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Received: 8 March 2016 Revised: 6 September 2016 Accepted: 22 September 2016 DOI: 10.1111/xen.12277
ORIGINAL ARTICLE
A comprehensive microbiological safety approach for agarose encapsulated porcine islets intended for clinical trials Lawrence S. Gazda1 | James Collins2 | Archie Lovatt3 | Robert W. Holdcraft1 | Merribeth J. Morin4 | Daniel Galbraith5 | Melanie Graham6,7 | Melissa A. Laramore1 | Christine Maclean3 | John Black3 | Euan W. Milne3 | Douglas G. Marthaler2,7 | Horatiu V. Vinerean8,9 | Michelle M. Michalak10 | Deborah Hoffer1 | Steven Richter11 | Richard D. Hall12 | Barry H. Smith13,14 1
The Rogosin Institute-Xenia Division, Xenia, OH, USA
2
Veterinary Diagnostic Laboratory, University of Minnesota, Saint Paul, MN, USA
3
SGS Vitrology, Glasgow, UK
4
Targeted BioStrategies, Springfield, MA, USA
5
Sartorius Stedim BioOutsource, Glasgow, UK
6
Department of Surgery, University of Minnesota, Saint Paul, MN, USA
7
Department of Veterinary Population Medicine, University of Minnesota, Saint Paul, MN, USA
8
Office of Laboratory Animal Research, Florida International University, Miami, FL, USA
9
Department of Surgery, Herbert Wertheim College of Medicine, Miami, FL, USA
10
Maria Stein Animal Clinic, Maria Stein, OH, USA
11
Avista Pharma Solutions, Inc., Agawam, MA, USA
12
Bob Evans Farms, LLC, New Albany, OH, USA
13
Department of Surgery, Weill Medical College of Cornell University and NewYork-Presbyterian Hospital, New York, NY, USA
14
The Rogosin Institute, New York, NY, USA
Correspondence Lawrence S. Gazda, The Rogosin InstituteXenia Division, Xenia, OH, USA. Email:
[email protected] Funding information This work was supported by Metromedia Bio-Science LLC.
Abstract Background: The use of porcine islets to replace insulin-producing islet β-cells, destroyed during the diabetogenic disease process, presents distinct challenges if this option is to become a therapeutic reality for the treatment of type 1 diabetes. These challenges include a thorough evaluation of the microbiological safety of the islets. In this study, we describe a robust porcine islet-screening program that provides a high level of confidence in the microbiological safety of porcine islets suitable for clinical trials.
Abbreviations: Viruses: BDV, Borna disease virus; BVDV-1, -2, Bovine viral diarrhea virus type 1 & 2; PAV, Porcine adenovirus; PCV, Porcine circovirus; PCMV, Porcine cytomegalovirus; PEV, Porcine enterovirus; PERV-A/B/C, Porcine endogenous retrovirus A B C; PHEV, Porcine hemagglutinating encephalomyelitis virus; PHoV, Porcine hokovirus; PLHV-1, -2 -3, Porcine lymphotropic herpesvirus type 1 2 & 3; PPV, Porcine parvovirus; PRRSV, Porcine reproductive & respiratory syndrome virus; PRCV, Porcine respiratory coronavirus; PRotA, Porcine rotavirus A; PRotC, Porcine rotavirus C; PRV, Pseudorabies virus; PTV, Porcine teschovirus; RV, Rabies virus; REO-1, -2 -3, Reovirus type 1 2 & 3; SEOV, Seoul virus; SIV, Swine influenza virus; SNV, Sin Nombre virus; EMCV, Swine encephalomyocarditis virus; HEV, Swine hepatitis E virus; SVDV, Swine vesicular disease virus; SWPOX, Swine pox virus; TTV, Torque tenovirus; TGEV, Transmissible gastroenteritis virus; EEEV, Eastern equine encephalomyelitis virus; VEEV, Venezuelan equine encephalomyelitis virus; WEEV, Western encephalomyelitis virus; VSV-IN, Vesicular stomatitis Indiana virus; VSV-NJ, Vesicular stomatitis New Jersey virus; WNV, West Nile virus. Cell Lines: BHK-21, Baby hamster kidney cell line; BT, Bovine turbinate cell line; MA104, African green monkey kidney cell line; MARC-145, Monkey kidney cell line; MDCK, Madin Darby canine kidney cell line; MRC-5, Human fetal lung fibroblast cell line; PAM, Pulmonary alveolar macrophages; PK-15, Porcine kidney cell line; PPK, Primary porcine kidney cell line; ST, Swine testicular cell line
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2016 The Authors Xenotransplantation Published by John Wiley & Sons Ltd Xenotransplantation 2016; 1–20 wileyonlinelibrary.com/journal/xen | 1
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Gazda et al.
