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)

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

31

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 32 JOURNAL OF ZOO AND WILDLIFE MEDICINE
three species: Partula suturalis vexillum, Partula tae-
niata nucleola, and Partula tohiveana were released
into a protected reserve in Moorea, French Poly-
nesia. Unfortunately, the reserve boundary failed
to exclude Euglandina, so the remaining Partula
had to be recaptured. However, the presence of
juvenile snails proved that reintroduction had
been partially successful.9
Before further reintroductions were attempted,
a disease risk analysis (DRA) was performed by
staff of the Zoological Society of London (ZSL)
according to IUCN guidelines.6,13,19 Relatively few
reports of pathologic investigations of Partula snails
have been published,2,5,11 but the DRA reviewed
these and other publications related to snail species
to decide whether translocations might risk transfer-
ring any pathogens they carry and whether sickness
and death in these species and native animals could
result in failure of the program. Captive breeding
in zoos makes it possible to rear large numbers of
individuals for maintenance of the species and the
return to natural habitat once any factors respon-
sible for decline have been removed or at least
reduced. However, even with good security, there
is a risk of crossover of pathogens from other cap-
tive species. The DRA identified microsporidia and
suspected cryptosporidia as potential pathogens
but not thought to be critically significant.5,8 How-
ever, health screening for infectious agents was rec-
ommended prior to translocation.
Following implementation of the DRA recom-
mendations, Partula snails were reintroduced to var-
ious sites in French Polynesia in 2015 and in each
of the four subsequent years. This study describes
the results of health screenings carried out at ZSL
London Zoo (LZ) and formed part of the evidence
that enabled the Official Veterinarian (OV) to sign
the Animal and Plant Health Agency Export Health
Certificate for export to French Polynesia and, ulti-
mately, release to the wild.
MATERIALS AND METHODS
Partula populations
In 2015, individuals of three Partula species
were sent to LZ from four zoos in the United King-
dom and two zoos in the United States and housed
in a biosecure quarantine room, with additional
individuals of two of these species from the zoo’s
own collection. All were destined for reintroduction
that year and were health screened together prior to
export. Partula snails are typically maintained and
bred in glass tanks;1 hence, screening was based on
testing each tank population. Imported and resident
tank populations were kept separate. Numbers of
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snails in each varied markedly (range: 12 [all adults]
to 205 [125 newborns, 30 juveniles, and 50 adults
but no subadults]), and in one case (the most popu-
lated), some individuals were subsequently moved
to a new tank. The following year, populations of
Partula species from four United Kingdom zoos
and one French zoo (similarly varied in numbers
of snails per tank) were again quarantined and
screened at LZ. However, instead of separating
those LZ tank populations destined for reintro-
duction and moving them to the quarantine room,
the whole zoo collection was screened. Two
additional zoos successfully carried out screen-
ing in 2016, and others did so in the three subse-
quent years. Therefore, from 2017 to 2019, LZ
only screened its own tank populations. Because
the entire LZ collection was tested from 2016 to
2019, some tank populations that were maintained
for over a year were checked twice or sometimes
more often; this is indicated in the results when
relevant.
Fecal screening
Three fresh fecal samples were obtained from
each tank. Upon receipt, samples were refrigerated
until examination but were always examined on the
day of collection to ensure freshness. All three sam-
ples were examined for the presence of parasites
due to the previous experience of the intermittent
appearance of various parasites (LZ, unpubl. data).
In 2018, some tanks (those not destined for export)
were only sampled once, whereas occasionally
fourth, and even fifth, samples from tanks destined
for export were collected if large numbers of para-
sites had been detected. In 2015, a bacteriologic cul-
ture was also attempted from all three samples, but
in view of the abundance of mixed bacteria, from
2016, only one sample from each tank was cultured.
Postmortem examinations
Dead snails were examined under three circum-
stances: 1) individuals found dead with no gross
signs of autolysis and on a day when they could be
submitted for immediate examination; 2) individ-
uals found dead, but not fresh, or on a day when
immediate examination was not possible; there-
fore, they were fixed in 70% ethanol and then sub-
mitted; and 3) healthy adult individuals randomly
selected by keepers for euthanasia (carried out by
exposure to an overdose of isoflurane for a mini-
