) Digestive
gland, Partula nodosa, wild-caught museum specimen, long-term formalin and ethanol storage. H&E, origi-
nal magnification 6003. Arrow, intracytoplasmic microsporidian spores. Inset: Luna stain, 1,000 3 oil
immersion. (d) Digestive gland duct epithelium, Partula taeniata nucleola, captive-bred specimen. H&E, orig-
inal magnification 6003. Arrowhead, cryptosporidial organism budding from apical epithelial surface. (e)
Digestive gland duct epithelium, Partula nodosa, wild-caught museum specimen, long-term formalin and
ethanol storage. H&E, original magnification 6003. Arrowhead, cryptosporidial organism budding from
apical epithelial surface. Note that the variations in tinctorial character and tissue preservation in the H&E
and Luna stains between Fig. 1a, b, d and Fig. 1c, e (and insets) are due to long-term storage of the wild-
caught specimens in formalin, with subsequent transfer to ethanol versus immediate fixation in formalin for
the captive-bred specimens. Staining procedures were identical.

Table 7. Microsporidial and Cryptosporidium-like infections reported histologically by species.
2015–2016 2017–2019
No. of No. of No. of No. of

No. of histology microsporidia histology microsporidia Cryptosporidium-like
Species examinations positive (%) examinations positive (%) positive (%)
Partula affinis 30 7 (23.3) 0 — —

Partula garrettii 4 0 (0.0) 3 0 (0.0) 2 (66.7)
Partula hebe bella 13 7 (53.8) 9 7 (77.8) 4 (44.4)
Partula hyalina 11 6 (54.5) 5 4 (80.0) 0 (0.0)
Partula mirabilis 0 — 2 2 (100.0) 0 (0.0)
Partula mooreana 27 5 (18.5) 16 1 (6.2) 9 (56.2)
Partula navigatoria 9 2 (22.2) 10 0 (0.0) 7 (70.0)
Partula nodosa 14 0 (0.0) 0 — —
Partula rosea 0 — 3 2 (66.7) 3 (100.0)
Partula suturalis vexillum 17 4 (23.5) 11 7 (63.6) 2 (18.2)
Partula taeniata nucleola 8 5 (62.5) 24 24 (100.0) 16 (66.7)
Partula tohiveana 30 6 (20.0) 46 0 (0.0) 3 (6.5)
Total 163 42 (25.8) 129 47 (36.4) 46 (35.7)
like infections were detected both histologically (albeit little histologic evidence was obtained from
and by the presence of presumptive oocysts in snails that were found dead), and the breeding,
MZN-stained apex smears. From 2017 to 2019, behavior, and mortality of exported tank populations
129 snail carcasses were examined by both meth- were assessed as within normal limits. Consequently,
ods, and although 46 were positive histologically, over this period, a total of 4,978 individuals of 12
only one of these individuals had detectable species were transported to French Polynesia for
oocysts (3–5 lm) in its apex smear. Four addi-
reintroduction.
tional snails had these oocysts in the apices, but
Although microsporidia are well recognized in
with no histologic evidence of infection.
invertebrates, a presumed Steinhausia species was
Possible microsporidial spores were occasion-
implicated in the deaths of the last-known speci-
ally detected in fecal samples stained with MZN
mens of Partula turgida (now reidentified as Partula
(2 lm diameter), but there was poor association
clarkei).4,21 It was present in the digestive gland
between the presence in feces and the presence of
of all individuals examined in association with
sporocysts by postmortem histology. For the 91
reported cytoplasmic vacuolation in the digestive
tank populations with both sets of results, only
cells. However, this was no proof of pathogenic-
54.9% agreed (nine dual positives and 41 dual
ity,18 and during the 5 yr of screening reported in
negatives), and 45.1% differed (eight just fecal
this study, microsporidial sporocysts were com-
positive, 33 just histology). For the Cryptosporidium-
monly seen in histologic sections, but never in
like oocysts, the agreement between tank fecal and
association with compelling pathology (such as
apex smear results was even lower at 37.4% (99
inflammation, necrosis, or digestive gland atrophy).
tanks: 19 dual positives, 18 dual negatives, 57 just
Cytoplasmic vacuolation was often present but con-
fecal positives, and five just apex smear positives).
sidered within normal limits.7 Four wild-caught,
However, fecal results and histology findings com-
museum-archived Partula affinis (collected on
pared better: 60.4% (48 tanks, 25 dual positives,
Tahiti, French Polynesia in 1909) and five Partula
four dual negatives, 15 just fecal positives, and
nodosa (also collected on Tahiti in 1909) that had
four just histology positives).
initially been fixed in formalin and subsequently
transferred to ethanol were donated by the Acad-
DISCUSSION
emy of Natural Sciences (Philadelphia, PA 19103,
Over 5 yr of screening, although acknowledging USA; Gerlach, pers. comm.). These were examined
the limited information about Partula medicine at histologically, and microsporidia were identified in
the time, no organisms were discovered that were the digestive cells and intestinal mucosal epithelial
deemed too great a risk to prevent the export and cells of two of the Partula nodosa in a similar appar-
release of the snails. It was impossible to quantify ently inert context to those in the snails included in
risks, but the focus was on the potential pathogenic the screening program (Fig. 1). Unfortunately, simi-
organisms discussed in the Partula DRA,6 and these lar wild-caught Partula hyalina were too autolyzed
are discussed in the following. In addition, no histo- for meaningful interpretation (LZ, unpubl. data).
logic changes of major concern were discovered Despite the prevalence, suspected microsporidial