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Methods: A four-checkpoint program systematically screens the donor herd (Large White – Yorkshire × Landrace F1 hybrid animals), individual sentinel and pancreas donor animals and, critically, the islet macrobeads themselves. Molecular assays screen for more than 30 known viruses, while electron microscopy and in vitro studies are employed to screen for potential new or divergent (emergent) viruses. Results: Of 1207 monthly samples taken from random animals over a 2-year period, only a single positive result for Transmissible gastroenteritis virus was observed, demonstrating the high level of biosecurity maintained in the source herd. Given the lack of clinical signs, positive antibody titers for Porcine reproductive and respiratory syndrome virus, Porcine parvovirus, and Influenza A confirm the efficacy of the herd vaccination program. Porcine respiratory coronavirus was found to be present in the herd, as expected for domestic swine. Tissue homogenate samples from six sentinel and 11 donor animals, over the same 2-year period, were negative for the presence of viruses when co-cultured with six different cell lines from four species. The absence of adventitious viruses in separate islet macrobead preparations produced from 12 individual pancreas donor animals was confirmed using validated molecular (n = 32 viruses), in vitro culture (cells from four species), and transmission electron microscopy assays (200 cell profiles per donor animal) over the same 2-year period. There has been no evidence of viral transmission following the implantation of these same encapsulated and functional porcine islets into non-immunosuppressed diabetic cynomolgus macaques for up to 4 years. Isolated peripheral blood mononuclear cells from all time points were negative for PCV (Type 2), PLHV, PRRSV, PCMV, and PERV-A, PERV-B, and PERV-C by PCR analysis in all six recipient animals. Conclusion: The four-checkpoint program is a robust and reliable method for characterization of the microbiological safety of encapsulated porcine islets intended for clinical trials. KEYWORDS
pancreatic islets, xenotransplantation, zoonoses
1 | INTRODUCTION
In another approach, the replacement of lost islet cells through human islet allotransplantation has established the clinical utility
The ultimate goal for the treatment of type 1 diabetes is the res-
of islet grafting.6,7 Although only 13% of human islet recipients re-
toration of physiological blood glucose regulation without the
main insulin-independent at 5 years, the majority of patients (67%)
requirement for lifelong therapy such as immunosuppression or im-
retain some graft function as evidenced by persistent C-peptide
munoregulation. There are numerous challenges for the attainment
secretion.8 Importantly, islet transplantation largely eliminates hy-
of this goal, and diverse approaches ranging from insulin pumps
poglycemic unawareness post-grafting. Further improvement in
to gene therapy to stem cell strategies are all being pursued.1–5
long-term insulin independence is likely; data from a few select
Other: ATMP, Advanced Therapy Medicinal Products; CBER, Center for Biologicals Evaluation and Research; CFR, Code of Federal Regulations; cGMP, Current Good Manufacturing Practices; CHMP, Committee for Medicinal Products for Human Use; cpe, Cytopathic effect; DPF, Designated pathogen-free; df, Dilution factor; ELISA, Enzyme linked immunosorbent assay; EMA, European Medicines Agency; FDA, Food and Drug Administration; HA, Hemadsorption test; HEPA, High-efficiency particulate arrestance; HI, Hemagglutination inhibition; IEQ, Islet equivalent; an islet of diameter 150 μm. ICH, International Council of Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; IHC, Immunohistochemistry; IND, Investigational new drug application; IXA, International Xenotransplantation Association; ISO, International Organization for Standardization; n.d., Not done; MVDL, University of Minnesota Veterinary Diagnostic Laboratory; NAHLN, National Animal Health Laboratory Network; NHP, Non-human primate; PAR-1, PERV-A receptor 1; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PCR, Polymerase chain reaction; qPCR, Quantitative PCR; Ph. Eur., European Pharmacopeia; PQA Plus®, Pork Quality Assurance Plus; PRNT, Plaque reduction neutralization test; RBC, Red blood cells; qRT-PCR, Quantitative reverse transcription PCR; SN, Serum neutralization; SOP, Standard operating procedures; SPF, Specific pathogen free; TEM, Transmission electron microscopy; USDA, United States Department of Agriculture; USP, United States Pharmacopeia; VI, Virus isolation; VN, Virus neutralization; WHO, World Health Organization
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Gazda et al.
