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Published quarterly by the Canadian Veterinary Medical Association Publication trimestrielle de l’Association canadienne des médecins vétérinaires JULY/JUILLET 2017, VOL. 81, NO. 3

ARTICLES Oxidative stress and food supplementation with antioxidants in therapy dogs Porcine reproductive and respiratory syndrome Sara Sechi, Filippo Fiore, Francesca Chiavolelli, virus (PRRSV) in pig meat Corrado Dimauro, Anna Nudda, Raffaella Cocco . . .206 Philippe Raymond, Christian Bellehumeur, Malliga Nagarajan, Diane Longtin, Evaluation of transmission infrared spectroscopy Alexandra Ferland, Peter Müller, and digital and optical refractometers to identify Rachel Bissonnette, Carole Simard...... 162 low immunoglobulin G concentrations in alpaca serum Development of porcine circovirus 2 (PCV2) open Ibrahim Elsohaby, Jennifer J. Burns, reading frame 2 DNA vaccine with different Christopher B. Riley, J. Trenton McClure...... 217 adjuvants and comparison with commercial PCV2 subunit vaccine in an experimental challenge Cystic duct pressures after ligation with a Changhoon Park, Jiwoon Jeong, Kyuhyung Choi, novel absorbable device in an ex vivo caprine Su-Jin Park, Ikjae Kang, Chanhee Chae...... 171 cholecystectomy model Andrea J. Sundholm Tepper, Odd V. Höglund, Retrospective study of the relationship of Torque Bonnie G. Campbell, Chi-Ya Chen, teno sus virus 1a and Torque teno sus virus 1b Boel A. Fransson...... 223 with porcine circovirus associated disease Alejandro Vargas-Ruiz, Hugo Ramírez-Álvarez, José I. Sánchez-Betancourt, Víctor Quintero-Ramírez, SHORT COMMUNICATIONS/COMMUNICATIONS BRÈVES Ignacio C. Rangel-Rodríguez, Joel A. Vázquez-Perez, Evaluation of serum symmetric dimethylarginine Lucia A. García-Camacho ...... 178 in dogs with heartworm infection Identification of the linear ligand epitope Bom-Sul Choi, Hyeongsun Moon, Sang-IL Suh, on classical swine fever virus that interacts Changbaig Hyun...... 228 with porcine kidney 15 cells Maternal and fetal arterial blood gas data in Yin Mei, Feng Yue, Hong-mei Ning, Juan-juan Zhou, normotensive, singleton, isoflurane anesthetized Xuan-nian Wang...... 186 sheep at 124–126 days of gestation Direct repeat unit (dru) typing and antimicrobial Claire M. Loughran, Matthew W. Kemp, resistance of methicillin-resistant Staphylococcus Gabrielle C. Musk...... 231 pseudintermedius isolated from dogs in Atlantic Towards an improved estimate of antimicrobial use Canada in animals: Adjusting the “population correction unit” Matthew E. Saab, J. Scott Weese, J.T. McClure...... 192 calculation The impact of carboplatin and toceranib phosphate Brian R. Radke...... 235 on serum vascular endothelial growth factor (VEGF) and metalloproteinase-9 (MMP-9) levels and survival in canine osteosarcoma Tracy L. Gieger, Julie Nettifee-Osborne, Briana Hallman, Chad Johannes, Dawn Clarke, Michael W. Nolan, Laurel E. Williams...... 199 Article

Porcine reproductive and respiratory syndrome virus (PRRSV) in pig meat Philippe Raymond, Christian Bellehumeur, Malliga Nagarajan, Diane Longtin, Alexandra Ferland, Peter Müller, Rachel Bissonnette, Carole Simard

Abstract Porcine reproductive and respiratory syndrome, caused by the porcine reproductive and respiratory syndrome virus (PRRSV), is an economically important disease in the swine industry. Previous studies demonstrated the presence of the virus in pig meat and its transmissibility by oral consumption. This study further analyzed the infectivity of PRRSV in commercial pig meat. Fresh bottom meat pieces (n = 1500) randomly selected over a period of 2 y from a pork ham boning plant located in Quebec, Canada, were tested by reverse transcriptase polymerase chain reaction (RT-PCR). Each trimmed meat was stored in the plant freezer, subsampled weekly for up to 15 wk, and tested with quantitative RT-PCR to determine the viral load. Meat infectivity was evaluated using specific pathogen-free piglets, each fed with approximately 500 g of meat at the end of the storage time. Genotype-specific RT-PCR confirmed the presence of PRRSV mainly during cold weather in 0.73% of the fresh meat pieces. Wild and vaccine strains of genotype 2 were detected. Porcine reproductive and respiratory syndrome virus nucleic acid was stable in meat stored at around 220°C during the 15 wk. Serological and molecular analysis showed the transmission of infection by a majority of PRRSV positive meat pieces (5/9) fed orally to naïve recipients. The results confirmed a low prevalence of PRRSV in market’s pig meat, and virus transmissibility by oral consumption to naïve recipients even after several weeks of storage in a commercial freezer. It occurred mainly with meat harboring the highest PRRSV RNA copies, in the range of 109 copies per 500 g of meat, with both wild type and vaccine-related strains.

Résumé Le syndrome reproducteur et respiratoire porcin, causé par le virus du syndrome respiratoire et reproducteur porcin (vSRRP), est une maladie ayant un impact économique important pour l’industrie porcine. Des études antérieures ont démontré la présence du virus dans la viande de porc ainsi que sa transmissibilité par ingestion. La présente étude poursuit l’analyse de l’infectiosité du vSRRP dans la viande commerciale de porc. Des coupes de fesses de porc fraîches (n = 1500) sélectionnées aléatoirement sur une période de deux ans dans une usine de désossage située au Québec (Canada), ont été testées en utilisant une transcription réverse suivie d’une amplification en chaîne par polymérase (RT-PCR). Chaque pièce de viande parée a été entreposée dans les congélateurs à l’usine, échantillonnée hebdomadairement pendant 15 semaines, et testée par RT-PCR quantitatif afin de calculer la charge virale. Le potentiel infectieux a été évalué sur des porcelets exempts d’agent pathogène spécifique qui ont été nourris avec approximativement 500 g de viande à la fin de la période d’entreposage. Une RT-PCR spécifique au génotype a confirmé la présence du vSRRP principalement durant les temps froids, dans 0,73 % des pièces de viandes fraîches. Des souches sauvages et vaccinales du génotype 2 ont été détectées. L’acide nucléique du virus du syndrome respiratoire et reproducteur porcin est demeuré stable dans la viande durant la période d’entreposage de 15 semaines à 220 °C. L’analyse sérologique et moléculaire a démontré la transmission de l’infection par une majorité des pièces de viande positives au vSRRP (5/9) chez les porcelets naïfs ayant consommé la viande. Les résultats confirment la faible prévalence du vSRRP dans la viande distribuée sur le marché ainsi que la transmissibilité du virus par consommation orale chez des hôtes naïfs même après plusieurs semaines d’entreposage dans un congélateur commercial. La transmission s’est produite surtout avec les viandes ayant un nombre de copies d’ARN de vSRRP plus élevés, environ 109 copies par 500 g de viande, associées à des souches de type tant sauvage que vaccinal. (Traduit par les auteurs)

Introduction 15 kb in length that encodes at least 9 open reading frames (ORF) (4). There are 2 recognized PRRSV genotypes: the North American Porcine reproductive and respiratory syndrome (PRRS) is among (genotype 2, VR-2332 prototype) and the European (genotype 1, the most economically significant swine infectious diseases (1,2). Lelystad prototype). Both have similar genomic organizations, but The role of pig meat and swill feeding in PRRS virus (PRRSV) trans- are genetically and antigenically distinct (5). In Canada, the North mission is questioned as international trade in pork expands (3). American genotype has been reported in lineages typical of vac- The enveloped virus is between 50 and 65 nm in diameter and is cines (Ingelvac PRRS ATP, Ingelvac PRRS MLV) and of the MN-184 classified in the Arteriviridae family within the order Nidovirales. It wild types (6,7). Porcine reproductive and respiratory syndrome is contains a single-stranded positive-sense RNA of approximately mainly characterized by reproductive failure in sows and respiratory

Canadian Food Inspection Agency, Saint-Hyacinthe Laboratory, Saint-Hyacinthe, Quebec J2S 8E3. Address all correspondence to Dr. Philippe Raymond; telephone: (450) 768-6808; fax: (450) 768-6767; e-mail: [email protected] Received July 7, 2016. Accepted January 17, 2017.

162 The Canadian Journal of Veterinary Research 2017;81:162–170 illness in pigs of all ages. These clinical signs vary markedly between in a labeled plastic bag and stored immediately in cardboard boxes herds and depend on the virulence of the infecting strain. In eastern among commercial meats in the company’s freezer, maintained Europe and China, new highly pathogenic subtypes of PRRSV have between 224°C and 221°C. Based on the reverse transcriptase been reported (8–10). polymerase chain reaction (RT-PCR) assays, 1 PRRSV non-confirmed, Porcine reproductive and respiratory syndrome virus is highly 9 PRRSV positive, and 2 RT-PCR PRRSV negative frozen meat pieces contagious in pig herds. It can be transmitted by direct contact or were selected for further analysis. In storage follow-up experiments, by infectious aerosols. Vaccines have been used to limit the propa- they were subsampled weekly at the PHBP from T1 up to 15 consecu- gation of the disease, but are often found inefficient (11). The virus tive weeks (Tmax). Subsamples were collected in each frozen meat can also persist for months in the target cells of the monocyte and piece near the first sampling locations using a clean and sterilized bit macrophage lineages of an infected animal, despite the immune and a drill. Approximately 2 g of frozen meat were subsampled and protection (11,12). Transient detection of PRRSV in pig meat follow- kept on ice until processed at the lab a few hours later. The bottom ing experimental transmission has been observed (12). The virus frozen meat pieces were immediately put back in their boxes in the was isolated in vitro from 6 out of 1049 sample pools of fresh meat company’s freezer, among commercial meats. At the last sampling

(13), while others failed to detect PRRSV in 472 pig carcasses tested time (Tmax), leftover frozen meats were transported in a frozen state by PCR (14). and stored at 270°C, until used in viral transmission bioassays. In countries currently free of PRRS, the risk of importing the PRRS virus in fresh pork is a concern. Some early import risk analyses for Viral transmission bioassays chilled or frozen meats have concluded that virally infected pig meat All animals were treated according to the policy and guidelines could represent a source for the introduction of the virus in PRRS- of the Canadian Council on Animal Care (CCAC) and the Animal free countries (15,16). These analyses were notably based on a study Care Committee of our laboratory. For the feeding trials, 9 PRRSV from Lelystad, the Netherlands, showing the transmission of PRRSV RT-PCR confirmed positive and one non-confirmed pig meat sample through oral uptake of infected porcine muscular tissues by naïve were fed to the piglets. These bioassays were adapted from Magar recipients (17,18). In 2003, Magar and Larochelle (6) led a study to and Larochelle (6) using the same facilities, biosecurity measures, investigate whether pig meat could harbor PRRSV and if so, whether and animal adaption protocol. Each meat sample was used to feed viruses in positive meats could infect the animals when fed to SPF 2 specific pathogen-free (SPF) piglets 5 to 6 wk of age. Each pig pigs. In their study, analyses and bioassays were performed with meat pair was housed in a separate cubicle. These piglets were provided conserved at ultra-low temperatures. It was argued that these experi- by the CFIA Ottawa Laboratory (Fallowfield, Ontario). The meat ments were performed in optimal conditions for virus survival in meat samples were thawed at 4°C overnight, weighed, cut into small (19–21). For instance, van der Linden et al (18) showed that freezing pieces (around 2 cm3) with sterile scalpels, and divided in 4 equal meat at 223°C for 10 d and then thawing it decreased the virus titers portions of approximately 250 g each. Piglets were fed a portion on in the majority of PRRSV infected muscle samples. The present study 2 consecutive days. The portion used to feed the pigs on the second was undertaken to reproduce conditions in commercial settings under day was kept at 4°C overnight. In an alternative protocol, 3 meat which meat would be cut, packaged, and stored in a frozen state for samples (ID 1311, 1332, and 1424) were also divided into 4 equal por- many weeks, similar to overseas import or export conditions. It pro- tions of around 250 g. While 2 portions were stored at 4°C overnight, vides additional knowledge on PRRSV prevalence, survival in pig the other 2 were stored at room temperature (RT, between 18°C to meat, meat viral load, and transmission from meat to naïve piglets. 23°C). A pig was fed on 2 consecutive days with the meat sample stored at 4°C while the paired animal, located in a separate cubicle, Materials and methods received the other part of the same meat sample stored at RT. Each trial included either 1 or 2 control pigs maintained on a standard pig diet with no thawed meat throughout the experiments. Pork samples Pork carcasses were from market pigs killed at 3 abattoirs from Enzyme-linked immunosorbent assay (ELISA) the province of Quebec, Canada. Carcasses were transported in Blood samples were collected on a regular basis from the jugu- refrigerated trucks and received at a pork ham boning plant (PHBP) lar vein of each piglet with a 20-G needle. Throat mucus samples located in St. Hyacinthe, Quebec, 2 to 5 d after slaughter. Between were collected with minitip flocked swabs (Millipore; Billerica, 5 and 17 fresh bottom meat pieces of around 900 to 1100 g each were Massachusetts, USA) to follow PRRSV infection in piglets. Both were selected randomly from the cutting and trimming lines 2 to 3 d a collected at arrival and at 0, 7, 14, 21, and 28 d after feeding. Only week (117 visits). The meat pieces could not be associated with a the throat samples from the first 3 bioassays were tested. Serum and specific abattoir once on the cutting and trimming line. Each meat throat samples were used to detect PRRSV by molecular assays. piece was identified using a unique identification number (ID). Serum samples were tested for the presence of antibodies to PRRSV Approximately 2 g of muscle tissue was subsampled from each in piglets via ELISA (HerdCheck; IDEXX Laboratories, Westbrook, meat piece with a sterile scalpel at 3 distant locations and pooled Maine, USA) as recommended by the manufacturer. in a 50 mL sterile tube. These subsamples corresponded to week 0 RNA extraction (T0). Meat subsamples were kept on ice until their arrival at the CFIA Saint Hyacinthe Laboratory for RNA extraction a few hours later. All meat subsamples from the PHBP were homogenized on the At the PHBP, each of the selected fresh bottom meat pieces was put same collecting day as previously described (22). Supernatant

2000;64:0–00 The Canadian Journal of Veterinary Research 163 ­aliquots were stored at 270°C until nucleic acid extraction. RNA was Mississauga, Ontario). The RNA transcript was quantified with the extracted from 100 mL of the meat homogenate using the RNeasy Nanodrop-1000. Mini Kit according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Total RNA was eluted with 40 μL of RNase-free water. SYBR Green quantitative Real-Time RT-PCR A Nanodrop-1000 (Thermo Scientific, Wilmington, Delaware, USA) (qRT-PCR) was used to quantify total RNA in each sample (OD 260 nM). For the To assess viral load before the transmission experiment at T10 or meat-fed piglets, RNA was extracted from 140 mL of serum sample Tmax in meats and to evaluate viral decay over time from T1 to Tmax, using the QIAamp Viral RNA Mini Kit (Qiagen). RNA was also viral RNA copies were estimated by one-step SYBR Green qRT-PCR extracted from throat swab samples using RNeasy Mini Kit with assay (ORF-7 qRT-PCR) using the QuantiTect SYBR Green RT-PCR 0.6 mL RLT buffer. All RNA extracts were stored at 270°C prior Kit (Qiagen) an the RNA transcript standard. The PR15M and PR16M to the analyses. The OR7 RT-PCR analyses were conducted within primers and the MX4000 or MX3005p real-time thermocyclers a week while samples from decay were combined on the same (Agilent Technologies, Santa Clara, California, USA) were used for plate and tested by OR7 qRT-PCR at the end of the storage period. these assays. The qRT-PCR included a reverse transcription step at Phylogeny analyses were conducted on RNA extract stored up to 3 y. 50°C for 30 min and a PCR amplification step with enzyme activa- tion at 95°C for 15 min. These were followed by 40 cycles of 95°C RT-PCR for 15 s, 57°C for 30 s, and 72°C for 30 s, and finally a dissociation

The RT-PCR for the detection of the ORF-7 gene of both American cycle. All samples from the same animal (T0 to Tmax) were analyzed and European strains was performed on 1500 fresh meat samples on the same plate to avoid inter-assay variability. In the presence of a collected randomly as a screen test (T0). A 3 mL volume of RNA specific melting temperature [Mt (78.5°C to 80.5°C)], the sample was extract was mixed with 0.48 mM of primers P1 and P2 developed declared positive. Samples were declared positive but not quantifi- previously and tested using the Qiagen One-Step RT-PCR Kit and able for PRRSV when non-specific Mt was detected in addition to the GeneAmp PCR System 9770 (Applied Biosystems, Carlsbad, the specific one. These results were indicative of a simultaneous non- California, USA) in the presence of RNasin (Promega, Madison, specific amplification and were detected only below 100 viral RNA Wisconsin, USA) (23). The RT-PCR included a reverse transcription copies/mL, which represents the limit of quantification (LOQ). No step at 50°C for 30 min and a PCR amplification step with enzyme specific amplification was detected more than 95% of the time below activation at 95°C for 15 min followed by 34 cycles of 94°C for 30 s, the limit of detection (LOD) of 10 viral RNA copies/mL. The number 57°C for 30 s, and 72°C for 30s, and a final elongation step at 72°C of viral RNA copies per mg of each quantifiable subsample tested by for 7 min. When analyzed on ethidium bromide agarose gel (2%) a qRT-PCR was calculated according to the standard curve multiplied specific band of 303 bp was expected for genotype 2, whereas a band by the RNA extract volume (40 mL), divided by the extraction effi- of 291 bp was expected for the genotype 1. A sample having a band cacy factor (7%) and by the weight of the meat homogenate sample of the expected molecular weight was declared PRRSV presumptive, (20 mg per extract), and normalized using the total RNA extracted. if the positive and negative controls were valid. Each presumptive positive RNA sample was retested with distinct ORF-7 RT-PCR Porcine circovirus (PCV) detection using similar RT-PCR mix and amplification conditions, but different For the detection of PCV nucleic acid in meat extract, DNA was primer sets. These tests were also done to determine the genotype of extracted using the DNeasy Tissue Kit (Qiagen) and recovered the virus. The primer set PR15M (GGTAAGATCATCGCTCAGCA) with 50 μL of elution buffer. A volume of 3 μL was tested using the and PR16M (GACACAATTGCCGCTCACTA) was developed in Platinum Taq DNA Polymerase Kit (Invitrogen), 1.5 mM MgCl2, this study and was specific to genotype 2 (ORF-7 RT-PCR) (data not and 0.2 mM of each VCP5F (AGTGAGCGGGAAAATGCA) and shown). This primer set amplified a region that corresponds to bp VCP6R (CACACAGTCTCAGTAGATCATCC) primers. This primer position 14969 to 15116 of NVSL (AY545985.1). To detect genotype 1, set amplified a region that corresponds to bp position 515 to 741 of P4 and P2 primers were used (23). Each presumptive positive was postweaning multisystemic wasting syndrome PCV (AF027217.1). also tested with PRRSV primers UN17F and UNI17R used in the The PCR was achieved after 2 min of incubation at 95°C followed by study of Magar and Larochelle (6). A presumptive meat was con- 35 cycles at 95°C for 1 min, 56°C for 1 min, and 72°C for 1 min. The firmed PRRSV positive using at least 2 distinct extracts tested with PCR products were kept at 4°C until agarose gel evaluation. Positive ORF-7 RT-PCR assays, otherwise it was declared non-confirmed. results for the PCR were further confirmed using the same PCR This approach was used to reduce the number of false positives. conditions with primer set VCP22F (TGGCCCGCAGTATTCTGATT) Meat harboring a virus level close to the LOD might have been and VCP23R (CAGCTGGGACAGCAGTTGAG) that amplified a classified as non-confirmed in this process as well as some cross- region that correspond to bp position 790 and 861 of postweaning contaminated pieces. multisystemic wasting syndrome PCV. RNA transcript Virus decay An RNA transcript was produced from the PRRSV NVSL strain To estimate virus decay over time, a ratio of viral RNA copy

97-7895 using the PR29F (TTAATACGACTCACTATAGGGTGGG (Cx/C1) was calculated for each quantifiable subsample of a storage

TAAGATCATCGCTCAGCA) and PR30R (TTGACGACAGACA follow-up series, where Cx represents the copy numbers/mL for a

CAATTGCCGCT) primer set as well as the MEGAshortscript T7 Kit, specific subsample from week 2 to week 10 (Tx), and C1 represents and purified with the MEGAclear Kit (Applied Biosystems/Ambion, the value obtained at week one (T1). The degradation rate “k” was

164 The Canadian Journal of Veterinary Research 2000;64:0–00 determined by plotting the natural logarithm transformation of the tive meats were detected in April (3/98), 2 in September (2/120), ratio of viral RNA copies (Cx/C1) versus time (weeks). Virus half- 2 in October (2/225), 1 for each January (1/91), February (1/123), life was then calculated using the equation virus half-life = (ln2)/k. May (1/112), and November (1/97), and none during the months Statistical analyses were performed on the linear regression model of March (0/86), June (0/120), July (0/194), August (0/184), and by ANOVA using the F statistic (Minitab release 16 software). The December (0/50).

T0 and T1 values of a same series were also compared using a paired t-test, with P , 0.05 being statistically significant. Estimation of PRRSV load and viral decay in pig meat Meat viral load To estimate viral decay, qRT-PCR was performed on meat sub-

The meat viral load was estimated at T10 or Tmax by multiplying the samples maintained in the PHBP freezer and collected weekly for up normalized PRRSV RNA copies per mg by the total meat quantity to 15 wk (Table I). During this follow-up series, only 4 meat samples given to 1 pig for the bioassay (around 500 g). had an average PRRSV RNA concentration above the LOQ. For meats ID 715 and 1311, the number of viral RNA copies in almost all Phylogeny analysis the subsamples could not be estimated (, LOQ) although specific Phylogeny analysis was done using the nucleotide sequence of RT-PCR products were detected for PRRSV (10/10 and 9/11, respec- an amplified PRRSV ORF-5 region of NVSL. The RT-PCR was done tively) confirming their positive status. For sample 1424, only 3 out using primer set P420 and P620 (24) and the One-Step RT-PCR Kit as of 10 subsamples were positive and values were below the LOQ. No described. The nested PCR was done using primer set 5FN and 5DN follow-up subsamples were positive for ID 574 and ID 597 from T1 to

(25) and Platinum TAQ DNA Polymerase Kit (Invitrogen, Carlsbad, Tmax. Sample ID 340 and both negative controls remained negative California, USA). The ORF-5 cDNA sequences were aligned using throughout the storage period. the neighbor-joining method with Clone Manager (Scientific & To estimate the variation of virus decay over time, a ratio of Educational Software, Cary, North Carolina, USA). An ORF-5 normalized viral RNA copies of each quantifiable subsample of sequence of 544 bp was used for the alignment, which corresponded a follow-up series was calculated for samples ID 84, 115, 319, and to bp positions 13811 to 14354 of NVSL. Genbank reference strains 1332 (Table I). No significant decay in viral RNA concentrations of used for the alignment were: 17198-6 (EF442776.1), 2000-54471A PRRSV was observed during the storage period. Subsamples from (EU556182.1), 34075-NE (U66380.1), 98-6470-1 (AF339493.1), CH-1a ID 84 were tested in 3 separate experiments to estimate the inter- (AY032626.1), FJ-1 (AY881994.1), HB-1 sh/2002 (DQ642048.1), IA-27 assay coefficient of variation (CV%) of the meat extract viral RNA (EU758940.1), IAF-EXP91 (L40898.1), IAF-Klop (U64928.1), Ingelvac copies/mL. It was estimated at 42%, showing significant variation ATP MLV (DQ988080.1), Ingelvac RespPRRS MLV (AF066183.4), from one qRT-PCR test to another for the same sample.