Figure 2. Histologic findings in Partula snails. H&E stain. (a) Digestive gland, Partula tohiveana. Digestive
gland from an active (euthanized) snail. Digestive gland tubules (asterisk) contain epithelial cells with abun-
dant granular cytoplasm and moderate numbers of larger globular protein inclusions (arrow; see panel b).
Lipid cells are present in the interstitium (arrowhead). Original magnification 2003. (b) Digestive gland,
Partula nodosa. Higher magnification of cytoplasmic protein inclusions often found alongside granules in
active snails. Original magnification 6003. (c) Digestive gland, Partula mooreana. Shrunken epithelial cells
with brown intracytoplasmic granules in excretory cells and dilated lumen within digestive gland tubule
(asterisk) of a snail with concurrent pathology (see panel f). Interstitial infiltrates of hemocytes are present
(arrow). Original magnification 2003. (d) Mantle and perivisceral connective tissue, Partula tohiveana.
Abundant lipid cells (asterisk) expand the connective tissues below the mantle and surrounded viscera.
Original magnification 1003. (e) Digestive gland, Partula garretti. Intense hemocytic infiltration (asterisk)
between digestive gland tubules (arrow). Original magnification 2003. (f) Foot process connective tissue,
Partula mooreana. Intense multifocal to coalescent infiltrates of brown-pigmented hemocytes among gland
cells (same animal as in panel c). Original magnification 2003. (g) Foot process, Partula hyalina. Solid granu-
loma-like nodular infiltrate of plump hemocytes (asterisk) with embedded nematode profile (arrowhead).
Original magnification 2003. From health screening at another institution but examined by one of the
authors (MFS). (h) Foot process, Partula hyalina. Embedded nematode profile (arrow) from panel g. Original
magnification 6003. (i) Digestive gland, Partula tohiveana. Oblique section through a migrating nematode
(arrow) without tissue reaction. Original magnification 2003.
sporocysts or spores were rarely detected in fecal investigations are required to clarify the molecular
samples and never in postmortem apex impression identities of the putative microsporidia and whether
smears. An earlier attempt to identify Microsporidium they are indeed species of Steinhausia.
DNA in fecal samples containing MZN acid-fast Cryptosporidium-like oocysts approximately 4 to
protozoal “cysts” was unsuccessful (Bass and Tro- 5 lm in diameter (but occasionally less than 4 lm,
man, pers. comm.), but these cysts most likely rep- hence, the 3 to 5 lm range used in the study) and
resented Cryptosporidium-like oocysts. Further staining acid-fast with MZN have been routinely