institutions indicate that an increasing number of islet recipients (approaching 50%) are able to maintain external insulin independence for 5 years.9 Nonetheless, the availability of suitable human pancreases for either whole organ or isolated islet transplantation is entirely inadequate as evidenced by the recovery of only 1293 donor pancreases in the United States in 2015.
10
2 | MATERIALS AND METHODS 2.1 | Source animal facility Donor pigs were Large White – Yorkshire × Landrace F1 hybrid animals obtained from well-characterized, non-genetically modified
The use of xenogeneic islets could provide a solution to the lim-
Choice Genetics (formerly Newsham) source animals. The Source
ited supply of human islets for transplantation. Porcine islets are
Animal Facility is an independently owned herd located in rural
particularly suitable as a source of insulin-producing cells because
Southwest Ohio. The breeding herd numbers approximately 2000
porcine insulin differs from human insulin by only a single amino
sows and utilizes a two-tiered quarantine testing system through a
acid and porcine insulin has been shown to provide reliable and safe
modern multisite production strategy. Three segregated facilities
glycemic control in human diabetic patients. Furthermore, only the
(nursery, gilt isolation, and farrowing and gestation) maintain data
transplantation of whole pancreas or intact islets of Langerhans can
records and follow standard operating procedures (SOPs) for animal
be expected to replicate normal and precise physiological glucose
husbandry, vaccination, housing, cleaning and waste management,
control, which is dependent on a variety of cell types and proteins
and biosecurity measures. Controlled transportation of animals, sup-
from pancreatic islets. This enormously complex regulatory ma-
plies and semen, feeding and watering, pest and rodent control, gravel
chinery is absent in all other approaches to the re-establishment of
barrier around facility perimeter, and caretaker qualifications including
normoglycemia.
routine health assessments are all components of the comprehensive
The use of animal-derived islet tissue, however, will necessitate
biosecurity program. Routine Quality Assurance site audits are used
either suppression of the patient’s immune system or the physical
to verify compliance. Corn-based pig feed was produced exclusively
isolation of the islets to prevent their rejection. We have previously
with corn grown and ground onsite at the source animal facility. Gilts,
described the ability of agarose-agarose encapsulated porcine islets
gestation sows, and lactating sows were given the same base feed,
to function in diabetic animal models.11–15 These encapsulated islet
consisting of corn with various supplements (eg, amino acids, vitamins,
macrobeads are at least partially immune isolated as an outer layer
minerals, protein), with slight variations based on the breeding status
of dense agarose prohibits direct cell access to the xenogeneic islets.
of the animal. All ingredients used in the production of complete feed
Although small immune mediators, such as cytokines, can still enter
were certified to be free of any restricted use protein products as de-
the macrobead, this encapsulation approach has been sufficiently ro-
fined in the FDA Code of Federal Regulation Title 21 Part 589.2001
bust to allow spontaneously diabetic BB rats to survive for more than
(21 CFR 589.2001) pertaining to cattle materials prohibited in animal
6 months without immunosuppression and without exogenous insulin
feed. Complete medical histories are archived for all source animals.
Ongoing studies in non-immunosuppressed diabetic
The facility holds Pork Quality Assurance Plus (PQA Plus®) certifica-
cynomolgus macaques also demonstrate porcine islet macrobead
tion, which is a producer-driven program to ensure U.S. pork products
function for more than 6 months in 5 of 6 animals (unpublished data,
are of the highest quality and safe.16 The PQA Plus program mandates
see Checkpoint 4 below).
Good Production Practices with an emphasis on biosecurity and prac-
administration.