Lelystad (M96262.2), Lena (JF802085.1), MD-001 (AF121131.1), The meat viral load was estimated at T10 or Tmax, based on the viral MN184 (EF442777.1), NADC-8 (AF396835.1), PA8 (AF176348.2), RNA copies detected per mg of meat (Table I). The extraction efficacy PrimePAC (AF066384.1), PRRSV0003749 (DQ477778.1), SDSU73 factor was found to be extremely low for the internal extraction con- (EF442775.1), and VR-2332 (AY150564.1). trol that was used. Only 7% of the input virus in the homogenized meat subsamples was recovered after RNA extraction of the muscle Results tissues. Recovery rates between 1% and 12% were also reported in previous studies using process control viruses with mouse norovirus, mengo, and hepatitis E virus (26). Numbers vary according to the PRRSV in pig meat method, the matrix, and the control virus. Pork meat usually has a Overall, 1500 fresh meat samples were collected randomly at the high fat content that can interfere with nucleic acid extraction and PHBP over a 2-year period. All meat samples were screened for inhibit the subsequent detection step. Nevertheless, low recovery the presence of PRRSV using the RT-PCR targeting both American yields are associated with high inter-assay variations. Based on these and European strains. Among meat samples, 16 (1.1%) were found results, each piglet consumed approximately 109.2 to 109.8 viral RNA presumptive positive for PRRSV with a typical amplified frag- copies of PRRSV for every 500 g of meat it was fed. This range was ment size of around 303 bp, a very faint band for some samples. estimated to be equivalent to 105.0 to 105.6 viral RNA copies per mL Presumptive positives were tested with distinct ORF-7 RT-PCR to after extraction. confirm the positive results and to define the PRRSV genotypes. Out of 16 presumptive PRRSV positive cases, 11 were confirmed positive Experimental transmission with at least a second set of ORF-7 primers. Those confirmed posi- Results of PRRSV and antibody detection from pigs fed with tives represented 0.73% of the overall meat tested. All confirmed PRRSV positive meat samples are summarized in Table II. Overall, positives were typical of the North American strains. From these, a total of 18 piglets were fed in 5 different bioassays using 2, 3, 1, 2, 9 were selected to further estimate the viral decay and to determine and 1 positive PRRSV meat samples, respectively. Two piglets were the infectivity of the stored meat at Tmax by feeding piglets during fed with a non-confirmed PRRSV meat sample. A total of 9 control bioassays. Two samples (ID 245 and 587) were not selected because piglets were included in the bioassays. Meat samples were gener- they were positive for PCV type 2 in RT-PCR (data not shown). A ally readily consumed after much chewing. Usually, all meat pieces non-confirmed and 2 RT-PCR negative meat samples were also kept were eaten by the pigs within 30 min to 2 h. At one occasion, the for the viral decay study. During the study, 3 confirmed PRRSV posi- sample was smaller and the pigs receiving the sample were fed

2000;64:0–00 The Canadian Journal of Veterinary Research 165 Table I. Variations of porcine reproductive and respiratory syndrome virus (PRRSV) concentrations in selected meat samples stored at 220°C over time Follow-up positive Meat Weeks at Normalized PRRS viral RNA copies per mg results a b b c ID 220°C T0 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 Tmax Average (T1 to Tmax) 84 15 104.8 104.5 104.1 104.5 104.3 104.5 104.7 104.5 104.7 104.4 104.4 104.1 104.5 11/11 115 13 103.7 104.0 103.9 104.0 104.4 103.8 103.6 103.5 1 103.7 103.5 1 103.9 11/11 319 11 103.5 103.7 103.5 103.2 1 103.8 103.9 103.5 103.4 104.1 103.7 103.9 103.7 11/11 340 11 0 0 NT 0 0 0 0 0 0 0 0 0 0 0/10 574 11 1 0 0 0 0 0 0 0 0 0 0 0 , LOQ 0/11 597 11 1 0 0 0 0 0 0 0 0 0 0 0 , LOQ 0/11 715 11 1 1 1 1 1 1 1 NT 1 1 1 1 , LOQ 10/10 1311 11 103.6 1 1 0 1 103.7 1 1 1 1 1 0 , LOQ 9/11 1332 11 103.9 103.5 103.7 103.5 103.7 1 103.8 104.0 103.8 103.7 103.8 103.7 103.7 11/11 1400* 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0/11 1414* 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0/11 1424 11 0 0 1 1 0 0 0 0 0 1 0 NT , LOQ 3/10 a PRRSV negative control (*). b The total period of time the meat was stored at 220°C (Tmax). c The average PRRS viral RNA copy per mg calculated from meat subsample collected from week 1 (T1) to the end of the storage period (Tmax) and normalized based on the RNA extract concentration. 1 — Positive samples that are detected but are below the limit of quantification in quantitative RT-PCR; , LOQ — Average value below the limit of quantification of 103.5 RNA copies per mg; ID — meat identification number; NT — samples not tested. with a total of 386 g instead of ~500 g (ID 715, Table II). During sequence remains low. Two viruses could not be amplified by nested the 28-day observation period, all pigs appeared healthy. Positive RT-PCR despite several attempts (ID 597 and 574). These difficulties transmission was observed with 5 confirmed PRRSV positive meat could have been attributed to the low concentration of PRRSV RNA samples (5/9, 55.6%). Transmission in PRRSV confirmed positive in meat samples (i.e., , LOQ) or strain variability. Based on the cases was detected at 7 d post-exposure (DPE) by PCR analysis of PRRSV ORF-5 sequence homology, 2 of the positive PRRSV transmis- the serum and throat samples and at 14 DPE by serology. None of sible meats (ID 319 and 1332) showed a close relationship (. 97%) the control piglets were shown to be infected. The meat viral load with modified live virus Ingelvac RespPRRS MVL vaccine and the was an important factor for transmission. Oral transmission was strains of lineage 5 over 544 bp (Figure 1) (27). The meat sample detected with all meat samples (4/4) with quantifiable viral load. ID 715, confirmed positive but negative in the transmission bioas- The pair of piglets fed with meat sample ID 1332 was found positive say, was also closely related to RespPRRS MVL vaccine type (98%) with both meat pieces stored at RT and at 4°C. Positive transmission and was 100% homologous to meat sample ID 1332 over a smaller was also detected with only 1 of the 2 piglets exposed to sample sequence stretch of 457 bp. Four confirmed positive PRRSV meats ID 1424. This sample remained non-quantifiable throughout the (ID 84, 115, 1311, and 1424) were related (~ 90%) to wild-type strains follow-up in the freezer and only a limited set of subsamples at T2, of lineage 1 such as MN184 and IAF-Klop (Figure 1). The meat ID 245

T3, and T9 were found to be qRT-PCR positive (, LOQ). For this was also closely related to the lineage 1 genotype. The meat ID 245 animal, viral transmission occurred only with the sample that was ORF-5 sequence was closely related to meat 84 (100% over 478 bp). thawed at 4°C. The pair of piglets that was fed with meat sample Sample IDs 245 and 715 were not included in the ORF-5 phylogram ID 1311 was also apparently exposed to a lower virus load and was because it was not possible to cover the same ORF-5 region with not infected. Average PRRSV RNA copies in meat sample ID 1311 these samples. The control strain used during the RT-PCR (NVSL) was below the limit of quantification except at T0 and T5. The pair did not show a high homology with the detected sequences (, 90%). of piglets that consumed the non-confirmed positive meat sample The current study confirmed the presence of 3 Ingelvac RespPRRS ID 340 was not infected either. MVL related vaccine-type and 5 wild-type strains in confirmed PRRSV positive meats. Homology of strains Strains from positive transmission bioassays were successfully Discussion sequenced and matched both before and after transmission (5/5). However, not all 11 PRRSV cases confirmed to be positive were The percentage of presumptive RT-PCR positive PRRSV meat successfully sequenced. The ORF-5 targeted sequence of 544 bp samples was very similar to the ones reported previously in meat was obtained for 6 isolates only. Two isolates were only partially samples from Quebec (1.07% versus 1.2%) (6). However, only 11/16 sequenced (ID 245 and 715). The quality of ID 587 ORF-5 virus were confirmed. In contrast, others have not found PRRSV by

166 The Canadian Journal of Veterinary Research 2000;64:0–00 Table II. Porcine reproductive and respiratory syndrome virus (PRRSV) detection in pigs experimentally fed with PRRSV positive meat samples Estimated Meat meat Meat weight viral load Bioassay RT-PCR/ELISAd (Days post exposure) Pig ID (g)b (PRRSV copy)c (trial #) °Ca 27 0 7 14 21 28 844-02 84 253/249 109.8 1 4 2/2 2/2 1/2 1/1 1/1 2/1 844-07 84 251/251 109.8 1 4 2/2 2/2 1/2 1/1 1/1 1/1 844-04 319 250/250 109.6 1 4 2/2 2/2 1/2 1/1 1/1 1/1 844-06 319 250/250 109.6 1 4 2/2 2/2 1/2 1/1 1/1 2/1 844-01 340 246/246 0 1 4 2/2 2/2 2/2 2/2 2/2 2/2 844-09 340 245/250 0 1 4 2/2 2/2 2/2 2/2 2/2 2/2 844-08 Ctrl 0/0 — 1 — 2/2 2/2 2/2 2/2 2/2 2/2 844-05 Ctrl 0/0 — 1 — 2/2 2/2 2/2 2/2 2/2 2/2 848-09 574 250/250 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2 848-02 574 250/250 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2 848-06 597 250/237 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2 848-07 597 250/233 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2 848-10 715 251/135 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2 848-04 715 251/136 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2 848-05 Ctrl 0/0 — 2 — 2/2 2/2 2/2 2/2 2/2 2/2 848-08 Ctrl 0/0 — 2 — 2/2 2/2 2/2 2/2 2/2 2/2 852-04 115 250/238 109.2* 3 4 2/2 2/2 1/2 NT NT 1/1 852-05 115 251/239 109.2* 3 4 2/2 2/2 1/2 1/1 NT 1/1 852-01 Ctrl 0/0 — 3 — 2/2 2/2 2/2 2/2 2/2 2/2 874-04 1311 250/250 1 4 4 2/2 2/2 2/2 2/2 2/2 2/2 874-02 1311 250/250 1 4 RT 2/2 2/2 2/2 2/2 2/2 2/2 875-01 1424 250/250 1 4 4 2/2 2/2 1/2 1/1 1/1 1/1 874-03 1424 250/250 1 4 RT 2/2 2/2 2/2 2/2 2/2 2/2 874-01 Ctrl 0/0 — 4 — 2/2 2/2 2/2 2/2 2/2 2/2 875-02 Ctrl 0/0 — 4 — 2/2 2/2 2/2 2/2 2/2 2/2 879-03 1332 250/250 109.5 5 4 2/2 2/2 1/2 1/1 1/1 1/1 879-02 1332 250/250 109.5 5 RT 2/2 2/2 1/2 1/1 1/1 1/1 878-07 Ctrl 0/0 — 5 — 2/2 2/2 2/2 2/2 2/2 2/2 879-01 Ctrl 0/0 — 5 2 2/2 2/2 2/2 2/2 2/2 2/2 a The meat was stored at 4°C or room temperature (RT) for 24 and 48 h after storage at the PHBP. b Each meat sample was divided in 2 equal parts The quantity of meat sample given on both days to each piglet. c The total PRRS viral load in equivalent viral copies calculated using the ORF7 qRT-PCR results from aliquots of the meat homogenate stored

at 220°C at Tmax. Value calculated using concentration at T10 (*). Confirmed positive samples that are below the limit of quantification in quantitative RT-PCR(1). d Serum test results from ORF7-RT-PCR genotype 2 assays and HerdCheck ELISA results either positive (1) or negative (2). ID — meat identification number; NT — not tested.

RT-PCR in carcasses at slaughter (14). The prevalence, the meat negligible in such cases (19). The aging of the screening ORF7 prim- sampling changes and the sensitivity of the RT-PCR assays could ers is probably not a major issue based on their homologies with the explain the PRRSV detection variation observed between studies. In reference PRRSV strains of the 2008 circulating lineages (27). Other addition, meat is a heterogeneous matrix composed of various ele- factors associated with the sampling scheme could have influenced ments such as fat, muscles, nerves, and vessels. In actively infected prevalence estimates, such as the type of meats, the handling of animals, PRRSV is not expected to be evenly distributed in meat the carcasses before packaging, and the seasons during which the but rather, located in residual blood (12,28). It is thus expected to samplings were performed. Despite having taken the necessary be unequally distributed among subsamples collected to conduct precautions, samples collected at the PHBP could have been cross- the studies, leading to significant variations. In the current study, contaminated in the processing line or during sample analysis. The the levels of viral RNA were relatively low in general and close to confirmation steps used herein reduce the probability of overestimat- the limit of detection of the molecular assays. The impact of assay ing the presence of the virus in pig meat and underestimating their sensitivity and low recovery yields on prevalence estimates is not transmission in the bioassays.

2000;64:0–00 The Canadian Journal of Veterinary Research 167 Figure 1. Phylogram of porcine reproductive and respiratory syndrome (PRRS) viral ORF-5 cDNA sequence and confirmed positive meat samples (in bold). The X-axis represents the percentage divergence. The numbered lineages follow the classification described previously (27). Internal labels represent the percentage of 1000 trees that support the clustering, only bootstrap values . 50% are shown.

No PRRSV positive meat was detected for 3 consecutive months stability of PRRS virions decreases when the temperature increases during summer, although 33% of the meat samples were collected (12,32–34), these trends seem to confirm the lower prevalence of during those months. Although more sampling would be needed PRRSV during the summer months, suggesting a lower risk in oral to confirm this trend, those results suggest that fewer pigs at the transmission. age of slaughter are infected with PRRSV or the level of infection is Similarly, the standard meat storage temperature at the packag- lower during summer. A study conducted by Larochelle et al (29) ing center might be too low to affect PRRS viral decay in frozen in Quebec reported that about 75% of field cases of PRRSV between meat within the study time frame. The 15-week follow-up study 1998 and 2002 were submitted from November to April, suggesting was designed to provide insight on the virus integrity in meat a higher prevalence of disease during colder months. Other studies throughout the period of time needed for transportation and storage have also shown more frequent mechanical transmissions of PRRSV during exportation overseas. No significant decay of viral RNA was in cold weather than in warm weather (30,31). Considering that the detected in our study, but that could be the result of the subsamples

168 The Canadian Journal of Veterinary Research 2000;64:0–00 heterogeneity, the variations in inter-assays, and the poor extraction meat samples with a high PRRSV viral load remained low, especially efficiency. It could also be the result of the study time frame and the when considering field strains only. RNA stability. Indeed, the data are compatible with the reported absence of PRRSV titer variation after 10 wk at 220°C in cell culture Acknowledgments media (12), while others have reported a decrease in viral infectiv- ity over time with storage temperature increase (220°C to 30°C) in This research was made possible through the financial support of absence of viral RNA concentration variation (34,35). the Canadian Food Inspection Agency, the Canadian Pork Council, Several studies have analyzed the oral transmission of PRRSV to and Olymel. We would like to acknowledge Dr. Bianca Morel and pigs in order to assess the risk associated with swill feeding (36). her team for their assistance in the serological assays and sampling, This study is the first to report on the PRRSV RNA concentrations Drs. Fatima Belayat and Yves Robinson for their assistance with in pig meat collected at the PHBP, that are associated with positive autopsies, M. René Mineau for his support in the animal facilities, transmission after storage in commercial conditions and thawing. Ms Sylvianne Paul for her technical assistance, the CFIA Ottawa For meat viral loads above 109 total PRRSV RNA copies/500 g of Fallowfield Laboratory and M. Steve Smith for providing SPF piglets, meat, the transmission rate was high (100%) but these samples were Dr. Carl Gagnon for consultation, and M. André Perron for revision still relatively rare in the survey. The transmission rate of positive of the manuscript. PRRSV meat samples was slightly lower than the one reported by Magar and Larochelle (6) (5/9 versus 7/11) in the same region. These References results are surprisingly similar considering the differences between both studies, including the meat cuts, the storage temperature, and 1. Holtkamp DJ, Kliebenstein JB, Neumann EJ, et al. Assessment the variation in viral load. of the economic impact of porcine reproductive and respiratory The transmission rates observed with frozen packaged meat syndrome virus on United States pork producers. J Swine Health from the PHBP were relatively low when taking into account the Prod 2013;21:72–84. prevalence of confirmed positive PRRSV meat products. The product 2. Music N, Gagnon CA. The role of porcine reproductive and respi- of the prevalence and the transmission rate in this study (0.4% for ratory syndrome (PRRS) virus structural and non-structural pro- 500 g) was in the same range as a model developed to describe the teins in virus pathogenesis. 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170 The Canadian Journal of Veterinary Research 2000;64:0–00 Article

Development of porcine circovirus 2 (PCV2) open reading frame 2 DNA vaccine with different adjuvants and comparison with commercial PCV2 subunit vaccine in an experimental challenge Changhoon Park, Jiwoon Jeong, Kyuhyung Choi, Su-Jin Park, Ikjae Kang, Chanhee Chae

Abstract The objective of this study was to compare the protection against challenge with porcine circovirus 2 (PCV2) induced by an experimental vaccine based on open reading frame (ORF) 2 of PCV2 DNA plus an adjuvant (aluminum hydroxide, cobalt oxide, or liposome) and a commercial PCV2 subunit vaccine. A total of 35 colostrum-fed, cross-bred, conventional piglets were randomly divided into 7 groups. The commercial vaccine was more efficacious against PCV2 challenge than the 4 experimental vaccines according to immunologic, virologic, and pathological outcomes. The pigs inoculated with the experimental vaccine containing the liposome adjuvant had significantly higher levels (P , 0.05) of neutralizing antibodies and interferon-g-secreting cells, and significantly lower levels (P , 0.05) of PCV2 viremia than the pigs inoculated with the other experimental vaccines. The pigs inoculated with the experimental vaccines containing either the liposome adjuvant or the cobalt oxide adjuvant had significantly lower lymphoid lesion scores (P , 0.05) than the pigs in the group inoculated with the PCV2 DNA vaccine dissolved in phosphate-buffered saline. Liposome proved to be a potent adjuvant that efficiently enhanced both humoral and cellular immune responses induced by the PCV2 DNA vaccine.

Résumé L’objectif de la présente étude était de comparer la protection contre une infection défi avec le circovirus porcin de type (CVP2) induite par un vaccin expérimental à base du cadre de lecture ouvert (ORF) 2 de l’ADN de CVP2 plus un adjuvant (hydroxyde d’aluminium, oxyde de cobalt, ou liposome) et un vaccin CVP2 sous-unitaire commercial. Un total de 35 porcelets croisés, conventionnels et nourris au colostrum ont été séparés de manière aléatoire en sept groupes. Le vaccin commercial était plus efficace contre l’infection par CVP2 que les quatre vaccins expérimentaux en fonction des résultats immunologiques, virologiques, et pathologiques. Les porcs inoculés avec le vaccin expérimental contenant l’adjuvant liposome avaient des titres significativement (P , 0,05) plus élevés d’anticorps neutralisants et un nombre significativement (P , 0,05) moindre de cellules secrétant de l’interféron-g et une virémie à CVP2 que les porcs inoculés avec les autres vaccins expérimentaux. Les porcs inoculés avec les vaccins expérimentaux contenant l’adjuvant liposome ou oxyde de cobalt avaient des scores de lésions lymphoïdes significativement (P , 0,05) plus faibles que les porcs dans le groupe inoculé avec le vaccin ADN CVP2 dissout dans de la saline tamponnée. Les liposomes se sont avérés être un adjuvant puissant qui a augmenté de manière efficace autant la réponse immunitaire humorale que cellulaire induite par le vaccin à ADN CVP2. (Traduit par Docteur Serge Messier)

Introduction Commercial PCV2 vaccines were introduced into the international market in 2006 (4). Their efficacy is evaluated on the basis of the Porcine circovirus 2 (PCV2), the smallest nonenveloped, single-­ induction of protective immunity and reduction of PCV2 viremia (4). stranded, circular DNA virus in nature, belongs to the genus High levels of PCV2 viremia have been shown to be associated Circovirus of the family Circoviridae (1). It has 2 major open reading with the development of PCVAD (5–8). The induction of a humoral frames (ORFs): ORF1 encodes the replication-associated protein, and response (neutralizing antibodies) and a cell-mediated response ORF2 encodes the capsid protein (2). This virus is considered a caus- [interferon-g-secreting cells (IFN-g-SCs)] contributes to reducing ative agent of several diseases and syndromes, collectively referred PCV2 viremia and subsequently controlling PCV2 infection (9). to as porcine circovirus-associated diseases (PCVAD) (3), among Experimental PCV2 vaccines have also been studied during which postweaning multisystemic wasting syndrome (PMWS) is the past 10 y (10). A PCV2 DNA vaccine holds promise for elicit- the most important (3). ing protective humoral and cell-mediated immune responses and

Seoul National University, College of Veterinary Medicine, Department of Veterinary Pathology, 1 Gwanak-ro, Gwanak-gu, 08826, Seoul, Republic of Korea. Changhoon Park and Jiwoon Jeong contributed equally to this work. Address all correspondence to Dr. Chanhee Chae; telephone: 182-2-880-1277; fax: 182-2-871-5821; e-mail: [email protected] Received October 7, 2016. Accepted January 11, 2017.

2017;81:171–177 The Canadian Journal of Veterinary Research 171 reducing PCV2 viremia in mouse models of PCV2 infection (11–15). nanoparticles in double-ionized water (500 mg/mL) were disrupted In contrast, a PCV2 DNA vaccine was not able to reduce PCV2 with a probe sonicator (220 to 240 V, 10.3A; Philip Harris Scientific, viremia efficiently in pigs (16), and protective immunity has not Hyde, England) at 40% power for 1 min to break up aggregation. been investigated. Then equal volumes of pORF2 DNA (2 mg/mL in 2 3 PBS) were Commercially available PCV2 subunit vaccines are still effective added and mixed by vortexing for 5 min. The hydrodynamic size in protecting against concurrent PCV2b and the emerging PCV2b and zeta potential were measured with a Zetasizer (Nano-ZS90; mutant strain (9,17). However, it is necessary to develop a vaccine Malvern, Malvern Hills, England). Finally, a 1-mL dose containing that is more cost-effective than the current PCV2 vaccines. Perhaps 500 mL of pORF2 DNA (2 mg/mL in PBS) and 500 mL of nanoparticle a DNA vaccine is the best candidate. Such vaccines have often been adjuvant (500 mg/mL) was prepared and stored at 4°C before use. disappointing when tested in pigs because of the relatively poor For the preparation of liposome nanoparticles to be used as an induction of protective immunity (18). One possible approach to adjuvant, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (Avanti improve the immune responses is the use of different adjuvants. Polar Lipids, Alabaster, Alabama, USA), dihexadecyl phosphate Therefore, the objective of this study was to compare the protection (Sigma-Aldrich, St. Louis, Missouri, USA), and cholesterol (Avanti against PCV2 challenge induced by a commercial PCV2 vaccine and Polar Lipids) were mixed at a ratio of 3.18:0.55:1.55 (w/w/w) accord- an experimental PCV2 ORF2 DNA vaccine containing an adjuvant: ing to techniques previously described (21). Briefly, lipid films were aluminum hydroxide, cobalt oxide, or liposome. created in a glass vial by evaporating the organic solvent [a mixture of chloroform and methanol at a ratio of 6.5:3.5 (v/v)] under a steady Materials and methods stream of nitrogen gas. Traces of organic solvent were removed by keeping the films in a vacuum desiccator overnight. The lipid films were then hydrated for 12 h by adding pORF2 DNA (1 mg/mL in Preparation of an ORF-2-based plasmid and DNA PBS). Next, the suspensions were sonicated in a bath-type sonicator vaccine (Branson Ultrasonics Corporation, Danbury, Connecticut, USA) for The entire ORF2 sequence was amplified from the genomic 10 min and then extruded through 400-, 200-, and 100-nm membrane DNA of PCV2b SNUVR000463 [GenBank (National Center for filters (Hamilton Company, Reno, Nevada, USA) and stored at 4°C Biotechnology Information, Bethesda, Maryland, USA) acces- before use. The hydrodynamic size and zeta potential were measured sion no. KF871068] with the use of specific primers as previously with a Zetasizer. described (14) and subcloned into the mammalian expression vec- tor pCI-neo (Promega, Madison, Wisconsin, USA) to construct the In-vitro transfection with the PCV2 molecular recombinant expression plasmid for ORF2 (pORF2). To ascertain DNA clone Cap protein expression in porcine cells, PCV-free porcine kidney To test the infectivity of the clone in vitro, PCV-free PK-15 cells (PK)-15 cells were transfected with pORF2 with the use of the were grown in 8-well chamber slides as previously described reagent Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) (22). Briefly, when the PK-15 cells were about 85% confluent they according to the manufacturer’s instructions. After a 48-hour incu- were transfected with the clone alone or with different adjuvants bation the cells were fixed and examined by the indirect immuno- (1:1 mixture) as described. Mock-transfected cells with empty pCI- fluorescent antibody (IFA) technique with the use of a mouse mono- neo vector were included as controls. Three days after transfection clonal antibody against porcine PCV2 ORF2 (Rural Technologies, the cells were fixed with a solution containing 80% acetone and 20% Brookings, South Dakota, USA) and goat IgG against mouse antigen methanol at 4°C for 20 min, and an IFA assay using a PCV2-specific conjugated with fluorescein isothiocyanate (Kirkegaard and Perry rabbit polyclonal antiserum (Veterinary Diagnostic Laboratory, Laboratories, Gaithersburg, Maryland, USA) as the primary and Iowa State University, Ames, Iowa, USA) was done to determine secondary antibodies, respectively. Finally, the pORF2 was purified the in-vitro infectivity of the molecular DNA clone. with an EndoFree Plasmid Giga Kit (Qiagen, Valencia, California, USA) for use as a DNA vaccine. Animals A total of 56 colostrum-fed, cross-bred, conventional piglets born Preparation of the various forms of experimental to PCV2-unvaccinated sows were purchased at 5 d of age from vaccine a commercial farm. All the piglets were seronegative for PCV2, Equal volumes of phosphate-buffered saline (PBS; 10 mM, pH 7.4) porcine reproductive and respiratory syndrome virus (PRRSV), and pORF2 DNA (2 mg/mL in 2 3 PBS) were mixed on a magnetic and Mycoplasma hyopneumoniae and did not have PCV2 or PRRSV plate stirred at 45 3 g for 24 h, and the resultant vaccine was admin- viremia according to real-time polymerase chain reaction (PCR). istered to pigs within 4 h of formulation. Twenty-one pigs were used for a negative-control study, and the Equal volumes of aluminum hydroxide gel (Rehydragel LV; remaining 35 were used as experimental subjects. All of the methods General Chemical, Berkeley Heights, New Jersey, USA) and pORF2 were approved by the Seoul National University Institutional Animal DNA (2 mg/mL in 2 3 PBS) were mixed on a magnetic plate stirred Care and Use Committee. at 45 3 g for 24 h, and the resultant vaccine was administered to pigs within 4 h of formulation. Negative-control study To add cobalt oxide as an adjuvant, we used a slight modification The 21 pigs were randomly divided into 7 groups (3 pigs/group) of previously described methods (19,20). Briefly, cobalt oxide (Co3O4) by means of the random-number generation function of Excel

172 The Canadian Journal of Veterinary Research 2000;64:0–00 Table I. Experimental design and scoresa for lymphoid lesions and porcine circovirus 2 (PCV2) antigen in the different groups of piglets Lymphoid PCV2 PCV2 lesion antigen Group PCV2 vaccineb challenge score score 1 DNA vaccine–PBS Yes 1.00 6 0.45c 10.2 6 3.6d 2 DNA vaccine–aluminum hydroxide Yes 0.71 6 0.70c,d 9.7 6 2.7d 3 DNA vaccine–cobalt oxide Yes 0.57 6 0.73d,e 9.2 6 1.7b 4 DNA vaccine–liposome Yes 0.43 6 0.49d,e 9.0 6 2.6d 5 Commercial vaccine Yes 0.29 6 0.49e 7.8 6 1.8d 6 No Yes 2.00 6 0.53f 24.7 6 8.7e 7 No No 0.11 6 0.26g 0d a Lymphoid lesions were scored as follows: 0 — no lymphoid depletion or granulomatous replacement; 1 — mild lymphoid depletion; 2 — moderate lymphoid depletion; and 3 — severe lymphoid depletion and granulomatous replacement. The PCV2 antigen score was determined by means of the Image J 1.45s Program of the US National Institutes of Health, Bethesda, Maryland, USA (http://imagej.nih.gov/ij/); the number of PCV2-positive cells per unit area (0.25 mm2) was counted. Different letters in superscript indicate statistically significant differences (P , 0.05) among groups. b DNA vaccine dissolved in phosphate-buffered saline (PBS) or prepared with an adjuvant; the commercial vaccine was CircoFLEX (Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA).