detected in fecal samples and apex impression previously classified as Flavobacterium and likely
smears for many years. They were prominent during were included as such in these reports.14 No clear
a period of Partula species high mortality between pathologic reactions were detected against bacteria
2006 and 2008,8 but corresponding organisms in tissues, and only one case had lesions associated
were never recognized in histopathologic sections, with the presence of nematodes. Nematodes may
albeit the snails were fixed in 70% ethanol. Some be pathogenic in mollusc hosts, but this is not
cyst-positive samples were processed for fluores- common.12
cent antibody testing against Cryptosporidium par- Although the role of infectious agents in causing
vum, but these were also negative. Following the disease and death in snails remains unproven, there
introduction of 10% buffered formalin as the fixa- is evidence that environmental factors (temperature,
tive prior to histopathologic examination, Crypto- humidity, and light) and diet (calcium concentration
sporidium-like parasitic bodies were detected and the addition of dog or cat vitamin supplements)
regularly. Thus, this is the fixative of choice for can affect reproductive success and mortality of cap-
molluscs, in contrast to most other invertebrates.18 tive Partula populations.11 Therefore, it is possible
Recent testing for Cryptosporidium DNA has also that sublethal environmental and dietary inadequa-
produced some positive results (Wigglesworth and cies could also lead to protozoal, and other, infec-
Blake, pers. comm.). As with the microsporidial tions becoming pathogenic. This is probably true of
infections, limited evidence of tissue reaction and other stressors such as increased population den-
no evidence of inflammation or ulceration were sity and transportation between tanks and between
noted with the Cryptosporidium-like organisms in collections.
the midgut. Interestingly, similar Cryptosporidium- Although this study did not identify any clear
like organisms were detected in one of the four association between the agents identified histo-
wild-caught Partula affinis and two of the five Par- logically and morbidity or mortality in the snail
tula nodosa mentioned earlier, suggesting that they populations, it remains important to understand
are common infections in wild Partula and also the expected parasite burdens and background
that long-term storage in formalin, with transfer changes found in captive Partula snail populations.
to ethanol, did not prevent histologic visualization This should provide a baseline for the consider-
of them. However, recent deaths in captive Partula ation of unexpected or unforeseen events in captiv-
rosea at LZ were associated with emaciation and ity and allow comparison with the (currently largely
large numbers of presumptive Cryptosporidium neglected) study of wild populations. Normal
(LZ, unpubl data), and similar observations were growth and longevity, plus physiology and his-
made by one of the authors (MFS) during a Par- tology across the life stages, need to be studied.10
tula rosea mortality event in 2016 at another col- Molecular studies should be used to characterize
lection. The identity of the organisms in the latter the microsporidia and Cryptosporidium-like organ-
case was further investigated with transmission elec- isms and investigate the normal microbiome of
tron microscopy and found to be consistent with a captive and, hopefully in the future, wild snails to
Cryptosporidium species. look for evidence of host specificity that might
The lack of agreement between the different indicate coevolution. This review has highlighted
diagnostic tests for the presence of microsporidia the difficulty of using just individuals found dead
and the Cryptosporidium-like protozoa was disap- to look at the possible causes of mortality in tank
pointing but may have several explanations, includ- populations. Therefore, targeted euthanasia should
ing irregular passage of spores and oocysts into the always be considered, especially for individuals
intestinal lumen, and hence the feces, innate low assessed as potentially unwell, and also for healthy
detection rates in feces and apex smears, and vari- individuals after careful consideration of the effect
able detection rates across the years. Hopefully, that loss of further snails may have on the captive
any correlation can be formally tested in the future, population. In addition, further research is required
and improvements made with increasing use of to identify the optimal method of euthanasia that is
molecular diagnostic testing. rapid and effective and causes no or minimal dis-
Nematodes and bacteria were commonly identi- tress. There were also variations in testing meth-
fied in fecal samples and histologic sections, pri- odology over the 5 yr, so future efforts should be
marily in the intestinal lumen, but occasionally in made to standardize all procedures.
the tissues in association with autolysis, suggesting
postmortem invasion. Similar findings have been Acknowledgments: The authors pay tribute to
reported previously with many of the same bacteria Dr. Trevor Coote who died in February 2021 and
identified after culture.2,5 Myroides species were was the Partula program field biologist and prime

mover in reestablishing viable populations of Partula 10. Goe A, Rodriguez C. Partula snail medicine. In:
species in the wild. The authors thank all colleagues Miller RE, Calle PP, Lamberski N (eds.). Fowler’s zoo
in the Zoological Society of London Wildlife Health and wild animal medicine, Volume 10, Current therapy.
Services and Animal Management teams for the Philadelphia (PA): W. B. Saunders Co; 2023. p. 381–385.
care and welfare of the zoo’s Partula and other 11. Gouveia AR. Investigation of the factors affect-
ing the population dynamics of captive Partula snails.
invertebrate species. The authors are also indebted
PhD Thesis, 2012. Imperial College, London (United
to colleagues at other collections involved in the
Kingdom).
reintroductions (Bristol Zoo, Chester Zoo, Detroit
12. Grewal PS, Grewal SK, Tan L, Adams BJ. Para-
Zoo, Edinburgh Zoo, Marwell Zoo, St. Louis Zoo, sitism of molluscs by nematodes: types of associations
Parc Zoologique de Thoiry, and Whipsnade Zoo). and evolutionary trends. J Nematol. 2003;35(2):146–156.
Thanks also to the Pathology Department at the 13. IUCN Species Survival Commission. Guidelines
Royal Veterinary College for processing snail tis- to reintroductions and other conservation transloca-
sues for histology, Dr. David Bass and Catherine tions. Version 1.0. Gland (Switzerland): IUCN Species
Troman at the Natural History Museum for con- Survival Commission; 2013.
ducting PCR testing, and Dr. Justin Gerlach for 14. Jooste PJ, Hugo CJ. The taxonomy, ecology and
entrusting the authors with precious wild-caught cultivation of bacterial genera belonging to the family
specimens and advising on Partula taxonomy. Flavobacteriaceae. Int J Food Microbiol. 1999;53(2–3):
81–94.
15. Murray J, Murray E, Johnson MS, Clarke B. The
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