13
The clinical use of islets from animals also requires a well-thought-
tices to reduce the risk of introducing and spreading of infectious
out strategy to assure the microbiological safety of the animal tissue.
agents. All retired breeding sows not used as pancreas donors are sent
A strategy to provide an aseptic product and to reduce the probability
to a local abattoir for pork products.
of inadvertent transmission of adventitious viruses is critical to the
Source animals are routinely vaccinated for various agents
successful implementation of this exciting therapy. Our approach
(Table 1). Killed vaccines are used when available. Modified live virus
takes advantage of the unique aspects of the islet macrobead, namely
vaccines are used with no evidence for reversion to virulence in back-
its long-term viability in culture and lack of patient immunosuppres-
passage studies (PARASAIL and ProSystem RCE) or when no USDA-
sion, to limit the microbiological safety risk of porcine islets for trans-
licensed killed vaccines are available (Enterisol Ileitis). The modified
plantation. Essential to our approach is the establishment of four
live virus vaccine Ingelvac PRRS is the exception to the above [vaccine
checkpoints that span the macrobead production and transplantation
virus has been found in one study with non-vaccinated pigs in con-
processes. The checkpoint approach is built upon an increasing level
tact with vaccinated animals17] and is alternated quarterly with a killed
of microbiological screening from the donor herd up to the islet prod-
PRRS vaccine.
uct, using multiple assay formats with the ability to screen for known
The facility operates a Source Animal Health Screening Program
and unknown pathogens. Using the checkpoint approach, we provide
to continuously assess the health status of the herd. Between January
evidence to support the use of donor animals housed in high hygienic
2013 and December 2014, the screening program was composed of
but not HEPA-filtered environments, not only for islet xenotransplan-
random antemortem monthly herd testing and a sentinel animal pro-
tation but also other xeno-cell therapies. In this study, we present and
gram that includes both antemortem and postmortem testing. Target
document the effectiveness of our microbiological safety strategy to
agents, test methods, and sample types for the monthly and sentinel
minimize the microbiological risks of agarose encapsulated porcine is-
antemortem screening are shown in Table 2. The sentinel animal pro-
lets for the treatment of type 1 diabetes.
gram consists exclusively of randomly selected breeding sows as they
PRRS
b
Porcine Reproductive and Respiratory Syndrome Virus
Porcine Reproductive and Respiratory Syndrome Virus
Ingelvac® PRRSc
d
Rotavirus G serotypes 5 and 4 of Serogroup A, C. perfringens Type C, E. coli (K99, K88, 987P, F41)
E. coli (K99, K88, 987P, F41), C. perfringens type C, Bordetella bronchiseptica, Pastruella multocidatypes A/D
ProSystem® RCEb
Prefarrow Shield 9
5 wk pre-farrowing. 2 wk pre-farrowing. c January and July. d April and October.
a
a
Parvo Shield® L5E
®
Lawsonia intracellularis
Parvovirus, 5 strains of Leptospira and Erysipelas
Enterisol Ileitis
M. hyopneumonia
SIV H1N1 and H3N2
®
Mycoplasma hyopneumonia
Circovirus
Pneumostar® SIV
Myco Shield
MycoFLEX®
CircoFLEX
Haemophilus parasuis
PARASAIL®
®
Target agent
Vaccine
T A B L E 1 Vaccine information and vaccination schedule
Killed
Modified live
Modified live
Killed
Killed
Attenuated
Killed
Killed
Killed
Killed
Avirulent live
Form
Newport
Boehringer Ingelheim
Merck
Novartis
Novartis
Boehringer Ingelheim
Novartis
Novartis
Boehringer Ingelheim
Boehringer Ingelheim
Newport Laboratories
Manufacturer
X
X
X
Weaning
X
X
3 wk X
6 wk
Post-weaning
X
X
Gilts
X
X
Pre- farrowing
X
Post- breeding
X
X
Xd
Whole herd
4
| Gazda et al.