(Microsoft Corporation, Redmond, Washington, USA). The pigs All pigs were commingled and randomly assigned to 1 of 2 rooms. were vaccinated with only null plasmid, only an adjuvant (alumi- At the time of challenge the negative-control group was moved to a num hydroxide, cobalt oxide, or liposome), or null plasmid plus separate, similar room. The pigs in each group were housed in the an adjuvant. The negative-control vaccine was prepared the same same pen within the same room. Blood samples were collected at as the PCV2 DNA vaccine but with a null plasmid instead of the −42 (7 d of age), −28 (21 d of age), 0 (49 d of age), 7, 14, 21, 42, and ORF2-based plasmid DNA. The pigs in each group were housed in 70 (119 d of age) d after challenge. All the pigs were euthanized for the same pen within the same room. Blood samples were collected necropsy at 70 d after challenge, and superficial inguinal lymph at −42 (7 d of age), −28 (21 d of age), 0 (49 d of age), 7, 14, 21, 42, and nodes were collected for histopathological and immunohistochemi- 70 (119 d of age) d after challenge. All the pigs were euthanized for cal study. necropsy 70 d after challenge, and superficial inguinal lymph nodes were collected for histopathological and immunohistochemical study. Quantification of PCV2 DNA in blood DNA was extracted from serum by means of the QIAamp DNA Experimental design mini kit (Qiagen, Crawley, England) and used to determine the PCV2 This study used a randomized, blinded, controlled design. The genomic DNA copy numbers by real-time PCR with PCV2b-specific 35 experimental pigs were randomly divided into 7 groups (5 pigs/ primers as previously described (23). group) by means of the random-number generation function of Excel (Table I). Groups 1 to 4 received the PCV2 ORF2 DNA vaccine Serum antibody assay dissolved in PBS or the PCV2 ORF2 DNA vaccine with aluminum The serum samples were assayed by a serum virus neutralization hydroxide, cobalt oxide, or liposome as adjuvant, respectively, as test (24). Neutralizing antibody titers were expressed as the recipro- two 1.0-mL doses injected intramuscularly in the right side of the cal of the highest serum dilution that completely blocked infection neck at 7 and 21 d of age. The dose (1.0-mL) of pORF2 (1 mg) in each of the PCV-free PK-15 cells with the challenge PCV2b strain. vaccine with an adjuvant was administered to the pigs as previously described (13). The pigs in group 5 were injected with a commercial Enzyme-linked immunospot (EliSpot) assay PCV2 subunit vaccine (CircoFLEX; Boehringer Ingelheim Vetmedica, The numbers of PCV2-specific IFN-g-SCs stimulated by the chal- St. Joseph, Missouri, USA) at 21 d of age (−28 d after challenge) lenge PCV2b strain were determined in peripheral blood mono- according to the manufacturer’s recommendations. At 49 d of age the nuclear cells (PBMCs) as previously described (25) with slight pigs in the vaccinated groups (groups 1 to 5) and the unvaccinated, modifications. Briefly, 100 mL containing 5 3 105 PBMCs in RPMI challenged group (group 6) were inoculated intranasally with 2 mL 1640 medium supplemented with 10% fetal bovine serum (HyClone each of PCV2b [strain SNUVR000463, 5th passage; 1.0 3 105 median Laboratories, SelectScience, Bath, England) was seeded onto plates tissue culture infectious doses (TCID50) per milliliter]. The pigs in that had been coated with IFN-g monoclonal antibody against group 7 remained unvaccinated and unchallenged and served as porcine antigen (5 mg/mL; MABTECH, Mariemont, Ohio, USA) negative controls. and incubated with the PCV2b challenge isolate at a multiplicity

2000;64:0–00 The Canadian Journal of Veterinary Research 173 of infection (ratio of the number of virions added per cell to the vaccine with liposome adjuvant had significantly lower (P , 0.05) number of cells) of 0.05, phytohemagglutinin (10 mg/mL; Roche copy numbers than the pigs receiving the other variations of the Diagnostics GmbH, Mannheim, Germany) as a positive control, or PCV2 DNA vaccine. At 21 d after challenge the pigs receiving the

PBS as a negative control for 20 h at 37°C in a 5% humidified CO2 vaccine with aluminum hydroxide adjuvant had significantly lower atmosphere. The wells were then washed 5 times with PBS (200 mL (P , 0.05) copy numbers than the pigs receiving the vaccine with PBS per well). Thereafter the procedure was conducted with the commer- (Figure 1). No genomic copies of PCV2 were detected in the serum cial EliSpot Assay Kit (MABTECH) according to the manufacturer’s from the unvaccinated, unchallenged pigs throughout the experi- instructions. Spots on the membranes were read by the automated ment or in the pigs receiving only the null plasmid, only an adjuvant, AID EliSpot Reader (AID GmbH, Strassberg, Germany). The results or the null plasmid plus an adjuvant in the negative-control study. were expressed as the numbers of IFN-g-SCs per million PBMCs. There was a significant difference (P , 0.05) in the log2-­ transformed neutralizing antibody titers (Figure 2A) among the Histopathological and immunohistochemical groups: the titers increased significantly in the pigs receiving the study PCV2 DNA vaccine with liposome or CircoFLEX from −28 to 0 d For morphometric analysis of histopathological lesions and after challenge, as well as in all the vaccinated pigs and the unvac- determination of the PCV2 antigen score in lymph nodes, sections cinated, challenged pigs from 0 to 7 d after challenge. Significantly of 3 superficial inguinal lymph nodes were examined blindly (22,26). higher titers were induced in the pigs receiving the PCV2 DNA The lymphoid lesion scores ranged from 0 to 3: 0 — no lymphoid vaccine with liposome or CircoFLEX than in all the other challenged depletion or granulomatous replacement; 1 — mild lymphoid deple- groups at 0, 7, and 14 d after challenge. At 21, 42, and 70 d after tion; 2 — moderate lymphoid depletion; and 3 — severe lymphoid challenge the titers were significantly higher in pigs vaccinated with depletion and granulomatous replacement. The PCV2 antigen CircoFLEX than in pigs receiving the PCV2 DNA vaccine and the scores were determined by means of the Image J 1.45s Program unvaccinated, challenged pigs. At 21 d after challenge the titers were of the US National Institutes of Health, Bethesda, Maryland, USA significantly higher in the pigs receiving the PCV2 DNA vaccine with (http://imagej.nih.gov/ij/); the number of PCV2-positive cells per liposome than in the pigs receiving the other PCV2 DNA vaccines unit area (0.25 mm2) was counted. and the unvaccinated, challenged pigs. No neutralizing antibodies were detected in the unvaccinated, unchallenged pigs throughout Statistical analysis the experiment or in any of the pigs in the negative-control study. Before statistical analysis the real-time PCR and neutralizing anti- The numbers of PCV2-specific IFN-g-SCs (Figure 2B) increased body data were transformed to log10 and log2 values, respectively. significantly (P , 0.05) in the pigs receiving the PCV2 DNA vac- The normality of the distribution of the data for the examined vari- cine with adjuvant or CircoFLEX from −28 to 0 d after challenge, ables was evaluated by the Shapiro–Wilk test. Continuous data (for as well as in the pigs receiving the PCV2 DNA vaccine with PBS PCV2 DNA, PCV2 serologic findings, and PCV2-specific IFN-g-SCs) and the unvaccinated, challenged pigs from 0 to 7 d after chal- were analyzed with 1-way analysis of variance (ANOVA) followed lenge. The numbers decreased significantly (P , 0.05) in all the by Tukey’s multiple-comparison test at each time point. Repeated- challenged groups from 42 to 70 d after challenge. The numbers measures ANOVA followed by a post-hoc test for least significant dif- were significantly higher (P , 0.05) in the pigs receiving the PCV2 ferences was used to assess the mean difference in continuous data DNA–­liposome and CircoFLEX vaccines than in the pigs in the other over time within the same group. Discrete data (lymphoid lesion vaccine groups as well as the unvaccinated, challenged group at 0, 7, score and PCV2 antigen score) were analyzed by Mann–Whitney and 14 d after challenge. At 0 and 14 d after challenge the numbers tests. A value of P , 0.05 was considered to be significant. were significantly higher (P , 0.05) in the pigs receiving the PCV2 DNA–aluminum hydroxide and PCV2 DNA–cobalt oxide vaccines Results than in the pigs receiving the PCV2 DNA–PBS vaccine and the unvaccinated, challenged pigs. At 0 d after challenge the numbers The IFA assay with PCV2-specific antibody confirmed that the were significantly higher (P , 0.05) in the pigs receiving the PCV2 molecular DNA clone alone and with different adjuvants was infec- DNA–PBS vaccine than in the pigs in the unvaccinated, challenged tious in vitro: about 10% to 15% of the PK-15 cells were transfected. group. At 7 d after challenge the numbers were significantly higher From −42 to 0 d after challenge PCV2 DNA was not detected in (P , 0.05) in the pigs receiving the PCV2 DNA vaccine–cobalt oxide any of the serum samples from the 7 groups. From 0 to 7 d after than in the pigs receiving the PCV2 DNA vaccine–PBS and the challenge the PCV2 genomic copy numbers in the serum increased unvaccinated, challenged pigs. No PCV2-specific IFN-g-SCs were significantly (P , 0.05) in the 5 vaccinated groups as well as the detected in the unvaccinated, unchallenged pigs throughout the unvaccinated, challenged group. From 21 to 42 d after challenge experiment. In addition, no PCV2-specific IFN-g-SCs were detected the copy numbers decreased significantly (P , 0.05) in the pigs vac- in any of the pigs in the negative-control study. cinated with CircoFLEX. From 14 to 70 d after challenge the copy The scores for lymphoid lesions and PCV2 antigen (Table I) were numbers were significantly lower (P , 0.05) in the vaccinated groups significantly lower (P , 0.05) in the vaccinated groups than in the than in the unvaccinated, challenged group. At 7, 42, and 70 d after unvaccinated, challenged group. The pigs in the CircoFLEX group challenge the pigs vaccinated with CircoFLEX had significantly had significantly lower (P , 0.05) lymphoid lesion scores than the lower (P , 0.05) copy numbers than the pigs vaccinated with PCV2 pigs in the PCV2 DNA vaccine groups and the unvaccinated, chal- DNA. At 7, 14, 21, and 42 d after challenge the pigs receiving the lenged group. The pigs in the PCV2 DNA vaccine–cobalt oxide

174 The Canadian Journal of Veterinary Research 2000;64:0–00 A PCV2 genomic copies/mL 10 PCV2-specific NA titers 2 Log Log

Days after challenge Days after challenge Figure 1. Mean numbers of genomic copies of porcine circovirus 2 (PCV2) B DNA in serum, transformed to log10, after challenge with a PCV2b strain (SNUVR000463) in groups of piglets receiving an experimental PCV2 DNA 

vaccine dissolved in phosphate-buffered saline ( ) or prepared with an PBMC adjuvant [aluminum hydroxide gel (), cobalt oxide nanoparticles (), or 6 liposome ()], receiving the commercial vaccine CircoFLEX (Boehringer -SC/10

Ingelheim Vetmedica, St. Joseph, Missouri, USA) (), or remaining g unvaccinated (), as well as in the serum of unvaccinated, unchallenged piglets (). Different superscript letters indicate statistically significant differences (P , 0.05) among groups. PCV2 specific IFN- and PCV2 DNA vaccine–liposome groups had significantly lower (P , 0.05) lymphoid lesion scores than the pigs in the PCV2 DNA Days after challenge vaccine–PBS group. In addition, no lymphoid lesions or PCV2 Figure 2. Humoral and cell-mediated immune responses in the groups. antigens were detected in any lymph nodes from the pigs in the A — titers of PCV2-specific neutralizing antibody (NA), transformed to log . B — numbers of PCV2-specific interferon-g-secreting cells negative-control study. 2 (IFN-g-SCs) per 106 peripheral blood mononuclear cells (PBMCs). Group symbols and superscript numbers as in Figure 1. Discussion In this study PCV2-specific neutralizing antibodies and IFN-g-SCs commercial vaccines in pigs challenged with PCV2 (25,28,29). The were induced only in pigs vaccinated with the PCV2 ORF2 DNA PCV2 DNA vaccine with liposome induced higher levels of neutral- vaccines with adjuvant and not in the pigs vaccinated with only izing antibodies and PCV2-specific IFN-g-SCs than the DNA vaccine null plasmid, only an adjuvant, or null plasmid and an adjuvant. alone or with other adjuvants and thus greater protective immunity Transfection efficiency of the plasmid is critical for the immunogenic- and a greater reduction in PCV2 viremia. Therefore, the liposome ity of a DNA vaccine. In the present study the adjuvants (aluminum adjuvant is particularly promising for PCV2 DNA vaccination. hydroxide, cobalt oxide, and liposome) could have affected the As expected, induction of protective immunity and reduction transfection efficiency of the DNA vaccine but did not. Therefore, of PCV2 viremia by the commercial PCV2 subunit vaccine based the differences in immunogenicity of the vaccines with the various on ORF2 was even better than with the ORF2-based DNA vaccine. adjuvants were due to the different effects of each adjuvant on the These results agree with previous findings in which a prototypic immune responses of the pigs. PCV2 subunit vaccine was more efficient in reducing PCV2 viremia Liposome is a potent adjuvant that could efficiently enhance in pigs than a PCV2 DNA vaccine (16). However, these results are in the neutralizing-antibody immune responses induced by a PCV2 contrast with a murine model in which protective humoral immunity DNA vaccine. Aluminum hydroxide remains the primary adjuvant induced by a PCV2 DNA vaccine was better than that induced by used in veterinary vaccines. As expected, in the present study the a PCV2 subunit vaccine (14). We have no clear explanation for this vaccine with aluminum hydroxide gel induced the lowest levels of discrepancy, but it may be due to a difference between mice and PCV2-specific IFN-g-SCs compared with the other vaccines with pigs in activation of dendritic cells by a PCV2 DNA vaccine, as has adjuvant. In addition, the vaccine with cobalt oxide nanoparticles previously been reported (18). was not able to induce higher levels of humoral and cell-mediated The PCV2 DNA vaccine used in this study was administered immune responses than the vaccine with the liposome adjuvant. twice to the experimental subjects, at 7 and 21 d of age, since the These results indicate that liposome may be a good candidate for first vaccination did not provoke sufficient immune response, but use as an adjuvant for a PCV2 DNA vaccine. the commercial vaccine was administered only once, at 21 d of age, Induction of humoral and cell-mediated immunity is likely to con- according to the manufacturer’s recommendation. It is not too early tribute to a reduction of PCV2 viremia (5,6,8,27), which plays a key to vaccinate at 1 wk, because a previous study showed immune role in the development of PCVAD (8,27). In this study the humoral responses to PCV2 vaccine in 5-day-old pigs (30). immunity induced by the PCV2 DNA vaccine with liposome con- Although DNA vaccines have generally been regarded as effective tributed to a reduction in PCV2 viremia, as has been reported for in smaller animals such as mice, but less efficient in larger species

2000;64:0–00 The Canadian Journal of Veterinary Research 175 such as pigs and humans (18), the contrary has been shown as well 11. An D-J, Song D-S, Park B-K. Systemic cytokine profiles of (31,32). DNA vaccination can stimulate both humoral and cellular mice vaccinated with naked DNAs encoding six open reading immune responses in pig models (14), and the potential utility of frame antigens of porcine circovirus type 2 (PCV2). Res Vet Sci plasmid DNA as a component of a PCV2 vaccine is currently an 2008;85:503–509. area of active investigation. An additional possible approach to 12. Dong B, Feng J, Lin H, et al. Immune responses of mice immu- improve immune responses is by the use of different adjuvants. In nized by DNA plasmids encoding PCV2 ORF 2 gene, porcine the present study the pigs vaccinated with the 1-dose commercial IL-15 or the both. Vaccine 2013;31:5736–5744. PCV2 vaccine exhibited significantly lower levels of PCV2 viremia 13. Kamstrup S, Barfoed AM, Frimann TH, Ladekjaer-Mikkelsen than the pigs vaccinated with the 2-dose experimental PCV2 DNA AS, Botner A. Immunisation against PCV2 structural protein by vaccine in this study. In the field, swine producers prefer to use DNA vaccination of mice. Vaccine 2004;22:1358–1361. a 1-dose PCV2 vaccine because it requires less labor and reduces 14. Shen H-G, Zhou J-Y, Huang Z-Y, et al. Protective immunity stress in the animals. Further work is needed to develop a 1-dose against porcine circovirus 2 by vaccination with ORF2-based experimental PCV2 DNA vaccine that is comparable in efficacy to DNA and subunit vaccines in mice. J Gen Virol 2008;89: the 1-dose commercial PCV2 vaccine. 1857–1865. 15. Sylla S, Cong Y-L, Sun Y-X, et al. Protective immunity conferred Acknowledgments by porcine circovirus 2 ORF2-based DNA vaccine in mice. Microbiol Immunol 2014;58:398–408. This research was supported by contract research funds from 16. Blanchard P, Mahe D, Cariolet R, et al. Protection of swine the Research Institute for Veterinary Science, College of Veterinary against post-weaning multisystemic wasting syndrome Medicine, Seoul National University, Seoul, Republic of Korea, (PMWS) by porcine circovirus type 2 (PCV2) proteins. Vaccine and by the BK 21 Plus Program (grant 5360-20150100) for Creative 2003;21:4565–4575. Veterinary Science Research, National Research Foundation of Korea. 17. Jeong J, Park C, Choi K, Chae C. Comparison of three commercial one-dose porcine circovirus type 2 (PCV2) vaccines in a herd References with concurrent circulation of PCV2b and mutant PCV2b. Vet Microbiol 2015;177:43–52. 1. Mankertz A, Persson F, Mankertz J, Blaess G, Buhk HJ. Mapping 18. McCullough KC, Summerfield A. Targeting the porcine immune and characterization of the origin of DNA replication of porcine system — Particulate vaccines in the 21st century. Dev Comp circovirus. J Virol 1997;71:2562–2566. Immunol 2009;33:394–409.

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2000;64:0–00 The Canadian Journal of Veterinary Research 177 Article

Retrospective study of the relationship of Torque teno sus virus 1a and Torque teno sus virus 1b with porcine circovirus associated disease Alejandro Vargas-Ruiz, Hugo Ramírez-Álvarez, José I. Sánchez-Betancourt, Víctor Quintero-Ramírez, Ignacio C. Rangel-Rodríguez, Joel A. Vázquez-Perez, Lucia A. García-Camacho

Abstract Genus Iotatorquevirus consists of 2 species, Torque teno sus virus 1a and Torque teno sus virus 1b, which are ubiquitous in swine populations, and are widely reported in association with porcine circovirus associated disease (PCVAD). To evaluate the relationship with PCVAD, 100 formalin-fixed paraffin-embedded tissue samples were used to detect both Iotatorquevirus species by nested PCR and sequencing. Sixty-eight PCVAD cases were selected as well as 32 porcine circovirus type 2 (PCV2) non-affected cases. Overall, 33 of the 100 cases were positive for Torque teno sus virus 1a and 8 of 100 were positive for Torque teno sus virus 1b. Only 24 of 68 (35%) PCVAD cases were positive for Torque teno sus virus 1a; 39% (9/23) of post-weaning multisystemic wasting syndrome, and 33% (15/45) of PCV2-associated reproductive failure cases. Among PCV2 non-affected cases, 28% were positive for Torque teno sus virus 1a and 6% were positive for Torque teno sus virus 1b. Torque teno sus virus 1b was not detected in PCV2-associated reproductive failure cases. Regardless of the PCV2-status, a lower frequency of both Iotatorquevirus species was found than depicted in other reports and there was no statistical relationship with PCVAD (x2 , 0.01). Given the worldwide genomic variability of Iotatorquevirus species, it is feasible that species prevalent in Mexico share a lower nucleotide sequence identity, leading to different pathogenic potential.

Résumé Le genre Iotatorquevirus consiste en deux espèces, le virus Torque teno sus 1a et le virus Torque teno sus 1b, qui sont ubiquitaires dans la population porcine, et couramment rapportés en association avec la maladie associée au circovirus porcin (MACVP). Afin d’évaluer la relation avec MACVP, 100 échantillons de tissus fixés dans la formaline et enrobés de paraffine ont été utilisés pour détecter les deux espèces de Iotorquevirus par réaction d’amplification en chaîne par la polymérase nichée et séquençage. Soixante-huit cas de MACVP ont été sélectionnés ainsi que 32 cas non-affectés d’infection par le circovirus porcin de type (CVP2). Globalement, 33 des 100 cas étaient positifs pour le virus Torque teno sus 1a et 8 des 100 étaient positifs pour le virus Torque tenos sus 1b. Seulement 24 des 68 (35 %) cas de MACVP étaient positifs pour le virus Torque tenos sus 1a; 39 % (9/23) du syndrome de dépérissement post-sevrage, et 33 % (15/45) des cas de problèmes reproducteurs associés au CVP2. Parmi les cas non-affectés de CVP2, 28 % étaient positifs pour le virus Torque teno sus 1a et 6 % étaient positifs pour le virus Torque tenos sus 1b. Le virus Torque tenos sus 1b n’a pas été détecté dans les cas de problèmes reproducteurs associés au CVP2. Indépendamment du statu vis-à-vis le CVP2, une fréquence plus basse des deux espèces d’Iotatorquevirus fut trouvée comparativement à ce qui est décrit dans d’autres études et il n’y avait pas de relation statistiquement significative avec MACVP (x2 , 0,01). Étant donné la variabilité génomique mondiale des espèces d’Iotatorquevirus il est possible que les espèces prévalentes au Mexique partagent une plus faible identité de séquences nucléotidiques, entrainant ainsi un potentiel pathogène différent. (Traduit par Docteur Serge Messier)

Introduction on Taxonomy of Viruses (2), all human and animal TTV belong to Anelloviridae family. Genus Iotatorquevirus comprises of 2 species, Torque teno virus (TTV) is a circular single-stranded negative TTSuV1a (former TTSuV1) and TTSuV1b (former TTSuV2). The sense DNA virus that enclosed 4 open reading frames (ORF); ORF1, phylogenetic relationship of incomplete sequences has proposed ORF2, ORF 1/1, and ORF 2/2 (former ORF3), and a GC-rich region 4 biotypes (a, b, c, and d) of TTSuV1a, and 2 biotypes (a and b) of within an un-translated region (UTR), which was first discovered TTSuV1b (3,4). Genus Kappatorquevirus, however, includes only one in human samples with post-transfusion hepatitis of unknown eti- species: Torque teno sus virus k2 (2). High prevalence of co-infection ology in 1997 (1). Currently, based on the International Committee between TTSuV1a and TTSuV1b has been documented worldwide.

Department of Biological Sciences, College of Superior Studies (FESC), National University of Mexico (UNAM), Carretera Cuautitlán– Teoloyucan Km 2.5, Cuautitlán Izcalli, 54714, Estado de México (Vargas-Ruiz, Ramírez-Álvarez, Quintero-Ramírez, Rangel-Rodríguez, García-Camacho); Department of Swine Production, College of Veterinary Medicine, National University of Mexico (UNAM), Av. Universidad 3000, Col. Cd. Universitaria, Del. Coyoacán, CP 04510, Distrito Federal (Sánchez-Betancourt); Infectious Diseases Research Center, National Institute of Respiratory Diseases, Mexico City, Mexico (Vázquez-Perez). Address all correspondence to Dr. Lucia A. García-Camacho; tel: 5623 1854; e-mail: [email protected] Received September 22, 2016. Accepted January 20, 2017.

178 The Canadian Journal of Veterinary Research 2017;81:178–185 Figure 1. Geographical distribution of porcine population in the Mexican Republic.  Mexican states with higher swine production (27).  States from which samples were submitted.

