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UNIVERSITY OF HAWA'" LIBRARY
SURVEY OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS IN CATTLE EGRETS (BUBULCUS IBIS) ON O'AHU
A THESIS SUBMITTED TO THE GRADUATE DMSION OF THE UNIVERSITY OF HAW AI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF
MASTER OF SCIENCE
IN
ANIMAL SCIENCES
NOVEMBER 2006
By Barbara Tang Maresca
Thesis Committee:
Ha1ina M. Zaleski, Chairperson James R. Carpenter
Joan C. Dobbs Larry C. RawSon
We certity that we have read this thesis and that, in OUT opinion, it is satisfactory in scope
and quality as a thesis fur the degree of Master of Science in Animal Sciences.
THESIS COMMITTEE
ii
ACKNOWLEDGMENTS
I thank:
HaIina M. Zaleski, Ph.D. for advice on the project and funding for travel to South Dakota
State University for education of the assays
Eric A Nelson, Neal H. Ferrin and South Dakota State University, Animal Disease
Research and Diagnostic Laboratory for the performance of the assays and review
of the thesis
James R. Carpenter, Ph.D., UHM, Department ofHNFAS, Graduate Conunittee Member
for advice on the project
Joan C. Dobbs, Ph.D. UHM, Department ofHNFAS, Graduate Conunittee Member for
advice on the project,
Larry C. Rawson, D.V.M, United States Department of Agricu1ture, Veterinarian for
advice on the project,
Bradley R. LeaMaster for the equipment and supplies necessary to collect and store
Samples
Arlene Buchholz for providing information on porcine reproductive and respiratory
syndrome virus
Stephanie L.K. Bennett for assistance with collection of insect samples from cattle egrets
Steve M Tanaka for the culling of cattle egrets at Lualualei
Michael Richardson for help in preservation of insect samples
Richard Tsuda for the identification of insects
Matt, my husband; his fimrily, and my fimrily for their support through the years
iii
ABSTRACT
Porcine reproductive and respiratory syndrome (PRRS) drastically affects the
profitability of pig production by causing reproductive and respiratory difficulties. The
virus can infect pigs directly and indirectly. Airbome transmission is suspected, but
controlled experiments have yielded mixed results. The ability of animals to transmit
virus bad been demonstrated in MaI1ard ducks, houseflies and mosquitoes. On some hog
farms worldwide and on O'ahu where PRRS occur despite strict biosecurity, the route of
transmission remains a mystery. The objective of this project was to determine the PRRS
status of cattle egrets, who are frequent visitors to livestock farms. Sera from 24 cattle
egrets and 2 spotted doves (incidentally) culled by the USDA in West O'ahu were tested
fur PRRS virus antibodies. Blocking enzyme-linked immunosorbent assay results fur 2S
serum samples were negative and I dove sample was borderline positive with percent
inhibition at 17.34% (positive ~17%). Fluorescent fucusing neutralization resu1ts on 21
serum samples yielded eight negatives «1:4 dilution), 10 borderlines (1:8 dilution), and
3 low positives (1: 16 dilution). Western blotting and reverse transcription polymerase
chain reaction were unable to detect viral antibodies or nucleic material, respectively.
Low positives in serological tests due to cross-reactions with other viruses and fulse
negatives in the polymerase chain reaction due to variation in strains ofPRRS virus
cannot be ruled out. These results do not provide evidence that cattle egrets are potential
carriers of the PRRS virus, but a controlled challenge study should be considered.
Identifying and controlling routes of transmission ofPRRS is vital in protecting
Hawai'i's pork industry.
iv
TABLE OF CONTENTS
Acknowledgments .......................................................................................................... iii Abstract ..............................................................................•............................•.............. iv List of Table ..................................................•.......................•..•..•.....•................•.......... vii
. f' .. List 0 Figures ............................................................................................................... V11
List of Abbreviations .................................................................................................... viii Chapter I: Literature Review .................•......................................................................... 1
Hawai'i Pork Industry .....................•............................................................................ 1 Porcine Reproductive and Respiratory Syndrome ................•...•.................................... 7
History .............................................................•........................................................ 7 Virus Classification .................................•..•..•...........•.................•................•............ 8 Virus Characteristics ................................................................................................. 9 PRRSV Strains ....................................................................................................... 11 Vaccines ................................................................................................................. 13 Clinical Signs ......................•..............................................................•.....•.............• 15 Effects on Sows and Newborns .........................................•..................................... 16 Atypical PRRS ........................................................................................................ 17 Effects on Boars .................•..........................•.....••......................•........•.••............... 18 PRRS and Immunology .••................................•..........................•..•.••.••.................. 18 Virus Research ........................................................................................................ 20 Serology ................................................................................................................. 20 Western Blotting ..................................................................................................... 22 RT -PCR and Swine Bioassay .................................................................................. 22 PRRS Virus Persistence .......................................................................................... 23 PRRS Virus Transmission ....................................................................................... 23
Direct Transmission ............................................................................................. 24 Vertical Transmission .........•..•.....................................................•....................... 27 Indirect Transmission .......................................................................................... 28 Airborne Transmission ........................................................................................ 30 Animal Transmission ........................................................................................... 31
Cattle Egrets (Bubulcus ibis) ...................................................................................... 33 Chapter IT: Materials and Methods .....•••..................................................................... 36 Permits ....................................................................................................................... 36
Sample Collection .•.................•..•..•..................................................•...................... 36 Blocking Enzyme-Linked Immunosorbent Assay .................................................... 40 Fluorescent Focus Neutralization ..................................................................•......... 41 Western Blotting ..................................................................................................... 43 Reverse Transcription Polymerase Chain Reaction ................................................. 43
Chapter III: Results and Discussion ............................................................................... 44 Cattle Egrets .............................................................................................................. 44 Insect Identification. .................•........•..•............................................•..•..•................• .45 Blocking Enzyme-linked Immunosorbent Assay and Fluorescent Focus Neutralization .................................................................................................................................. 47
Western Blotting & Reverse Transcription Polymerase Chain Reaction ..................... 51 Technical Limitations .......................................•...........•..............•.............•................ 54
v
Flll1her Studies 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 •• 00 ..................... 0 •• 0 •••••••••••••••••••••••••••••• 0 ... 0 •••••• 0 ••••••••••••••••• 55 Chapter IV: COnclusionlImplications ............................................................................. 57
Economic Impacts ....••..•................................•..•........•..•......................•.••..•............. 57 Control Measures .................................................................................................... 58
Appendix ...................................................................................................................... 61 References .........••....•.......................•..•......................................•................................... 62
vi
LIST OF TABLES
Table l. The prevalence of porcine reproductive and respiratory ..................................... 6 Table 2. Porcine reproductive and respiratory syndrome virus proteins. Open reading
frames (ORFs) dictate the ....•........................................................................... 10 Table 3. Cattle egret and spotted dove weights .............................................................. 44 Table 4. Orders of insects identified from 19 cattle egrets •••••.•••••.•.•••••••.•••••••••••.••.•.••.••.• 46 Table 5. Scientific names of insects identified from two cattle egrets by Richard ........... 46 Table 6. Results for blocking enzyme-linked immunosorbent assay (bELlSA) and
fluorescent focus neutralization (FFN) •..•..•....•..••••••••••••.••••••••.................•...•••• .49 Table 7. SlImmaryofresults .......................................................................................... 52 Table 8. Comments on avian dissection. .........•..•................................•.....•.................... 61
LIST OF FIGURES
Figure l. Hawai'i swine inventory from 1984-2003 ....•.........................•..•.•.................... 3 Figure 2. Map ofO'ahu with inset showing infected areas of West O'ahu in 1996 .......•.. 5 Figure 3. Prevalence of porcine reproductive and respiratory syndrome (PRRS) in the
State ofHawai'i 2000-2001 ............................................................................. 7 Figure 4. Porcine reproductive and respiratory syndrome virion ................................... 11 Figure 5. Location of bird collection ...........................................................................•. 37 Figure 6. Destination of cattle egret and spotted dove crop contents ............................. 39 Figure 7. Use of avian serum samples in various assays .............••.•.........................•..... 52
vii
LIST OF ABBREVIATIONS
ABTS-2, 2' -azino-bis(3-ethylbenzthiazoJine-6-sulfonic acid ACB-antigen coating buffer ATCC-American Type Cuhure Collection BSA-bovine serum albumin bELISA-blocking enzyme-linked immunosorbent assay bp-base pairs C-Celsius CA-Califurnia CD-cluster of differentiation DNA-deoxyribonucleic acid ELISA-enzyme-1inked immunosorbent assay EVA-equine vira1 arteritis FFN-fluorescent fucus neutra1ization FFU-fluorescent fuci unit (s) g-gram(s) GP-glycoprotein h-hour (s) HASS-Hawai'i Agricuhural Statistics Service HDOA-Hawai'i Department of Agriculture IACUC- Institutional Anirnal Care and Use Committee IF A-indirect fluorescent assay kb-kilobase kDa-ki1oda1ton LDV -lactate dehydrogenase-elevating virus M-membrane M-Molar MD-Maryland ME-Maine MEM-Minimum Essential Medium min-minute MLV-modified live virus mRNA-mitochondria1 noonucleic acid MSD-mystery swine disease N-nucleocapsid NCTAMS-Naval Computer and Telecommunications Area Master Station run-nanometer OD-optica1 density OlE-Office Internationale des Epimoties ORF-open reading frame PBS-phosphate buffer solution PCR-polymerase chain reaction PI -percent inhibition PRRS-porcine reproductive and respiratory syndrome PRRSV-porcine reproductive and respiratory syndrome virus
viii
RNA-nbonucleic acid SDS-PAOE-sodium dodecyl sulfute polyacrylamide gel electrophoresis SDSU-South Dakota State University SNA-serum neutralizing antibody TOE-transmissible gastroenteritis TCID-tissue culture infectious dose TNF-tumor necrosis fuctor p.l-microliter VI-virus isolation VT-Vermont WB-Western blotting wtlvol-weightlvolume
ix
ClIAPfERI LITERATURE REVIEW
Hawai'i Pork Industry
The pork industry has tremendous cultural, environmental, and economic impacts
on the state ofHawai'i Since the Polynesians introduced pigs to Hawai'i around 300
A.D. (Abbott, 1992), fresh pork have been the main dish for many local traditional
celebrations including baby's first birthday and Chinese New Year parties.
The pork industry is also important to the local environment. Unmarketable
produce from supermarkets and fuod waste from restaurants may be recycled by pig
farmers as feed. Pig manure, along with urine, has the potential to be composted into
fertilizer (Sharma et al., 1996).
Most producers in Hawai'i raise their swine from birth to market but a few have
only feeder or only finisher pigs (Sharma et al., 1996). Hawai'i's pork producers
primarily use a system of confinement, with a few open pastures, or a combination of
both to manage their swine. Due to the high cost ofland in Hawai'i, most producers do
not have the space to house animals of different ages in different units. As a resuh, most
producers are furced to practice a continuous flow system which often leads to the
housing pigs of various ages closely together (H. M Zaleski, personal communication).
Although land issues exist, the industry can still bring in an annual income of$4 to 5
million to the state ofHawai'i in farm gate value [Hawai'i Agriculture Statistics Service
(HASS), 2006a] and provide direct employment as well as related opportunities in
veterinary medicine, pork processing and marketing, feed sales, and other areas (Sharma
et al., 1996).
I
Despite the opportunities generated through the selling of pigs, Hawai'i's swine
industry is struggling to compete with mainland pork. The local contnbution of swine to
Hawai'i's market has been slowly decreasing since 1970 (HASS, 2006b) while in the 48
contiguous states there has been a steady but very small increase in production (Pork
Checkoff; 2006). As seen in Figure 1, there are three dips in the years that are clearly
attnbutable to Hurricane 'Iniki that hit Kaua'i in 1992, a Young Brothers freight rate
increase and porcine reproductive and respiratory syndrome virus (PRRS) outbreak on
O'ahu in 1996 and a transmissible gastroenteritis outbreak on O'ahu in 2000 (H.M
Zaleski, personal communication; HASS, 1998). Sharma et a1. (1996) found that not
only the competitiveness of the market but also high land and feed costs, waste and
environmental regulations and swine diseases, especially true for porcine reproductive
and respiratory syndrome, are added deterrents to potential swine producers.
Porcine reproductive and respiratory syndrome arrived to the state of Hawaii on
two separate occasions. In 1992, PRRS was diagnosed on the island ofMaui based on
symptoms observed during the time of outbreak. In July and August of 1996, a storm of
abortions on O'ahu prompted ajoint investigation among the University ofHawai'i.
Cooperative Extension Services, Hawai'i Department of Agriculture (HDOA) and private
veterinarians to determine the cause. In both incidences, it was found that PRRS arrived
via untested breeding stock (LeaMaster and Zaleski, 1996; Zaleski et at, 1996).