Quarterly sentinel
Monthly Herd
2013
Serum Serum
Culture TEM qPCR qPCR HI ELISA ELISA ELISA ELISA SN
Enteric viruses
Influenza A*
PCMV
PPV*
PRCV
PRRSV*
PRV
TGEV
BVDV-1. -2
PRNT PRNT
WEEV
WNV
Serum
PRNT PRNT
EEEV
VEEV
Serum
SN
VSV-NJ
Serum
Serum
Serum
Serum
SN SN
EMCV
VSV-IN
Serum
Serum
Serum
Serum
Serum
Buffy coat
Nasal swab
Feces
Feces
Culture
Feces
Serum
Salmonella spp.
ELISA
PRRSV*
Serum
Serum
Sample type
Brachyspira sp.
ELISA ELISA
TGEV
Test method
PRCV
Target agent
T A B L E 2 Monthly herd and quarterly sentinel antemortem screening
32/49
44/49
0/49
JAN
0/10
0/10
1/10j
2/10j
0/11
3/11
3/11j
2/11
1/10
1/10j
0/10 2/10j
0/11
2/11
i
0/11
0/11
0/11
9/11
10/11
11/11
f
0/11
0/11
2/8
j
0/10
0/10
0/10
0/10
0/10
9/10
10/10
10/10
e
0/10
4/10
10/10
b
0/11
36/50
34/50
1/50
AUG
a
29/56
55/56
0/56
JUL
0/11
27/50
50/50
0/50
JUN
0/10
n.d.
30/50
46/50
0/50
MAY
j
47/55
50/55
0/55
APR
0/11
48/50
47/50
0/50
MAR
0/10
0/10
0/10
2/10
h
0/10
0/10
0/10
9/10
10/10
10/10
d
0/10
0/10
0/10
2/10
n.d.
26/50
49/50
0/50
FEB
32/51
25/51
0/51
SEPT
26/50
29/50
0/50
OCT
0/10
1/10j
1/10j
0/10
0/10
0/10
0/10
0/10
0/10
0/10
10/10
2/10
9/10g
0/10
0/10
10/10c
0/10
0/10
48/50
10/50
0/50
NOV
(Continues)
45/50
16/50
0/50
DEC
Gazda et al. 5
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HI ELISA ELISA ELISA ELISA SN SN SN SN PRNT PRNT PRNT PRNT
PPV*
PRCV
PRRSV*
PRV
TGEV
BVDV-1, -2
EMCV
VSV-IN
VSV-NJ
EEEV
VEEV
WEEV
WNV b
qPCR
PCMV
a
TEM qPCR
Enteric viruses
Culture
Salmonella spp.
Influenza A*
Culture
ELISA
PRRSV*
Brachyspira sp.
ELISA ELISA
TGEV
PRCV
Test method
Serum
Serum
Serum
Serum
Serum
Serum
Serum
Serum
Serum
Serum
Serum
Serum
Serum
Buffy coat
Nasal swab
Feces
Feces
Feces
Serum
Serum
Serum
c
Sample type
37/51
22/51
0/51
JAN
d
0/10
0/10
0/10
1/10j
3/10j 0/10
0/10 0/10
1/10j
0/10
0/10
0/10
0/10 0/10
3/10p
0/10
0/10
8/10
8/10
0/10
0/10
0/10
9/10
0/10
0/10 10/10l
9/10k
0/10
0/10
0/10
10/10 0/10
0/10
25/50
23/50
0/50
MAY
2/10
31/50
33/50
0/50
APR
c
35/49
14/49
0/49
MAR
0/10
0/10
45/50
0/50
0/50
FEB
46/50
20/50
0/50
JUN
48/50
14/50
0/50
JUL
e
0/10
2/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
8/10
4/10
10/10n
0/10
0/10
0/10
0/10
0/10
25/50
11/50
0/50
NOV
5/10j
30/51
15/51
0/51
OCT
j
21/50
6/50
0/50
SEPT
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
9/10
4/10
10/10m
0/10
0/10
10/10
c
0/10
0/10
38/47
9/47
0/47
AUG
19/48
7/48
0/48
DEC
*Vaccination, n.d. (not done), bacteriophage, few picornaviridae (n = 2), caudovirales, dilution factor (df): 128 (n = 1), 512 (n = 2), 1024 (n = 4), 2048 (n = 3), df: 512 (n = 3), 1024 (n = 1), 2048 (n = 3), 4096 (n = 3), fdf: 64 (n = 1), 1024 (n = 2), 2048 (n = 6), 4096 (n = 1), 8192 (n = 1), gdf: 256 (n = 1), 1024 (n = 1), 2048 (n = 3), 4096 (n = 2), 8192 (n = 2), hdf: 64, idf: 64 (n = 1), 128 (n = 1), jdf: 10, kdf: 256 (n = 2), 512 (n = 3), 1024 (n = 1), 2048 (n = 2), 8192 (n = 1), ldf: 256 (n = 2), 1024 (n = 3), 2048 (n = 3), 4096 (n = 2), mdf: 64 (n = 1), 512 (n = 1), 1024 (n = 1), 2048 (n = 2), 4096 (n = 1), 8192 (n = 1), ndf: 64 (n = 1), 256 (n = 1), 512 (n = 2), 1024 (n = 2), 2048 (n = 2), 4096 (n = 2), pdf: 32.