mum of 1 h; the standard method used at ZSL and
found to be rapid and effective) and submitted for
immediate, fresh examination. The aim was to
examine approximately 10% of the population
 FLACH ET AL—PARTULA 5-YR HEALTH SCREENING REVIEW
of adults in each tank, with at least some snails
examined fresh after euthanasia from tanks with
sufficient numbers. Exceptions were made if the
number of adults in a tank population was too low
to risk removing any individuals (generally assessed
as less than 10, but dependent on additional factors,
such as the number of adults of the same species in
other tanks).
Snails presented fixed in ethanol were not exam-
ined grossly, but a proportion were submitted for
histology. All others were weighed and measured
as previously described.16 If the snail did not easily
slide from the shell when traction was applied,
heavy-duty scissors were used to open the shell to
ensure minimal crush and stretching artifacts. The
extracted body was weighed and examined exter-
nally. The tip of the apex was removed, and impres-
sion smears of the cut surface made on a microscope
slide for parasitologic examination (see the follow-
ing). The body was then opened with a sterile scalpel
blade, a swab taken for bacteriologic culture, and the
internal tissues examined grossly. A small piece of
foot tissue was removed and frozen as a source of
DNA, and finally, the remaining carcass, plus the
amputated apex, were fixed for histologic exami-
nation. In 2015 and 2016, this was achieved by
immersion in 70% ethanol, but from 2017, 10% buff-
ered formalin was used, as this was demonstrated
to yield better quality fixation. Fixed snails were
bisected longitudinally and processed into forma-
lin-fixed and paraffin-embedded sections by stan-
dard techniques. Slides were stained with H&E,
modified Ziehl-Neelsen (MZN), Gram–Twort, and
periodic acid–Schiff (PAS) using standard labora-
tory protocols,20 plus the Luna stain specifically
for microsporidia,17 and were reported by an ana-
tomic veterinary pathologist (MFS).
Parasitologic testing
Feces underwent direct microscopy of a wet prep-
aration and microscopy of a dry smear stained with
MZN stain. Apex smears were likewise examined
microscopically after staining with MZN. Wet prep-
arations were prepared by mixing a small amount of
feces (a bacteriologic Nichrome wire loopful) in two
drops of sterile physiologic saline and examined
microscopically for the presence of any parasites
or ova, with particular emphasis on helminths (often,
but not always, identified further as adult or larval
nematodes), flagellated protozoa, and ciliated pro-
tozoa. The MZN-stained smears were prepared
and examined microscopically under oil immer-
sion for the presence of acid-fast bacilli (possible
Mycobacterium species) and spores and oocysts
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33
(microsporidian or protozoal or both). Size (greatest
diameter) was measured with an eyepiece graticule
that had been calibrated from a stage micrometer
slide and was then reported as having diameters of
approximately 2 lm (suspect microsporidial spores)
or in the range of 3 to 5 lm (presumptive crypto-
sporidial oocysts).
Bacteriologic testing
Fecal samples and coelomic swabs were inocu-
lated onto 5% horse blood agar plates, incubated at
25°C for 48 h. Single bacterial colonies of predomi-
nant types were subcultured onto further plates.
Resulting pure cultures were then identified by
standard methods: colony morphology; appearance
and Gram-staining characteristics; and biochemical
reactions (API commercial test kits and online
software, bioMérieux UK Limited, Basingstoke,
Hampshire RG22 6HY, United Kingdom), follow-
ing an initial oxidase test. A 20NE API test identi-
fied oxidase-positive organisms, a 20E test detected
negative bacteria, and a 10S test showed any atypi-
cal reactions.
Other evidence
Although the majority of the screening was for
the presence of potential pathogens and abnormal
histologic findings, the OV also considered the
keepers’ assessments of the tank populations, in
particular, evidence of successful breeding, normal
feeding and activity, and the absence of excessive
mortality (above that expected with normal aging).
RESULTS
Over 5 yr, 12 species of Partula snails, origi-
nally derived from nine contributing zoos but
managed as a single contributing feeder popula-
tion at LZ, were screened (Table 1). These were
maintained as 129 different tank populations,
and some of which (at LZ) were screened on
more than one occasion.
Fecal sampling
Of the 535 fecal samples examined (Table 2), 45%
contained flagellated protozoa, 35.5% had MZN
acid-fast oocysts with a diameter between 3 and
5 lm, 25.0% had ciliated protozoa, while helminths
(adults or larvae or both), smaller (2 lm) MZN
acid-fast-suspect spores, and MZN acid-fast
bacilli were noted less frequently (11.6%, 5.2%, and
11.8%, respectively). Interestingly, although hel-
minths and 3 to 5 lm oocysts were observed each
year and flagellated protozoa in four of the 5 yr, the
 34 JOURNAL OF ZOO AND WILDLIFE MEDICINE