2
Swine Inventory ofStllte of Hawai'i Counties
40 PR:RS
35 '" 0.0 30 0 = ... 25 0
'" 'C 20 = os '" 15 = 0 ..c
10 ... 5
Jb
/\ Freight Tr. '"
"'\ --O'ahu
'-.. .I>--.. --MauiIMoloka'ilLana'i 'flniki \ r --- Hawai ' i
~ ~ ----.- Kaua'i
... '\.- .. ~ ~
r ..... ~~ 0
1980 1985 1990 1995 2000 2005
Years
Figure I. Hawai'i swine inventory from 1984-2003. Hurricane 'Iniki, porcine reproductive and respiratory syndrome (PRRS), a freight increase and transmissible gastroenteritis (TGE) all had adverse effects on the islands' swine inventory. * Adapted from graph by H.M. Zaleski based on Hawai'i Agriculture Statistics 1988, 1994, 1996, 1998, 2000, 2003.
During the 1996 outbreak, 18 out of approximately 20 swine farms heavily
concentrated in Mikilua Valley on West O'ahu were infected with PRRS (Nakatan~
2002; Bakutis, 1996) (Figure 2). Approximately 90% of the 2,500 sows in the valley
were infected. Farmers recorded losing one-third to all of their newborns in a litter
during the peak of infection in July and August (L.C. Rawson, personal communication;
Zaleski et al. , 1996). Pigs were reported to be feverish, lethargic, had difficulty breathing
and succumbed to secondary infections (Bakutis, 1996; Zaleski et a1., 1996).
Reproductive failures included stillbirths, abortions, premature farrowing and preweaning
mortality (Moniz, 1996). About 5% of the infected sows died, with fevers reported as
high as 41. 7°C (Zaleski et aI. , 1996). This outbreak was estimated to cost the swine
3
producers $2.75 million, not including the loss due to persistent and secondary infections
in weaned and growing pigs, feed costs and vaccinations. Several hog farms have closed
due to the PRRS outbreak (L.C. Rawson, personal communication).
The movement of contaminated air and infected animals throughout the valley
were the suspected furms of transmission (Moniz, 1996). Despite the adaptation of strict
biosecurity practices, farms within close proximity of one another were still affected
(Zaleski et a1., 1996), prompting the state veterinarian to restrict the movement of swine
from O'ahu in September (Lum, 1996).
4
E=:::::::r~1 1 0 km 15mi C!) 2 006 Y~hoo! Inc
S .. y
,-00-.--
M .. un .. luil S .. y
©2006 N A.VTEQ
Figure 2. Map ofO'ahu with inset showing infected areas of West O'ahu in 1996. Red clouds highlight areas ofheavy porcine reproductive and respiratory syndrome infection in 1996. Longitude: 1580 10' West; Latitude: 21 0 23' North. *The map and its contents are used with permission from MapQuest and NAYTEQ.
Despite assistance provided by HDOA and private veterinarians to install a
modified-live PRRS virus (PRRSY) vaccination program, many producers still felt
(
hopeless and were emotionally, as well as financially, exhausted from battling PRRS
(Moniz, 1996; Rodrigues, 1996; Zaleski et aI., (996). Although the pounds of pigs sent
to market in 1998 indicated a financial rebound from the PRRS outbreak (HASS, 1998),
the state ofHawai' i has not been PRRS-free since 1996 (H.M. Zaleski, personal
5
communication). A 2000-2001 serological survey done by HOOA reported that the
highest prevalence ofPRRS was on O'ahu where 59% of the farms and 35% of the swine
tested positive. PRRS was least prevalent on East Hawai'i where 6% of the farms and
3% of swine tested positive (Table I). Kaua'i remained the only island PRRS-free
(Nakatani, 2002; L.C. Rawson, personal communication) (Figure 3). As a result, in
December 2001, HDOA implemented a new quarantine for Kaua'i. The new quarantine
requires approval from HOOA prior to the movement of swine to Kaua'i (FoppoJi, 2001).
Table 1. The prevalence of porcine reproductive and respiratory syndrome (PRRS) in the state ofHawai'i in 2000-2001.
% % Fanns Fanns Fanns Swine Swine Swine Tasted Positive Posltiva Tasted Positive Posltiva
O'ahu 69 41 59 1480 524 35 Maul 24 10 42 457 103 23 west Hawal'l 11 4 36 195 53 27 Moloka'i 23 2 9 220 18 8 East Hawarl 18 1 6 289 8 3 Kaua'i 27 0 0 313 0 0
*Dsts from Hawai'i State Department of Agriculture, Anima1 Industry Division, Vector Control Branch from 2000-2001.
6
Kaua'j
O'ahu • H6nolulu Moloka'j
• Maui
Hawai'i Porcine Reproductive and
Respiratory Syndrome 2000-2001 .. • State Survey
Positive Locations
E==~~'1 00 km ' 50mi
@2000 Yah oo! Inc @2006 NAVTEQ Figure 3. Prevalence of porcine reproductive and respiratory syndrome (PRRS) in the State ofHawai'i 2000-2001. Red areas were PRRS positive. Longitude: 1540 47' West; Latitude: 21 0 18' North. ·Data from Hawai'i State Department of Agriculture, Animal Industry Division, Vector Control Branch. The map and its contents are used with permission from Yahoo! and NAVTEQ.
Porcine Reproductive and Respiratory Syndrome
History
PRRS was initially called Mystery Swine Disease (MSD) due to a worldwide
ignorance of its origin, effects, causative agent, treatment, and transmissibility. The first
cases of PRRS were documented in 1987 in the United States and in 1990 in Europe
(Zimmerman, 2003a). The emergence ofthese two isolates is hypothesized to be from
the same ancestors but have long diverged into independent lines (Plagemann, 2003).
According to the Office Intemationale des Epizooties (OlE), by 1991, MSD spread
7
quickly throughout EW'Ope: the Netherlands in January; Belgium in March; Great Britain
in May; Spain in October; and France in November (Zimmerman, 2003a; Wensvoort,
1994).
As the disease spread, many names evolved. The mystery swine disease was also
known as blue ear disease, blue-eared pig disease, mystery pig disease, new pig disease,
porcine epidemic abortion and respiratory syndrome, and porcine reproductive and
respiratory syndrome (PRRS). In 1991, DIE acknowledged porcine reproductive and
respiratory syndrome as the official name for the disease (Zimmerman, 2003a;
Wensvoort, 1994).
The mystery ofPRRS began to unravel in 1991 when Wensvoort et al., (1991)
isolated the Lelystad virus, a EW'Opean strain, as the cause. Collins et al. (1992) later
isolated the American strain [ATCC (American Type Culture Collection)] VR-2332) of
the PRRS virus and Dea et al. (1992) isolated another strain in Canada antigenically
similar to PRRS viruses from EW'Ope.
Virus Classification
Porcine reproductive and respiratory syndrome virus is a single-stranded.
positive-sense nbonucleic acid virus belonging to Order Nidovirales and Family
Arteriviridae in the genus Arterivirus (Meulenberg et al., 1993). It is classified in the
same filmilyas lactate dehydrogenase-elevating virus (LDV) in mice, equine viral
arteritis (EVA) virus and simian hemorrhagic fever virus (Conzelmann et al., 1993;
Meulenberg et al., 1993). The classification was based on similar genetic expression,
molecular structures and organization, host specificity, replication strategies and
pathology (Conzelmann et al., 1993; Dee et al., 2000; Nelson et al., 1993; Rossow, 1998;
8
Meulenberg et ai, 1993; Dea, et ai, 2000). This family ofvirus is known to replicate in
host monocytes, especially macrophages, and cause reproductive and/or respiratory
failures (Meulenberg et ai, 1993).
Virus Charaeterlsties
Like other arteriviruses, the PRRS virus has open reading frames (ORFs) that
control the expression of various viral proteins (Table 2) (Meulenberg et ai, 1993;
Conzelmann et ai, 1993). PRRSV has a total of eight ORFs. ORFs 1a and 1 b code fur
replicases and polymerases while the other six codes fur structural proteins. ORFs 2-4
express minor glycosyIated membrane proteins (GP24); ORF 5 codes fur a major 25 kDa
glycosyIated membrane protein (GPs); ORF 6 is responsible fur the expression ofan 18-
19 kDa nonglycosyIated membrane (M) protein; and ORF 7 codes fur a 15 kDa
hydrophilic, nucleocapsid (N) protein (Meu1enberg et ai, 1993; Dea et ai, 2000;
Conzelmann et ai, 1993).
Generally, the PRRS virus (Figure 4) consists of a lipid bilayer surrounding a
nucleocapsid core (Conzelmann et ai, 1993; Wensvoort, 1994; Mardassi et ai, 1994).
The PRRS virus has a diameter ranging from 48-83 nm and a core from 25-30 nm. The
major proteins M and GPs and the minor proteins GP2 and GP4 furm the lipid bilayer.
The N proteins are the major proteins that dimerize around a 15 kb nbonucleic acid,
funning an icosahedral core (Conzelmann et ai, 1993; Allende et ai, 1999). The
function of the minor protein GP3 is uncertaio but studies indicate a possible neutralizing
antibody-stimulating effect. High genetic variation in the ORF3 is a possible reason fur
the protein's immunogenic properties (Dea et ai, 2000).
9
Table 2. Porcine reproductive and respiratory syndrome virus proteins. Open reading frames (ORFs) dictate the expression of replicase and polymerase, glycoproteins 2-4 (GP2-4), two minor membrane proteins (GP, and M) and a hydrophilic nucleocapsid protein (N).
Type Functional Structural Proteins
Proteins
Minor Proteins Major Proteins
ORF la I Ib 2 3 4 5 6 7
Codes for Replicase & 29-30kDa 42-50kDa 31-35 kDa 24-26kDa 18-19kDa 14-15kDaN polymerase GPz GP3 GP4 GP, Mproteins proteins
Importance RNA Uncertain, Possibly Possibly Potential to Possibly PRRS replication perhaps stimulate stimulate make DNA stimulate Diagnostic
for viral neutralizing neutralizing vaccine & neutralizing assays; replication antibodies antibodies stimulate antibodies Vaccine;
effect in pig effect in pig neutralizing effect in pig Possibly antibodies stimulate
effect in pig neutralizing antibodies
effect in pig
"'Adapted from Meulenberg et 81, 1993; Conzelmann et 81, 1993; Dea et 81, 2000; Wootton et 81, 1998; Yang et 81, 2000.
10
I MProtan ~~I---~-'
N p'orein
Figure 4. Porcine reproductive and respiratory syndrome virion. Its components are rnRNA, glycoproteins 2-5 (GP2-5), two membrane proteins (GP5 and M), and a nucleocapsid (N) protein. • Adapted from Dea et a!., 2000.
Research on PRRS viral proteins provides insight into the different techniques
that may be used in the diagnosis and treatment of pigs infected with PRRS. The N, M
and GPJ-5 proteins have the potential to elicit neutralizing antibody effects in pigs (Dea et
aI. , 2000; Yang et aI. , 2000). GP) proteins also have the potential to be used in DNA
vaccines through the use of plasm ids and Escherichia coli (Dea et al., 2000; Kwang et aI.,
1999).
PRRSV Strains
Similarity in PRRS strains is due to the conservation of protein and genetic
structures. Nelson et al. (1993) noticed that the N protein is one of the most conserved
proteins amongst the strains ofPRRSV. They were ab le to produce two monoclonal
antibodies, SDOW I2 and SDOWI7, which recognized both the American and European
epitopes of the conserved N, a protein that has 58% similarity in genetic sequences
(Meng et al., 1994). Other studies have demonstrated similarity in protein and genetic
11
structures that can serve as the fuundation fur an effective PRRS vaccine. (Wootton et al.,
1998; Verheije et al, 2001; Meng et al., 1994; Yang et al., 1999; Meu1enberg et al.,
1998).
Diversity in protein and genetic structures can lead to many different strains, even
within a small population. Three different strains have been fuund on one farm with
1750 sows (Dee et al., 2001). In Japan, 37 different strains were fuund affecting only one
prefecture (Itou et al., 2001). Despite originating from the same ancestors, the North
American and the European strains have demonstrated high percentages of genetic,
antigenic and structural differences (Nelsen et al., 1999; Stadejek et al., 2002; Murtaugh
et al., 1995). For example, Allende et al.'s (1999) study showed the genetic differences
between the North American strain 16244B and Lelystad virus, one of the European
strains, by demonstrating a 53% and 31.7% difference in the amino acid sequences of
their ORFI a and ORF2a proteins, respectively. The differences in genetic structures are
likely to lead to different effects. Differences of 47-72% in the amino acid sequences of
ORF2_7'S may have led to a more virulent American strain, ATCC VR 2385 (Meng et al.,
1994).