Quarterly sentinel
Monthly herd
2014
Target agent
T A B L E 2 (Continued)
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Gazda et al.
are the only animals housed long term (>2 years) at the facility. Blood,
of viral infection. The PRNT testing for EEEV, VEEV, WEEV, and WNV
nasal swabs, and fecal samples are collected from sentinel animals for
occurred at the National Veterinary Service Laboratory (Ames, IA, USA).
microbiological screening. Gilts selected from the nursery for breeding are first quarantined and qualified for introduction to the gestation facility. Once inside the breeding facility, breeding sows of all ages
2.3 | Porcine islet isolation and encapsulation
are candidates for the sentinel program. Trained boars used for heat-
Donor pancreases were procured from sows that were over 2 years
checking are not part of the sentinel program as they are not eligible
of age with a history of multiple parities. Following electrical stunning
for pancreas donation, although these animals are part of the routine
and exsanguination of the donor, abdominal viscera were retrieved
herd health screening performed monthly.
by a sterile-gowned technician and transferred to a sterile container for transport. The viscera were then placed in a custom built, HEPA-
2.2 | Postmortem microbiological screening of sentinel and donor animals
filtered, laminar flow work station for tissue retrieval. Surface monitoring was performed on the work surface prior to retrieval, and settle plates were placed inside the workstation during dissection to moni-
Following electrical stun and exsanguination at a USDA-regulated
tor for microbiological contamination. Intact pancreas was dissected
abattoir in compliance with all federal regulations concerning animal
from the viscera by a sterile-gowned technician and immediately
handling and welfare, numerous tissues including brain, heart, lung,
transferred to cold HBSS (CellGro, Manassas, VA, USA). The tissue
liver, tonsil, lymph node, spleen, ileum, kidney, pancreas, bone marrow,
was then surface-rinsed by submersion in cold povidone-iodine 10%
PBMCs, feces, and serum were collected and archived as fixed and fro-
solution (VetOne, Boise, ID, USA), followed by two submersion rinses
zen aliquots at the time of pancreas procurement. Aliquots of isolated
in cold HBSS. The pancreas was transported to the islet isolation labo-
islets were also frozen and archived. Donor animal tissue samples were
ratory in a sterile container submerged in cold HBSS. The container
sent to the University of Minnesota, Veterinary Diagnostic Laboratory
was then transferred to a biological safety cabinet where the pancreas
(MVDL) to screen for the bacterial and viral pathogens listed in Table 3.
was removed and a transport media sample collected for total biobur-
The MVDL is one of the three major swine diagnostic laboratories
den testing (Avista Pharma Solutions, Inc., Agawam, MA, USA). Warm
in the United States, and swine veterinarians use the MVDL’s assays
ischemia times (beginning of exsanguination to pancreas immersion in
to monitor heard health and identify new outbreaks of infectious
cold transport solution) ranged from 10 to 15 minutes. Cold ischemia
disease. The laboratory is a member of the National Animal Health
times (pancreas immersion in cold transport solution until placement
Laboratory Network (NAHLN) of the USDA. A complete qualification
in digestion chamber) ranged from 42 to 57 minutes.