Iotatorquevirus species are ubiquitous in domestic and wild pigs, Materials and methods and have been identified in Europe (Hungary, Italy, France, and Spain), Asia (China, Korea, Japan, and Thailand), and North America (Canada and USA). Case selection Transmission among pigs is horizontal and mainly via fecal-oral, Archived formalin-fixed paraffin-embedded porcine tissues but transmission through other routes may be important (3). It is (lymph node, spleen, tonsils, and fetal hearts) from 2001 to 2009 unknown whether TTSuV1a and TTSuV1b infection promotes a were selected from 100 swine cases. Sixty-eight cases were from specific disease as a primary agent or in co-infection with other non-vaccinated swine with confirmed PCVAD on the basis of clinical pathogens (5). However, it has been suggested that in co-infections signs, characteristic microscopic lesions, and in situ hybridization with other viruses, TTSuV1a might promote increased disease (11). The cases were subdivided as follows: 23 PMWS-affected tis- severity or virulence and TTSuV1b might be associated with repro- sues (lymph nodes and spleen), depicting severe lymphoid deple- ductive failure (1). In this scenario, several studies have suggested tion and granulomatous inflammation, and a diffuse pattern of an involvement of both species with porcine circovirus associated PCV2-positive in situ hybridization (ISH) and 45 cases of PCV2-RF, disease (PCVAD) since cases of post-weaning multisystemic wasting consisting of fetal hearts with non-suppurative myocarditis as well syndrome (PMWS) have shown a high prevalence of Iotatorquevirus as a random ISH pattern positive for PCV2. The PCVAD-positive species (6,7). Moreover, clinical manifestations and characteristic cases were submitted from 13 states of the Mexican Republic with lesions of porcine dermatitis and nephropathy syndrome (PDNS) a high-density swine population (Figure 1). Additionally, 32 PCV2 have been reproduced by TTSuV1a inoculation in gnotobiotic pigs non-affected cases were evaluated and consisted of 12 tissues (lymph (8). In fact, TTSuV1a has been proposed as the additional factor node and tonsil) negative for PCV2 by ISH from age-matched, clini- (X-factor) for the development of PCVAD (9). Porcine circovirus cally normal, non-vaccinated pigs from a PCV2 non-affected farm associated disease is economically important to the swine industry according to clinical criteria (12) and 20 hearts from PCVAD non- since it has an impact on production and reproduction parameters. infected aborted fetuses submitted for diagnosis. Each tissue was Although all clinical presentations of PCVAD [PMWS, PDNS, por- tested individually. cine circovirus 2-associated reproductive failure (PCV2-RF), and granulomatous enteritis] in Mexico have been confirmed by in situ Primer design hybridization (10), the prevalence of Iotatorquevirus species or their Due to genomic variability among available sequences, degen- possible association with PCVAD has not been recorded. The aim erate primers that target ORF1 of TTSuV1a and TTSuV1b were of the present work was to identify TTSuV1a and/or TTSuV1b from designed using computer software [Primer3 imput program (v.3.0.0; well-documented cases of PCVAD in order to assess their potential Institute for Biomedical Research, Boston, Massachusetts, USA) (13) relationship with the occurrence of PCVAD in an unvaccinated and Bioedit software program (v7.2.5; Ibis Bioscience, Carlsbad, population. California, USA) (14)]. Ten TTSuV1a sequences from different­

2000;64:0–00 The Canadian Journal of Veterinary Research 179 Table I. Sequences of the primers utilized for nested polymerase chain reaction (PCR) Species Sequence Primer Length TTSuV1a 59-AACTGGCAGGACCACCTATG-39 Forward 481 bp 59-AGTGTBACHTCHCCACTYC-39 Reverse 59-AAAGAGACGCTATGGCTGGA-39 Forward nested 255 bp 59-TGYTTTTCWGTGTCCCAYTGC-39 Reverse nested TTSuV1b 59-ATGCCTTACAGACGCTATC-39 Forward 605 bp 59-TGTGATGTTAATTTTGGTGGA-39 Reverse 59-AAGCTCCGGTCATACAATG-39 Forward nested 211 bp 59-GCTGTCCATATATTTCTCCAG-39 Reverse nested Sequences of the primers utilized the detection to the TTSuV1a and TTSuV1b for nested PCR. Bold letters indicate degenerate base. All primers were synthesized by another source (Integrated DNA Technologies, Coralville, Iowa, USA).

Figure 2. Iotatorquevirus species nested polymerase chain reaction (PCR) from porcine circovirus associated disease (PCVAD) positive cases. A — TTSuV1a nested PCR from FR-PCV2 positive cases. One hundred base pairs molecular weight marker (MWM). Lanes 1 positive control, lane 2 negative control, line 3, 4, and 5 showing 255 bp amplified products. B — TTSuV1b nested PCR from post weaning multisystemic wasting syndrome (PMWS) positive cases. One hundred base pairs MWM. Lanes 5 positive control, lane 7 negative control, line 8, 9 and 10 display 211 bp amplified products, 2% agarose gel.

­countries available in the NCBI database were used to design polymerase (GoTaq Flexi DNA polymerase; Promega Corporation, the primers (GenBank accession numbers: HM633249, HM633253, Madison, Wisconsin, USA) PCR buffer 13, magnesium chloride HM633258, AY823990, HM633257, AB076001, GU188045, GU456383, 2.25 mM (TTSuV1a)/1.5 mM (TTSuV1b), 0.2 mM of each deoxy- GU456384, GQ120664). For TTSuV1b, 12 sequences available in the nucleotide (dNTP), 100 pmol of each primer, and 20 ng of template. NCBI database from different countries were used to design the prim- The following thermal cycle was as follows: the initial activation ers (GenBank accession numbers: HM633230, JX173484, HQ204188, step at 94°C for 3 min followed by 40 cycles of 1 min at 94°C, 1 min GU376737, KC461227, JQ782385, HM633218, GU456386, GU188046, at 56°C (TTSuV1a) or 53°C (TTSuV1b) and 1 min at 72°C, finally last GU570207, AY823991, NC014092). First-round reverse primer contains extension step of 10 min at 72°C. The PCR products were electro- only 1 degenerate base. Primers were synthesized commercially (IDT phoretically separated in a 1.5% agarose gel stained with ethidium Integrated DNA Technologies, Coralville, Iowa, USA). bromide. The gel was visualized under ultraviolet light (Apollo Instrumentation, Claremont, California, USA) and photodocumented Nested polymerase chain reaction (PCR) (Doc-It System; UVP BioImaging Systems, Cambridge, UK). DNA extraction from all tissues was done separately using com- mercial kits according to the manufacturer’s instructions (QIAamp Sequencing DNAFFPE Tissue kit; Qiagen, Germany). Briefly, DNA was eluted in Two amplified products from each species were randomly a volume of 200 mL molecular grade water, and stored at 220°C. The selected and purified from agarose gel using a commercial kit (Min lowest limit of detection was determined by serial dilution (1:2) and Elute Gel Extraction kit; Qiagen) following the manufacturer’s was of 12.5 ng/mL. Nested 50-mL polymerase chain reaction (PCR) instructions. The sequencing of the purified PCR products was were done using the sets of degenerate primers (Table I) in a thermo- done using high fidelity, processing, and specificity enzyme kits cycler (Eppendorf, Hamburg, Germany) containing 2.5 UTaq DNA (Taq Platinum Polymerase; High Fidelity, Carlsbad, California,

180 The Canadian Journal of Veterinary Research 2000;64:0–00 Figure 3. Phylogenetic tree of the TTSuV1a showing the nucleotide Figure 4. Phylogenetic tree of the TTSuV1b showing the nucleotide sequences of different identified Torque teno virus with GenBank sequences of different identified Torque teno virus with GenBank accession number, and country of origin. The sequences were distrib- accession number, and country of origin. The sequences were dis- uted in 3 TTSuV1a sequence groups ( Canadian, Chinese, American, tributed in 2 TTSuV1b sequence groups ( Chinese, American, and Brazilian, and Hungarian;  Mexican, German, and Chinese;  Chinese Mexican;  German, Spanish, Brazilian, Chinese, and Mexican). TTSuV1a and American;  Spanish). TTSuV1b sequences ( Canadian, Chinese, sequences ( American, German, and Canadian) and TTV human American, and Brazilian), and TTV human sequences ( Italian and sequences ( Italian) are included. The Mexican sequences ( KU891808 Japanese) are included. The Mexican sequences ( KU891810 and and  KU891809) belong to the 2 TTSuV1b positive cases described KU891811) belong to the 2 TTSuV1a positive cases described in this in this work. The length of the branch represents the genetic distance work. The length of the branch represents the genetic distance among among the sequences. the sequences.

USA). Internal primers were used to sequence partial sequences of conserved among available sequences. The topography of the phy- ORF1, using a sequencer (Model 3100; Applied Biosystems, Foster, logenetic tree of TTSuV1a (Figure 3) revealed that the amplified California, USA). sequences belong to the species 1a, conforming a well-defined cluster (Mexican, German, and Chinese sequences) standing supported Data analysis with bootstrap values of 89. Conversely, the phylogenetic tree of Nucleotide sequences were edited, aligned, and analyzed using TTSuV1b (Figure 4) showed that one Mexican sequence clustered computer software (Bioedit software v7.2.0) (13). Phylogenetic with American and Chinese sequences, but another sequence was analysis were done using molecular evolutionary genetics analysis located in a different clade, revealing a major phylogenetic rela- version 6 (MEGA6) (15). The maximum likelihood tree was com- tionship with TTSuVk2 Brazilian sequences (GenBank accession puted using Tamura-Neg Gamma distance. The test of phylogeny numbers: AY823991, NC014092). was carried out through bootstrap method with 1000 number of In the current retrospective study, 100 cases were used (68 PCVAD- replication and the gaps/missing data treated with pairwise deletion. affected, and 32 PCVAD-non affected). Overall, 33% (33/100) were Two by four contingency frames were made based on PCR results positive to TTSuV1a (TTSuV1a1) and 8% (8/100) were positive to to evaluate the relationship of TTSuV1a and TTSuV1b with PCVAD TTSuV1b (TTSuV1b1). From all the PCVAD cases, 35% (24/68) were using a Chi-squared test at a trust interval of 99% (P , 0.01). TTSuV1a1 and 9% (6/68) were TTSuV1b1, whereas 28% (9/32) and 6% (2/32) of the non-affected cases were positive to TTSuV1a and Results TTSuV1b, respectively (Table II). The frequency of PMWS-affected cases compared to age-matched clinically normal piglets is shown in The PCR protocols using specific degenerate primers to amplify Table III. Thirty-one percent (11/35) of total cases were TTSuV1a1 TTSuV1a and TTSuV1b were optimized to obtain of 255 bp and 22.8% (8/35) were TTSuV1b1. Thirty-nine percent (9/23) of (Figure 2A), and 211 bp (Figure 2B), respectively, from PCVAD PMWS-affected cases were positive for TTSuV1a, 26% (6/23) were cases. The sequences of 2 nested products of each virus species TTSuV1b1. Of the clinically normal pigs evaluated, 17% (2/12) proved to be specific (GenBank accession numbers: KU891810 and were TTSuV1a1 and 17% (2/12) were TTSuV1b1. Co-infection KU891811 TTSuV1a, KU891808 TTSuV1b and KU891809 TTSuVk2). was found only in PMWS cases, 3% of the total cases, and 9% (3/35) At alignment, the amplified regions of each species were highly of PMWS cases. Concerning cases of reproductive failure in sows

2000;64:0–00 The Canadian Journal of Veterinary Research 181 Table II. Overall nested polymerase chain reaction (PCR) results for open reading frame (ORF1) region of TTSuV1a and TTSuV1b (n = 100) TTSuV1a1 TTSuV1a1 TTSuV1a2 TTSuV1a2 TTSuV1b1 TTSuV1b2 TTSuV1b1 TTSuV1b2 Total PCVAD affected 3 21 3 41 68 PCVAD non-affected 0 9 2 21 32

Totala 3 30 5 62 100 a Not statistically different between porcine circovirus-associated disease (PCVAD)-affected and PCVAD non-affected at P-value 0.01 (X2 test).

Table III. Post-weaning multisystemic wasting syndrome (PMWS) nested polymerase chain reaction (PCR) results for open reading frame (ORF1) region of TTSuV1a and TTSuV1b TTSuV1a1 TTSuV1a1 TTSuV1a2 TTSuV1a2 TTSuV1b1 TTSuV1b2 TTSuV1b1 TTSuV1b2 Total PMWS-affected 3b 6 3 11 23 Age-matched clinically normal piglets 0 2 2 8 12

Totala 3 8 5 19 35 a Not statistically different between PMWS-affected and age matched healthy piglets at P-value 0.01 (X2 test). b Co-infection only in PMWS-affected.

Table IV. Reproductive failure nested polymerase chain reaction (PCR) results for open reading frame (ORF1) region of TTSuV1a and TTSuV1b TTSuV1a1 TTSuV1a1 TTSuV1a2 TTSuV1a2 TTSuV1b1 TTSuV1b2 TTSuV1b1 TTSuV1b2 Total PCV2-RF 0 15 0 30 45 non PCV2-RF 0 7 0 13 20

Totala 0 22 0b 43 65 a Not statistically different between porcine circovirus type 2-associated reproductive failure (PCV2-RF) and non-PCV2-RF at P-value 0.01 (X2 test). b Not TTSuV1b positive cases.

(Table IV), 34% (22/65) of cases and 33% (15/45) of PCV2-RF cases pig models, TTSuV1a was proposed to act as an aggravating factor were TTSuV1a1. Similarly, 35% (7/20) of the reproductive failure of PMWS (7) and characteristic PDNS lesions were reproduced in cases not associated with PCV2 were positive for TTSuV1a1. No PCV2-negative pigs after inoculation with TTSuV1a and porcine case of reproductive failure was positive for TTSuV1b. There was reproductive and respiratory syndrome virus (8). In PMWS-affected no statistical relationship between the manifestation of PCVAD and pigs, higher prevalence and increase viral load have been found the presence of TTSuV1a or TTSuV1b by Ji2 test. with TTSuV1b than with TTSuV1a (6,22). In addition, a possible association of TTSuV1b with reproductive failure in sows has been Discussion proposed (1,24). Taken altogether, current information not only reveals high worldwide prevalence of Iotatorquevirus species but also Several reports have suggested that co-infection of Iotatorquevirus a close association of its occurrence in cases of PCVAD. However, species with other viruses might increase severity of disease as a a relationship of both TTSuV1a and TTSuV1b with development of result of synergy (1,5,16). Consequently, both species have been the PCVAD is still unclear. target of research for the study of multifactorial diseases, such as In the present study, the degenerate nested PCR proved to amplify porcine respiratory disease complex (17–20) and PCVAD (6–8,21–23). TTSuV1a and TTSuV1b ORF1 specific sequences. The nested PCR In natural cases of PMWS, a high frequency of co-infection results revealed that TTVSuV1a and TTSuV1b are widely distrib- between TTSuV1a and PCV2 has been described (6). In gnotobiotic uted in Mexican states with a high cluster of pig population, as it

182 The Canadian Journal of Veterinary Research 2000;64:0–00 is described for PCV2 (25). Also, both species were amplified from that the prevalence is noticeably lower with PCVAD, however, cases of PCVAD. In the current scenario, only 35% of total cases though the same trend was noted, it is not statistically significant. were positive to TTSuV1a. Similar findings were obtained regard- Co-infection of both species in PMWS cases was much lower than less of presentation, 39% (9/23) of PMWS cases and 33% (15/45) of that reported in Spain (76%) (22), but similar to that reported in PCV2-RF cases (Table II). The global frequencies of the present work Japan (10%) (18). However, in the current work, co-infection of age- are far lower than reported in other countries, such as Spain (90%), matched clinically normal pigs was not observed (1,22,24). Korea (85%), and China (80%), but comparable to frequencies in Immune suppression caused by PCV2 infection has been sug- Thailand and the United States, which are reported to be 40% and gested as a predisposing factor for the finding of TTSuV1b in PMWS 33%, respectively (26). In the same case series, frequencies in Canada cases because an increased viral load of TTSuV1b was seen in were found to be highly variable (46% to 100%); it was suggested PMWS-affected pigs compared to the viral load of clinically normal that differences in pig density might influence TTSuV1a prevalence pigs. Thus, clinically normal pigs might restrain infection with both (26). However, we observed a low prevalence of TTSuV1a from Iotatorquevirus species and PCV2 (28). Such statements are in agree- Mexican states with the highest proportion of swine farms (27). With ment with the data presented herein since co-infection, though in regard to TTSuV1b, the gathered data of the present work showed low proportion, was only found in PMWS-affected cases. more disparity since European countries have reported higher fre- The role of Iotatorquevirus in reproductive failure has been pro- quencies (6,21,22,28). posed. To the authors’ knowledge, there are no reports of TTSuV1a Detection of TTSuV1a and/or TTSuV1b has been strongly associ- nor TTSuV1b in cases of PCV2-RF. A higher seroprevalence of ated with cases of PMWS, particularly in Europe. Among PMWS- TTSuV1a (60% to 75%), compared to TTSuV1b (30% to 34%), has affected pigs, TTSuV1a seroprevalence of 66% to 76% has been been detected in healthy sows (24,33). Litters from clinically nor- reported in Spain (6,22,23,28). Likewise, TTSuV1a frequencies of 77% mal sows infected with TTSuV1a or TTSuV1b showed that 43% and 71.4% were reported in Sweden (21) and Slovakia (29), respec- and 19% of piglets were TTSuV1a1 and TTSuV1b1, respectively tively. Whereas TTSuV1a detection was 41% and 58% in Great Britain (34). Likewise, 50% of stillbirths were TTSuV1a1 but 7% of still- from fresh tissues and sera, respectively (30). Frequency of TTSuV1b births were TTSuV1b1, suggesting that both species may cause in among PMWS-affected cases, however, showed even more discrep- utero infection (33). Viremia in sows is a prerequisite for vertical ancy since European countries, such as Spain, have reported frequen- transmission, with the heart being a frequently affected tissue (17). cies of 91% (6,28) and 100% (22) that are consistent with the Sweden Histopathology of fetal hearts from cases of reproductive failure in prevalence of 94% (21). Moreover, TTSuV1b occurrence of 71% and sows showed both non suppurative myocarditis and PCV2-specific 64.3% were found in Great Britain (30) and Slovakia (29), respectively. ISH (35) in 39% of PCV2-RF and 47% of transplacental transmission. Altogether, PMWS-affected findings of the present work are These tissues were included in the present work, revealing a low not consistent with prevalence in Europe, but are comparable to frequency of TTSuV1a and an absence of TTSuV1b with no signifi- TTSuV1a prevalence of 48%, 40%, and 30% reported in Brazil (31), cant statistical relation between the occurrence of PCV2-RF and the Cuba (16), and Japan (18), respectively. However, the latter 2 studies presence of TTSuV1a. were performed on emaciated pigs without further laboratory confir- Using tissue pools from aborted fetuses, the frequency rates of mation of PCVAD status in affected tissues by ISH or immunohisto- TTSuV1a and TTSuV1b were 17% and 30%, respectively. Heart tissue chemistry. An explanation might be linked to geographic relationship was part of this tissue pool, but the precise site of infection could within Asia and North America, but TTSuV1b prevalence of 94.7% not be identified (36). Fetal hearts were one of the individual tissues found in Brazil (30) from PCV2-affected pigs is not in agreement evaluated in the current study, but other additional tissues that could with that hypothesis. Prevalence rates of 37.5% and 31% reported in potentially harbor Iotatorquevirus species were not evaluated, thus Cuba (16) and Japan (18), respectively, are still closer to our findings. Iotatorquevirus species could not be definitively ruled out in cases of Differences in Iotatorquevirus species prevalence rates among stud- PCV2-RF. Nevertheless, since the heart is the main target organ for ies might be related to target tissue. For instance, lower frequencies vertical transmission, preliminary findings suggest that participation were found (41% for TTSuV1a and 79% for TTSuV1b) using pools of TTSuV1b regarding PCV2-RF is unlikely. of fresh lung, liver, kidney, spleen, and lymph node, but higher The current retrospective work is noteworthy because a non- prevalence of both species (77% for TTSuV1a and 94% for TTSuV1b) vaccinated porcine population was evaluated from a time period was obtained from fresh lymph nodes (21). Such differences are most prior to the start of through and worldwide immunization programs. likely associated with PCV2-target tissues because lymph nodes Therefore, it may elucidate distinct viral dynamics as influenced and spleen are regarded as the main target of PMWS-affected pigs, by changing immune status and add to the understanding of the displaying higher PCV2 loads (32). Therefore, testing fresh lymph evolving nature of viruses. In addition, a TTSuVk2 sequence was node samples alone might increase the likelihood of detecting TTSuV amplified based on a new taxonomy (2). Thus, further classification species (6,22). In the current study, results were considerably lower of TTSuV1b is essential to separate it from TTSuVk2 species. compared to most reports despite the fact that severely and dif- Taken together, the data reported herein are in agreement with a fusely PCV2-affected lymphoid tissues were used. Consequently, lack of relationship between Iotatorquevirus species and occurrence the prevalence of TTSuV1a and TTSuV1b from PMWS-affected pigs of PCVAD. Currently, a broad phylogenetic study is being done to appears to be low in Mexico. ascertain prevailing swine Anelloviridae species in Mexico as well as Prevalence of Iotatorquevirus species in cases of PMWS is reported its genomic variability that might account for a distinctive patho- as higher than that of clinically normal pigs (1,21). Results indicate genic potential.

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2000;64:0–00 The Canadian Journal of Veterinary Research 185 Article

Identification of the linear ligand epitope on classical swine fever virus that interacts with porcine kidney 15 cells Yin Mei, Feng Yue, Hong-mei Ning, Juan-juan Zhou, Xuan-nian Wang

Abstract Binding of the viral ligand to a specific receptor is the first step of virus entry into target cells. The envelope proteins Erns, E1, and E2 of classical swine fever virus (CSFV) are involved in the interaction with host cell receptors to mediate CSFV infection. The aim of this investigation was to identify epitopes that bind to porcine kidney (PK)-15 cells to prevent CSFV infection. Ten peptides representing Erns, E1, and E2 were synthesized. Immunohistochemical study showed that the SE24 peptide, which is derived from the E2 amino acid sequence, could effectively bind to PK-15 cells. Similarly, a flow cytometry assay demonstrated that SE24 binding to PK-15 cells could be blocked by CSFV. The binding of SE24 with PK-15 cells leads to decreased CSFV infection of PK-15 cells in a dose-dependent manner. These results suggest a potential new strategy for the prevention and control of CSFV infection that requires further investigation.

Résumé La liaison d’un ligand viral à un récepteur spécifique est la première étape de l’entrée du virus dans les cellules cibles. Les protéines Erns, E1, et E2 de l’enveloppe du virus de la peste porcine classique (VPPC) sont impliquées dans l’interaction avec les récepteurs de la cellule hôte afin de médier l’infection par le VPPC. L’objectif de la présente étude était d’identifier les épitopes qui se lient aux cellules de rein de porcs (PK-15) afin de prévenir l’infection par le VPPC. Dix peptides représentant Erns, E1, et E2 ont été synthétisés. Des études immunohistochimiques ont montré que le peptide SE24, qui est dérivé de la séquence des acides aminés d’E2, pouvait effectivement se lier aux cellules PK-15. De manière similaire, une épreuve par cytométrie en flux a démontré que la liaison de SE24 aux cellules PK-15 pouvait être bloquée par le VPPC. La liaison de SE24 avec PK-15 amène une diminution de l’infection des cellules PK-15 d’une manière dose dépendante. Ces résultats suggèrent une nouvelle stratégie potentielle pour la prévention et la maîtrise de l’infection par le VPPC qui nécessite de plus amples études. (Traduit par Docteur Serge Messier)

Introduction glycoproteins E1 and E2 form a heterodimer that mediates invasion of CSFV into host cells (12,13). The structural protein E2 is a key A viral ligand generally is an epitope molecule that binds to a host determinant and major immunogen for viral entry and immunity. cell receptor; namely, the virus attachment protein (1). This receptor Surprisingly, the cellular membrane protein annexin 2 as the host is a molecule on the surface of the host cell that recognizes the viral protein can bind to CSFV E2 and promote viral growth in porcine ligand and interacts with it to mediate viral invasion into the host kidney (PK)-15 cells (14). cell (2,3). Binding of the viral ligand to its receptor is the decisive Blocking the interaction of viral ligand and receptor could inhibit factor in virus–host specificity and tissue tropism. Therefore, full virus infection. However, the amino acid sequence of the viral ligand understanding of this interaction is critical to clarifying the patho- responsible for receptor binding is unknown. The purpose of this genic mechanisms of virus infection. Previous studies have reported study was to provide a theoretical basis for further revealing the on some classical swine fever virus (CSFV) receptors, such as heparin mechanism of CSFV infection, as well as for screening antiviral drugs sulfate, mannose-6-phosphate, b-actin, low-density lipoprotein, and designing new vaccines from the perspective of the interaction 50-kDa membrane protein, and 60-kDa actin-binding membrane between viral ligand and receptor. protein, that are involved in the process of CSFV infection (4–8). Research on CSFV ligands has confirmed that envelope glycoprotein Materials and methods Erns is a disulfide-linked homodimer known to possess ribonuclease activity. It lacks a typical transmembrane region and is found both on the surface of infected cells and in the culture medium. The Peptide synthesis interaction of Erns with cell surface glycoaminoglycans contributes in The bioinformatics software DNASIS MAX (Version 2.5; Informer part to binding of the virus to susceptible cells (9–11). The envelope Technologies, Madrid, Spain) was used to analyze the amino acid

College of Veterinary and Animal Sciences, Henan Institute of Science and Technology, Xinxiang, Henan, 453003, China (Yin Mei, Hong-mei Ning); College of Life Science and Technology, Xinxiang University, Xinxiang, Henan, 453003, China (Feng Yue, Juan-juan Zhou, Xuan-nian Wang). Address all correspondence to Dr. Xuan-nian Wang; telephone: 186-373-3682111; fax: 186-373-3683344; e-mail: [email protected] Received September 20, 2016. Accepted February 13, 2017.