Genetic and antigenic changes leading to new strains have been observed in
multiple pig-ta-pig passages over a period of367 days (Chang et al., 2002). Antigenic
changes are not uncommon to the PRRS virus. Monoclonal antibody studies have shown
that certain regions of the viral proteins are extremely mutational and are the basis fur
new strains when under pressure. As new strains evolve due to divergent genetic
changes, new strains can also emerge due to recombination of genetic material. Given the
opportunity to coexist, different strains with common ancestors are more likely to
12
recombine their genetic makeup than strains with unrelated ancestors (van Vugt et aI.,
2001). The diversity in PRRS strains makes it extremely difficult to devise a broad and
effective vaccine against all strains ofPRRS (Nelson et aI., 1993; Drew et aI., 1997;
Rowland et aI., 1999).
In 2001, the University of Minnesota confirmed that O'ahu farms were affected
by an American strain and Maui farms were affected by a Emopean strain of virus
(which came to be called "EmoPRRS") with the occurrence of the latter strain in the
United States being extremely rare. The American PRRS strain on O'ahu, predominately
in Mikilua Valley, caused severe outbreaks while the EmoPRRS strain caused little
disturbance in production on Maui (HDOA, 2001; L. C. Rawson, personal
communication). A study by Halbur et aI. (1995) noted that pigs infected with the
Lelystad virus, a European strain, displayed milder and more transient clinical signs in
comparison to an American strain.
Vaccines
Different strains ofvirus can lead to different efficacies of vaccines, making the
use of monoclonal antibody vaccines controversial (Dee et aI., 2001; Itou et aI., 2001;
Bmner et aI., 1997; Meng, 2000). An inactivated-virus vaccine derived from a Spanish
strain was able to show a marked difference in healthy piglets born; a 70% piglet survival
rate from intranasally vaccinated sows in comparison to a 10"10 piglet survival rate from
unvaccinated sows (Plana-Durin et aI., 1997). In the Netherlands, a survey showed that
some breeders were dissatisfied with a modified-live virus (ML V) vaccine because they
did not notice a significant difference in sow performance with the use of vaccines
(Bouwkamp, 1999). The vaccination of sows with a ML V vaccine during gestation was
13
linked to stillborns, mummified pigs, and lower birth rates and pigs weaned per litter
(Deweyet al., 1999; Mengeling et al., 1998b). Modified-live virus PRRS vaccines,
derived from the American PRRS strain, have been known to have amino acid changes or
structura1 changes in their proteins that resulted in a virulent vaccine virus (Yang et al.,
1998; Keyet al., 2001). In 1996, previously PRRS-negative sows and herds were
detected with the North American PRRS virus after vaccination with an American-based
ML V (ATCC VR-2332) vaccine (Blllner et al., 1997; Nielsen et al., 2001, 2002;
Storgaard et al., 1999; Mortensen et al., 2002). Sows with low titers of antibody against
PRRSV placed together with vaccinated sows were also susceptible. Vaccinated boars
would shed the vaccine virus in their semen and infect sows that were inseminated with
that semen (Blllner et al., 1997). Despite this modified-live American PRRS virus's
reversion to virulence, Blllner et al. (1999) have shown that it can help to boost the
antibody level of a pig infected with the European strain. Christopher-Hennings et al.
(1997) noted that the best response to a vaccine is one that has a viral genetic base
homologous to that of the infecting virus.
Deoxyribonucleic acid vaccines have also been investigated in attempts to combat
PRRS. With DNA vaccines, specific genetic sequences would be injected into the animal
via a plasmid vector to generate a humoral or cell-mediated immunity (Kwang et al.,
1999; Donnelly et al., 1997). Kwang et al. (1999) used genetic primers ofORF's 4,5,6
and 7 to express antibodies against PRRS in young pigs. Despite the success ofKwang
and his team, DNA vaccines still require more detailed research since their use can lead
to dramatic, unwanted and even futal cell replicating consequences (Kwang et al., 1999;
Pir7.adeh and Dea, 1998).
14
In 1993, the first ML V vaccine was developed and approved fur the use on pigs
infected with PRRS virus (Dee, 1996). Ingelvac® PRRS ML V vaccine, based on an
American strain ofPRRS virus, is a vaccine currently sold by NOBL
Laboratories/Boehringer Ingelheim, the initial producer of the vaccine (Dee, 1996;
Boehringer Ingelheim, 2006). The vaccine is currently approved fur pigs 3 weeks of age
or old, but not boars. Christopher-Hennings et al. (1997) showed that the use ofMLV
vaccines could resu1t in the shedding of the vaccine virus in semen although shedding of
the wild-type virus was reduced.
Clinic:al Signs
The PRRS virus has different effects on swine herds depending on the strain of
virus, time of infection, housing and external environments, degree of herd immunity and
herd size. Different strains can cause different clinical signs ofpRRS. Mengeling et al.
(1996) noted that the less virulent strains such as the Lelystad virus and theATCC YR-
2431 strain produced mild fevers, and abnormal rapid and labored breathing. The more
virulent American strains such as ATCC YR-2385 result in additional clinical signs such
as high fevers, lethargy, anorexia, and bluing of the skin (Mengeling et al. 1996). Even
the strain used to make vaccines can emerge into a new straio that leads to different
clinical signs ofPRRS (Mengeling et al., 1999). In general, PRRS elicits the common
signs of a viral infection in pigs such as lethargy, anorexia, depression, and fever
(Wensvoort, 1994; Collins et al., 1992). PRRS virus destroys alveolar macrophages,
phagocytic cells that are part of the body's immune system. The destruction leads to
impaired immunity and secondary infection by opportunistic bacteria such as
Haemophilus parasuis, Streptococcus suis, Pasteurella multocida, and Actinobacillus
15
pleuropneumoniae (Goldberg et a1., 2000). Pulmonary problems such as rapid and
labored breathing and, in severe cases, lesions of the lungs have been noted (Wensvoort,
1994; Collins et a1., 1992). Infected boars can experience reduced h"bido and shed the
virus in their semen (Christopher-Hennings and Nelson, 1996; Christopher-Hennings et
a1., 1997; Swenson et a1., 1994; Wills et a1., 199780 1997b), while infected pregnant sows
can have very complicated and costly reproductive fuilures (Mengeling et a1., 1995b;
Collins et a1., 1992).
Effects on Sows and Newborns
In addition to the common signs of a virus infection, sows can exhibit skin lesions
and transient bluing or reddening in the vulva and around the ears. Pregnant sows are
affected with different reproductive failures, depending on the stage of pregnancy. Sows
in early gestation (about 45 days) may seem unaffected by a moderate PRRS infection
(Betner, et a1., 1994; Mengeling and Lager, 1992). The fuiled pregnancy might go
unnoticed because the body will probably reabsorb the dead fertilized eggs during the
embryonic phase. Infected sows will probably show a delayed return to estrus
(Mengeling et a1., 1995b, I 998b). On the other hand, infections during late gestation
(about 90 days) by the same strain ofvirus can lead to late term abortions and/or cause
the sow to give birth to a weak, dead and/or mummified litter. Newborns that survive can
have persistent infections that can result in delayed weaning. A delayed or increased
return to estrus as the virus persists in the herd further disrupts the production cycle
(Mengeling and Lager, 1992; Mengeling et a1., 1995b; Done and Paton, 1995).
Neonates born to infected sows are often weak and can have swelling around the
eyes, rough hair coats, diarrhea, nervousness and splayed legs. Death rates with
16
newborns showing these clinical signs can reach as high as 80-100% (Done and Paton,
1995). Under these viremic conditions, growth rates are also compromised. The
neonate's health can be further debilitated with secondary or coexisting infections,
especially if the weakened newborn is unable to suckle and get colostrum from the sow
(Greiner et aI., 2000). Ahhough Chung et aI. (1997) reported sows could provide
protection to their newborns via maternal neutralizing antibodies, the protection is
temporary. Neutralizing antibody levels usually drop between week 6 and 8, coinciding
with an increase ofvirus level.
Infected newborns that survive are weaned later than normal because average
daily gains are reduced by 50 to 75%. Decreased daily gains in weaned and finishing
pigs can be further complicated with secondary or concurrent infections (Kay et aI.,
1994). Surviving weaned pigs can be a major source ofvirus fur the herd, capable of
shedding the virus fur up to 30 weeks (Zimmerman, 2003b; Albina et aI., 1994). In a
study of eight farrow-to-finish farms, Chung et aI. (1997) concluded that six- to nine
week old pigs were the major source of re-infection fur the herd.
Atvoieai PRRS
Vaccination can be effective against mild to moderate PRRS infections but is not
always effective against more virulent strains ofPRRS viruses. In 1996, a more virulent
strain ofPRRS virus, unaffected by vaccination at that time, began surfilcing in Danish
herds (Mengeling et a1, 1996, 199811, 1999; Blltner et aI., 1997; Nielsen et aI., 2001, 2002;
Mortensen et aI., 2002; Storgaard et aI., 1999). Atypical PRRS is characterized by
sudden onset of greater than 1 (101o abortion rates that lasts two to fuur weeks and more
than 5% of sow and boar deaths (Mengeling et aI., 1998a). Sows with atypical PRRS can
17
experience reproductive firilure in early or late gestation, with the sows in later gestation
experiencing more severe losses. Late-term gestational sows can experience increased
stillborns, mummified births, abortions and even death (Blltner, 1997; Mengeling et aI.,
1998a). Studies have suggested that the viral strain responsible fur atypical PRRS is
probably a modified-live vaccine virus (Blltner et aI., 1997; Nielsen et a1, 2001, 2002;
Mortensen et aI., 2002; Storgaard et aI., 1999). Mengeling et aI. (1998a, 1999) noticed
that new and more severe cases ofPRRS with atypical clinical signs have been sur:liu:ing
on vaccinated farms.
Effects on Boars
Boars exhibit less common clinical virus signs than sows. Adult boars may
display signs of decreased libido, fever, depression and anorexia ahhough the signs can
be short-lived (Yaeger et aI., 1993). In a study by Prieto et aI. (1996), clinical signs were
not obvious in 26 out of29 PRRSV inocuIated adult boars. Adult boars can eliminate the
virus from their bodies relatively quickly. The virus is generaI1y not detectable after
post-inocuIation day 23 via serological techniques and day 31 via RT -PCR (Cluistopher
Henniogs et aI., 1995a.) The absence of clinical signs coupled with a short viremia can
make diagnosis difficult fur swine producers (Bouma, 2000).
PRRS and Immunology
The production of antibodies is vital to normal body functions. In the case of a
PRRS viral invasion, the immune system stimu1ates the production of antibodies to
protect the body against the viruses. The virus suppresses the immune system by
invading and replicating in the monocytes (especially macrophages) of the spleen, lungs
and lymph nodes. After replication, the viruses lyse their host macrophages and invade
18
more macrophages via the bloodstream. After a series of invasions and replications, the
immune system is weakened (Aiello, 1998). Y oon and Stevenson (2002) recorded the
production of non-neutralizing antibodies, depending on the type of assay used, as early
as five days, peaking at approximately 30 to 50 days and declining to undetectable levels
at 4 to 12 months after infection. Neutralizing antibodies are general antibodies that
render a virus ineffective by binding to it. These neutralizing antibodies, detectable by
the serum neutralization assay, are fuund as early as 9 to 28 days, peaking at 10 to II
weeks and then declining to undetectable levels by approximately 356 days after
inocuIation.
Macrophages work together with cytokines in a cell-mediated defense against
viruses that have entered the cells. The attack on macrophages prevents the stimulation
and production ofTNF-~ one of two cytokines that further stimulate the production of
more macrophages (L6pez-Fuertes et aI., 2000). Nielsen and Botner (1997) fuund that
PRRSV could suppress the production ofT-cell subpopulations, a group of white blood
cells that are important in the attack against virus infections. CD2, CD4 and CD8 levels
were fuund to drop a few days after infection but quickly returned to pre-infection levels
by 8 to 10 days after infection. Shimizu et aI. (1996) did not notice any significant
adverse effects PRRSV might have on T-cell populations.
The PRRSV not only suppresses the pig's immune system but also, to some
extent, uses the pig's own protective antibodies to enhance its survival, a process known
as antibody dependent enhancement. Antibody dependent enhancement is particularly
noticeable during the use of vaccines or during transplacental infections. The antibodies
19
stimulated by vaccines or of maternal origin tend to enhance the movement ofPRRSV
into host cells, exacerbating the PRRS infection (Cancel-Tirado et at, 2004).
Virus Research
In order to study the virus, researchers must be able to grow the virus. Porcine
alveolar lung macrophages and MARC-145 cells, a population of cells derived from the
MA-I04 monkey kidney cells, are the best media for virus replication purposes (Kim et
at, 1993; Wensvoort et al., 1991). Kim et al. (1993) found that 11 strains ofPRRS virus
prefer to replicate in cultured monkey kidney (MARC-145) cell populations. In this
study, the MARC-145 cell populations supported virus numbers up to 1085 tissue culture
infective dose 50 (TClDso)/O.1 ml Appropriate methods of collecting tissue samples are
vital in maintaining virus viability for the experiments (Mengeling et al., 1995a).