report for all assays within the Herd Health Program, “Performance
Pancreases were perfused with 7.5 Wunsch U/g collagenase
Characteristics of Diagnostic Assays Used to Screen Source Animals
(Collagenase MA; Vitacyte, Indianapolis, IN, USA), 12 000 Neutral
for Xenotransplantation,” has been submitted to the FDA as part of
Protease U/g pancreas (BP Protease; Vitacyte), and 2.5 mg/pancreas of
an Investigational New Drug (IND) application for the use of agarose-
Pulmozyme (Genentech, South San Francisco, CA, USA) solution pre-
agarose encapsulated porcine islets to treat patients with type 1 dia-
pared in Cold Storage Purification Stock Solution (CellGro). Islet counts
betes. The qualification report contains detailed information on all
were expressed as islet equivalents (IEQ), based on a standard islet
diagnostic assays used to screen source animals and includes assay
size of 150 μm, and 500 IEQ were encapsulated in agarose-agarose
protocols and data regarding sensitivity, specificity, and repeatability.
macrobeads as previously described.18 Following quantification, islets
The MVDL assays are validated according to guidelines established
were separated into 2000 IEQ aliquots before being resuspended in
by the American Association of Veterinary Laboratory Diagnosticians,
0.5 mL of 0.8% (w/v) SeaKem Gold agarose (derived from Gelidium
and Standard Operating Procedures are available from the MVDL upon
species of seaweed; Lonza Rockland, Inc., Rockland, MD, USA) pre-
request (
[email protected]). Detection of the different target agents in-
pared in Minimal Essential Medium plus 25 m/mol L HEPES buffer
cluded the following methods: Bacterial Culture, Transmission Electron
(Sigma-Aldrich, St. Louis, MO, USA). The islet-agarose suspension was
Microscopy (TEM), Immunohistochemistry (IHC), conventional or
expelled beneath the surface of sterile mineral oil (Sigma-Aldrich) to
quantitative Polymerase Chain Reaction (PCR or qPCR, respectively)
produce four 0.125-mL spherical beads of approximately 5-6 mm in
and quantitative Reverse Transcription (qRT-PCR), Hemagglutination
diameter, each containing 500 IEQ. The 1st coat macrobeads were cul-
Inhibition (HI), Serum Neutralization (SN), Virus Isolation (VI), and
tured at 37°C in a humidified atmosphere of 5% CO2. After 3-5 days,
Plaque Reduction Neutralization Test (PRNT). In addition, virus isola-
a second coat of 5% SeaKem Gold agarose was applied, giving each
tion assays were performed on the infection susceptible cells Madin-
macrobead a final diameter of 8-9 mm. The determination of the av-
Darby canine kidney (MDCK) cells, porcine alveolar macrophages
erage pore size of the agarose macrobeads is difficult at best, given
(PAM), primary porcine kidney cells (PPK), porcine kidney cells (PK-15),
the gelation mechanism of random polymer coils forming aggregated
African green monkey kidney cells (MARC-145), and bovine turbinate
double helices, which results in pores of various diameters with ill-
cells (BT) to look for unknown porcine viruses. Each cell type was cul-
defined walls. For this reason, the macrobead has not been considered
tured in the presence of tissue homogenate (brain, ileum, kidney, lung,
to provide absolute viral sequestration although the encapsulated is-
liver, mesenteric lymph node, spleen, and tonsil) from sentinel animals
lets are protected from direct immune cell contact. Agarase, the en-
or donor animals and monitored for morphological changes indicative
zyme necessary to break down agarose, is abundant in ocean-dwelling
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Gazda et al.
8
T A B L E 3 Sentinel and pancreas donor postmortem screening 2013
2014
Target agent
Test method(s)
Sample type
Sentinels
Pancreas donors
Sentinels
Pancreas donors
Aerobic
Culture
Lung
0/3
0/6
n.d.
0/2
Brachyspira sp.
Culture
Feces
0/3
0/7
n.d.
0/1
Salmonella spp.
Culture
Feces, mesenteric lymph node, spleen, tissue poola
0/3
0/7
n.d.