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
years. Also, flagellated protozoa were extremely
abundant in 2017 and 2019, reasonably so in 2018,
and very rare in 2015. The results for the different
Partula species were also very variable (Table 3).
Mixed bacteria were grown from all 199 fecal
samples cultured. Myroides species were present,
and often predominant, in almost 90% of these
(Table 4). The second most common identification
was Aeromonas hydrophila/caviae (species indistin-
guishable using current methods), but these bacte-
ria were only present in 15.6% of samples and
found in only 3 yr. Another 15 bacterial types
were identified to genus or species level but were
only found infrequently.
Postmortem examinations
Over 5 yr, 479 snails were examined postmortem,
with the majority having died, but with an increas-
ing percentage euthanized so that they could be
examined fresh (Table 5). No 2 lm diameter spores
were observed in the MZN-stained apex impression
smears, whereas 3 to 5 lm oocysts were detected in
Table 2. Fecal parasite screening 2015–2019 by year.
each year but in low numbers. They were most
commonly detected in Partula mooreana (12.0% of
samples), followed by Partula navigatoria (11.6%),
Partula hebe bella (10.5%), and Partula hyalina
(10.0%), with low numbers in Partula suturalis vexil-
lum, Partula taeniata nucleola, and Partula tohiveana,
and none in the other species. Acid-fast bacilli
were only observed in these smears in 2017 (four
cases) and 2019 (one case). Bacterial culture of the
coelomic swabs invariably yielded a mixture of
species, with Myroides species again being predom-
inant (63.5% of all cultures), especially in snails
that had died (122 of 151 [80.8%] compared with 69
of 150 [46.0%] euthanized snails). However, in 2015,
no Myroides species were identified. Aeromonas
hydrophila/caviae were the second most common
but only cultured from 4.6% of swabs. Twenty-six
other bacterial genera and species were identified
but none consistently.
Histology findings
In total, 292 snail carcasses (61.0% of postmor-
tem examinations) were examined histologically

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)

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 FLACH ET AL—PARTULA 5-YR HEALTH SCREENING REVIEW 35

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-
ysis was a regular feature of Partula histologic sec-
tions (86 cases; 29.5%) and was much more
common in snails that had died (79 of 88; 89.8%)
than those euthanized (7 of 204; 3.4%). Hence, it
also declined over 5 yr, as the proportion of eutha-
nized snails examined increased (2015, 23 of 49;
2016, 44 of 114; 2017, 14 of 53; 2018, 2 of 43; and
2019, 3 of 33). In cases that were relatively unaf-
fected by autolysis, the tissues routinely identified
were radula, foot process, kidney, ganglion, ovotes-
tis, reproductive ducts, digestive gland, salivary
gland, shell gland, lung and pulmonary cavity, heart
and gastrointestinal tract, but in addition, the albu-
men gland, pedal mucus gland, and eyestalk were
occasionally seen.7,22
Microsporidial sporocysts were detected in all
5 yr (Table 6 and Fig. 1) and in all except two spe-
cies (Partula nodosa and Partula garrettii; Table 7). In
contrast, apical, extracytoplasmic, intracellular
protozoal stages consistent with developing Crypto-
sporidium oocysts were only detected in 2017 and
subsequent years, following the change of fixative
from 70% ethanol to 10% buffered formalin. They
were then reported each year and in eight of the
10 species examined. The percentage of cases posi-
tive for microsporidial sporocysts also increased in
the years following this change (Table 6), although
this was not consistent across species (Table 7).
The abundance of microsporidial sporocysts in
examined sections was most commonly described
as occasional or rare (68.6%), followed by moderate
numbers (29.1%) and large numbers (2.3%), with
the majority present in the digestive gland acinar
epithelial cells (digestive cells; 71.0%). Others were
observed in mucosal epithelial cells of the intestine
(39.5%). Sometimes, they were present in both sites.
The Cryptosporidium-like organisms were most com-
monly present in large numbers (65.2%), followed
by occasional (19.6%) and moderate numbers
(15.2%), typically affecting focally extensive or
extensive populations of epithelial cells of the
digestive gland duct (73.9%) or midgut intestine
(65.2%), and commonly in both. Occasionally, free
presumptive oocysts were reported in the digestive
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)