Serology
By 1995, Joo (1994,1995) noted several serological tests available for PRRS
testing: indirect fluorescent antibody (IFA), immunoperoxidase monolayer assay, serum
neutraIizing antibody (SNA), enzyme-linked immunosorbent assay (ELISA) and Western
blotting (WB). These assays can determine the absence or presence ofPRRS antibody
but not the strain ofvirus that induced the positive antibody response. In serology, a
negative response can have several interpretations: I) the host of the sample is truly
negative and has never been exposed to the virus; 2) the host of the sample was once
exposed but no longer has antibodies or has titers that are too low to detect; or 3) the
sample was contaminated with other microscopic biological material and is actuaIIy
positive ( fhlse negative) (Dee et al., 1996). Indirect fluorescent antibody usually detects
the presence of antibodies in blood between 7 and 14 days after inoculation and can do so
20
for up to 3 to 5 months. The ability ofIFA to detect antibody diminishes as antibody
titers in serum decrease. In order to obtain 95% oonfidence in detecting a 10% level of
PRRS infection in a herd, at least 30 samples must be oollected (Joo, 1994, 1995).
Serum neutralizing antibody assay specifically detects neutralizing antibodies that
appear later in a PRRS infection, making it a poor assay to diagnose the onset of
infection. Serum neutralization anttbody can detect PRRS neutralizing anttbodies as
early as 9 to 11 days after infection (Joo, 1995). The fluorescent focus neutralization
(FFN) assay used in this study is similar to a serum neutralizing antibody assay and virus
neutralization assay except that microtiter plates are fixed and stained with a fluorescein
oonjugated monoclonal anttbody (Nelson, 2001).
Enzyme-linked immuoosorbent assay was developed to detect antibodies in
serum. The oommercial IDEXX ELISA detects both the U.S. and the European strain of
the virus. The resu1ts are expressed in sample to positive ratios and optical density (00)
values. The 00 values are read directly from the machine; the values <0.2 are negative
and values ;a().3 are positive. IDEXX ELISA HerdChek@, the former oommercially
available ELISA, was recently disoontinued and ELISA (2xR) is now oommercially
available. ELISA (2xR) is the primary serological test and is often used with the
polymerase chain reaction procedure for oonfirmation. Enzyme-linked immuoosorbent
assay is a good test to determine herd infection status while RT -PCR can determine
individual PRRS status (Batista et aL, 2004; Cho et aL, 1997). Enzyme-linked
immuoosorbent assay is cheap and efficient in terms of high specificity and sensitivity for
herd diagnosis but borderline resu1ts should be oonfirmed with IF A, SNA, RT -PCR or
virus isolation (VI) (Batista et aL, 2004; Cho et aL, 1997; Sl!feIlSen et aL, 1997, 1998;
21
Bfiltner, 1997). This study used a blocking ELISA (bELISA) that had a 97.8% diagnostic
sensitivity and 100% diagnostic sensitivity (Ferrin et al., 2004). Due to the blocking or
competitive furmat of the assay, this type of ELISA is not species dependant. This was
important as this study attempted to detect antibodies of a porcine disease in avian serum.
Western Blotting
Western blotting (WB) involves the use of electrophoresis to separate PRRSV
proteins and then transferring thern onto nitrocellulose membranes where they can be
detected and visualized. The appearance of specific bands in reference to the known
protein indicates a positive response. The procedure is laborious and requires detailed
handling to prevent fillse responses but it can help confirm confuunding results from
other serological tests. In this study, both WB and polymerase chain reaction were done
to further elucidate the results from bELISA and FFN (Nelson, 2001).
RT-PCR and Swine Bioassay
Reverse transcription polymerase chain reactions (RT -peR) use genetic primers
to detect specific nbonucleic acid (RNA) sequences. Serological diagnoses require the
time fur antibody titers to reach a level detectable by the assays. As a result, the
diagnosis ofPRRS cannot occur at the onset of infection. Reverse transcription
polymerase chain reaction can detect the presence ofPRRS virus nbonucleic acid as soon
as 24 hours after infection (Spagnuolo-Weaver et a1., 1998; Christopher-Hennings and
Nelson, 1996). Early diagnosis can help prevent the spread of the virus to uninfected
pigs and lead to a more effective control and treatment program. This study added a
nested procedure to RT -PCR to increase the diagnostic sensitivity although RT -PCR is
known to be highly sensitive and specific (Christopher-Hennings et a1., 1995b; Horter et
22
at, 2002; E. A. Nelson, personal communication). Other versions ofPCR have been
used to detect PRRS viral genetic material for diagnostic purposes (Horter et at, 2002).
Reverse transcription polymerase chain reaction is often used with a swine
bioassay, an assay that requires the inoculation of a live animal to determine the
infectiousness of the virus in question. In this case, swine are inoculated with the virus in
question to determine if the virus is still infectious (Swenson et at, 1994). Despite all
these available assays. BfJtner (1997) noted that no one test can detect all strains ofPRRS
virus.
PRRS Virus Persistence
The PRRS virus can persist over a period of time after ioitia! infection in a herd.
Nursery and growing pigs are the main sources of the herd's endemic infections. Surveys
found that PRRS viruses can exist in herds at subclinica1levels for years (Baysinger et
at, 1997; Chung et at, 1997; Dee et at, 1996). In an infected breeding herd, Dee et at
(1996) noted that there might be up to 15% of the herd that remains seronegative after an
outbreak. The presence of such a subpopu1ation of naive pigs can leave the entire herd
susceptible to re-infection if persistently infected sows begin to shed virus again. The
infection in this naive subpopu1ation could be passed on to newly born piglets, other
naive pigs or even to pigs in another fimn when the animals are sold (Dee et at, 1996;
Bilodeau et at, 1994). Other studies have also shown that pigs with persistent infections
are a threat to swine and herd health (Allende et at, 2000; Albina et at, 1994; Wills et at,
1997b).
PRRS Virus Transmission
The pig can come into direct contact with the virus in the environment {paton and
23
Drew, 1995; Dee et al., 1996). The PRRS virus dies easily from dehydration; however, it
can survive fBirly long in the environment wtder certain specific conditions. At 21°C at
pH 7.5, Bloemraad et al. (1994) reported the virus' half-life (time in which the virus
population is decreased by bait) at 20 h. At 4°C at the same pH, the virus' half-life
increased to 139 h. Zimmerman et al. (2003b) noted that the study by Benfield and his
team in 1992 showed that the infectivity of the virus remained wta1tered after one month
at 4°C and four months at -700C. Zimmerman et al. (2003b) also noted that a study by
Pirtle and Beran in 1996 recorded the survival ofPRRS virus up to 8 days in well water
and up to 11 days in city water.
Direct Transmission
Direct pig-to-pig transmission of the PRRS virus has been demonstrated in the
laboratory and in the field (paton and Drew, 1995; Collins et al., 1992; Dee et al., 1996;
Bierk et al., 2001; Wills et al., 1997a; Wensvoort et al., 1991; Wensvoort, 1994). An
infected pig can infect a healthy pig via intranasal, oral and/or vaginal contact. Wills et
al. (1997a) noticed the persistence ofPRRS virus in macrophages of urine up to day 14,
in serum up to day 21, in saliva up to day 42 and in oropharyngeal samples up to day 84
after inoculation. Infected pigs shedding the PRRS virus through their nasa\ mucus can
infect healthy pigs with a viral dose as low as 103.5 TClDso by normal nose-to-nose
rubbing (Christianson et al., 1992; Collins et al., 1992). Teuffert et al. (1998) showed
that PRRS virus could be isolated from nasa\ swabs for up to 40 days in nasa\ mucus and
Rossow et al. (1994) fowtd that infected pigs can shed the virus in their urine 28 days
after inoculation. Infected pigs shedding the virus via their nasa\ mucus, feces and/or
urine can contaminate feeds and sleeping quarters, giving the virus opportunities to enter
24
through the oral passages. With the virus present in the saliva for such a long period of
time, it is likely that boar-to-boar aggressive behavior can have a dramatic impact on the
spread of the virus (Wills et aL, 1997a).
Virus shedding in semen is a problem to producers. The duration ofvirus
shedding is important to producers, considering the widespread use of artificial
insemination. Studies have shown virus shedding in semen to be inconsistent (Swenson et
aL, 1994; Christopher-Hennings et aL, 1995a, 1995b, 2001; Wills et aL, 1997a).
Christopher-Hennings et aL (1995a) found that boars began shedding PRRS virus in their
semen as early as three days after inoculation and continued for 25, 56 and 92 days after
inoculation, depending on the boar. Although Teuffert et aL (1998) found the virus in
semen of an inoculated boar only on day 19 after inoculation, Shin et aL (1997) reported
the shedding as long as 50 days after infection, peaking at day 7 after inoculation. In
addition to sporadic viral shedding in semen, the ability of adult boars to shed virus in
semen without viremia and in the presence of neutralizing antibodies further complicates
the diagnosis and spreads the virus (Christopher-Henniogs et aL, 1995a, 2001).
Christopher-Hennings et aL (2001) looked at the correlation between boar breeds
and susceptibility to virus shedding. They noticed a slight but not statistically significant
trend that linked the duration ofvirus shedding in semen to different breeds, noting that
Yorkshire boars tend to shed less or for a shorter period than Landrace boars.
As the use of artificial insemination grows in the swine industry, it is important to
understand the effects ofvirus on semen and sperm and the potential for transmission.
PRRS viruses reach sperm in the boar genitals via macrophages (Christopher-Hennings et
aL, 1994). Experimental studies have detected PRRS virus in preputial swabs (Teuffert et
25
aL, 1998) and have found the virus live and replicating in seminiferous tubules, primarily
in spermatids, spermatocytes and interstitial macrophages (Sur et aL, 1997).
PRRS can have various effects on seminal quality although studies have varied in
their findings. Studies have shown that infected boars can have increased sperm
abnormalities (Shin et aL, 1997) although it has little to no effect on conception and
fertilization rates in sows (Prieto et aL, 1997). However, insemination of infected semen
still can result in transp1acental infection and lead to newborn deaths (Prieto et aL, 1997).
A study by Yaeger et aL (1993) observed a decrease in semen volume but normal sperm
morphology. Sows inseminated with infected semen have been known to show clinical
signs ofPRRS (Shin et aL, 1997; Prieto et aL, 1997; Yaeger et aL, 1993). Sows can be
infected with PRRS by contamioated semen either by way of artificial insemination or
direct copulation (Prieto et aL, 1996). A study by Teuffert et aL (1998) demonstrated,
despite noticeable adverse effects on ejaculate quality and volume, an inability for the
infected semen to cause infection. reproductive firilure, or seroconversion in inoculated
sows. Prieto et aL (2005) compared the aforementioned studies and concluded that
perhaps the different results in sow infections are due to individual sow susceptibility and
various infectious concentrations.
Non-domestic pigs can also be a source ofPRRSV. A serological survey in
France showed that 33 out of909 wild swine samples were positive for the PRRS virus
antibodies, indicating a possible reservoir for this disease in the wild (Albina et aL, 2000).
A PRRS outbreak in Hawai'i's feraJ pig population could seriously threaten the domestic
population.
26
Vertical Transmission
PRRS can be transmitted from mother to young. Experiments have demonstrated
that the American strain ofPRRS virus is capable of crossing the placenta and infecting
fetuses and newborns, resuhing in mummified fetuses, stillborns, and debilitated live
piglets (Christianson et at, 1992; Wensvoort et al, 1991). It is still unknown exactly how
the virus crosses the p1acenta and reaches the embryos and fetuses, but Mengeling et at
(1995b) suggested that the PRRS virus used macrophages to cross the placenta and infect
individual pigs. This hypothesis is supported by the observation that the PRRS virus has
an ability to thrive in macrophages and other monocytes (Voicu, 1994). Mengeling et at
(I99Sb) thinks that the idea that the virus may cross the placenta via direct infection of
the blood during increased arterial connections between the sow and the piglets is
possible but not probable, especially since litters can be born with just a few infected
individuals. If the placental blood were viremic, it is likely that the infection would be
more widespread instead of affecting just a few individual newborns.
Prieto et at (1997) noticed that exposure to the PRRS virus at the onset of
gestation did not affect conception and fertilization rates of 1 O-day old IUIattached
embryos but did cause the death of infected 20-dayold attached embryos. The death of
20-dayold embryos reached 35.4%, three times more than the deaths of the controls,
which was only 9.8%. The ability of the viruses to tenaciously grip onto the embryo's
external barrier could lead to contamination of naive sows during embryo transfer.
Studies have shown that exposure at approximately 50 days of pregnancy led to infected
fetuses and stillborns (Christianson et at, 1992; Mengeling et at, I 995b) but Mengeling
et at (1995b) reported that exposure at 90 days of gestation is probably the most
27
significant, resulting in a significantly greater number of fetal and newborn infections.
The increased rate of infection during this period of gestation may be dependent on both
the degree of protection the placental barrier provides at that time and the ability of the
fetus to support viral replication at that time.