186 The Canadian Journal of Veterinary Research 2017;81:186–191 Table I. Characteristics of the synthetic peptides Molecular Peptide Number of weight name Amino acid sequence Gene sequence amino acids (kDa) SE01 LATDTELKEGQGMMDASEGTNYTCCKLQRHEW ctggccacagacacggaactgaaagaaatacagggaatgatgga 47 5.82 NKHGWCNWYNIDPWI tgccagcgaggggacaaactatacgtgctgtaagttacagagaca tgaatggaacaaacatggatggtgtaactggtacaatatagaccc ctggata SE02 LMNRTQANGAEGPPGKECAGTCRYDKDADING ttgatgaatagaacccaagcaaacttggcagaaggccctccggc 52 5.71 GTQARNRPTTLTGCKKGKNF caaggagtgcgctgtgacttgcaggtacgataaagatgctgacat caacgtggtcacccaggccagaaacaggccaacaaccctgacc ggttgcaagaaaggaaaaaatttt SE03 TNTGENARQGAARGTSWLGRQLSTAGKRLEGR accaacactatagagaatgccagacagggagcagcgagggtaa 37 4.25 SKTWF catcttggctcgggaggcaactcagcactgccgggaagaggttgg agggtagaagcaaaacctggttt SE11 YTNNCTPACLPKNTKGIGPGKFDTNAEDGKILHE tacactaacaactgcaccccggcttgcctccccaaaaatacaaag 38 4.17 MGGH ataataggccccggaaaatttgacactaacgcggaagacggaaa gattctccatgagatggggggtcac SE12 IPQSYEEPEGCDTNQLNLTGELRTEDGIPSSVWN attcctcaatcccatgaagaacctgaaggctgcgacacaaaccag 50 5.83 GGKYGCGRPDWWPYET ctgaatctaacagtggaactcaggactgaagacgtaataccgtcat cagtctggaatgttggcaaatatgtgtgtgttagaccagactggtgg ccatatgaaacc SE21 RLACKEDYRAISSTNEIGLLGAGGLTTTWKEYSH cggctagcctgcaaggaagattacaggtacgcaatatcatcgacc 45 5.16 DLQLNDGTGKA aatgagatagggctactcggggccgaaggtctcaccaccacctgg aaagaatacaaccacgatttgcaactgaatgacgggaccgttaag SE22 FDGTNPSTEEMGDDFGFGLCPFDTSPGGKGKY ttcgacgggaccaacccatcaactgaggaaatgggagatgacttc 40 4.39 NTTLLNGS gggttcgggctgtgcccgttcgatacgagtcctgttgtcaagggaaa gtacaatacaaccttgttgaacggtagt SE23 PTTLRTEGGKTFRREKPFPHRMDCGTTTGENED ccaacaactctgagaacagaagtggtaaagaccttcaggagaga 51 5.86 LFYCKLGGNWTCGKGEPV caagccctttccgcacagaatggattgtgcgaccaccacagtgga aaatggagatttattctactgtaagttggggggcaactggacatgtg tgaaaggtgaaccagtg SE24 VHASDERLGPMPCRPKEIGSSAGPVRKTSCTF gtgcatgcatcagatgaaagactgggccctatgccatgcagaccc 50 5.73 NYAKTGKNKYYEPRDSYF aaagagattgtctctagtgcaggacctgtaaggaaaacttcctgta cattcaactacgcaaaaactttgaagaacaagtactatgagccca gggacagctacttc SE25 QYMLKGEYQYWFDLDVTDRHSDYFAEFVVLVVV caatatatgcttaagggcgagtatcagtactggtttgacctggacgt 46 4.95 ALLGGRYVLWLIV gactgaccgccactcagattacttcgcagaatttgtcgtcttggtagt ggtagcactgttaggaggaagatatgtcctgtggctaatagtg

sequences of CSFV proteins Erns, E1, and E2. With standard operat- After a wash with cold diethyl ether (at 4°C) to remove impurities, ing procedures 10 peptides to cover parts of these sequences were the peptide was dissolved in acetonitrile–water [1:1 (v/v)], which synthesized by the solid-phase peptide synthesis method with the contained 1% acetic acid, and freeze dried. The characteristics of the 9-fluorenylmethyloxycarbonyl (Fmoc) amino acids attached to Wang 10 preliminarily purified synthetic peptides are provided in Table I. resin (Gill Biochemical, Shanghai China). The Cys residue was added at its N terminal to couple the bifunctional reagent sulfosuccinimi- Cells and viruses dyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Each Porcine kidney-15 cells were maintained in Dulbecco’s modified synthesized peptide was cleaved from the resin with trifluoroacetic Eagle’s medium (DMEM; Gibco ThermoFisher Scientific) supple- acid, precipitated with cold ether, and recovered by centrifugation. mented with 10% fetal bovine serum (FBS) and incubated at 37°C

2000;64:0–00 The Canadian Journal of Veterinary Research 187 Figure 1. Binding of peptide SE24 to 100% confluent porcine kidney (PK)-15 cells when incubated at peptide concentrations of 0.2 (A), 0.1 (B), 0.05 (C), and 0.025 (D) mmol/L. Biotin-labeled bovine serum albumin (BSA) was used as a negative control (E) and Dulbecco’s modified Eagle’s medium as a blank control (F). The immunohistochemical staining procedure is detailed in Materials and methods. The staining was gradually lighter with the decrease in peptide concentration, which indicates that the capacity of peptide SE24 to bind with PK-15 cells is strong.

in 5% CO2. These cells are often used to propagate CSFV in vitro. by 0.3% H2O2 in methanol (Sinopharm Chemical Reagent Company, The CSFV used was the Shimen strain, the standard virulent strain Ningbo, China), 50 mL/well, for 10 min at 4°C, then were washed isolated from China in 1945 and grown in PK-15 cells. 3 times with PBST and blocked with 10% nonfat milk (Yili Industrial Group Company, Hohhot, China) in PBST, 200 mL/well, for 1 h at Biotin labeling of the peptides 37°C. The cells were again rinsed in PBST 3 times, and avidin labeled Sulfo-NHS (hydroxysulfosuccinimide)–biotin (ThermoFisher with horseradish peroxidase (Boster Biological Engineering, Wuhan, Scientific) was dissolved in double distilled water for a concentra- China) in 10% nonfat milk in PBST (1:500), 50 mL/well, was added. tion of 10 mmol/L. The peptides were dissolved in dimethylsulf- After incubation for 1 h at 37°C the supernatant was decanted and oxide (Aladdin Company, Shanghai, China) for a concentration of the plate washed with PBST 3 times. The AEC staining kit (Sigma- 40 mg/mL. A peptide–biotin solution was prepared by mixing 80 mL Aldrich, St. Louis, Missouri, USA) was used to detect binding of of sulfo-NHS–biotin and 50 mL of the peptide at room temperature peptide to cell: 50 mL of substrate was applied to each well for 10 min for 30 min. After overnight incubation at 4°C the solution was at room temperature, and the cells were then observed through a adjusted to a concentration of 1 mg/mL with DMEM containing microscope. 10% FBS and frozen for storage. Peptide-binding and virus-blocking assays Binding assay Three groups were set up. In group A, biotin-labeled peptide, The PK-15 cells were incubated in 96-well plates at 37°C in 5% 0.2 mmol/L, was used to bind to PK-15 cells. In group B, biotin-

CO2 until 100% confluent and then incubated at 4°C for 1 h with labeled BSA was used as a negative control with PK-15 cells. In the biotin-labeled peptides at concentrations of 0.2, 0.1, 0.05, and group C, the biotin-labeled peptide was used to bind to PK-15 cells 0.025 mmol/L in aliquots of 100 mL per well. Three replicates of after CSFV adsorption to the PK-15 cells. The biotin-labeled peptides, each peptide at each dilution ensured full binding of the peptide biotin-labeled BSA, and CSFV, 100 3 the median tissue culture infec- 6 with the PK-15 cells. Biotin-labeled bovine serum albumin (BSA) tious dose (TCID50), were incubated with 1 3 10 PK-15 cells for 1 h was used as a negative control, and DMEM without biotin–peptide on ice. Three replicates were done in each group. In all 3 groups, was used as a blank control. Unbound peptide was washed away 100 mL of avidin labeled with fluorescein isothiocyanate (1:500) was with precooled 0.01 M phosphate-buffered saline (PBS; 0.02% added and the mixture incubated for 30 min on ice. The supernatant

KH2PO4, 0.29% NH2PO4, 0.02% KCl, and 0.8% NaCl), pH 7.4, contain- was decanted, and the PK-15 cells were resuspended with 0.5 mL ing 0.05% Tween 20 (PBST) (Aladdin). The PK-15 cells were fixed of prechilled PBS. After being mixed, the samples were assayed by

188 The Canadian Journal of Veterinary Research 2000;64:0–00 64 64 Gm:23.77 A 64 Gm:17.58 B Gm:20.70 C CV:24.12 CV:28.72 CV:25.42 [251-1023]5103 (51.0%) [251-1023]1465 (14.7%) [251-1023]3939 (39.4%) M2 M2 M1 M2 M1 M1 Events Events Events

0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FL1-H FL1-H FL1-H

Figure 2. A — binding of biotin-labeled peptide SE24 to PK-15 cells. B — binding of biotin-labeled BSA to PK-15 cells. C — binding of biotin-labeled SE24 to PK-15 cells pretreated with classical swine fever virus (CSFV). Avidin labeled with fluorescein isothiocyanate was applied and flow cytometry used to detect the binding. See Materials and methods for details. The mean proportion of positive cells (6 standard deviation) decreased from 50.86% 6 3.30% to 39.43% 6 1.35% after CSFV pretreatment, indicating that the virus can block the binding of peptide SE24 to PK-15 cells.

Figure 3. Rate of CSFV infection of PK-15 cells incubated with peptide SE24 at concentrations of 0.2 (A), 0.1 (B), 0.05 (C), 0.025 (D), 0.0125 (E), and 0.00625 (F) mmol/L. G — BSA (unrelated protein control). H — negative serum (isotype control). See Materials and methods for details. With a decrease in peptide concentration, the rate of CSFV infection gradually increased.

2000;64:0–00 The Canadian Journal of Veterinary Research 189 Table II. Inhibition by peptide SE24 of infection of porcine kidney 15 cells by classical swine fever virus 2.4 Infected cells; Peptide concentration mean 6 standard deviationa 2.2 (mmol/L) Number Percentageb 0.2 0c 0c 0.1 260.00 6 9.00d 3.71 6 0.12d 2.0 0.05 286.00 6 10.53d 4.08 6 0.15e logy 0.025 566.33 6 22.30e 8.09 6 0.32f 1.8 0.0125 684.33 6 14.01f 9.78 6 0.20g 0.00625 874.67 6 21.39g 12.49 6 0.30h 1.6 a Different superscript letters in the same column indicate a significant difference (P , 0.05). b The total number of cells per well was 7.0 3 103. 1.4

Attune Acoustic Focusing Cytometer (Applied Biosystems, Waltham, 25.0 24.5 24.0 23.5 23.0 22.5 22.0 21.5 Massachusetts, USA); 1 3 104 cells were counted for each sample. logx Infection-inhibition assay Figure 4. Graphic representation of the decrease in the rate of CSFV infection of PK-15 cells when incubated with increasing concentrations The PK-15 cells were cultured in 96-well plates until 70% to 80% of peptide SE24, until none of the cells were infected at a peptide confluent and then incubated with peptide SE24 at concentrations of concentration of 0.2 mmol/L. The infection rate (logy) and the peptide concentration (logx) were natural log-transformed. 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625 mmol/L, for 100 mL/well, in 10% DMEM. Three replicates were done. For controls, BSA was used as an unrelated protein and negative serum was used as an isotype. (Figure 1, panel E)] nor the DMEM [blank control (Figure 1, panel F)] After incubation at 37°C for 1 h the supernatant was decanted, and showed binding to the PK-15 cells. Peptide SE24, whose amino acid

10 3 TCID50 of CSFV was added to infect the cells. After 3 washes sequence is shown in Table I, is located on the E2 protein of CSFV with 10% FBS in DMEM and refreshment with 5% FBS in DMEM, and has a molecular weight of 5.73 kDa. Its binding occurred in a the cells were cultured at 37°C for 24 to 48 h. dose-dependent manner and was mainly on the cell membrane. For immunohistochemical staining the cells were rinsed in PBST The ability of CSFV to block the binding of peptide SE24 to 3 times for 5 min each time, then fixed by being incubated with 4% PK-15 cells was measured by flow cytometry (Figure 2). The mean paraformaldehyde for 10 min at 4°C, and then rinsed again in PBST proportion of positive cells and the mean fluorescence intensity 3 times for 5 min each time. Nonspecific binding sites were then of the binding were 50.86% 6 3.30% and 23.52% 6 1.24%, respec- blocked by incubation of the cells with 10% skimmed milk powder tively, but when the PK-15 cells were pretreated with CSFV the for 1 h at 37°C followed by rinsing in PBST 3 times for 5 min each means were significantly lower (P , 0.05), at 39.43% 6 1.35% and time. Next, rabbit anti-CSFV serum (1/200) was added and the mix- 20.26% 6 0.58%, respectively. The means for the negative-control ture incubated for 30 min at 37°C and then rinsed in PBST 3 times group were significantly lower still (P , 0.05), at 14.50% 6 1.21% for 5 min each time. Then goat IgG against rabbit antigen (1/40) and 17.47% 6 0.71%, respectively. The infection-inhibition assay was added and the mixture incubated for 30 min at 37°C and then showed that peptide SE24, 0.2 mmol/L, could completely inhibit rinsed in PBST 3 times for 5 min each time. Finally, the AEC kit was CSFV infection of PK-15 cells (Figure 3, panel A). With a decreasing used again, with substrate applied for 10 min at room temperature. concentration of the peptide, the number of infected cells increased The number of infected cells was counted through a microscope. (Figure 3, panels B to F), as did the infection rate (Table II). The infec- tion of PK-15 cells by CSFV was not inhibited by BSA, the negative Statistical analysis control, at any concentration, and the cells stained reddish brown Data were analyzed with the paired-samples t-test by means of (Figure 3, panel G). With the blank control, no PK-15 cells stained IBM SPSS Statistics for Windows, Version 19.0 (IBM Corporation, reddish brown (Figure 3, panel H). With the SE24 concentration as Armonk, New York, USA) and are presented as mean 6 standard the independent variable and the inhibition rate as the dependent deviation. P-values of less than 0.05 were considered statistically variable, and log transformation of the data, Figure 4 shows graphi- significant. cally that with a decrease in peptide concentration, the infection rate increased gradually. Results Discussion The binding-assay results showed that peptide SE24 could interact with PK-15 cells at all the tested concentrations. Data for the other According to previous studies, the envelope glycoproteins of peptides are not shown. Positive cells stained brownish red (Figure 1, CSFV — namely, Erns, E1, and E2 — participate in virus adsorption panels A to D). Neither the biotin-labeled BSA [negative control and invasion (9–13). On this basis, 10 peptides were synthesized to

190 The Canadian Journal of Veterinary Research 2000;64:0–00 cover parts of the amino acid sequences of these glycoproteins. The 4. Hulst MM, van Gennip HG, Vlot AC, Schooten E, de Smit AJ, infection-inhibition assay confirmed the inhibitory effect of peptide Moormann RJ. Interaction of classical swine fever virus with SE24 on CSFV infection of the target cells, which was consistent membrane-associated heparin sulfate: Role for virus replication with the blocking by CSFV of peptide SE24 binding to PK-15 cells. in vivo and virulence. J Virol 2001;75:9585–9595. Surprisingly, our study showed that peptide SE24 and CSFV com- 5. Kornfeld S. Structure and function of the mannose 6-phosphate/ petitively bind to the same receptor on the membrane of PK-15 cells. insulinlike growth factor II receptors. Annu Rev Biochem 1992; The binding of peptide SE24 to PK-15 cells can block CSFV adsorp- 61:307–330. tion but also suppress PK-15 cells already infected with CSFV. These 6. Li D, Dong H, Li S, et al. Hemoglobin subunit beta interacts with findings suggest that peptide SE24 is a potential drug target for the the capsid protein and antagonizes the growth of classical swine prevention and treatment of classical swine fever. fever virus. J Virol 2013;87:5707–5717. The highest concentration of peptide SE24 used in the infection- 7. He F, Ling L, Liao Y, et al. Beta-actin interacts with the E2 protein inhibition assay, 0.2 mmol/L, almost entirely inhibited CSFV infec- and is involved in the early replication of classical swine fever tion of PK-15 cells. Water solubility and hydrophilicity can affect virus. Virus Res 2014;179:161–168. peptide activity. Thus, improving the solubility and hydrophilicity 8. Schelp C, Greiser-Wilke I, Moennig V. An actin-binding pro- of the peptide would be very important in development of a peptide tein is involved in pestivirus entry into bovine cells. Virus Res drug. Currently, the biologic activity and function of the polypep- 2000;68:1–5. tide, and shortening of its length, are being studied in our labora- 9. Hulst MM, Moormann RJ. Inhibition of pestivirus infection in tory. Now that we have shown in vitro that peptide SE24 effectively cell culture by envelope proteins Erns and E2 of classical swine binds to target cells and inhibits infection of those cells by CSFV, fever virus: Erns and E2 interact with different receptors. J Gen further study is needed to verify the binding function and infection- Virol 1997;78:2779–2787. inhibition activity of the peptide in vivo. 10. van Gennip HG, Hesselink AT, Moormann RJ, Hulst MM. The peptide SE24 consists of 50 amino acids located on the CSFV Dimerisation of glycoprotein Erns of classical swine fever virus E2 protein (amino acids 955 to 1004). Many experimental features is not essential for viral replication and infection. Arch Virol of SE24 are consisted with E2 function. For instance, E2 is a deter- 2005;150:2271–2286. minant of cell culture tropism, and the E1 and E2 proteins formed a 11. Langedijk JP, van Veelen PA, Schaaper WM, de Ru AH, heterodimer that mediated invasion of host cells by CSFV (9,12,13). Meloen RH, Hulst MM. A structural model of pestivirus The E2 glycoprotein is likely to be involved in the host specificity of Erns based on disulfide bond connectivity and homology mod- pestiviruses at their point of uptake into cells (15), and the E2 protein eling reveals an extremely rare vicinal disulfide. J Virol 2002; of CSFV can interact with swine host factors by the yeast 2-hybrid 76:10383–10392. system (16). According to the prediction of E2 structure (17), peptide 12. Liang D, Sainz IF, Ansari IH, Gil LH, Vassilev V, Donis RO. The SE24 is located close to the transmembrane region rather than the envelope glycoprotein E2 is a determinant of cell culture tropism antigen region. In theory, the viral ligand should be in the antigen in ruminant pestiviruses. J Gen Virol 2003;84:1269–1274. region or the extracellular domain, which facilitates binding with the 13. Wang Z, Nie Y, Wang P, Ding M, Deng H. Characterization viral receptor. Whether peptide SE24 can be used as a viral ligand of classical swine fever virus entry by using pseudotyped is unknown. The process by which viruses invade cells is complex. viruses: E1 and E2 are sufficient to mediate viral entry. Virology This study has suggested a novel approach for the prevention and 2004;330:332–341. treatment of viral diseases: inhibiting viral infection of host cells by 14. Yang Z, Shi Z, Guo H, Qu H, Zhang Y, Tu C. Annexin 2 is a blocking the virus receptor–ligand interaction. host protein binding to classical swine fever virus E2 glyco- protein and promoting viral growth in PK-15 cells. Virus Res Acknowledgments 2015;201:16–23. 15. Asfor AS, Wakeley PR, Drew TW, Paton DJ. Recombinant pes- This work was supported by the National Natural Science tivirus E2 glycoproteins prevent viral attachment to permissive Foundation of China (grant 31201877) and the Foundation of the and non permissive cells with different efficiency. Virus Res Henan Educational Committee (grant 16A230001). 2014;189:147–157. 16. Gladue DP, Baker-Bransetter R, Holinka LG, et al. Interaction References of CSFV E2 protein with swine host factors as detected by yeast two-hybrid system. PLoS One 2014;9:e85324. 1. Rajcáni J. Molecular mechanisms of virus spread and virion 17. van Rijn PA, Bossers A, Wensvoort G, Moormann RJ. Classical components as tools of virulence. Acta Microbiol Immunol Hung swine fever virus (CSFV) envelope glycoprotein E2 containing 2003;50:407–431. one structural antigenic unit protects pigs from lethal CSFV 2. Norkin LC. Virus receptor: Implications for pathogenesis and challenge. J Gen Virol 1996;77:2737–2745. design of antiviral agents. Clin Microbiol Rev 1995;8:287–293. 3. Chakraborty S, Veettil MV, Chandran B. Kaposi’s sarcoma associ- ated herpesvirus entry into target cells. Front Microbiol 2012;3:6.

2000;64:0–00 The Canadian Journal of Veterinary Research 191 Article

Direct repeat unit (dru) typing and antimicrobial resistance of methicillin-resistant Staphylococcus pseudintermedius isolated from dogs in Atlantic Canada Matthew E. Saab, J. Scott Weese, J.T. McClure

Abstract There are few reports investigating the characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs in Canada and none from Atlantic Canada. The objectives of this study were to strain type MRSP isolates cultured at a regional diagnostic laboratory using direct repeat unit (dru) typing and to describe their antimicrobial resistance profiles. Ninety-four isolates recovered from dogs between 2010 and 2012 had dru typing, cluster analysis, and antimicrobial susceptibility testing done. The majority of isolates belonged to type dt11a (30.9%), dt10h (24.5%), dt9a (18.1%), and dt11af (10.6%) with the remaining 15.9% of isolates distributed among 13 dru types. The predominant dru types identified were similar in Ontario; however, cluster 9a appears to be less common in Atlantic Canada. A significant difference in the distribution of clusters among Atlantic provinces was detected (P = 0.01). Resistance to $ 2 non-b-lactam antimicrobials was observed in 71.4% of the isolates. The MRSP isolates from this study were notably less resistant than those reported in the literature. A more comprehensive study of the MRSP dru types could help further elucidate the distribution of this pathogen in Canada.

Résumé Il y a peu de publications rapportant la caractérisation des isolats de Staphylococcus pseudintermedius résistant à la méticilline (SPRM) chez les chiens au Canada et aucun ne provenant des provinces maritimes. Les objectifs de la présente étude était de typer les isolats de SPRM cultivés à un laboratoire de diagnostic régional en utilisant le typage direct des unités répétées (dur) et de décrire les profils de résistance antimicrobienne. Quatre-vingt-quatorze isolats canins obtenus entre 2010 et 2012 furent soumis au typage du dur, une analyse de regroupement, et un antibiogramme. La majorité des isolats appartenait au type dt11a (30,9 %), dt10h (24,5 %), dt9a (18,1 %), et dt11af (10,6 %) et le 15,9 % des isolats restant étaient distribués parmi 13 types dur. Les types prédominants de dur identifiés étaient similaires à ceux de l’Ontario, Canada, bien que le regroupement 9a semble moins fréquent dans les provinces maritimes. Une différence significative dans la distribution des regroupements entre les provinces maritimes a été détectée (P = 0,01). Une résistance à $ 2 antimicrobiens n’appartenant pas à la classe des b-lactames fut observée chez 71,4 % des isolats. Les isolats de SPRM de la présente étude était sensiblement moins résistant que ceux rapportés dans la littérature. Une étude plus complète des types dur des SPRM pourrait aider à élucider un peu plus la distribution de cet agent pathogène au Canada. (Traduit par Docteur Serge Messier)

Introduction nary hospitals, and the potential of hospital-wide outbreaks should not be underestimated (1,4). Not only has MRSP been reported in Over the last decade, methicillin-resistant Staphylococcus pseudin- the canine population, but also there have been several reports of termedius (MRSP) has emerged as a major opportunistic pathogen humans infected or colonized with MRSP (5–11). Understanding in dogs, and as the leading cause of skin disease, otitis, surgical the diversity and dissemination of MRSP is essential for develop- site infections, and wound infections (1,2). Methicillin-resistance in ing mitigation and control strategies and for future epidemiologic MRSP is mediated by the mecA gene, which codes for the expres- studies. Currently, there is no study characterizing MRSP isolates sion of a modified penicillin-binding protein, PBP29, and resistance in Atlantic Canada. to most b-lactam antimicrobials (2). Increasing antimicrobial resis- A number of molecular tools have been reported to genetically dis- tance to antimicrobials other than b-lactams, has been reported in criminate among staphylococci. Most of these tools were first devel- MRSP isolates, leaving few therapeutic options available in many oped and standardized for methicillin-resistant Staphylococcus aureus instances (1–3). Methicillin-resistant Staphylococcus pseudintermedius (MRSA), but few have been further refined for discriminating MRSP has been implicated in hospital-acquired infections within veteri- (1,12–16). While pulsed-field gel electrophoresis (PFGE) remains the

Diagnostic Services, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island (Saab); Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island (Saab, McClure); Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario (Weese). Address all correspondence to Mr. Matthew Saab; e-mail: [email protected] Received December 20, 2016. Accepted February 9, 2017.