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 36 JOURNAL OF ZOO AND WILDLIFE MEDICINE

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

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 FLACH ET AL—PARTULA 5-YR HEALTH SCREENING REVIEW 37

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-
tions, absent or scant mucoid intestinal content,
or increased numbers of yellow- and brown-pig-
mented connective tissue hemocytes frequently
had dilated digestive gland acini, small flattened
digestive cells containing brown granules and vac-
uoles, and more conspicuous excretory cells. The
latter appearance was interpreted as digestive gland
inactivity or atrophy. There was no correlation
between the presence or absence of microsporidia or
Cryptosporidium-like organisms and these changes.
Comparisons between parasitologic findings from
different tests
At postmortem examination, microsporidial spo-
rocysts were only detected histologically; no
microsporidial spores were observed in MZN-
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.

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 38 JOURNAL OF ZOO AND WILDLIFE MEDICINE

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

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 FLACH ET AL—PARTULA 5-YR HEALTH SCREENING REVIEW 39

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
samples and never in postmortem apex impression
smears. An earlier attempt to identify Microsporidium
DNA in fecal samples containing MZN acid-fast
protozoal “cysts” was unsuccessful (Bass and Tro-
man, pers. comm.), but these cysts most likely rep-
resented Cryptosporidium-like oocysts. Further
investigations are required to clarify the molecular
identities of the putative microsporidia and whether
they are indeed species of Steinhausia.
Cryptosporidium-like oocysts approximately 4 to
5 lm in diameter (but occasionally less than 4 lm,
hence, the 3 to 5 lm range used in the study) and
staining acid-fast with MZN have been routinely