Sows exposed to the virus during late gestation have been found to shed the virus
in their mammary secretions. Although the mechanism by which the virus reaches the
secretions is Wlcertain, it has been suggested that the virus might be invading
macrophages that are on their way to the mammary glands (Wagstrom et al, 2001). The
level of infective virus foWld in the mammary secretions exceeded the minimum infective
viral particles of 10 or less for intranasal and intramuscular infections as reported by
Yoon et al (1999), suggesting that suckling on the infected mammary secretions may
lead to PRRS infection in newborns (Wagstrom et al, 2001). The entire litter may
become infected by directly suckling on infected milk or by coming into direct contact
with infected littermates (Wagstrom et al, 2001). A sow vaccinated with the MLV
vaccine RespPRRS® from NOBL LaboratoriesIBoehringer Ingelheim may have reduced
shedding in her following lactations (Wagstrom et al, 2001).
Indirect Transmission
Humans can also contnbuteto the spread of the PRRSV. Hands that are not
washed properly after exposure to swine feces, blood, saliva and urine can transmit
PRRSV. Exposed clothing materials such as boots, gloves and coveralls have also been
foWld to carry the PRRS virus. A standard sanitation protocol of changing coveralls,
boots and washing hands with soap and hot water (lO seconds under hot water, 30
seconds with soap, and another 10 seconds Wlder hot water) was sufficient to prevent the
28
transmission ofPRRS from one room to the next. Stricter protoaJ1s such as showering
and waiting for 12 hours have been effective in killing the virus (Otake et al., 2002b). It
is important to be wary of other equipment (such as needles and medicine boxes) that can
come into aJntact with infected swine fluids (Otake, 2002c).
Dee et al. (2004) fuund several sanitation protoaJ1s and fium traffic management
useful. Foot traffic from one farm onto another is a way PRRSV can be transmitted from
one farm to the next. Dee et al. (2004) showed that driving from one farm to the next
with aJntaminated boots aJuld transmit viruses. Dee et al. (2004) were able to infect
swine with PRRS viruses swabbed from aJntaminated shoes. The practice of using
disposable boots, and/or immersing fuotwear in boot baths containing undiluted Clorox
bleach (6% sodium hypochlorite) fur a minimum offive seaJnds after stepping on urine
or feces were shown to be effective in killing the virus.
Dee et al. (2004) also noticed that shipping aJntamers aJuld bring viruses onto a
farm. Shipping aJntainers in the wintertime can be left on aJntaminated snow and then
transported onto the fiIrm, where the snow melts. Such situations can be avoided if the
aJntamer is enclosed in a bag, is never set down and the consignee removes the container
straight from the bag.
Dee et al. (2002, 2003) have also tested the survival ofPRRSV on various
surfuces under cold (less than O"C) and warm (20"C) weather aJnditions. Plastic, metal,
cardboard, Styrofuam, concrete, rubber and linoleum were fuund to harbor PRRSV
during both cold and warm weather although PRRSV survival during the warmer weather
on those surfaces were shorter. Dee et al. (2002, 2003) also tested the possibility of
mechanical transmission from the field into enclosed buildings in these two different
29
temperature conditions and fuund that warm weather was much less conducive to PRRSV
transmission. On the other, cold weather conditions permitted the mechanical
transmission of the infectious virus from various outside (e.g. the field) and inside (e.g.
trucks and buildings) areas 8 out oflO times (Dee et a1., 2002). It is important fur
farmers to make note of where they are coming from and where they are going to in order
to reduce the transmission ofPRRSV, especially in the winter time.
Improperly sanitized animal transportation vehicles are also capable of infecting
swine from different:litrms. The virus can infect pigs contained in a vehicle
contaminated with a virus dose greater than or equal to loJ TCIDso fur up to two hours.
Sanitation protocols such as removing soiled material only from the transportation
vehicle, using a I :256 dilution of phenol fur 10 minutes to clean the vehicle interior or a
combination of both were ineffective in disinfecting the contaminated interior of the
transportation vehicle. The use of37% furmalin solution was also ineffective in
disinfecting the interior and preventing infection of naive swine. The most effective
sanitation methods were the use of a fugger to apply g1utaraldehyde-quaternary
ammonium chloride and/or allowing the interior of the vehicle to dry (Dee et a1., 2004).
Airborne Transmission
Airborne transmission of the virus has been suspected (Mortensen et a1., 2002) but
researchers have not been able to experimentally confirm airborne transmission.
Otake et aI. (2002a) was not able to show aerosol transmission in a controlled
experiment. Aerosol transmission was not observed at distances 30 m and 80 m away
from a room of infected hogs. However, pigs housed in the same bam but 2.5 m away
from each other and not allowed direct contact became infected. It is suspected that
30
insects or rodents might bave transmitted the virus or the nasal, oral and other bodily
fluids of the infected pigs reached the uninfected pigs' pens. In 2004, Trincado et aI.
(2004a) conducted an experiment similar to Otake et aI. (2002a). In this experiment,
Trincado et aI. (2004) decreased the distance from 30 m and 80 m to 15 m between the
infected and uninfected holding facility and extended the period of exposure to the
infected pigs. Despite these changes to increase air transfer between the infected and
uninfected filcilities, aerosol transmission was not observed. Kristensen et aI. (2003)
found that with 1 %, 10010 and 70010 air exchange, the virus could be transmitted by aerosol
over a distance of 1 m, consistent with Torremorell et aI. (1997).
Animal Transmission
Transmission of the virus by animals has been studied. Zimmerman et aI. (1997)
demonstrated in the laboratory that the PRRS virus isolated from infected pigs could
infect Mallard ducks with PRRS. The virus can be carried in the Mallards for up to 38
days and be shed in Mallard feces for up to 25 days. Subsequently, PRRS virus isolated
from Mallard feces were capable of infecting healthy pigs, indicating a possible
transmission route between ducks and pigs in an ecosystem. The team also showed that
the virus isolated from the pig that was infected via Mallard feces could be used to infect
another pig. Contrary to Zimmerman's findings (1997), Trincado et aI. (2004b)
performed a similar study using the same challenge dose and methods of detection but
were not able to infect mallards with the PRRSV. The main differences between the two
studies were the different strain used for inocu1ation and the age of the mallards used.
These two major differences may bave accounted for the antithetical results. Guinea
fowls displayed a slight susceptibility to PRRS although the results were not statistically
31
significant (Zimmerman et aI., 1997). Studies on rats, mice and other avian species such
Muschovy ducks and chickens yield no significant results (Hooper et aI. 1994;
Zimmerman et aI., 1997). Wills et aI. (2000) studied dogs, cats, rats, mice, skunks,
sparrows and starlings but fuund them all to be negative via VI and RT -PCR methods.
They did find two opossums and one raccoon to be positive fur PRRS viral RNA but the
sample size was very small.
Insect transmissions could explain what seem to be apparent airborne
transmissions. Recently, the transmission ofPRRSV has been demonstrated with
houseflies (Otake et aI., 2003b, 2004) and mosquitoes (Otake et aI., 2002d, 2003c). A
housefly (Musca domestica) was able to transmit the virus by feeding on the scoured
back of an infected pig and then on that of an uninfected pig. The uninfected pig was
detected with PRRS antibodies after 14 days (Otake et aI., 2003b).
Mosquitoes (Aedes verans) could be mechanical (Otake et aI., 2002d), but not
biological vectors (Otake et aI., 2003c). Otake et al. (2002d) showed that mosquitoes
feeding on an infected pig could mechanically transmit the PRRS virus to an uninfected
pig. A subsequent study (Otake et al., 2003c) showed that the mosquitoes could carry the
PRRSV in the intestinal tract up to 6 b. Within the 6 hours, the carrier mosquitoes, if
alIowed to subsequently feed on uninfected pigs, could transmit the virus. The study
showed that the mosquitoes did not carry the virus on their external surfiwes or in their
salivary glands. The detection ofPRRSV particles in a mosquito thorax suggested that
mosquitoes might be able to carry the virus in the thorax but the suthors stated that virus
detection at this anatomical site was probably due to contamination during insect
dissection.
32
With flies and mosquitoes, PRRSV was fuund more frequently in the gut of the
insect than on its exterior (Otake et al., 2003a, 2003b, 2004). The virus probably survives
longer in the gut of houseflies and mosquitoes (Aedes verana) because it is shehered from
the elements (Otake et al., 2004; 2003b). Otake et al. (2003a) showed that the virus could
survive in the gut of houseflies as long as 12 hours after its ingestion from an infected
pig. Fighting between boars can lead to open wounds that would allow flies and
mosquitoes to land and feast on, thus transmitting the PRRSV.
Cattle Egrets (Bubulcus ibis)
A study done by Adames et al (1993) fuund cattle egrets to have antibodies to the
Saint Louis encephalitis virus although no viruses were isolated, suggesting the egret may
be a host to the virus sometime in its life. With a possible avian host fur the PRRS virus,
there is reason to suspect that birds that frequent piggeries may playa vital role in
transmitting the virus from one piggery to another. Cattle egrets are often seen near
livestock furms worldwide; and in the state ofHawai'~ cattle egrets have been observed
to feed on pig furms on a daily basis and thus are potential transmitters of the PRRS virus
(S. M. Tanaka, personal communication).
Cattle egrets (Bubulcus ibis) are in the class Aves, order Cociniifurmes, and the
family Ardeidae. The cattle egret is a migratory white bird native to Africa but had
established colonies naturally in North and South America, Australia and Europe. Cattle
egrets stand about 43.2 em and weigh about 300 to 500 g. Ahhough males and females
have no significantly different features, breeding aduhs tend to have buff-colored crests,
chests, bills, and legs. The juveniles have completely white plumages with black legs
and yellowish bills. Cattle egrets are seasonally monogamous and lay two to three bluish
33
eggs per clutch in stick nests. Often. only two chicks will fledge in about a month or two.
B. ibis mate during the summer months but migratory species could mate year round
when exposed to a tropical climate (Telfair, 1983; Telfair, 2002; Gill, 1995). Cattle egrets
are very prolific breeders with a possible lifespan of seven to eight, and sometimes as
high as 20 years (Telfair, 2002). Telfair (2002) stated thst the Texas population increased
180% every year between 1959 and 1972 but tapered off to 120"'{' every year between
1979 and 1990.
Cattle egrets are very opportunistic feeders. They are often seen around livestock
because they use cattle and other livestock to flush the insects out of their hiding places in
the field. Csttle egrets are also seen around airports because the roar of airplane engines
drive insects out of their hiding places in nearby fields. Although egrets often prey on
insects such as grasshoppers, centipedes, flies, and crickets, they have also been fuund to
prey on amphibians and native Hawaiian chicks (Telfair, 2002; S. M Tanaka, personal
communication; Singh et at, 1988).
Under the authority of the State Agriculture Board, cattle egrets were introduced
to Kaua'i in 1959 and subsequently to MaW, Hawai'i, Moloka'i and O'ahu from a
population in Florida. A group of biologists, led by the State Department of Agriculture,
approved the introduction ofcatt1e egrets in 1958 for the purpose of pest control (Thistle,
1964). Hawai'i is the only place in the world where the population of cattle egrets was
not naturally established (Telfair, 1983).
Cattle egrets often leave their rookeries during the day to feed on insects and
return to the same rookery to rest at night (Telfair, 2002; S. M. Tanaka, personal
communication). In Hawai'i, cattle egrets have often been observed to feed on pig farms
34
(S. Tanaka, personal communication). Their frequent visits to pig fiInns make them a
suspected carrier of the PRRS virus. The socialization of egrets in one central area after a
day of visiting various fiInns could lead to limn-to-limn transmission. Controlling and
identifying the routes of transmission is a vital step in protecting the pork industry in
Hawai'i and worldwide. In Hawai'i, direct pig-to-pig transmission cannot explain all
incidences of infection because closed herds were infected, and the spread was within
neighboring fiInns rather than following live pig sales (Figure 2; Zaleski et a1., 1996).
The objective of this study was to determine whether antibodies to the porcine
reproductive and respiratory syndrome virus can be detected in the cattle egret population
at the Naval Computer and Telecommunications Area Master Station (NCI'AMS),
Pacific Site, in Wai'anae, Hawai'i
35
CHAPTER II MATERIALS AND METHODS
Permits
The project was approved by the University ofHawai'i's Institutional Animal
Care and Use Committee (project protocol number 00-059) and permits were obtained
from the state Department of Land and Natural Resources (permit number WL02-03) and
Fish and Wildlife Services (permit number MB042489-0) to possess avian carcasses in
accordance to the United States' Migratory Bird Act. Prior to getting approval from
lACUC, an internet training course on the Regulations for the Care and Use of Vertebrate
Animals must be reviewed and completed on the website
http://webct2.hawaii.edu:8900/public/uhmsylviakl /.