192 The Canadian Journal of Veterinary Research 2017;81:192–198 gold standard for MRSA outbreak investigations, it has been shown penicillin (10 mg), and trimethoprim-sulfamethoxazole (SXT; 25 mg). to be less useful for MRSP investigations (12,14,17,18). Historically, Methicillin resistance was detected by oxacillin (1 mg) disk diffusion, multi-locus sequence typing (MLST) has been the primary tool for and confirmed by mecA expression using the PBP29 latex agglutina- investigating the population genetic structure of MRSP, and 2 major tion test (PBP29 Latex Agglutination Test; Oxoid Company, Nepean, sequence types (ST) have been identified (1,16,19). Both PFGE and Ontario). All isolates identified presumptively as MRSP were placed MLST are laborious, costly, and require the use of reference strains, in a medium containing 15% glycerol and frozen at 280°C. thus making them impractical tools for implementation in smaller Patient province of residence, submitting clinic, anatomical diagnostic laboratories. source/site of specimen, and antimicrobial susceptibility data were The use of a single-locus marker for isolate discrimination has extracted from the laboratory data management system and descrip- also been explored for MRSA, and provides a less expensive and tive statistics were computed. Only 1 isolate from each patient was less laborious approach to strain typing (20,21). Sequence analysis of included for analysis in most patients. If multiple MRSP isolates on the staphylococcal protein A region (spa typing) has been shown to repeated lab submissions were recovered from a patient but had the have greater discriminatory power than MLST and PFGE for MRSA, same dru type, only the first isolate was included for analysis. If dif- but it is less successful for strain typing all MRSP isolates (14,20–22). ferent dru types were isolated from the same patient on different lab The mec-associated direct repeat unit (dru) typing was first demon- submissions, then both isolates were retained for analysis. strated to be useful in discriminating highly-clonal MRSA isolates in Scotland (13), and has more recently been successfully applied Molecular identification and typing to MRSP isolates from Canada, the United States (US), Europe, Genomic material was extracted using a resin matrix (InstaGene and Australia (22–24). In a previous study, significant associations Matrix; Bio-Rad Laboratories, Montreal, Quebec). Manufacturer’s between dru cluster and MLST were established, in which cluster guidelines were followed except a large loopful of bacterial colo- 9a was associated with ST71 (the “International clone”) and cluster nies were suspended in 1.0 mL of polymerase chain reaction (PCR) 11a was associated with ST68 (the “North American clone”) (1,22). water, and the incubation time at 56°C was increased from 30 min This single-locus sequence-based method is rapid, standardized, to 1 h. Isolated DNA was frozen at 220°C until use. A multiplex and cost-effective, making it an ideal candidate for use in small-scale PCR reaction was used to identify coagulase-positive staphylococci laboratories (22,23). to the species level based on partial amplification of the nuc gene In a previous study, significant differences have been reported locus (26). Staphylococcus pseudintermedius isolates were character- in the distribution of the 2 main dru clusters, 9a and 11a, between ized using the dru typing method, as previously described (17). A Canada, the US, and Europe (22). The report also inferred that varia- 40-mL reaction was prepared containing 4 mL of template DNA, tion within a country may exist, as some dru types were detected 0.8 U of DNA polymerase, buffer mixture containing 1.5 mM in some US states, but not the others included in the study. To date, MgCl2 and 200 mM of each dNTP (KAPA2G Fast Hot Start DNA there has been only one study investigating dru types in Canada, polymerase; KAPA Biosystems, Boston, Massachusetts, USA), addi- which was limited to Ontario; therefore, genetic information on tional 0.3 mM MgCl2, and 0.8 mM of the forward primer druGF MRSP isolates from Canadian regions other than Ontario is needed. (59-GTTAGCATATTACCTCTCCTTGC-39) and the reverse primer Thus, the primary objective of this current study was to explore the druGR (59-GCCGATTGTGCTTGATGAG-39). Reaction mixtures strain type diversity of MRSP isolates from dogs using the dru typ- were thermally cycled for an initial denaturation step of 94°C for ing method from submissions to a regional diagnostic laboratory in 2 min followed by 30 cycles at 94°C for 1 min, 52°C for 1 min, and Atlantic Canada. A secondary objective was to describe the antimi- 72°C for 1 min (17). The PCR products were purified using a kit crobial resistance (AMR) pattern of these MRSP isolates. (QIAquick PCR Purification Kit; Qiagen, Mississauga, Ontario) prior to sequencing. Materials and methods The dru repeats and types were assigned using a website (www.dru-typing.org) following the previously described nomen- clature (17). Cluster analysis was used to compare the relatedness Isolate screening and collection among dru types, and a minimum spanning tree (MST) was gener- Isolates were recovered from canine specimens submitted ated. Distance intervals (or similarity values) were created using to the Atlantic Veterinary College (AVC) Diagnostic Services a bin distance of 1.0%, in which dru types separated by an MST Bacteriology Laboratory for routine culture and susceptibility test- distance of # 2 repeats (. 98.5% similarity) were considered closely ing. Staphylococci were identified by colony morphology, including related and assigned to the same cluster. Root nodes were assigned hemolysis, a positive tube coagulase test and matrix-assisted laser to the dru type with the greatest number of isolates. Cluster analysis desorption/ionization time-of-flight (MALDI-TOF) mass spec- was completed using the TRST plug-in tool (BioNumerics version trometry. Antimicrobial susceptibilities to the following drugs were 6.6; Applied Maths, Austin, Texas, USA), as described previously for determined using the Kirby-Bauer disk diffusion method, following MRSP dru typing (22–24,27). Clinical Laboratory Standards Institute (CLSI) standard M31-A3 (25): ampicillin (10 mg), amikacin (30 mg), amoxicillin-clavulanic Statistical analysis acid (30 mg), cephalexin (30 mg), cefovecin (30 mg), chlorampheni- Multi-drug resistant (MDR) MRSP were defined as being resistant col (30 mg), clindamycin (30 mg), doxycycline (30 mg), enrofloxacin to $ 2 antimicrobials classes in addition to b-lactams. Dru clusters (5 mg), erythromycin (15 mg), fusidic acid (10 mg), gentamicin (10 mg), were used for comparisons to decrease the number of tested groups.

2000;64:0–00 The Canadian Journal of Veterinary Research 193 Table I. Number (and percent) of antimicrobial resistant methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates overall and by dru cluster, following Clinical Laboratory Standards Institute (CLSI) guidelines. Multidrug resistance (MDR) in each cluster is also reported Overall 9a 10h 11a No cluster Antimicrobial (n = 94) (n = 18) (n = 27) (n = 45) (n = 4) Amikacin 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0%) Chloramphenicol 23 (24.5%) 3 (16.7%) 0 (0%) 19 (42.2%) 1 (25%) Clindamycin 54 (57.5%) 16 (88.9%) 7 (25.9%) 29 (64.4%) 2 (50%) Doxycycline 14 (14.9%) 1 (5.6%) 7 (25.9%) 4 (8.9%) 2 (50%) Erythromycin 63 (67.0%) 17 (94.4%) 11 (40.7%) 32 (71.1%) 3 (75%) Enrofloxacin 46 (48.9%) 16 (88.9%) 0 (0%) 29 (64.4%) 1 (25%) Fusidic acid 2 (2.1%) 1 (5.6%) 1 (3.7%) 0 (0%) 0 (0%) Gentamicin 34 (36.2%) 10 (55.6%) 2 (7.4%) 20 (44.4%) 2 (50%) SXT 70 (74.5%) 18 (100%) 23 (85.2%) 27 (60.0%) 2 (50%) MDR 66 (70.2%) 17 (94.4%) 15 (55.6%) 31 (68.9%) 3 (75%) SXT — trimethoprim-sulfamethoxazole.

Unconditional associations between dru cluster and patient data and Table II. Frequency of methicillin-resistant Staphylococcus dru cluster and resistance to the non-b-lactam antimicrobials were pseudintermedius dru types from 94 isolates collected at a assessed using Chi-squared or Fisher’s exact tests, where appropri- regional diagnostic laboratory between 2010 and 2012 ate, with significance set at P # 0.05. Subtables were explored for Frequency any significant association to determine where the differences were. dru type (n = 94) Cluster All statistical computations were done using computer software dt11a 29 (30.9%) 11a (Stata/IC 13.1 for Mac; StataCorp, College Station, Texas, USA). dt10h 23 (24.5%) 10h dt9a 17 (18.1%) 9a Results dt11af 10 (10.6%) 11a dt10as 3 (3.2%) 10h Isolate collection dt10cca 1 (1.1%) 10h dt10cja 1 (1.1%) 11a The diagnostic laboratory collected 129 isolates from 90 dogs dt11bn 1 (1.1%) 11a between January 2010 and December 2012. Of those, 98 isolates were dt11caa 1 (1.1%) 11a retained for analysis. Twenty-three patients had multiple submis- dt11v 1 (1.1%) 11a sions. Isolates were collected from various specimens (n = 98): skin dt11y 1 (1.1%) 11a (n = 52, 53.1%), ears (n = 18, 18.4%), wounds (n = 8, 8.2%), surgical dt5ka 1 (1.1%) — sites (n = 6, 6.1%), urine (n = 5, 5.1%), abscesses (n = 2, 2.0%), and dt6ta 1 (1.1%) — other (n = 7, 7.1%). Most of the patients were seen by private vet- dt8aga 1 (1.1%) — erinary clinics in the region (71.4%) compared to being seen by the dt9baa 1 (1.1%) 9a AVC Veterinary Teaching Hospital (AVC-VTH) (28.6%). dt9bda 1 (1.1%) — Antimicrobial resistance patterns dt11cma 1 (1.1%) 11a a Novel dru types identified in this study. Only 8.2% (n = 8) of isolates were susceptible to all tested drugs with the exception of b-lactams (Table I). Another 20.4% (n = 20) of isolates were resistant to 1 non-b-lactam antimicrobial class, while 71.4% (n = 70) were resistant to $ 2 non-b-lactam antimicrobial Direct repeat unit typing classes. The most common resistance was to SXT (74.5%), followed The dru types were determined for 94/98 isolates, because by erythromycin (68.4%), clindamycin (55.1%), enrofloxacin (46.9%), 4 isolates were not available for typing. From the 94 isolates, 18 dru gentamicin (34.7%), chloramphenicol (23.5%), doxycycline (15.3%), types were recovered with 3 predominant dru types contributing and fusidic acid (3.1%). In isolates that displayed multi-drug resis- more than 70% of the distribution: dt11a (30.9%), dt10h (24.5%), tance (n = 70), 68.6% of isolates were resistant to SXT, erythromycin, and dt9a (18.1%). The frequency of each dru type can be found in and clindamycin, while 55.7% (n = 70) of those were also resistant Table II. Nine of the dru types were previously unidentified at the to enrofloxacin. Amikacin resistance was not detected in any of the time of analysis and are represented by one isolate each: dt5k, dt6t, isolates. dt8ag, dt9bd, dt10cc, dt10cj, dt11ca, and dt11cm.

194 The Canadian Journal of Veterinary Research 2000;64:0–00 dt6t

3

dt10cc dt10as

dt10h dt9bd dt10cj

2 2 dt11bn dt5k 2

2 dt8ag 2 dt11af dt9a dt11ca dt11a dt9ba

dt10v

dt11v dt11y

dt11cm

Figure 1. Minimum spanning tree (MST) of methicillin-resistant Staphylococcus pseudintermedius in Atlantic Canada. Numerical values on the branches indicate the similarity (MST distance) among different dru types. The dru types separated by an MST distance of # 2 ($ 98.5% similar) were considered closely related and assigned to the same cluster.

A minimum spanning tree (MST) was constructed to identify The dru cluster was not significantly different among the AVC- relatedness among dru types, and to determine the predominate VTH and private practice (P = 0.18) or specimen types (P = 0.91). clusters (Figure 1). The cluster analysis identified 3 main dru clusters Significant associations among AMR and dru clusters were detected that included 90/94 typed isolates (Figure 1). Cluster 11a contained for all antimicrobials except fusidic acid (P = 0.31; Table I). For the most diversity, comprising 9 dru types and 47.9% of the typed clindamycin, erythromycin, enrofloxacin, and SXT, cluster 9a had isolates. Cluster 10h contained 3 dru types and 28.7% of the isolates, a significantly higher proportion of resistant isolates (P # 0.01). while cluster 9a contained 2 dru types and 19.2% of the isolates. Cluster 10h had significantly higher proportion of isolates resistant Table II contains the dru type and cluster association. to doxycycline (P = 0.04), while cluster 11a had a significantly higher proportion of chloramphenicol resistance (P , 0.001). The propor- Associations among dru clusters, patient tion of MDR isolates was significantly different among dru clusters demographics, and AMR (P = 0.03), with cluster 9a having the highest proportion of MDR Significant differences were determined among the distribu- (94.4%), followed by 10h (55.6%) and 11a (68.8%). tions of dru clusters in the 4 Canadian Atlantic provinces (P = 0.01; Table III). In Newfoundland and Labrador (NL), cluster 9a was Discussion over-represented with 5/7 strains belonging to dt9a. Cluster 10h was present in Nova Scotia (NS; 15/52) and New Brunswick (NB; 5/23), The majority of isolates (73.5%) collected as part of this con- but not NL, and was the predominant cluster from Prince Edward venience sampling at the AVC Diagnostic Services Bacteriology Island (PEI; 7/12) isolates. Both mainland provinces, NB and NS, Laboratory were represented by dru types dt11a, dt10h, and dt9a, shared the same predominant cluster, 11a, which comprised over which is similar to the findings in a previous multi-national study 50% of the isolates from each province. (22). However, in the current study, clusters 11a and 10h comprised

2000;64:0–00 The Canadian Journal of Veterinary Research 195 Table III. Number (and percent) of methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates in each province overall and by dru cluster dru cluster NB NL NS PEI Overall (n = 94) 23 (24.5%) 7 (7.4%) 52 (55.3%) 12 (12.8%) 9a (n = 18) 6 (33.3%) 5 (27.8%) 6 (33.3%) 1 (5.6%) 10h (n = 27) 5 (18.5%) 0 (0.0%) 15 (55.6%) 7 (25.9%) 11a (n = 45) 12 (26.7%) 2 (4.4%) 28 (62.2%) 3 (6.7%) No cluster (n = 4) 0 (0.0%) 0 (0.0%) 3 (75.0%) 1 (25.0%) NB — New Brunswick; NL — Newfoundland and Labrador; NS — Nova Scotia; PEI — Prince Edward Island. more than 75% of the isolates collected, whereas in the Ontario testing can be from cases proving difficult to treat, with the primary study, cluster 11a (including dt10h) and 9a were evenly distributed reason being non-response to antimicrobial therapy. It is likely that (22). The distribution of MRSP dru clusters in Atlantic Canada was diagnostic laboratory submissions are biased to be more resistant. more similar to what has been reported in California, Illinois, North Interestingly, cluster 9a isolates had the highest proportion of Carolina, Tennessee, and Texas (22). Differences among geographic MDR at 94.4%, and also had a significantly higher proportion of regions within the US were also noted in the previous study (22). resistant isolates for each antimicrobial except fusidic acid, chloram- Significant differences in the diversity of strains were detected phenicol, and doxycycline. Strong associations between cluster 9a among provinces of Atlantic Canada (Table III). It should be noted and ST71 have been shown (22), and reports have also shown ST71 to that this study was not designed for prevalence estimation because have an increased antimicrobial resistance compared with other STs of the convenience sampling approach. Although it may appear that (1,29). MLST was not done on the isolates in this study, but the clus- the incidence of MRSP is higher in certain provinces, these numbers ter 9a isolates in this study likely belong to ST71. Although this study are reflective of the sample submission demographics from these was not designed for prevalence estimation, the ST71 clone associ- regions (i.e., the laboratory receives approximately 45% of their ated dru cluster 9a seems to have not disseminated into Atlantic canine samples from NS, but only 5% are from NL). These provin- Canada (18.1%), with the possible exception of Newfoundland, to cial differences in the distribution of dru types could be attributed the degree that it has in Europe (90%), the US (66%), and Ontario, to geographic separation, since differences among Newfoundland Canada (47%) (22). and Labrador and the Maritime Provinces (NB, NS, and PEI) exist, as Resistance of the MRSP isolates to the non-b-lactam antimicrobi- well as population density, since provinces with larger populations als in this study was lower than in previous reports of multi-drug have greater dru type diversity. The small sample size of isolates resistance in MRSP, which could be explained by the low preva- from Newfoundland and Labrador may bias its estimate. It is pos- lence of cluster 9a isolates. However, caution should be taken when sible that the true diversity of the isolates from some provinces is comparing studies because of differences in testing methodology. underestimated, since each province except PEI has its own animal Specifically, it was reported that 62% (n = 107) of study isolates health microbiology laboratory, decreasing submissions from these were resistant to doxycycline in Ontario (24), while in our study provinces to the AVC diagnostic laboratory. doxycycline resistance of isolates was 15%. One study in Australia This study detected 9 dru types that had not been previously iden- and another in the United Kingdom (UK) found similarly high tet- tified (Table II), and required entry into the dru database. These new racycline resistance at over 50% and 35%, respectively (23,30). Since dru types are results of a random single-nucleotide polymorphism the time of this study, canine specific doxycycline breakpoints for S. in one of the 40bp dru repeat sequences, subsequently creating a pseudintermedius were proposed and implemented by CLSI (31,32). new dru repeat and thus new dru type. Novel dru types have also This change in zone diameter breakpoints would likely increase the been identified in a previous study (22). Although there were no number of doxycycline resistant isolates in both this study and the significant differences in the distribution of the new dru types, most Ontario study. Similar trends can be observed where the proportion of the novel dru types were isolated from Nova Scotia (5/9), and of resistant isolates in Atlantic Canada is much lower than those from skin samples (5/9). reported in Australia and the UK. A recent study completed at a More than 70% of the isolates recovered in this study were resis- Texas (USA) Veterinary Medical Teaching hospital reported ami- tant to $ 2 non-b-lactam antimicrobial classes, highlighting the clini- kacin resistance in 36% of their MRSP isolates from dogs, whereas cal concerns regarding management of MRSP infections. Multi-drug in our study amikacin resistance was not detected (33). A system- resistance has been frequently reported in MRSP isolated from canine atic literature review of antimicrobial resistance in MRSP isolates specimens (1,28). The isolates recovered in this study were from reported individual antimicrobial resistance ranging between 0% submissions to a regional diagnostic bacteriology laboratory that and 100%, with most studies reporting AMR estimates . 50% for also is the diagnostic laboratory for the AVC-VTH, thus a selection most antimicrobials, except for chloramphenicol and amikacin (28). bias is possible, based on submission of specimens from complicated Thus, it can be inferred that the MRSP isolates in Atlantic Canada are referral cases from the AVC-VTH. However, most samples in this typically less resistant, specifically to doxycycline and amikacin, than study were from primary care practices. Information about prior their counterparts in some other regions, even though multi-drug antimicrobial exposure was not always available, but submissions resistance is still common. A possible explanation for this low-level to a diagnostic laboratory for bacterial culture and susceptibility resistance could be the low population density of the region as a

196 The Canadian Journal of Veterinary Research 2000;64:0–00 whole, when compared with larger, more densely populated regions, matology practice staff and their respective pets. Vet Dermatol which could mean less exposure to AMR organisms and less total 2010;21:400–407. antimicrobial use. 8. Paul NC, Moodley A, Ghibaudo G, Guardabassi L. Carriage of The overall picture of MRSP in Atlantic Canada is similar to methicillin-resistant Staphylococcus pseudintermedius in small reports elsewhere in Canada and the world. The distribution of animal veterinarians: Indirect evidence of zoonotic transmission. the dru types reported in this study is similar to other reports from Zoonoses Public Hlth 2011;58:533–539. North America; however, the distribution is more similar to what 9. Sasaki T, Kikuchi K, Tanaka Y, Takahashi N, Kamata S, was observed in the US versus Ontario, Canada. The international Hiramatsu K. Methicillin-resistant Staphylococcus pseudinter- MRSP clone, ST71, which is disseminated throughout Europe and medius in a veterinary teaching hospital. J Clin Microbiol 2007; made up almost half the isolates in Ontario, Canada, was less com- 45:1118–1125. mon in Atlantic Canada. This confirms that dru type distributions 10. Starlander G, Börjesson S, Grönlund-Andersson U, Tellgren- can vary significantly across the same country, and a larger, more Roth C, Melhus Å. Cluster of infections caused by methicillin- comprehensive study of the dru types in Canada could help further resistant Staphylococcus pseudintermedius in a human hospital. clarify the dissemination of this pathogen. Multi-drug resistance in J Clin Microbiol 2014;52:3118–3120. these isolates is common, especially within cluster 9a isolates, but 11. Stegmann R, Burnens A, Maranta CA, Perreten V. Human infec- resistance to the non-b-lactam antimicrobials is still considerably tion associated with methicillin-resistant Staphylococcus pseudin- lower than has been previously reported. termedius ST71. J Antimicrob Chemother 2010;65:2047–2048. 12. Bannoehr J, Guardabassi L. Staphylococcus pseudintermedius in Acknowledgments the dog: Taxonomy, diagnostics, ecology, epidemiology and pathogenicity. Vet Dermatol 2012;23:253–266, e51–52. The authors thank AVC Diagnostic Services Bacteriology 13. Black CC, Solyman SM, Eberlein LC, Bemis DA, Woron AM, Laboratory for their valuable work with sample and isolate collec- Kania SA. Identification of a predominant multilocus sequence tion; Joyce Rousseau, Ontario Veterinary College, for assistance with type, pulsed-field gel electrophoresis cluster, and novel staphy- molecular techniques; Spencer Greenwood, AVC Lobster Science lococcal chromosomal cassette in clinical isolates of mecA- Centre, for use of equipment; Henrik Stryhn, Atlantic Veterinary containing, methicillin-resistant Staphylococcus pseudintermedius. College, for assistance with statistical analyses; and Robert Page, Vet Microbiol 2009;139:333–338. University of Prince Edward Island, for laboratory data extraction. 14. Moodley A, Stegger M, Ben Zakour NL, Fitzgerald JR, This study was financially supported by the Atlantic Veterinary Guardabassi L. Tandem repeat sequence analysis of staphylo- College Companion Animal Trust Fund and the Sir James Dunn coccal protein A (spa) gene in methicillin-resistant Staphylococcus Animal Welfare Centre. pseudintermedius. Vet Microbiol 2009;135:320–326. 15. Murchan S, Kaufmann ME, Deplano A, et al. Harmonization of References pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: 1. Perreten V, Kadlec K, Schwarz S, et al. Clonal spread of A single approach developed by consensus in 10 European methicillin-resistant­ Staphylococcus pseudintermedius in Europe laboratories and its application for tracing the spread of related and North America: An international multicentre study. strains. J Clin Microbiol 2003;41:1574–1585. J Antimicrob Chemother 2010;65:1145–1154. 16. Solyman SM, Black CC, Duim B, et al. Multilocus sequence typ- 2. Weese JS, van Duijkeren E. Methicillin-resistant Staphylococcus ing for characterization of Staphylococcus pseudintermedius. J Clin aureus and Staphylococcus pseudintermedius in veterinary medi- Microbiol 2013;51:306–310. cine. Vet Microbiol 2010;140:418–429. 17. Goering RV, Morrison D, Al-Doori Z, Edwards GFS, Gemmell 3. van Duijkeren E, Catry B, Greko C, et al. Review on methicillin-­ CG. Usefulness of mec-associated direct repeat unit (dru) typing resistant Staphylococcus pseudintermedius. J Antimicrob Chemother in the epidemiological analysis of highly clonal methicillin- 2011;66:2705–2714. resistant Staphylococcus aureus in Scotland. Clin Microbiol Infect 4. Boerlin P, Eugster S, Gaschen F, Straub R, Schawalder P. 2008;14:964–969. Transmission of opportunistic pathogens in a veterinary teach- 18. Strandén A, Frei R, Widmer AF. Molecular typing of methicillin- ing hospital. Vet Microbiol 2001;82:347–359. resistant Staphylococcus aureus: Can PCR replace pulsed-field gel 5. Boost MV, So SYC, Perreten V. Low rate of methicillin-­resistant electrophoresis? J Clin Microbiol 2003;41:3181–3186. coagulase-positive staphylococcal colonization of veteri- 19. Bannoehr J, Ben Zakour NL, Waller AS, et al. Population genetic nary personnel in Hong Kong. Zoonoses Public Hlth 2011;58: structure of the Staphylococcus intermedius group: Insights into 36–40. agr diversification and the emergence of methicillin-resistant 6. Gerstadt K, Daly JS, Mitchell M, Wessolossky M, Cheeseman SH. strains. J Bacteriol 2007;189:8685–8692. Methicillin-resistant Staphylococcus intermedius pneumonia fol- 20. Koreen L, Ramaswamy SV, Graviss EA, Naidich S, Musser JM, lowing coronary artery bypass grafting. Clin Infect Dis 1999;29: Kreiswirth BN. Spa typing method for discriminating among 218–219. Staphylococcus aureus isolates: Implications for use of a single 7. Morris DO, Boston RC, O’Shea K, Rankin SC. The prevalence of marker to detect genetic micro- and macrovariation. J Clin carriage of methicillin-resistant staphylococci by veterinary der- Microbiol 2004;42:792–799.

2000;64:0–00 The Canadian Journal of Veterinary Research 197 21. Shopsin B, Gomez M, Montgomery SO, et al. Evaluation of pro- pseudintermedius of canine origin: Literature review from 1980 tein A gene polymorphic region DNA sequencing for typing of to 2013. Vet Microbiol 2014;171:337–341. Staphylococcus aureus strains. J Clin Microbiol 1999;37:3556–3563. 29. Osland AM, Vestby LK, Fanuelsen H, Slettemeås JS, Sunde M. 22. Kadlec K, Schwarz S, Goering RV, Weese JS. Direct repeat unit Clonal diversity and biofilm-forming ability of methicillin-­ (dru) typing of methicillin-resistant Staphylococcus pseudinterme- resistant Staphylococcus pseudintermedius. J Antimicrob dius from dogs and cats. J Clin Microbiol 2015;53:3760–3765. Chemother 2012;67:841–848. 23. Siak M, Burrows AK, Coombs GW, et al. Characterization of 30. Maluping RP, Paul NC, Moodley A. Antimicrobial susceptibil- methicillin-resistant and methicillin-susceptible isolates of ity of methicillin-resistant Staphylococcus pseudintermedius iso- Staphylococcus pseudintermedius from cases of canine pyoderma lated from veterinary clinical cases in the UK. Br J Biomed Sci in Australia. J Med Microbiol 2014;63:1228–1233. 2014;71:55–57. 24. Weese JS, Sweetman K, Edson H, Rousseau J. Evaluation of 31. Maaland MG, Papich MG, Turnidge J, Guardabassi L. Pharmaco­ minocycline susceptibility of methicillin-resistant Staphylococcus dynamics of doxycycline and tetracycline against Staphylococcus pseudintermedius. Vet Microbiol 2012;162:968–971. pseudintermedius: Proposal of canine-specific breakpoints for 25. Clinical Laboratory Standards Institute (CLSI). Performance stan- doxycycline. J Clin Microbiol 2013;51:3547–3554. dards for antimicrobial disk diffusion and dilution susceptibility 32. Clinical Laboratory Standards Institute (CLSI). Performance tests for bacteria isolated from animals; approved guideline. standards for antimicrobial disk and dilution susceptibility Wayne, Pennsylvania: CLSI, 2008. CLSI Document No.: M31–A3. tests for bacteria isolated from animals; approved guideline. 26. Sasaki T, Tsubakishita S, Tanaka Y, et al. Multiplex-PCR method 3rd Edition. Wayne, Pennsylvania: CLSI, 2015. CLSI Document for species identification of coagulase-positive staphylococci. No.: VET01S. J Clin Microbiol 2010;48:765–769. 33. Gold RM, Cohen ND, Lawhon SD. Amikacin resistance in 27. Bartels MD, Boye K, Oliveira DC, Worning P, Goering R, Staphylococcus pseudintermedius isolated from dogs. J Clin Westh H. Associations between dru types and SCCmec cassettes. Microbiol 2014;52:3641–3646. PLoS ONE 2013;8,e61860. 28. Moodley A, Damborg P, Nielsen SS. Antimicrobial resistance in methicillin susceptible and methicillin resistant Staphylococcus

198 The Canadian Journal of Veterinary Research 2000;64:0–00 Article

The impact of carboplatin and toceranib phosphate on serum vascular endothelial growth factor (VEGF) and metalloproteinase-9 (MMP-9) levels and survival in canine osteosarcoma Tracy L. Gieger, Julie Nettifee-Osborne, Briana Hallman, Chad Johannes, Dawn Clarke, Michael W. Nolan, Laurel E. Williams

Abstract In this pilot study, 10 dogs with osteosarcoma (OSA) were treated with amputation and subsequent carboplatin chemotherapy (300 mg/m2 IV q3wk 3 4 doses) followed by toceranib phosphate (2.75 mg/kg PO q48h starting at day 14 post carboplatin). Monthly clinical monitoring and serum measurements of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were acquired. No dogs were removed from the study due to toxicity. Levels of VEGF and MMP-9 did not change over time. Seven dogs died related to local recurrence and/or pulmonary or bone metastasis and the remainder died of other causes. Median OSA-free survival was 238 d with 34% 1-year progression-free survival. Median overall survival was 253 d with 30% alive at 1.5 y and 10% alive at 2 y. Although this regimen was well-tolerated, survival times did not exceed previously published data from dogs treated with amputation plus chemotherapy alone.