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 40 JOURNAL OF ZOO AND WILDLIFE MEDICINE
detected in fecal samples and apex impression
smears for many years. They were prominent during
a period of Partula species high mortality between
2006 and 2008,8 but corresponding organisms
were never recognized in histopathologic sections,
albeit the snails were fixed in 70% ethanol. Some
cyst-positive samples were processed for fluores-
cent antibody testing against Cryptosporidium par-
vum, but these were also negative. Following the
introduction of 10% buffered formalin as the fixa-
tive prior to histopathologic examination, Crypto-
sporidium-like parasitic bodies were detected
regularly. Thus, this is the fixative of choice for
molluscs, in contrast to most other invertebrates.18
Recent testing for Cryptosporidium DNA has also
produced some positive results (Wigglesworth and
Blake, pers. comm.). As with the microsporidial
infections, limited evidence of tissue reaction and
no evidence of inflammation or ulceration were
noted with the Cryptosporidium-like organisms in
the midgut. Interestingly, similar Cryptosporidium-
like organisms were detected in one of the four
wild-caught Partula affinis and two of the five Par-
tula nodosa mentioned earlier, suggesting that they
are common infections in wild Partula and also
that long-term storage in formalin, with transfer
to ethanol, did not prevent histologic visualization
of them. However, recent deaths in captive Partula
rosea at LZ were associated with emaciation and
large numbers of presumptive Cryptosporidium
(LZ, unpubl data), and similar observations were
made by one of the authors (MFS) during a Par-
tula rosea mortality event in 2016 at another col-
lection. The identity of the organisms in the latter
case was further investigated with transmission elec-
tron microscopy and found to be consistent with a
Cryptosporidium species.
The lack of agreement between the different
diagnostic tests for the presence of microsporidia
and the Cryptosporidium-like protozoa was disap-
pointing but may have several explanations, includ-
ing irregular passage of spores and oocysts into the
intestinal lumen, and hence the feces, innate low
detection rates in feces and apex smears, and vari-
able detection rates across the years. Hopefully,
any correlation can be formally tested in the future,
and improvements made with increasing use of
molecular diagnostic testing.
Nematodes and bacteria were commonly identi-
fied in fecal samples and histologic sections, pri-
marily in the intestinal lumen, but occasionally in
the tissues in association with autolysis, suggesting
postmortem invasion. Similar findings have been
reported previously with many of the same bacteria
identified after culture.2,5 Myroides species were
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previously classified as Flavobacterium and likely
were included as such in these reports.14 No clear
pathologic reactions were detected against bacteria
in tissues, and only one case had lesions associated
with the presence of nematodes. Nematodes may
be pathogenic in mollusc hosts, but this is not
common.12
Although the role of infectious agents in causing
disease and death in snails remains unproven, there
is evidence that environmental factors (temperature,
humidity, and light) and diet (calcium concentration
and the addition of dog or cat vitamin supplements)
can affect reproductive success and mortality of cap-
tive Partula populations.11 Therefore, it is possible
that sublethal environmental and dietary inadequa-
cies could also lead to protozoal, and other, infec-
tions becoming pathogenic. This is probably true of
other stressors such as increased population den-
sity and transportation between tanks and between
collections.
Although this study did not identify any clear
association between the agents identified histo-
logically and morbidity or mortality in the snail
populations, it remains important to understand
the expected parasite burdens and background
changes found in captive Partula snail populations.
This should provide a baseline for the consider-
ation of unexpected or unforeseen events in captiv-
ity and allow comparison with the (currently largely
neglected) study of wild populations. Normal
growth and longevity, plus physiology and his-
tology across the life stages, need to be studied.10
Molecular studies should be used to characterize
the microsporidia and Cryptosporidium-like organ-
isms and investigate the normal microbiome of
captive and, hopefully in the future, wild snails to
look for evidence of host specificity that might
indicate coevolution. This review has highlighted
the difficulty of using just individuals found dead
to look at the possible causes of mortality in tank
populations. Therefore, targeted euthanasia should
always be considered, especially for individuals
assessed as potentially unwell, and also for healthy
individuals after careful consideration of the effect
that loss of further snails may have on the captive
population. In addition, further research is required
to identify the optimal method of euthanasia that is
rapid and effective and causes no or minimal dis-
tress. There were also variations in testing meth-
odology over the 5 yr, so future efforts should be
made to standardize all procedures.
Acknowledgments: The authors pay tribute to
Dr. Trevor Coote who died in February 2021 and
was the Partula program field biologist and prime
 FLACH ET AL—PARTULA 5-YR HEALTH SCREENING REVIEW
mover in reestablishing viable populations of Partula
species in the wild. The authors thank all colleagues
in the Zoological Society of London Wildlife Health
Services and Animal Management teams for the
care and welfare of the zoo’s Partula and other
invertebrate species. The authors are also indebted
to colleagues at other collections involved in the
reintroductions (Bristol Zoo, Chester Zoo, Detroit
Zoo, Edinburgh Zoo, Marwell Zoo, St. Louis Zoo,
Parc Zoologique de Thoiry, and Whipsnade Zoo).
Thanks also to the Pathology Department at the
Royal Veterinary College for processing snail tis-
sues for histology, Dr. David Bass and Catherine
Troman at the Natural History Museum for con-
ducting PCR testing, and Dr. Justin Gerlach for
entrusting the authors with precious wild-caught
specimens and advising on Partula taxonomy.
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Accepted for publication 13 November 2023
Supplemental Table. Histologic examinations
performed by species and year compared with
postmortem examinations.