Sample Collection
The birds were collected around a small wetland located at the Naval Computer
and Telecommunications Area Master Station (NCTAMS), Pacific Site, in Wai'anae,
Hawai ' i (Figure 5). The site is a breeding ground for native Hawaiian birds and situated
near cattle egret rookeries (S. M. Tanaka, personal communications). The United States
Department of Agriculture, Animal and Plant Healthy inspection Services, Wildlife
Services, Pest Control Branch, contracted with the Navy, were culling cattle egrets at
NCT AMS because the egrets were threatening the native birds breeding in that area. The
Navy decided to halt the culling of cattle egrets at NCT AMS indefinitely because 1)
cattle egret population was reduced to a non-threatening level to the native birds and 2)
native birds were beginning to breed. As a result, collection of cattle egret blood samples
was done on only two days.
36
Figure 5. Location of bird collection. Naval Computer and Telecommunications Area Master Station, Pacific site, is located in Wai ' anae, Hawai'i, near the area of the 1996 porcine reproductive and respiratory syndrome outbreak. Longitude: 1580 10' West; Latitude: 21 0 23' North.
Serum Samples
Seventeen and 13 blood samples from cattle egrets were collected on April 23 and
July 27 of2000, respectively, between 4:00 pm and 7:00 pm at NCT AMS, Lualualei.
Two spotted doves (Streptopelia chinensis) (Stone and Pratt, 1994) blood samples were
collected on July 27, 2000. The dead birds were examined for any external lesions or
abnormalities. A lateral incision was made on each side of the keel musculature. The
keel musculature was then reflected caudocranially to expose the cardiac cavity for blood
collection. Blood was either aspirated with a 14-gauge needle from the cardiac cavity, or
37
the heart was removed and blood was manually expressed into serum separator tube
(SST) Vacutainers™ with separator gels.
The collected avian blood samples were stored in a cooler with ice and
transported to the University ofHawai'i at Manoa where they were transferred to a non
fuod refrigerator to clot overnight. The samples were then centrifuged (model HN-SII,
International Equipment Company) the next day fur 15 min at 312g and the sera
transferred to 1.5 ml micro-centrifuge tubes, labeled and stored in the freezer at -20"C.
Six samples did not have adequate separation and were disposed of In December 2001,
a total of30 egret serum samples and two dove serum samples were packed into a
Styrofoam cooler with ice packs and shipped to the Animal Disease Research and
Diagnostics Laboratory at South Dakota State University (SDSU), fur the perfurmance of
serological and molecular diagnostic methods fur porcine reproductive and respiratory
syndrome virus (PRRSV). Blocking enzyme-linked immunosorbent assay (bELISA),
fluorescent fucus neutra1ization assay (FFN) and Western blotting (WB) were fur the
detection ofPRRS antibodies and reverse transcription polymerase chain reaction (RT -
PCR) procedures were used fur the detection ofPRRS vira1 RNA.
Crop ContentslInseet Identlfic:adon
The birds were then taken back to the University ofHawai'i at Manoa to be
weighed with an OHAUS GT -4100 digital scale, dissected and to have their crop contents
collected. Seventeen and II crop contents from cattle egrets were collected on Apri123
and July 27 of2000, respectively, at the University ofHawai'i at Manoa after each field
collection. The first set of cattle egret crop contents were examined and identified to
order using the National Audubon Society Field Guide to Insects & Spiders (Milne and
38
Milne, 2000). The second set of cattle egret crop contents were stored in a non-food
freezer at -20"C until they were transferred into various sizes of plastic and glass jars
filled with 70% isopropyl rubbing alcohol Two of the II samples were given to Mr.
Richard Tsuda at University ofHawai'i at Miinoa, Department of Plant and
Environmental Protection Sciences, Insect Diagnostic Laboratory for identification to
species. Nine of the samples were disposed of before they could be identified (Figure 6).
The crop contents of the spotted doves contained no insects. Hawai'i State Department
of Agriculture personnel incinerated all animal carcasses after all the collections and
examinations were done.
Figure 6. Destination of cattle egret and dove crop contents.
Collected: 30 Cattle Egrets
No identification 2 Spotted Doves because no insect
in crop: 2 Spotted Doves
Disposed before identification:
11 Cattle Egrets Insect identified: 19 Cattle Egrets
/ ~ Kept and identified Gave to Richard
to Order: Tsuda for 17 Cattle Egrets identification to the
species level: 2 Cattle Egrets
39
Blocking Enzyme-Linked Immunosorbent Assay
Twenty-fuur cattle egret and two dove serum samples were assayed fur PRRS
virus antibodies using the blocking ELISA (bELISA) descnbed by Ferrin et at. (2004).
This assay was designed fur detection ofPRRSV antibodies in pig serum. However, due
to the blocking or competitive furmat of the assay, it is not species dependent. All
samples were run singly in all assays because of insufficient amounts.
A purified recombinant open reading frame 7 (ORF7) nucleocapsid peptide
(Michael Murtaugh, University of Minnesota) was used fur the test or sample wells of an
Immulon 2 HB 96-well microtiter plate (Dynatecb, Chantilly, Virginia). An antigen
coating buffer (ACB) (0.015 M NaC0)-0.035 M NaHC0), pH 9.6, was used fur the
background control wells of the sample plate. The recombinant ORF7 nucleocapsid was
diluted 1 :2000 with ACB and 1 00 ~l of the dilution was pipetted into the test wells. One
hundred microliters of ACB was pipetted into the background control wells. The
microtiter plate was incubated at 37"C fur 1 h and then kept at 4°C overnight.
The next day, 200 ~l of phosphate buffer solution (PBS) containing 2% (wtlvol)
bovine serum albumin (BSA) was added to all the wells and then the microtiter plate was
incubated at 37"C for 1 h. The plate was then washed six times with at least 300 ~l of
PBS containing 0.05% Tween 20 (PBS-TweeIl2o) (SIGMA Chemical, St. Louis. MO)
using a Nunc-Immuno Wash eight-channel manifuld (Zymed, South San Francisco, CAl.
Sample and control sera were then diluted 1:4 with PBS-Tween20 containing 0.1 % BSA
(SMD) and 100 ~l of the dilution fur each sample was pipetted into both a nucleocapsid
antigen-coated well and a control well After all the dilutions were added, the microtiter
plate was returned fur incubation at 37"C fur 1 h.
40
After incubation, 100 III of SO OW 17 (South Dakota State University, Brookings,
South Dakota) biotinylated monoclonal antibody diluted 1 :200 with SMD was added to
all the wells. The microtiter plate was mixed and returned to the incubator fur 30 min at
37°C. The wells were then washed six times with at least 300 111 of PBS-Tweetl2o, and
after washing, 100 III of I :8000 strepavidin-horseradish peroxidase conjugate (Zymed,
San Francisco, CA) was added and the microtiter plate was returned to the incubator fur 1
h at 37°C. After incubation, the plate was again washed six times with at least 300 !1l of
PBS-TWeetl20 solution.
After the final washing, 100 I11of2, 2'-azino-bis (3-ethylbenztbiazoIine-6-
sulfunic acid) (ABTS) peroxidase substrate (Kirkegaard and Perry, Gaithersburg, MD)
was added to all the weIIs fur color development. The microtiter plate was incubated fur
exactly 15 min at room temperature. After incubation, 100 III of ABTS stop solution
(Kirkegaard and Perry, Gaithersburg, MD) was added to stop further color development.
The color was read at 405 nm with an EL800 microplate reader (BioTek Instruments Inc.,
Winooski. Vt.) and controIIed by XChek Software (lDEXX Laboratories, Westbrook,
ME). The color intensity was recorded as optical density (00) and the raw data were
transferred to a Microsoft Excel spread sheet to determine percent inhibition (PI) using
the equation:
PI = 100 - I 00 test !!8!llJIle antigen 00 buffer I X 100 I 00 negative serum antigen - 00 negative serum buffer I
Fluoreseent Focus NeutraUzadon
After runniog the blocking ELISA, 20 egret samples and one dove sample had
sufficient sera left fur FFN. The FFN assay is a virus neutralization assay to determine 41
the amowrt and presence or absence of neutralizing antibodies to certain PRRS virus
strains. In this case, because of an insufficient amowrt of serum left in each sample, only
dilutions ofl:4, 1:8, and 1:16 were assayed to detect neutralizing antibodies. The FFN
procedure used in this study was previously described by Wu et al. (2001) for the
quantification ofPRRSV neutralizing anttoodies in porcine serum. The avian serum
dilutions were made with 100 111 of Eagle's Minimum Essential Medium (MEM)
containing 2% horse serum on a 96-microtiter plate. An equal amowrt ofSD 23983 virus
(SDSU, Brookings, SD) dilution (I :50 with MEM containing 2% horse serum) was added
to all the wells. The plate was incubated for 1 h of incubation at 37"C and its contents
were then transferred to a new 96-well tissue culture microtiter plate with MARC-I 45
cells (SDSU, Brookings, SD). The MARC-145 cell microtiter plate was incubated
overnight for at least 18 h at 37"C. After incubation, the plate was fixed with 300 111 of
80% acetone for 15 min at room temperature, followed by the addition of30 111 of
fluorescein isothiocynate-conjugated anti-PRRSV monoclonal anttDody, SOOW-17
(1: 1 00 dilution with PBS containing 5% horse serum). The plate was incubated for
another 30 min at 37"C. The readings were done using an inverted fluorescent
microscope. The test fields were read in comparison to the control fields. Positive
results were reported as the highest dilution that had at least 90% inhibition of fluorescent
foci Ninety percent neutralization of fluorescent foci units at 1:4 dilutions or less was
negative; 1:8 dilutions were borderline; and 1: 16 were weak positive. In general, SDSU
considers a 1: 16 neutralizing titer to be a clear positive result. However, in this study,
some hemolysis of the serum samples can cause some inhIbition ofPRRSV infectivity.
As a resu1t, 1: 16 neutralizing titers were considered weak positives.
42
Western Blotting
Western blotting teclmique was used to detect PRRSV antibodies in 20 cattle
egret serum samples. The teclmique was previously descnbed by Wu et al. (2001).
Briefly, proteins from purified PRRSV were separated by SDS-PAGE on 17%
acrylamide gels, and then transferred to nitrocellulose membranes. Membranes were
blocked, cut into strips, each containing a normal host cell control lane as well as a
PRRSV protein lane, then probed with sample and control serums. Antibodies binding to
immobilized PRRSV proteins were detected with protein A and G horseradish peroxidase
(Sigma), fullowed by 4-chioro-l-naphthol
Reverse Transcription Polvmerase Chain Readion
After WB, the remainder of the egret serum samples were pooled into one sample
and used in a nested reverse transcription PCR (RT -nPCR) assay previously described by
Christopher-Hennings et al. (2001). The primers used were based on ORF7 from the
PRRS virus. RIbonucleic acid from egret serum samples was extracted using the
guanidinium isothiocyanate-phenol-chlorofurm method (Christopher-Hennings et al.,
2001). A GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CAl was used fur
the reverse transcription of RNA and done according to manufacturer's instructions. The
products ofRT -PCR were subject to gel electrophoresis and visualized with an ethidium
bromide-stained agarose gel A positive test sample band would have migrated to the
position of the positive control fragment at 236 bp (Christopher-Hennings et aL, 2001).
43
CHAPTERm RESULTS and DISCUSSION
Cattle Egrets
The weight of28 egrets at Lualualei ranged from 223 to 478 grams (g) and
averaged 345 g (Table 3). Cattle egrets 16 and 14 from April's and July's collections,
respectively, were not weighed because they were wet.
Table 3. Cattle egret and spotted dove weights.
April 13, 1000 JuIy7,lOOO
Egret Weight
Egret Weight
Dove Weight
Identification Identification Identification Number
(g) Number
(g) Number
(g)
I 341 1 279 9 163
2 354 2 343 11 166
3 473 3 223
4 329 4 367
5 285 5 316
6 271 6 350
7 376 7 349
8 370 8 368 9 434 10 400 10 263 12 316
11 478 13 360 12 381 15 345
13 304
14 316
15 383
17 296
Average 353 Average 335 Average 165 Average Egret Weight: 345 g
No obvious externa1lesions other than the gun wound were observed on any of
the cattle egrets harvested fur sampling. Upon close examination of the 30 cattle egrets 44
during dissection, one individual had a liver that was abnorma1ly more yellow than the
others. Six cattle egrets were fuund to have more abdominal tat than the other egrets.
Cattle egret 3 collected on April 23, 2000 was the heaviest and had noticeably more keel
muscle. No other obvious internal abnormalities were observed fur the rest of the cattle
egrets or doves. The observations made on the cattle egrets and spotted doves in this
study were compared to each other and not to any published reference.
The sample size was based on the assumption that there was an overall PRRS
seroprevalence of 10% in the cattle egret popu1ation of300 individuals at Lualualei
(HDOA, 2001; Joo, 1994, 1995; S. M. Tanaka, personal communication). Under this
assumption, a sample size of30 egrets would give us a 95% chance ofidentiiYing an
infected egret population (HDOA, 2001). Although there were 30 cattle egret sennn
samples collected, only 24 samples had sufficient sennn to run bELISA The sample size
continued to decrease as other assays were done. Initially, another opportunity to collect
more samples was planned but native birds were beginning to breed. To reduce
disturbances, the culling of egrets was cancelled indefinitely.