Résumé Dans cette étude pilote, 10 chiens avec un ostéosarcome (OSA) ont été traités par amputation et chimiothérapie subséquente avec du carboplatin (300 mg/m2 IV q3sem 3 4 doses) suivi de phosphate de toceranib (2,75 mg/kg PO q48h débutant au jour 14 suivant le carboplatin). Un suivi clinique mensuel et une mesure des taux sériques du facteur de croissance de l’endothélium vasculaire (FCEV) et de la matrice de la métalloprotéinase-9 (MMP-9) ont été obtenus. Aucun chien ne fut retiré de l’étude pour cause de toxicité. Les niveaux de FCEV et MMP-9 n’ont pas changé dans le temps. Sept chiens sont décédés dues à une rechute locale et/ou des métastases osseuses et les autres sont morts d’autres causes. La durée médiane de survie libre d’OSA était de 238 j avec 34 % de survie sans progression de la condition après 1 an. La durée de survie médiane était de 253 j avec 30 % en vie après 1,5 an et 10 % en vie après 2 ans. Bien que ce traitement ait été bien toléré, les temps de survie n’ont pas excédé les résultats publiés antérieurement pour des chiens traités par amputation et chimiothérapie uniquement. (Traduit par Docteur Serge Messier)

Introduction veterinary medicine. Three drugs have been identified as having significant activity against osteosarcoma: cisplatin, carboplatin, An estimated 10 000 cases of canine osteosarcoma (OSA) are and doxorubicin (2–4). Median survival times ranging from 290 to diagnosed in pet dogs annually in the United States (1). It is a locally 324 d and 1-year survival rates of 45.5% have been reported with the invasive and highly metastatic disease, with micrometastasis present administration of adjuvant cisplatin chemotherapy (3,4). Carboplatin in approximately 75% to 95% of dogs at the time of diagnosis, based chemotherapy is comparable, with a median survival of 321 d and on studies of survival following amputation (2,3). While surgery is 1-year survival of 35.4% (5). Similarly, the median survival time fol- indicated to remove the painful and functionally limiting primary lowing a course of doxorubicin chemotherapy is 366 d with 1- and tumor, amputation is considered palliative therapy and does not 2-year survival rates estimated at 50.5% and 9.7%, respectively (6). result in prolonged survival. Median survival times following ampu- A variety of strategies have been attempted in an effort to improve tation alone range from 102 to 168 d, with 1- and 2-year survival outcomes with chemotherapy. These include altering the time at rates of 11% to 21% and 2% to 4%, respectively. The administration which chemotherapy is initiated and using cisplatin or carboplatin of adjuvant chemotherapy in an effort to delay or prevent metas- in combination with doxorubicin (7,8). Unfortunately, these efforts tasis and improve the outcome, is the current standard of care in have failed to demonstrate significant improvements in median

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 1052 William Moore Drive, Raleigh, North Carolina 27607, USA (Gieger, Nettifee-Osborne, Hallman, Johannes, Clarke, Nolan, Williams); Iowa State University College of Veterinary Medicine, 1600 S 16th Street, 1569 LVMC, Ames, Iowa 50011, USA (Johannes); University of Georgia College of Veterinary Medicine, 2200 College Station Road, Athens, Georgia 30602, USA (Clarke); Veterinary Specialty Hospital of the Carolinas, 6405 Tryon Road, Cary, North Carolina 27518, USA (Williams); Comparative Medicine Institute, North Carolina State University, 1052 William Moore Drive, Raleigh, North Carolina 27607, USA (Gieger, Nettifee-Osborne, Nolan). Address all correspondence to Dr. Tracy L. Gieger; telephone: (919) 513-6690; e-mail: [email protected] Received September 4, 2016. Accepted December 20, 2016.

2017;81:199–205 The Canadian Journal of Veterinary Research 199 survival times over those seen with conventional single-agent treat- inhibition of KIT signaling in OSA (14). While initially developed ment protocols. Novel therapeutic approaches are needed to control for its activity against KIT — a receptor mutated in approximately metastatic disease and improve survival. 30% to 50% of canine mast cell tumors (MCT) — the activity of Angiogenesis is the process by which tumors induce new blood TP against other members of this receptor tyrosine kinase family, vessel formation and is essential for continued tumor growth and such as VEGF-R, suggests a potentially wider spectrum of activity. metastasis. In the absence of angiogenesis, tumors are restricted to Anti-tumor responses are reported in MCT lacking a KIT mutation, sizes ranging from several microns up to 1 to 2 mm (9). Endogenous providing support for this theory and suggesting that other inhibited angiogenesis inhibitors have been detected in the urine of OSA receptors, such as VEGF-R, play an important role in tumor progres- tumor-bearing dogs and these factors demonstrated potent inhibi- sion and regression (15). Toceranib phosphate exerts antiproliferative tion of endothelial cell proliferation (10). These endogenous factors effects on endothelial cells in vitro, suggesting a role in angiogenesis, were absent in urine collected from the same dogs 1 to 12 wk after and produces clinically meaningful disease stabilization in vivo in surgical amputation of the limb bearing the primary tumor. The pro- dogs with osteosarcoma (16–18). As part of a larger study of dogs angiogenic shift that occurs after primary tumor removal suggests with solid tumors being treated with TP, 11/23 (48%) dogs with that a strategy designed to pharmacologically control angiogenesis metastatic OSA had stabilization of their disease (18). The oral dos- should be evaluated for its capacity to suppress the rapid progres- ing of TP on an alternate-day schedule ideally lends itself to chronic sion of microscopic metastases to life-threatening metastatic disease. inhibition of tumor-promoted angiogenesis to prevent the progres- The use of angiogenesis inhibitors has emerged as a clinically valid sion of OSA micrometastases. therapeutic approach in human oncology. This strategy may also The goals of this study are: i) to evaluate the impact of mainte- have applicability to veterinary oncology, and is particularly appeal- nance antiangiogenic therapy using TP as a sole agent following ing for OSA where death is the result of progressive enlargement amputation and carboplatin chemotherapy as a means of improving of metastatic foci. outcome for dogs with OSA, and ii) to measure circulating levels Anti-angiogenic treatment strategies fall into 2 categories: i) those of endogenous pro-angiogenic factors before and during treatment that are directly cytotoxic to endothelial cells, and ii) those acting with TP, which may serve as biomarkers for survival and disease indirectly by eliminating critical pro-angiogenic factors [e.g., vascular progression. We hypothesize that the administration of TP following endothelial growth factor (VEGF)] and promoting anti-angiogenic amputation and carboplatin chemotherapy will inhibit the progres- factors. Vascular endothelial growth factor is a 45-kDa homodimer sion of metastasis through disruption of receptor tyrosine kinases with pluripotent effects in angiogenesis — stimulating the prolifera- with subsequent inhibition of angiogenesis. This will lead to pro- tion, invasion, and migration of endothelial cells. In addition, VEGF longed remission duration and survival in dogs with appendicular is a key target of several angiogenic factors and proteases such as OSA compared to rates previously achieved in a group of dogs matrix metalloproteinases (MMPs), with roles in tumor metastasis treated with amputation and carboplatin alone (5). We hypothesize (11). Matrix metalloproteinases are enzymes that degrade structural that the administration of TP following amputation and carboplatin elements outside of cells and play a critical role in tissue remodel- chemotherapy will decrease circulating levels of pro-angiogenic ing during tumor invasion, angiogenesis, and metastasis. Among MMP-9, a downstream factor and potential surrogate marker of MMPs, MMP-9 is of particular interest. Matrix metalloproteinase-9 VEGF-R inhibition in dogs with appendicular OSA. is induced via VEGF receptor (VEGF-R) activation and levels of this downstream factor may be useful as a surrogate marker of VEGF-R Materials and methods activity (12); however, there have been no studies directly correlat- ing serum MMP-9 levels with VEGF-R activity in serum or tissues Ten dogs with histologically confirmed appendicular OSA with of OSA-bearing dogs. no radiographic evidence of pulmonary metastasis at the time of Vascular endothelial growth factor is expressed by a variety of enrollment and for whom owner consent was given, were prospec- tumors, and therapies designed to decrease production or otherwise tively enrolled. Institutional IACUC approval was obtained. All block VEGF activity are being explored as a means of inhibiting dogs were evaluated with a physical examination, complete blood tumor proliferation. In an evaluation of 24 dogs with OSA, pretreat- (cell) count (CBC), serum biochemistry panel, urinalysis, 3-view ment platelet-corrected serum VEGF levels correlated significantly thoracic radiographs, and a quality-of-life (QOL) survey completed with disease-free interval (13). Results of this study and similar by the owner prior to treatment. Two weeks following amputation, human studies support an important role for VEGF in the develop- carboplatin (Paraplatin; Bristol Myers Squibb, New York, New York, ment and progression of metastatic disease in OSA. We hypothesize USA) chemotherapy was administered intravenously at a dosage that the inhibition of VEGF activity will delay the progression of of 300 mg/m2 at 3-week intervals for a total of 4 treatments. A CBC OSA micrometastases. was performed immediately before each carboplatin treatment and Toceranib phosphate [TP (Palladia; Zoetis, Durham, North 7 d and 14 d following the first 2 treatment cycles to monitor for Carolina, USA)], an FDA-approved drug for use in dogs, is a selec- potential hematologic toxicity. A 25% dosage reduction was pre- tive inhibitor of several members of the receptor tyrosine kinase fam- scribed for any patient developing grade 3 or higher bone marrow ily, including vascular endothelial growth factor receptor (VEGF-R), or gastrointestinal toxicity as defined by the Veterinary Co-operative KIT, FLT-3, and platelet-derived growth factor receptor (PDGF-R). Oncology Group Common Terminology Criteria for Adverse Events c-KIT wild type receptor overexpression was identified in a study of (VCOG-CTCAE) v1.0 (19). Three-view thoracic radiographs were 74 human pediatric high-grade OSA, indicating a potential role of the repeated prior to the third carboplatin chemotherapy treatment and

200 The Canadian Journal of Veterinary Research 2000;64:0–00 immediately before initiation of TP. Two weeks following the final uted to serum VEGF levels, serum VEGF levels were corrected for carboplatin chemotherapy treatment, patients began TP therapy at platelet counts as follows: serum VEGF (pg/mL)/platelet count a dosage of 2.75 mg/kg body weight (BW), PO, q48h. This drug was (3 106/mL) (20,21). to be continued indefinitely unless evidence of toxicity, development of concurrent co-morbidities, death, or owner withdrawal of the pet Statistical analysis from the study, occurred. Dogs that developed local recurrence or A sample size of 10 dogs provides a power of 80% and a 50% metastasis were censored from statistical analysis but were allowed level of significance to detect a 47% increase in 1-year survival (i.e., to continue to receive TP if the owner elected to do so; owners were increase the probability of survival at 1 y, from 34.5% to 50.7%) also counseled about all other potential treatment options outside in dogs with appendicular OSA treated with carboplatin chemo- of the study. To minimize the likelihood of toxicity, all dogs received therapy followed by TP following amputation. The historical control omeprazole (Prilosec 0.5 to 1.0 mg/kg BW, PO, q24h; AstraZeneca consisted of a previously reported group of 48 dogs treated with Pharmaceuticals, Stockholm, Sweden) or famotidine (Pepcid 0.5 to amputation and 4 cycles of carboplatin chemotherapy with a median 1.0 mg/kg BW, PO, q12-24h; McNeil Consumer Pharmaceuticals, survival of 321 d and 1-year survival of 34.5% (5). Osteosarcoma-free Fort Washington, Pennsylvania, USA) and, if needed, metoclo- survival time was defined as the date of amputation to the date of pramide (0.2 to 0.4 mg/kg BW, PO, q8h) and/or maropitant (Cerenia documentation of local recurrence or OSA metastasis. Overall sur- 2 mg/kg BW, PO, q24h 3 consecutive 5 d each week; Zoetis, vival (OS) was defined as the date of amputation until death from Parsippany-Troy Hills, New Jersey, USA). To avoid confounding any cause. A commercially available statistical program was used variables, NSAIDs were discontinued for the duration of the study (GraphPad Prism 6; San Diego, California, USA). The Kaplan-Meier period. Patients requiring ongoing analgesia received tramadol (1 to method was used to calculate OSA-free survival and overall sur- 4 mg/kg BW, PO, q6-12h), amantadine (3 to 5 mg/kg BW, PO, q24h), vival (OS). A Friedman’s test was used to compare VEGF and MMP and/or gabapentin (5 to 10 mg/kg BW, PO, q8-12h). values at baseline, pre-TP, and end of study. A Kruskal-Wallis test of repeated measures was used to compare platelet-corrected VEGF Sample collection values at baseline, pre-TP, and end of study. P , 0.1 was considered Serum samples for enzyme-linked immunosorbent assay (ELISA) significant. A P-value = 0.1 was selected due to the small sample size evaluation of VEGF and MMP-9 were collected for analysis: i) imme- to try to avoid type II error. diately before initiation of carboplatin chemotherapy, ii) immediately prior to initiation of TP, and iii) 3 and 6 wk after starting TP, and Results iv) every 8 wk thereafter for 18 mo or until the time of disease pro- gression if this occurred at less than 18 mo. Median age was 9.5 y (range: 5 to 11 y). Median body weight was 31 kg (range: 19 to 48 kg). Seven were spayed females and Patient monitoring 3 were castrated males. There were 3 golden retrievers, 1 mixed Disease status was monitored by a physical examination and breed dog, and 1 of each of the following breeds: Belgian tervu- 3-view thoracic radiographs at 8-week intervals until disease pro- ren, great Dane, border collie, Rottweiler, Labrador retriever, and gression was documented. To monitor for potential TP-related St. Bernard. Tumor locations included the distal radius (n = 5), distal toxicity, CBC and body weight were evaluated weekly for the first femur (n = 2), distal tibia (n = 2), and proximal humerus (n = 1). 6 wk and a chemistry panel and urinalysis were also performed at No patients had CBC abnormalities prior to treatment. Serum bio- weeks 1, 3, and 6. Subsequently, a CBC, body weight, chemistry chemical abnormalities included increased alkaline phosphatase in panel, and urinalysis were performed at 8-week intervals. Quality-of- 2 dogs [726 and 164 IU/L (normal , 140)] and in one dog, increased life surveys were also submitted by pet owners at each of these time BUN [67 mg/dL (normal , 26)], creatinine [3 mg/dL (normal points. Toxicity was recorded at each time point. A 1-week cessation , 1.5 mg/dL)], phosphorous [6.1 mg/dL (normal , 5.6 mg/dL)], of TP therapy followed by incremental dosage reductions (0.5 mg/kg and potassium [5.8 mmol/L (normal , 5.6 mmol/L)] (in this dog, BW increments to a minimum of 2.2 mg/kg BW) and/or decreased the urine specific gravity was 1.018). Seven dogs were treated with TP administration frequency (from q48h to Monday, Wednesday, and NSAIDs prior to being enrolled in the study for a variable duration Friday schedule) was instituted for any dog experiencing . grade 2 of time; these drugs were discontinued on the first day of chemo- adverse event(s). therapy in 3 dogs and 3 to 35 d prior to initiation of chemotherapy in the remaining dogs. Sample analysis Approximately 7 to 10 mL of whole blood was collected into Chemotherapy administration serum and plasma separator tubes by jugular venipuncture. Carboplatin chemotherapy was started a median of 16 d post- Immediately after collection, blood samples were centrifuged and the amputation (range: 11 to 30 d). Eight dogs received the first dose serum frozen at 270°C until assayed. Commercially available ELISA of carboplatin at 300 mg/m2; 1 dog received 239 mg/m2 (unknown test kits were used for VEGF (CAVEOO Canine VEGF Quantikine reason; given at RDVM) and the dog with renal insufficiency ELISA Kit; R&D Systems, Minneapolis, Minnesota, USA) and MMP-9 received 150 mg/m2 (arbitrary dose reduction due to renal insuf- (SEA553CA ELISA Kit for MMP9 Organism species; Cedarlane Labs) ficiency). Four dogs required dose reductions of carboplatin due to and tests were performed according to the manufacturers’ directions. neutropenia; no dogs required hospitalization for supportive care. To minimize the impact that VEGF released from platelets contrib- The carboplatin dose was reduced from 300 mg/m2 to 225 mg/m2

2000;64:0–00 The Canadian Journal of Veterinary Research 201 Overall survival time Osteosarcoma-free survival time

100 100

50 50 Percent survival Percent evidence of osteosarcoma Proportion surviving without Proportion 0 0 0 200 400 600 800 0 200 400 600 800 1000 Time (days) Time (days)

Figure 1. Median osteosarcoma-free survival time was 238 days with Figure 2. Median overall survival time was 253.5 days with 30% alive at 34.3% event-free at 1 year. 1.5 years and 10% alive at 2 years. after the first dose in 3 dogs and after the second dose in 1 dog (who 3 dogs at the owners’ request after metastasis was detected (dogs also had a 1-week treatment delay due to prolonged neutropenia). were censored from analysis on the date metastasis was diagnosed) and these dogs remained on the drug until death. An additional dog Toceranib phosphate administration received treatment with doxorubicin after pulmonary metastasis was Eight dogs received TP at doses ranging from 2.2 to 2.9 mg/kg detected. All dogs were followed until the time of their death and (median dose: 2.7 mg/kg). After starting TP, 1 dog had two 1-week all are deceased. Only 1 dog underwent necropsy. drug holidays related to grade I diarrhea and 1 dog had dose reductions and an extended dosing interval due to persistent, non- Survival data progressive grade I neutropenia. The median OSA-free survival was 238 d (range: 50 to 575 d) with 34% progression-free survival at 1 y (Figure 1). Overall survival was Toxicity outcomes 253 d with 30% alive at 1.5 y and 10% alive at 2 y (Figure 2). No dogs were removed from the study due to toxicity or declining QOL. Quality-of-life surveys were subjectively scored, and scores VEGF and MMP-9 data remained stable throughout the study (scores did not decline from Pre-treatment serum VEGF levels ranged from 0.8 to 20.4 baseline after starting carboplatin or TP) until the patient experi- (median: 6.5) and MMP-9 levels ranged from 8.9 to 25.2 (median: 12), enced a clinical decline typically related to disease progression. with wide variations in intra- and inter-patient variability. There was no significant difference in MMP (Figures 3 and 5) or VEGF [absolute Tumor-related outcomes (Figures 4 and 6) or platelet-corrected] values from baseline and pre-TP, Seven dogs developed metastasis as the first event. Pulmonary baseline and TP discontinuation, or pre-TP to TP discontinuation. metastasis was suspected based on thoracic radiographs in 6 dogs 60 to 240 d post-amputation (median 180 d) and osseous metasta- Discussion sis to the vertebrae and ribs was suspected in one dog based on radiographs at 575 d after amputation. One dog that developed In this study, continuous administration of TP was well-tolerated pulmonary metastasis also concurrently developed an amputation- in dogs with OSA treated with amputation and adjuvant carboplatin site stump recurrence 180 d post-amputation. Two of 10 (20%) dogs chemotherapy, although a survival benefit was not shown. In the developed pulmonary metastasis while receiving carboplatin che- present study, OS was 253 d with 30% alive at 1.5 y post-amputation, motherapy and did not continue on to the TP phase of the study. which is comparable to a historical group treated with amputation These 2 dogs were included in outcome analysis on an intent-to-treat and adjuvant carboplatin chemotherapy in which OS was 321 d basis. Of the 8 dogs that received TP, 3 died of metastasis (pulmo- with 35% alive at 1 y (5). To date, 2 previously published studies of nary in 2 dogs, osseous in 1 dog), 1 died of concurrent pulmonary dogs with OSA treated with carboplatin chemotherapy and metro- metastasis and stump recurrence, 3 died of other causes [lymphoma nomic chemotherapy (MC) have failed to demonstrate improved that was diagnosed during the study while the dog was receiving survival times versus historical publications of dogs treated with TP (n = 1), metastatic hemangiosarcoma that developed while the carboplatin alone. In one publication, as part of a larger study of dog was receiving TP (n = 1), and progressive spinal pain with no dogs with OSA treated with chemotherapy 1 MC [piroxicam and definitive diagnosis despite and extensive workup (n = 1)] and oral cyclophosphamide (10 to 12 mg/m2 per day)], 14 dogs were 1 dog was withdrawn from the study per the owner’s request with treated with carboplatin concurrent with MC (22). Three dogs were no evidence of OSA (n = 1). Toceranib phosphate was continued in removed from the study due to toxicity (gastrointestinal). The overall

202 The Canadian Journal of Veterinary Research 2000;64:0–00 40 40

30 30

20 20 MMP-9 (ng/mL) MMP-9 (ng/mL) 10 10

0 0

Baseline Baseline End of study End of study Pre-toceranib Pre-toceranib

Figure 3. Metalloproteinase-9 concentrations at baseline, before tocera- Figure 5. Individual patient metalloproteinase-9 concentrations at nib phosphate, and at time of discontinuation of toceranib phosphate. baseline, before toceranib phosphate, and at time of discontinuation of toceranib phosphate.

200 200

150 150

100 100 VEGF (pg/mL) VEGF (pg/mL) 50 50

0 0

Baseline Baseline End of study End of study Pre-toceranib Pre-toceranib

Figure 4. Vascular endothelial growth factor concentrations at baseline, Figure 6. Individual patient vascular endothelial growth factor concentra- before toceranib phosphate, and at time of discontinuation of toceranib tions at baseline, before toceranib phosphate, and at time of discontinu- phosphate. ation of toceranib phosphate. disease-free interval was 192 d, with a median survival time of 217 d. Although MC administration has not yet been shown to improve The lack of improvement with post-chemotherapy MC (including outcomes in dogs with OSA when used in combination with tradi- TP in some dogs) was also confirmed in a recent, larger study (23). tional maximally tolerated dose (MTD) chemotherapy, its potential One hundred twenty-six dogs with appendicular OSA were treated usefulness in highly metastatic diseases such as OSA, continues with amputation followed by 4 doses of carboplatin chemotherapy to be explored. In an attempt to further elucidate the tolerability and then randomized to receive MC [piroxicam/cyclophospha- and mechanism of action of MC, a recent study evaluated toxicity mide (10 mg/m2 q48h)] with or without addition of TP [MC 1 TP and lymphocyte profiles in tumor-bearing dogs treated with MTD (2.75 mg/kg PO q48h)]. Thirty-two dogs developed metastasis before doxorubicin (MTD-dox) only and those treated with MTD-dox the onset of MC or MC 1 TP, and a total of 35 received MC and 46 alone and in combination with metronomic cyclophosphamide received MC 1 TP. Toxicities were greater in the MC 1 TP group, but (MCTX) (24). A phase I study was performed and established that most were mild and resolved with supportive care. The median DFI the standard doxorubicin dose of 30 mg/m2 (IV q21d) and a previ- was 215 and 233 d and the OS was 242 and 318 d, respectively, for ously published MC dose of cyclophosphamide of 15 mg/m2 per the MC versus MC 1 TP groups, which did not differ significantly. day was tolerable when administered concurrently. Dogs were The 1-y survival rate was 35% and 38% for the MC versus MC 1 TP then randomized to receive MTD-dox (n = 8) or MTD-dox 1 MCTX groups, respectively. (n = 8). Both treatment groups showed declines in both total and

2000;64:0–00 The Canadian Journal of Veterinary Research 203 T-regulatory lymphocytes (Tregs) by day 7 after MTD-dox, and To our knowledge, this is the first study evaluating the use of there was no statistically significant difference between the groups single-agent TP (without other concurrent MC drugs such as NSAIDs in either total lymphocyte count or Tregs. In contrast to previous and/or cyclophosphamide) after amputation and carboplatin che- studies demonstrating that Tregs were selectively inhibited by CTX motherapy in dogs with OSA. Although there was no improvement (25), there was no selective depletion of Tregs. The authors theo- in outcome compared to historical controls, further work evaluating rized that concurrent administration of MTD-dox and MC may not the use of TP in dogs with OSA should be conducted. Since it has be beneficial if the MTD chemotherapy dampens the inhibition of been shown that dogs with gross metastasis from OSA can have the selective Treg depletion seen with MCTX alone. Although this stable disease and a clinical benefit from treatment with TP (18), it is study used doxorubicin and the current study as well as a previously likely that using TP in a microscopic disease setting before the onset published larger study used carboplatin, this theory could explain of visible metastasis may be beneficial. why MTD carboplatin 1 MC did not yield prolonged survival times In conclusion, the combination of carboplatin followed by TP was in dogs with OSA. Other potential mechanisms of efficacy of MC well-tolerated by dogs with appendicular OSA. There was inter- and include other immunomodulatory effects (e.g., restoring NK effec- intra-patient variability of VEGF and MMP-9 levels at all time points tor function), anti-angiogenic effects [including decreased tumor and survival times did not differ from previously published data microvessel density and decreased mobilization of circulating from dogs treated with amputation and adjuvant chemotherapy. endothelial precursors (CEPs)], inhibition of normal stromal cells Further larger studies to devise more effective therapeutic options that support growing cancer cells, and possibly direct anti-tumor for this disease are warranted. effects. The use of NSAIDs in MC can serve as a confounding factor when comparing studies since they inhibit COX-2, which may result Acknowledgments in an inability of CEPs to survive and proliferate within the tumor microenvironment (24); therefore, the contribution of metronomic This study was supported, in part, by a grant from Bone Cancer chemotherapy drugs such as cyclophosphamide to the outcome can Dogs and provision of drugs and finances from Zoetis. Preliminary be difficult to determine when NSAIDs are used concurrently. It is results were presented as an abstract at the American College of likely that the full potential of MC in addition to MTD chemotherapy Veterinary Internal Medicine Annual Forum, Denver, Colorado in for OSA will only be realized with clinical trials that compare use of June 2016. standardized MTD chemotherapy and MC regimens both concur- rently and sequentially. References One aspect of the present study that is unique, is the attempt to quantify the degree of antiangiogenesis via measurement of serum 1. Withrow SJ, Powers BE, Straw RC, Wilkins RM. Comparative MMP-9, VEGF, and platelet-corrected VEGF levels at baseline (post- aspects of osteosarcoma. Dog versus man. Clin Orthop Relat amputation), pre-TP (after 4 cycles of carboplatin), over time during Res 1991;270:159–168. TP administration, and at the time when TP was discontinued. We 2. Spodnick GJ, Berg J, Rand WM, et al. Prognosis for dogs with anticipated changes in VEGF and MMP-9 levels concomitant with appendicular osteosarcoma treated by amputation alone: changes in disease status. If changes were repeatable and consistent 162 cases (1978–1988). J Am Vet Med Assoc 1992;200:995–999. among the study dogs, these serum markers may have served a sur- 3. Thompson JP, Fugent MJ. Evaluation of survival times after rogate markers for the remission status of OSA. In a study of dogs limb amputation, with and without subsequent administra- with OSA treated with amputation, pre-amputation platelet-corrected tion of cisplatin, for treatment of appendicular osteosarcoma VEGF was significantly correlated with DFI (13). In a study of dogs in dogs: 30 cases (1979–1990). J Am Vet Med Assoc 1992;200: treated with metronomic chemotherapy (NSAID 1 cyclophospha- 531–533. mide) (26) for various metastatic tumors, VEGF levels were measured 4. Berg J, Weinstein MJ, Schelling SH, Rand WM. Treatment of dogs before (but not during) treatment, and dogs with lower VEGF levels with osteosarcoma by administration of cisplatin after amputa- had longer survival times and were more likely to respond to treat- tion or limb-sparing surgery: 22 cases (1987–1990). J Am Vet Med ment than dogs with levels above the median. Unfortunately, in the Assoc 1992;200:2005–2008. current group of dogs, the MMP-9 and VEGF (absolute or platelet- 5. 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This was a pilot study, and as a result, conclu- Cancer 1997;79:1343–1350. sions are inherently limited due to small sample size and lack of a 8. Bailey D, Erb H, Williams L, Ruslander D, Hauck M. Carboplatin contemporaneous control group. Failure to reliably assay angiogen- and doxorubicin combination chemotherapy for the treatment of esis biomarkers in this study show that other measures are needed to appendicular osteosarcoma in the dog. J Vet Intern Med 2003;17: determine subclinical implications of anti-angiogenic therapy in dogs. 199–205.