0/2
Leptospira*
IHC
Kidney (formalin-fixed)
0/3
0/7
0/3
0/4
L. intracellularis*
IHC
Ileum (formalin-fixed)
0/3
0/7
0/2
0/4
TGEV
IHC
Ileum (formalin-fixed)
0/2
0/7
0/3
0/3
b
c
Enteric viruses
TEM
Feces
2/3
1/7
0/3
1/3d
Influenza A*
qRT-PCR
Lung
0/3
0/7
0/3
0/4
PCMV
qPCR
Buffy coat
0/3
0/7
0/3
0/4
BVDV-1, -2
qRT-PCR
Tissue homogenatee
0/3
0/6
0/3
0/4
BVDV-1, -2
VI (culture: BT)
Tissue homogenatee
0/3
0/7
0/3
0/4
EMCV
VI (culture: BHK)
Serum, heart
0/3
0/7
0/3
0/4
PHoV
qPCR
Pancreas
n.d.
2/7
0/1
1/1
PHoV
qPCR
Mesenteric lymphnode
1/3
2/7
1/3
3/3
e
PERV-A
qRT-PCR
Tissue homogenate
3/3
7/7
3/3
4/4
PERV-B
qRT-PCR
Tissue homogenatee
3/3
7/7
3/3
4/4
qRT-PCR
e
2/3
5/7
3/3
4/4
0/7
0/3
0/4
PERV-C
Tissue homogenate
e
PRRSV
qRT-PCR
Serum, tissue homogenate
0/3
PRRSV*
ELISA
Serum
2/3
7/7
3/3
3/4
PRCV
ELISA
Serum
2/3
6/7
2/3
2/4
PRV
ELISA
Serum
0/3
0/7
0/3
0/4
TGEV
ELISA
Serum
0/3
0/7
1/3
0/3
VSV-IN
SN
Serum
0/3
0/7
0/3
0/4
VSV-NJ
SN
Serum
0/3
0/7
0/3
0/4
EEEVf
PRNT
Serum
1/3g
0/7
1/3g
0/4
PRNT
Serum
0/3
1/7g
1/3g
0/4
g
0/3
0/4
VEEVf WEEV
f
f
PRNT
Serum
0/3
2/7
PRNT
Serum
0/3
0/7
0/3
0/4
PPV*
HI
Serum
3/3h
6/6i
3/3j
4/4k
PHEV
HI
Serum
WNV
Porcine viruses
Culture: MDCK
2/2l
3/3m
n.d.
n.d.
n
0/3
0/7
0/3
0/4
n
Tissue homogenate
Porcine viruses
Culture: PAM
Tissue homogenate
0/3
0/7
0/3
0/4
Porcine viruses
Culture: PPK
Tissue homogenaten
0/3
0/7
0/3
0/4
n
Porcine viruses
Culture: PK-15
Tissue homogenate
0/3
0/7
0/3
0/4
Porcine viruses
Culture: MARC-145
Tissue homogenaten
0/3
0/7
0/3
0/4
n
0/3
0/7
0/3
Porcine viruses
Culture: BT
Tissue homogenate
a
b
0/4 c
*Vaccination, [n.d. (not done)], colon, ileum, liver, spleen, lung, and mesenteric lymph node, coronavirus (n = 1), caudovirales (n = 1), small round viral particle, ddilution factor (df): 10, ekidney, lung, mesenteric lymph node, tonsil, and spleen, fTesting performed at National Veterinary Service Laboratory, g bacteriophage, hdf: 512 (n = 1), 1024 (n = 2), idf:1024 (n = 2), 2048 (n = 2), 8192 (n = 2), ≥8192 (n = 1), jdf: 1024 (n = 1), 2048 (n = 1), 4056 (n = 1), kdf: 512 (n = 2), 2048 (n = 2), ldf: 20 (n = 1), 40 (n = 1), mdf: 40 (n = 1), 320 (n = 1), 640 (n = 1), nheart, intestine, liver, pancreas, kidney, lung, mesenteric lymph node, tonsil, and spleen.
bacteria but has not been found in mammals.19 The macrobeads, in the
Macrobeads were then cultured at 37°C in a humidified atmosphere
absence of trauma, remain intact indefinitely following implantation in
of 5% CO2 until collection for microbiological screening or implanta-
the abdominal cavity.
tion. Culture medium (RPMI 1640; Life Technologies, Grand Island,
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9
Gazda et al.
NY, USA; pre-screened for endotoxin