Insed Identification
The insects fuund in the crop of 19 cattle egrets were classified into six different
orders (Table 4). Insects from the crop of two of the 19 cattle egret samples were further
classified into species (Table 5). Diptera and Blattodea occurred most frequently in the
cattle egret crops while Dermaptera and Hymenoptera were the least. One cattle egret
had what seemed to be Diptera larvae, which, due to a lack of experience in insect
identification, were not classified in any of the orders. Within the order Diptera, two
species (Chrysomya megacephala and Chrysomya rufifactes) from the family
45
Calliphoridae and one species (Volucella obesa) from the fiunily Syrpbidae were
identified. Dermestes maculatus DeGeer and Dermestes ater DeGeer were from the
order Coleoptera, family Dermestidae. Within the order B1attodea, Pycnoscelus indicus
from the family Blaberidae was the one species identified (Table 5). The two spotted
doves have grit-like material in their crops. There were no insects collected fur them.
Table 4. Orders of insects identified from 19 cattle egrets.
Order Group Common Name Frequen~
Diptera Flies 10 B1attodea Cockroaches 8
Coleoptera Beetles 5 Orthoptera Grasshoppers, Crickets 4 Dennaptera Earwigs 1
Hymenoptera Bees, Wasps 1
0[< Frequency is the number of egrets where these insect orders were detected.
Table 5. Scientific names of insects identified from two cattle egrets by Richard Tsuda of the Insect Diagnostic Laboratory of the University of Hawai'i at Manoa.
Order Family Scientific Name Common Name
Diptera Calliphoridae Chrysomya megacephala Oriental Blow Flies
Diptera Calliphoridae Chrysomya nffifacies Hairy Maggot Blow Flies
Diptera Syrpbidae Volucella obesa Hover Flies
B1attodea Blaberidae Pycnosce/us indicus Burrowing Roaches
Coleoptera Dermestidae Dermestes ater DeGeer Black Lardon Beetles
Coleoptera Dermestidae Dermestes maculatus DeGeer Hide Beetles
46
The study recorded that the cattle egrets ate insects such as tlies, cockroaches,
beetles, crickets, earwigs, wasps, and maggot-like larvae but there was no evidence of
birds or frogs. The cattle egrets appeared to be opportlmistic feeders with a tendency to
feed on tlies and cockroaches. The Naval Computer and Telecommunications Area
Master Station at Lualualei is in a livestock-producing community. According to
Toyama and Ikeda (1976), Chrysomya megacephala and Chrysomya rufifacies, two of
the species oftlies fuund in the examined cattle egrets, were abundant and thriving on
Wai'anae hog and poultry farms. west O'abu's hog, dairy and poultry farms were homes
to Volucella obeaa, another fly species eaten by the Lualualei cattle egrets. DuPonte et
al (2003) also fuund Chrysomya megacephala, Chrysomya rufifacies and Volucella
oheaa living on swine farms on the islands ofHawai'~ Kaua'i and Maui. The crop
contents of this study's cattle egrets ate consistent with the egrets feeding on hog farms.
Visits to hog farms could have exposed the cattle egrets to the PRRS virus.
Blocking Enzyme-Hoked Immunosorbent Assay & Fluorescent Focus NeutraUzation
For bELISA, positive results have at least 17 percent inhibition (PI) (Ferrin et al,
2004). The negative controls averaged 0.16% inhibition and the positive controls
averaged 77.33 PI. The PI of24 egret samples ranged from 2.42 to 15.23 and all were
negative (Table 6). The PI fur one dove sample was 3.31 % and the other dove sample PI
was 17.36%, borderline positive.
For FFN, if neutralizing antibodies were absent or insufficient in the serum
samples and neutralized less than 90% ofvira1 antigens on the microtiter plate, then the
result was negative. Thus, 1:4 orless than 1:4 dilutinns were considered negative, 1:8
47
dilutions were borderline and 1: 16 were considered weak positives (Nelson, 2001; E. A.
Nelson, personal communication).
Fluorescent fucus neutralization was not perfurmed on one dove and five egret
samples due to insufficient amounts of serum. Eight cattle egret samples were shown to
have no neutraIizing effects at 1:4 or less than 1:4 dilutions. Ten samples were borderline
at 1:8 and 3 samples were weak positive at 1: 16 (Table 6).
48
Table 6. Results for blocking enzyme-linked immunosorbent assay (bELISA) and fluorescent focus neutralization (FFN) .
Sample No. bELISA FFN Sample Buffer PI(%) Results DButions Results OD OD
Control-l 2.351 0.066 0.66 Negative <1:4 Negative
Nwative Control-2
2.378 0.07 -0.34 Negative <1:4 Negative Negative Positive
0.257 0.069 91.83 Positive Not Done Not Done Control
Control-3 0.925 0.07 62.83 Positive 1:16 WeakPos
Positive L000423-02 2.36 0.086 1.14 Negative 1:8 Borderline LOO0423-04 2.458 0.102 -2.42 Negative <1:4 Negative LOO0423-09 2.258 0.116 6.88 Nwative 1:16 WeakPos LOO0423-1O 2.216 0.277 15.7 Negative Not Done Not Done LOO0423-11 2.337 0.27 10.14 Nwative Not Done Not Done L000423-12 2.239 0.289 15.23 Negative 1:8 Borderline L000423-13 2.315 0.069 2.36 Negative 1:8 Borderline L000423-14 2.337 0.102 2.84 Nwative 1:8 Borderline L000423-15 2.254 0.277 14.05 Negative 1:8 Borderline L000423-16 2.242 0.245 13.18 Nwative Not Done Not Done LOO0423-17 2.279 0.077 4.27 Nwative <1:4 Negative LOOO727-01 2.333 0.072 1.71 Nwative 1:4 Nwative LOOO727-02 2.329 0.267 10.36 Nwative 1:4 Nwative LOOO727-03 2.329 0.27 10.49 Nwative 1:8 Borderline LOOO727-04 2.345 0.28 10.23 Nwative 1:16 WeakPos LOOO727-05 2.399 0.32 9.62 Nwative 1:4 Negative LOOO727-06 2.283 0.277 12.79 Nwative 1:8 Borderline LOOO727-07 2.243 0.081 6.01 Negative 1:8 Borderline LOOO727-08 2.226 0.157 10.05 Negative 1:8 Borderline "'LOOO727-09 2.149 0.248 17.36 Borderline Not Done Not Done LOOO727-10 2.238 0.254 13.75 Negative 1:16 WeakPos "'LOOO727-11 2.303 0.079 3.31 Nwative 1:4 Negative LOOO727-12 2.244 0.291 15.1 Negative Not Done Not Done LOOO727-13 2.413 0.071 -1.82 Nwative <1:4 Nwative LOOO727-14 2.286 0.119 5.79 Nwative <1:4 Negative LOOO727-15 2.315 0.143 5.58 Nwative 1:8 Borderline . . . . .
OD IS optical density and PI IS percent inhibitIOn. "Weak Pos" IS weak positive . "Positive Contror' is strong positive. "Control-3 Positive" is weak positive. "'Dove samples.
49
The bELISA and FFN resuhs may be true negatives because the specific and
neutralizing antibodies may have left the bird's system or decreased to undetectable
levels. Studies (Y oon and Stevenson, 2002) in pigs showed that specific antibodies are
usually detected by ELISA by 7 to 14 days post-infection, peak by 30 to 50 days, and
approach levels beyond detection from 4 to over 10 months. For FFN, studies in pigs
showed that neutralizing antibodies can be detected as early as 9 to 28 days, peaking by
60 to 90 days, and decline to undetectable levels in 12 months. Although the antibodies
can remain detectable from 4 to over 10 months, the titer usually begins to drop unless
the body encounters another viral challenge (Y oon and Stevenson, 2002). Although we
assume that the avian immune system could respond to the PRRS virus as in the pig, the
peak infection of 1996 that would give us the highest level of cattle egret exposure to the
virus was long past. In other words, even if the egrets were present in 1996 to be
subjected to a high level ofPRRS virus, the specific and neutralizing antibodies may
have long disappeared from their system. Nevertheless, HDOA's 2001 survey found that
a European strain and an American strain ofPRRSV were still circulating in the state of
Hawai'i, though at a much lower level It is possible, though not probable, that this low
dose ofvirus may have stimulated neutralizing antibodies in the weak positive egrets of
the neutralization assay.
The modified-live vaccine virus, derived from an American PRRS virus, has been
known to circulate in unvaccinated animals and could be a source ofvirus for the Mikilua
area (B0tner et aL, 1997). Vaccination for PRRS is an ongoing practice in Mikilua
Valley and could perpetuate the circulation ofa vaccine virus (H. M. Zaleski, personal
communication; B0tner et aL, 1997; Christopher-Hennings et aL, 1997; HDOA, 2001).
50
The vaccine virus, along with the American strain ofvirus on O'aho, may still be present
and could have stimulated neutralizing antibodies in three weak positive individuals of
the FFN assay (H. M. Zaleski, personal communication).
Fluorescent fucus neutralization results showed 10 borderlines and three weak
positives. Generally, SOSU considers a I: 16 neutralizing titer to be a clear positive
result. However, in this study, some hemolysis of the serum samples (which is common
with birds) can cause some inhIbition ofPRRSV infectivity. Although 1:16 neutralizing
titers were considered weak positives, they may not be true weak positives (E. A. Nelson,
personal communication).
Western Blotting & Reverse Transeription Polymerase Chain Reaetion
The inconsistent results between bELISA and FFN prompted the perfurmance of
WB and RT -PCR in attempts to detect PRRS antibodies and PRRS viral RNA,
respectively. Western blotting did not detect any PRRS antibodies and RT-PCR resulted
in no detection ofPRRS viral RNA. Ifviral RNA had been present, the test sample band
would have migrated to the position of the positive control (PRRS virus 23983) fragment
at 236 bp (Nelson, 2001). Table 7 summarizes results fur all fuur assays and Figure 7
snmmarizes the number of avian serum samples used fur the specific assays.
51
Table 7. Summary of results. Blocking enzyme-linked immunosorbent assay (bELISA), fluorescent fucus neutralization (FFN) and Western blotting (WB) were fur the detection of porcine reproductive and respiratory syndrome viral antibodies. Reverse transcription polymerase chain reaction (RT -PCR) was fur the detection ofviral ribonucleic acid.
Antibodies Viral RNA
Assay bELISA FFN WB RT-PCR Negative Control <17% inhtbition <1:4 - -Positive Control >17% inhtbition 1:811:16 + + Weak Positive: ElUet 0 13
Weak Positive: Dove 1 0 Not Done Not Done Negative: Egret 24 7 20 20
Negative: Dove I I Not Done Not Done
NO DATA 6 11 12 12
Figure 7. Use of avian serum samples in various assays.
Start: 30 Cattle Egrets
Samples not done, low 2 Spotted Doves
sera 6 Cattle Egrets
... Blocking Enzyme-linked Immunosorbent Assay:
24 Egrets Samples not done, 2 Spotted Doves
low sera: .. 4 Cattle Egrets
Fluorescent Focus Neutralization: 1 Spotted Dove 20 Cattle Egrets I Spotted Dove
Samples not done, low .. sera: I Spotted Dove Western Blotting:
20 Cattle Egrets
! Reverse-Transcription Polymerase
Chain Reaction: 20 Cattle Egrets
52
Western blotting and RT-PCR confirmed the negative ELISA and FFN resu1ts
although the sample size had been reduced to only 20 samples fur both assays. Despite
the small sample size, PCR is known to detect sma1l amounts of genetic material
(Spagnuolo-Weaver et a1., 1998). Although there is a chance that the RT -PCR used in
this study may not recognize the various strains possibly circulating in Hawai'~ it is
highly unlik:ely. Based on the lack of suscepttbility of various species studied by
Trincado et aI. (2004b), Wills et a1., (2000), and Zimmerman et a1., (1997) and the
bELISA results from this study, it is more likely that both RNA and antibodies were not
present in the cattle egrets.
This study assayed serum samples in cattle egrets using bELISA, FFN, WB and
RT -PCR with negative results while Zimmerman et aI. (1997) assayed young mallard
fecal samples via VI using porcine alveolar macrophages with positive results. Bcx:snse
little is known about the avian immune system's response to PRRS virus, VI from fecal
samples may provide more accurate results than pig serology on avian serum. Trincado
et aI. (2004b), though, did assay adult mallard serum using the neutralization test and
avian feces using RT -PCR, VI and swine bioassay. All resu1ts were negative, though the
two studies used different strains of virus. Wills et aI. (2000) tested starling and sparrow
feces using VI and had negative resu1ts except fur one sparrow three days after virus
inoculation. It is possible that different avian species are susceptible to different strains
ofvirus at different stages of their lives and that antibodies are detectable at different
times after inocu1ation (Trincado, et a1., 2004b; Hooper et a1., 1994; Wills et a1., 2000).
53
Technical Limitations
The collector's lack of blood collecting experience from birds was a mctor,
resulting in hemolysis in the collected avian blood samples. Based on personal field
observations, the egret blood clotted fuirly quickly after death. In attempts to get fresh
liquid blood into the Serum Separator Tube Vacutainers™, the collector rushed the rate at
• which the blood was aspirated from the bird and transferred into the vials. After
centrifugation, some of the serum samples exhibited pink hues, indicative of hemolysis.
Hemolysis, along with the sticky gel in the Vacutainers™, was a source of microscopic
debris that could cause fulse negatives or fuIse positives in bELISA The presence of
biological agents could have interfered with the specificity of bELlS A, FFN and RT -PCR
and obscured the interpretation ofresuIts (N. H. Ferrin and E. A Nelson, personal
communications). Collection of blood from a dove was much more difficult than from a
cattle egret fur an inexperienced collector simply because the dove was much smaller.
The borderline positive (17.36 PI) bELISA resuh for the dove might have been caused by
intetfetence in the antibody-antigen interaction (N. H. Ferrin and E. A Nelson, personal
communications). Viruses from another disease could have caused the fulse weak
positive in the dove (N. H. Ferrin, personal communication), although all the birds
seetned, to the best of my knowledge, physically heahhy during the examination.
Zimmerman's (1997), Wills' (2000), Hooper's (1994) and Trincado's (2004b)
teams all had immediate access to better storage fucilities and more advanced techniques
than this study. In Zimmerman's study (1997) with maIIards, the tissue and fecal samples
collected were either stored at -7f1'C or assayed soon after collection without
compromising the quality of the samples. Hooper et at. (1994) stored their homogenized
54
tissue samples at -80"C befure VI using porcine alveolar macrophages was done.
Zimmerman et al. (2003b) noted that a study by Benfield and his team in 1992 showed
that the PRRS virus can survive at least one month at 4·C and at least fuur months at
-70"C. This study stored the centrifuged egret serwn in a -20·C freezer fur more than a
year befure they weremailedtoSDSUfurserology.Itis not sure how the length of
storage at this temperature affects the PRRS virus but it is more likely to be better
preserved at -70·C. Bloemraad et al. (1994) showed that the lower the temperature, the , longer the storage without damage to the virus. The long delay was partly due to the wait
on confirmation of a third collection date and partly due to the search fur a diagnostic
facility that would have an assay that will test avian samples fur PRRS anttbodies.
Although there were fuur different assays done fur this study, the relatively warmer
storage conditions could have led to a negative PRRS antibodies and virus detection.
Degradation of the PRRS virus was a possibility.
Further Studies
Virus inoculation of the cattle egrets could help us better conclusively decide
whether or not cattle egrets were susceptible to PRRS. Zimmerman et al. (1997), Wills et
al. (2000), Hooper et al. (1994) and Trincado et al. (2004) were able to ensure that the
experimental animals in question were exposed to the virus either orally, intranasally,
and/or by intramuscular injection. Access to PRRS virus stock would allow us to do
controlled inoculation experiments to gusrantee the test subject's exposure to the virus.
Sampling of organ tissues in cattle egrets might have led to positive detection of
the virus, although studies have fuund negative results in animal tissues from opossums,
raccoons, dogs, cats, skunks, rats and mice via VI and RT -PCR (Hooper et al., 1994;
ss
Wills et at., 2000). Otake et at. (2003a, 20003c) were able to detect PRRS viral particles
in the guts of houseflies up to 12 h and mosquitoes up to 6 h after ingestion from a
PRRS-infected pig.
Zimmerman et at. (1997) and Trincado et at. (2004) looked at the infectivity of
viruses in feces and organ tissues via swine bioassay. Swine bioassay is usually done
rollowiog a positive RT -peR result. Since PCR detects the presence ofviral DNA or
RNA, the pmpose of swine bioassay is to assess the infectivity of the viral particle round.
This study did not have the fucility, finances or reason to do such an assay. If ever there
were an opportunity to do a controlled inoculation study with cattle egrets, a swine
bioassay would be extremely useful.
Mechanical transfer ofPRRS virus in cattle egrets cannot be ruled out. Otake et
at. (2002<1; 2004) showed that an individual housefly and mosquitoes could mechanically
carry the PRRS virus in their gut and transmit it from one pig to another. Feces can also
be transmitted mechanically. As opportunistic feeders on flies that breed in pig manure
(Toyama and Ikeda, 1976), egrets could step on PRRS infected pig manure and carry the
PRRS virus from pig fimn to pig fimn during their search ror rood Since cattle egrets
congregate in rookeries at the site of collection (S. M Tanaka, personal communication),
it is likely that they could pass the infected manure to each other, and carry the virus to
other pig limns. Future studies should consider sampling bird feces and intestine.
56
CHAPTER IV CONCLUSIONIIMPLICATIONS
With conflicting results from the tests done, it is impossible to conclude that cattle
egrets are carriers of the PRRS virus. Low positive results suggest further research
should be done on cattle egrets and spotted doves. A virus challenge study to both cattle
egrets and spotted doves may answer the question why certain biosecured tiums are
getting the virus in Hawai'i and worldwide. Nevertheless, taking measures to prevent
any unknown vectors is better than getting a PRRS infection and suffering significant
economic losses.
Economic Impacts
The impact of economic loss is substantial with PRRS. The obvious costs are the
loss of sales of animals such as piglets and sows due to death; the low production rates
due to decreased bbido, low conception rates, anestrus, subclinically infected animals and
loss in average daily gains and feed efficiencY. Preventative measures can also be costly.
Diagnosis of the disease and vaccination of the pigs can be very costly, especially if a
diagnosis is wrong and requires retesting or the vaccine does not prevent future outbreaks
(Holck and Polson, 2003, Dee and Joo, 1994). In a farm with 1,000 sows, Pejsak and
Markowska-Daniel (1997) recorded 60"A. higher costs than the previous year in attempts
to treat and prevent secondary infections on a Polish fium. Kerkaert, et al. (1994)
recorded a 70% decrease in total profits, taking into consideration such filctors as sow
inventory, pigs weaned, reproductive performance, mortality rates, average daily gain,
feed conversion, labor, medication, vaccination and various supplies. Because costs in
Hawaii are higher than on the mainland (Sharma et al., 1996), losses due to filctors such
as poor feed efficiency have a larger effect. The immediate use of medication and 57
vaccinations to remedy the disease can lead to costly false hopes; sometimes, herd
management procedures must be evaluated to determine and control the initial source of
infection. Holck and Polson (2003) noted that Hoe1t1ing estimated an average of$302
per breeding female for four different herds.
Control Measures
On the U.S. mainland, there are several ways to manage PRRS in a herd. Nursery
depopulation is recommended for persistent populations. Before depopulating a nursery,
a serological profile should be done to determine the status and source of infection.
Nursery depopulation requires the permanent removal of seropositive pigs, a source of
infection. Seronegative animals are moved into a PRRS-:free environment while the
previous housing is washed and disinfected over a two-week period. All items that can
possibly be a PRRSV reservoir should be removed and/or washed and disinfected before
the seronegative animals are reintroduced into the area (Dee and Joo, 1994; Dee et al.,
1994; Dee and Joo, 1997). Nursery depopulation may not be effective if depopulated
herds are house in close proximity to the cleaned facility (Dee et al., 1997). Other similar
test and removal methods have shown promise (Dee et al., 2000; Vansickle, 2004; Le
Potier et al., 1997).
If a herd is infected, it is important to achieve complete herd immunity in order to
successfully get rid ofPRRS. Uninfected naive pigs in an infected herd can serve as the
victim for the next wave ofPRRS if the cohabiting carrier pigs begin to shed the virus
(Dee and Joo, 1994). To accomplish complete herd immunity, one might even consider
purposely exposing the naive animals to PRRSV by injection of a live virus vaccine. The
use of this method allows the producer to control the time and dose ofinjection
S8
(preferably 3-5 months befure breeding) and type ofvirus to use. In contrast, an
unexpected exposure to an unknown PRRS virus can lead to a more devastating
uncontrolled epidemic (Daniels and FitzSimmons, 2002; Dee and Joo, 1994). Any new
pig entering one's fimn should go through a series of tests to prevent PRRS outbreaks.
Since Hawai'i utilizes a continuous system of swine production. swine of
different ages are often housed together and infect each other easily. As a resuh,
depopulation can be a very costly option. The achievement of complete herd immunity
may be a better and more effective choice (H. M. Zaleski, personal communication). If
all fimns on O'ahu could achieve complete herd immunity, the virus can be kept at a
minimum, or possibly eradicated (S. A Dee and H. M. Zaleski, personal
communication). Pig fimns ofMikilua Valley in close proximity will not have to worry
about PRRS infection from their neighbors. Prior to entry onto these farms, pigs should
always be tested to determine whether or not the animal is negative or positive; and if
positive, which strain is involved (HDOA, 2001; H.M. Zaleski, personal communication).
Vaccination with the homologous strain should be fullowed to prevent pockets of low
immunity where the virus can thrive and cause outbreaks (Christopher-Hennings et al.,
1997).
Swine producers should also practice aseptic procedures and keep insect
infestations at a minimum. IfPRRS is not present on a farm, it is extremely important to
apply preventative biosecurity measures to keep it out (HDOA, 2001). In recent years,
many PRRS preventative biosecurity measures have been atudied. Studies have
suggested important sanitation procedures that include disinfecting shoes in 6% bleach
fuot baths, changing clothes and gloves frequently, washing hands, disinfecting all
S9
medical equipment and contacted surfaces and any other possible fumites often when
working around infected pigs and between farms (Dee, 2002; Dee et al., 2002, 2003,
2004; Otake et al., 2002b, 2002c). One must remember that although the frequency of
mechanical transmission in the warm weathers ofHawai'i are low, vira1 transmission is
not entirely impossible (Dee et al., 2003).
Otake et al. (2002d, 2003c, 20031, 2003b, 2004) have recently cited flies and
mosquitoes as concerns fur PRRSV transmission. Toyama and Ikeda (1976) have shown
that local pig can be infested with flies. With this knowledge, farmers might consider the
use of insect traps and/or electrical bug zappers to keep tIying insect populations low.
The use of screens is recommended where possible to reduce insect-pig contact. The risk
of avian and mammalian transmission is still questionable (Zimmerman et al., 1997;
Trincado, et al., 2004b; Hooper et al., 1994; Wills et al., 2000) although reducing contact
between outside animals and production pigs can prove to be beneficial in more ways
than one.
Further transmission studies can elucidate the mystery behind PRRS area spread
in Hawai'i Egret organ tissues and feces could be collected and assayed according to
Zimmerman et al. (1997) with a sufficient sample size. Inoculation studies with controls
could be designed to achieve more definitive answers. Based on the reports by Toyama
and Ikeda (1976) and Otake et al. (2002d, 20031, 2003b, 2003c, 2004), insects should be
thoroughly investigated. The PRRS virus is a dynamic virus (Change et al., 2002) and
studies should be continuous to keep ahead of the change.
60
APPENDIX
Table 8 Comments on avian dissection. . BirdID Wt.(g) Comments
L000423-01 341.1 Flies in crop L000423-02 353.9 Crop (crickets)
AduItlBreeding plllmmage; Very muscu1ar, gonads LOOO423-03 472.5 (male?); Crop (roaches)
L000423-04 329 Fat; Yellowed liver; Crop (maggots) LOO0423-05 284.6 Fats; Crop (wasp, flies) LOO0423-06 270.5 Crop (roaches)
Testicle in body cavity? Male?; Comb with colored LOOO423-07 376.1 feathers; Breeding plummage; Crop (lots of
roaches) L000423-08 369.5 Crop( beetles) L000423-09 434.1 Breeding plummage; Fats; Crop (flies) L000423-10 263.3 Crop( flies)
L000423-11 478.3 Crest w/colored feathers, lots of tilt, gonads; Crop (crickets/grasshoppers)
LOO0423-12 381.2 Fats; Crop (roaches); no gonads fuurul. LOO0423-13 303.7 Crop (roach, earwigs) L000423-14 316.2 Crop (mix of roach, flies, beetle?) L000423-15 383.1 Fat; Crop (roach, grasshopper/crickets) L000423-16 Wet, no weight L000423-17 296.1 Crop flies; Some fatty tissues LOOO727-01 279.2 LOOO727-02 343.1 LOOO727-03 223.3 Fats LOOO727-04 367.2 Breeding plumage LOOO727-05 315.9 Male? LOOO727-06 349.8 L000727-07 349.4 Breeding plumage LOOO727-08 367.7 LOOO727-09 163 Dove; grits, no insects LOOO727-10 400.4 LOOO727-11 166.3 Dove; no insects, grits LOOO727-12 315.6 LOOO727-13 359.6 LOOO727-14 Wet, no weight LOOO727-15 345.3
61
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