204 The Canadian Journal of Veterinary Research 2000;64:0–00 9. Folkman J. What is the evidence that tumors are angiogenesis 19. Veterinary co-operative oncology group (VCOG). Veterinary dependent? J Natl Cancer Inst 1990;82:4–6. co-operative oncology group — Common terminology criteria 10. Pirie-Shepherd SR, Coffman KT, Resnick D, et al. The role of for adverse events (VCOG-CTCAE) following chemotherapy angiostatin in the spontaneous bone and prostate cancers of pet or biological antineoplastic therapy in dogs and cats v1.0. Vet dogs. Biochem Biophys Res Commun 2002;292:886–891. Comp Oncol 2004;2:195–213. 11. Bergers G, Brekken R, McMahon G, et al. Matrix metal­- 20. Sendur MA, Askoy S, Yaman S, Arik Z, Ozdemir NY, Zengin N. loproteinase-9­ triggers the angiogenic switch during carcino- Plasma VEGF levels may not accurately reflect the truth all the genesis. Nat Cell Biol 2000;2:737–744. time. Med Oncol 2012;29:1403–1404. 12. Hiratsuka S, Nakamura K, Iwai S, et al. MMP9 induction by 21. George ML, Eccles SA, Tutton MG, Abulafi AM, Swift RI. vascular endothelial growth factor receptor-1 is involved in Correlation of plasma and serum vascular endothelial growth lung-specific metastasis. Cancer Cell 2002;2:289–300. factor levels with platelet count in colorectal cancer: Clinical evi- 13. Thamm DH, O’Brien MG, Vail DM. Serum vascular endothelial dence of platelet scavenging? Clin Cancer Res 2000;6:3147–3152. growth factor concentrations and postsurgical outcome in dogs 22. Bracha S, Walshaw R, Danton T, Holland S, Ruaux C, with osteosarcoma. Vet Comp Oncol 2008;6:126–132. Obradovich J. Evaluation of toxicities combined metronomic 14. Entz-Werle N, Gaub MP, Lavaux T, et al. KIT gene in pediatric and maximal-tolerated dose chemotherapy in dogs with osteo- osteosacomas: Could it be a new therapeutic target? Int J Cancer sarcoma. J Small Anim Pract 2014;55:369–374. 2007;120:2510–2516. 23. London CA, Gardner HL, Mathie T, et al. Impact of toceranib/ 15. London CA, Malpas PB, Wood-Follis SL, et al. Multi-center, piroxicam/cyclophosphamide maintenance therapy on outcome placebo-controlled, double-blind, randomized study of oral toc- of appendicular osteosarcoma following amputation and car- eranib phosphate (SU11654), a receptor tyrosine kinase inhibitor, boplatin chemotherapy: A multi-institutional study. PLoS One for the treatment of dogs with recurrent (either local or distant) 2015;10:e0124889. mast cell tumor following surgical excision. Clin Cancer Res 24. Rasmussen RM, Kurzman ID, Biller BJ, Guth A, Vail DM. Phase I 2009;15:3856–3865. lead-in and subsequent randomized trial assessing safety and 16. Mendel DB, Laird AD, Xin X, et al. In vivo antitumor activity of modulation of regulatory T cell numbers following a maximally SU11248, a novel tyrosine kinase inhibitor targeting vascular tolerated dose doxorubicin and metronomic dose cyclophospha- endothelial growth factor and platelet-derived growth factor mide combination chemotherapy protocol in tumour-bearing receptors: Determination of a pharmacokinetic/pharmacody- dogs. Vet Comp Oncol 2015:1–10. namic relationship. Clin Cancer Res 2003;9:327–337. 25. Burton JH, Mitchell L, Thamm DH, Dow SW, Biller BJ. Low-dose 17. London CA, Hannah AL, Zadovoskaya R, et al. Phase I dose- cyclophosphamide selectively decreases regulatory T cells and escalating study of SU11654, a small molecule receptor tyrosine inhibits angiogenesis in dogs with soft tissue sarcoma. J Vet kinase inhibitor, in dogs with spontaneous malignancies. Clin Intern Med 2011;25:920–926. Cancer Res 2003;9:2755–2768. 26. Marchetti V, Giorgi M, Fioravanti A, et al. First-line metronomic 18. London C, Mathie T, Stingle N, et al. Preliminary evidence for chemotherapy in a metastatic model of spontaneous canine biologic activity of toceranib phosphate in solid tumors. Vet tumours: A pilot study. Invest New Drugs 2012;30:1725–1730. Comp Oncol 2012;10:194–205.

2000;64:0–00 The Canadian Journal of Veterinary Research 205 Article

Oxidative stress and food supplementation with antioxidants in therapy dogs Sara Sechi, Filippo Fiore, Francesca Chiavolelli, Corrado Dimauro, Anna Nudda, Raffaella Cocco

Abstract The objective of this study was to evaluate the ability of a long-term antioxidant-supplemented diet to regulate the oxidative stress and general health status of dogs involved in animal-assisted intervention (AAI) programs. Oxidative stress is a consequence of the accumulation of reactive oxygen species (ROS). Exercise-induced oxidative stress can increase muscle fatigue and fiber damage and eventually leads to impairment of the immune system. A randomized, placebo-controlled, crossover clinical evaluation was conducted with 11 healthy therapy dogs: 6 females and 5 males of different breeds and with a mean age of 2.7 6 0.8 y (mean 6 SEM). The dogs were divided into 2 groups, 1 fed a high quality commercial diet without antioxidants (CD) and the other a high quality commercial diet supplemented with antioxidants (SD) for 18 wk. After the first 18 wk, metabolic parameters, reactive oxygen metabolite-derivatives (d-ROMs), and biological antioxidant potential (BAP) levels were monitored and showed a significant reduction of d-ROMs, triglycerides, and creatinine values in the SD group (P , 0.05) and a significant increase in amylase values in the CD group (P , 0.01). At the end of this period, groups were crossed over and fed for another 18 wk. A significant decrease in amylase and glutamate pyruvate transaminase (GPT) values was observed in the CD and SD group, respectively (P , 0.05). In conclusion, a controlled, balanced antioxidant diet may be a valid approach to restoring good cell metabolism and neutralizing excess free radicals in therapy dogs.

Résumé L’objectif de la présente étude était d’évaluer la capacité d’une diète long-terme supplémentée en antioxydant à réguler le stress oxydatif et l’état de santé général de chiens impliqués dans des programmes d’intervention avec assistance animale (IAA). Le stress oxydatif est une conséquence de l’accumulation d’espèces oxygène réactive (EOR). Le stress oxydatif induit par l’exercice peut augmenter la fatigue musculaire et les dommages aux fibres et éventuellement mener à un mauvais fonctionnement du système immunitaire. Une évaluation clinique croisée, randomisée, et avec groupe témoin-placebo a été menée avec 11 chiens d’assistance en santé : 6 femelles et 5 mâles de races différentes et d’un âge moyen de 2,7 6 0,8 ans (moyenne 6 écart-type). Les chiens ont été divisés en deux groupes, un premier groupe nourri avec une diète commerciale de haute qualité sans antioxydant (DC) et l’autre groupe avec une diète commerciale de haute qualité supplémentée avec des antioxydants (DS) pour 18 semaines. Après les premières 18 semaines, les paramètres métaboliques, les métabolites dérivés d’oxygène réactive (MDOR), et les niveaux de potentiel antioxydant biologique (PAB) ont été surveillés et ont montré une réduction significative des valeurs des MDOR, des triglycérides et de la créatinine dans le groupe DS (P , 0,05) et une augmentation significative des valeurs de l’amylase dans le groupe DC (P , 0,01). À la fin de cette période, les groupes ont été croisés et nourris pour 18 semaines supplémentaires. Une diminution significative des valeurs de l’amylase et de la glutamate pyruvate transaminase (GPT) a été obtenue dans les groupes DC et DS, respectivement (P , 0,05). En conclusion, une diète contrôlée, balancée en antioxydant pourrait être une approche valide pour restaurer un bon métabolisme cellulaire et neutraliser les radicaux libres excédentaires chez les chiens d’assistance. (Traduit par Docteur Serge Messier)

Introduction causes oxidative stress (2–4). It has been shown that exercise-induced oxidative stress contributes to increased muscle fatigue, muscle fiber Oxidative stress is a consequence of the accumulation of reac- damage (5), and eventually leads to immune system impairment (6). tive oxygen species (ROS), reactive nitrogen species (RNS), such as Both muscles and blood are endowed with antioxidant enzymes, hydroxyl radicals (OH-), superoxide anion radicals (O2), lipid per- such as superoxide dismutase (SOD), glutathione peroxidase (GPx), oxyl radicals (LOO-), and nitrate radicals (NO-) (1). Factors involved catalase (CAT), and non-enzymatic antioxidants, such as vitamins A, in the production of free radicals and reactive metabolites include C, and E, to counteract damage caused by free radicals (2,7,8). physical exercise, characterized by an increase in the body’s oxygen Oxidative damage induced by physical exercise has been reported consumption and correlated to an increase in ROS production, which in horses (9–11) and dogs (12,13). One study investigated the

Department of Veterinary Medicine, Pathology and Veterinary Clinic Section (Sechi, Fiore, Cocco) and Department of Agriculture, Livestock Sciences Section (Dimauro, Nudda), University of Sassari, Sassari, Italy; Department of Diagnostic Medicine, Clinical and Public Health, University of Modena and Reggio Emilia, Modena, Italy (Chiavolelli). Address all correspondence to Dr. Raffaella Cocco; telephone: (139) 079 229520; fax: 139079228211; e-mail: [email protected] The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Received November 7, 2016. Accepted January 4, 2017.

206 The Canadian Journal of Veterinary Research 2017;81:206–216 ­oxidative status of 9 dogs after 20 min and 4 h of aerobic exercise, file, urine analysis, and serum protein electrophoresis) and serological after 30 d of rest (14). Results showed a significant oxidative status tests (Ehrlichia canis, Rickettsia spp., Leishmania infantum, Anaplasma due to the high level of reactive oxygen metabolite-derivatives phagocytophilum, Bartonella spp., Toxoplasma gondii, Neospora caninum, (d-ROMs) and the low level of biological antioxidant potential (BAP) and Borrelia burgdorferi) were conducted on all dogs to confirm their in relation to the values before starting the exercises (P , 0.01) (14). good health status. Owner consent was obtained for each dog. Ethane exhaled in the breath may be considered a marker of oxi- All dogs were involved in 5 weekly (30 min each) animal-assisted dative stress as it is a product of free radical-mediated oxidation of intervention (AAI) sessions in the rehabilitation or social care of cell membrane lipids (15). The authors evaluated oxidative stress in humans. All sessions were held in the same place and at similar tem- terms of exhaled ethane in 8 human, 12 dog, and 11 equine athletes perature and humidity conditions. Patients involved in AAI sessions after a physical performance (15). A significant increase in exhaled consisted of 55 children with pervasive developmental disorders. ethane was observed in all species after training and was associated Each 30-minute session consisted of 15 min of active playing, such with oxidative stress. as ball throwing or running, 10 min of collaborative activities, such Evaluation of oxidative status with specific parameters could as the child and the dog looking for a hidden object together, and become an interesting and practical method of monitoring the activ- 5 min of dog-care activities, such as feeding and brushing the dog. ity of working dogs. Indeed, it is difficult to quantify ROS in practice because of their very short half-life and it requires complex tech- Diets niques over a long period of time. Due to their high reactivity, ROS The dogs were fed 2 varieties of dry dog food: Natural Trainer react with practically every organic molecule they meet, producing Adult Medium and Personal Trainer Long Life Adult Medium, which reactive oxygen metabolites (ROMs), which are more stable than the is supplemented with antioxidants (both from Nova Foods, Vicenza, ROS and are therefore easier to quantify. Conversely, BAP matches Italy). The Natural Trainer Adult Medium diet consisted of fresh the total antioxidant capability of plasma and includes either exog- chicken and turkey meat, corn, rice, dehydrated chicken and turkey enous (ascorbate, tocopherols, carotenoids) or endogenous (protein, meat, lard, dehydrated pork meat, flax seed, beet, corn oil, dehy- GPx, SOD, CAT) components that can oppose the oxidant action of drated fish flour, chicory extract, fructooligosaccharides, brewer’s reactive species (16). yeast, pea fiber, Spirulina algae (Arthrospira platensis), ribonucleotides In conditions of oxidative stress, a controlled, balanced antioxidant extract from yeast, green-lipped mussels (Perna canaliculus), apple diet may be a valid approach to restoring good cell metabolism and dried extract, black raspberry extract, salt, chloride-choline, methio- neutralizing excess free radicals. Two studies in dogs and rabbits nine, vitamin E, vitamin A (18 000 UI/kg), vitamin D3 (1.350 UI/kg), demonstrated an increase in tissue oxidative stability by supple- vitamin E (265 mg/kg), and copper (20 mg/kg). menting with a diet enriched with different antioxidants (17,13). The Personal Trainer Long Life Adult Medium dog food consisted The effect on exercise-induced oxidative stress of supplementing of corn, dehydrated chicken and turkey meat, fresh chicken and tur- with oral vitamin E (11 to 14 mg) for 10 wk was studied in 8 dogs key meat, rice, corn oil, lard, hydrolyzed proteins of chicken, beet, and induced a significant reduction in paroxonase-1 activity, eryth- corn gluten, flax seeds, dehydrated fish flour, brewer’s yeast (0.50%), rocyte membrane fluidity, and vitamin E and a significant increase sodium chloride, pea fiber, wood cellulose, calcium carbonate, fruit in plasma malondialdehyde levels (18). After 10 d of vitamin E oligosaccharides, broccoli dried extract, green-lipped mussels (Perna supplementation, these modifications were significant in the placebo canaliculus) (0.05%), Vitaberry Plus (grape seed extract, quercetin, group, but not in the supplemented group. blueberry dried extract, resveratrol, and strawberry and blackberry The objective of this study was to ascertain the ability of a long- dried extracts), and silicon dioxide (0.025%). term antioxidant-supplemented diet to regulate the oxidative stress and general health status of working dogs involved in animal- Experimental design assisted intervention (AAI) programs. Dogs were randomly divided into 2 groups using a dedicated software (Research Randomizer) (20). The first group (n = 5) received Materials and methods a high quality commercial control diet (CD) without antioxidant (Natural Trainer Adult Medium, Nova Foods), while the second Operative procedures and animal care were carried out in compli- group (n = 6) received a high quality commercial diet (SD) sup- ance with national and international regulations (Italian regulation plemented with antioxidants (Personal Trainer Long Life Adult D.L. vo 116/1992 and European Union regulation 86/609/EC). Medium, Nova Foods) for 18 wk. At the end of this period, a cross- The recommendations of the Consolidated Standards of Reporting over design of the same duration was conducted. (CONSORT) 2010 statement on randomized controlled trials were also consulted and considered (19). Sample collection Blood samples were collected from each dog at the beginning of Subjects the evaluation and at the end of the first experimental period after Eleven therapy dogs (6 females and 5 males) of different breeds 18 wk. The same procedure was carried out before and at the end of (1 Cirneco dell’Etna, 6 Labrador retrievers, 3 mixed breed, and 1 pit- the crossover period. Blood samples were collected from the cephalic bull), aged 2.7 6 0.8 y and with a body weight of 22.12 6 8.43 kg vein and stored in 2 tubes, one with heparin and the other without (mean 6 SEM), were enrolled in this clinical evaluation. Before the anticoagulant. Heparinized plasma samples and serum samples were recruitment, laboratory examinations (hemogram, biochemical pro- obtained by blood centrifugation at 6000 3 g for 1.5 min at 37°C.

2000;64:0–00 The Canadian Journal of Veterinary Research 207 Table I. Reference values of dROMs (1 U Carr = 0.08 mg H2O2/dL) and BAP test dROMs reference values BAP reference values Normal values 50 to 90 U CARR Optimal values 2400 to 2200 mmol/L Threshold borderline 92 to 95 U CARR Threshold borderline 2200 to 2000 mmol/L Mild oxidative stress 100 to 120 U CARR Slight deficiency 2000 to 1800 mmol/L Oxidative stress 140 to 200 U CARR Deficiency 1800 to 1600 mmol/L Strong oxidative stress 220 to 300 U CARR Strong deficiency 1600 to 1400 mmol/L Strong oxidative stress Over 300 U CARR Very strong deficiency , 1400 mmol/L dROMs — reactive oxygen metabolites-derivatives; U Carr — Carratelli units; BAP — biological antioxidant potential.

Metabolic profile evaluation Results Ten milliliters of blood were collected from the cephalic vein Both diets were similar in chemical composition and fatty acid for the oxidative stress assessment. After blood centrifugation in profile. The predominant fatty acids of the lipid fraction in both heparinized test tubes, the plasma was separated from the serum diets were palmitic acid (C16:0) and oleic acid (C18:1 c9), which and analyzed. Oxidative stress was measured with a spectro- were present in similar proportions (22% for palmitic acid and 35% photometric point-of-care assay (Free Radical Analytical System for oleic acid). The amount of linolenic acid (C18:3 n3) was higher FRAS 4; Evolvo, Langhirano, Italy). The dROMs test and the BAP (128%) in the experimental than in the control diet. test were completed. In the dROMs test, reactive oxygen metabo- The metabolic profile trend of the CD and SD group during the lites (primarily hydroperoxides) of the sample generated alkoxyl evaluation period is shown in Figure 1. Amylase concentration sig- and peroxyl radicals, in the presence of iron released from plasma nificantly increased from a T0 value of 778 6 32.10 U/L to 1059 6 proteins by an acidic buffer, according to the Fenton reaction. Such 82.75 U/L at T1 in the CD group (P , 0.01) (Figure 1C). Conversely, radicals then oxidized an alkyl-substituted aromatic amine (N,N- creatinine and triglycerides decreased significantly in the SD group dietylparaphenylendiamine), thus producing a pink-colored deriva- from a T0 value of 1.22 6 0.06 mg/dL to 1.00 6 0.06 mg/dL at T1 tive, which is photometrically quantified at 505 nm. The concentra- and from a T0 value of 164.6 6 55.73 mg/dL to 60.20 6 9.51 mg/dL tion of the dROMs is directly proportional to the color intensity and at T1, respectively (P , 0.05) (Figures 1H to P). is expressed as U CARR (Carratelli units). One U CARR corresponds The concentration of dROMs and BAP decreased significantly to 0.08 mg/dL hydrogen peroxide. In normal dogs, dROM values from a T0 value of 142.6 6 13.86 U CARR to 111.4 6 7.97 U CARR at range from 50 to 90 U CARR. T1 in the SD group and from a T0 value of 3332 6 177.5 mMol/L to In the BAP test, the plasma samples were mixed with a colored 2954 6 58.14 mMol/L at T1 in the CD group, respectively (P , 0.05) solution obtained by mixing a ferric chloride solution with a thio- (Figure 2). cyanate derivative solution that causes a discoloration, the intensity At the end of this period, groups were crossed over and fed for of which is measured photometrically at 505 nm and is proportional another 18 wk and the metabolic profile was reassessed (Figure 3). to the ability of the plasma to reduce ferric ion (16). The results are Amylase and GPT concentration significantly decreased from a expressed as mMol/L of reduced ferric ions. In normal dogs, BAP T0 value of 1059 6 82.75 U/L to 859.2 6 61.02 U/L at T1 in the values range from 2400 to 2200 mMol/L. The reference values of CD group and from a T0 value of 70.20 6 10.83 U/L to 50.60 6 dROMs and BAP test were validated for canine species (14,21) and 5.38 U/L at T1 in the SD group, respectively (P , 0.05) (Figures 3C are summarized in Table I. to L). Although no significant variation of dROMs was observed, A metabolic profile was also investigated for each dog, includ- a decrease in the CD group and an increase in the SD group was ing albumin, alkaline phosphatase (ALP), total bilirubin, calcium, observed at the end of the crossover period, probably due to the cholesterol, creatine phosphokinase (CPK), creatinine, phosphorus, antioxidant activity of the diet (Figure 4A). Similar to what was gamma-glutamyl transferase (GGT), glucose, aspartate aminotrans- observed in the first evaluation, a decrease in BAP concentration ferase (GOT), glutamate pyruvate transaminase (GPT), total proteins, was observed in the CD group (Figure 4B). triglycerides, and urea (Dimension RxL Max Integrated Chemistry System; Siemens Healthcare Diagnostics, Milan, Italy). Discussion Statistical analysis The objective of this study was to evaluate the potential ability Data were analyzed using GraphPad Prism 6 software (GraphPad of a long-term antioxidant-supplemented diet to control the oxida- Software, La Jolla, California, USA). All data are presented as the tive stress and general health status of therapy dogs involved in means 6 SEM and were first checked for normality using the AAI programs. Environmental stress, such as temperature, work- D’Agostino-Pearson normality test. Differences in dROMs, BAP, and ing, and dog competitions may induce psychological and physical metabolic profile parameters between the 2 diets before (T0) and stress, which could also influence the food intake of dogs, whether at the end (T1) of the first evaluation and crossover were analyzed it is excessive or not enough. using a 2-way analysis of variance (ANOVA), followed by Sidak’s An unbalanced diet lacking in essential nutrients weighs on the multiple comparisons test. general health status of dogs and may represent a risk factor for

208 The Canadian Journal of Veterinary Research 2000;64:0–00 Figure 1. The metabolic profile trend of the commercial diet (CD) and supplemental diet (SD) groups during the evaluation period (continues on next page).

2000;64:0–00 The Canadian Journal of Veterinary Research 209 Figure 1. The metabolic profile trend of the commercial diet (CD) and supplemental diet (SD) groups during the evaluation period (continues on next page).

210 The Canadian Journal of Veterinary Research 2000;64:0–00 Figure 1. The metabolic profile trend of the commercial diet (CD) and supplemental diet (SD) groups during the evaluation period.

thus indicating a positive trend towards the normal values observed for healthy dogs (14). Conversely, the significant reduction observed in the CD-fed group confirmed the lack of any antioxidant effect related to the diet. It is worth noting that, while a long period of washout was not observed, the overall trend of BAP and dROMs improved after SD supplementation, although slightly influenced by the previous diet assumption. On the other hand, the reduction of the amylase enzyme after the crossover could be related to the inhibitory activity of antioxidant molecules on key enzymes linked to type-2 diabetes (a-amylase and a-glycosidase) as previously observed in in-vitro studies by using flavonoid, anthocyanin, and other polyphenol compounds (22–25). The decrease in GPT level after the crossover suggests that the SD diet is endowed with liver-protective properties as reported in studies where different polyphenol extracts were tested in laboratory animals (26,27). As a result, the beneficial effects of antioxidant supplementation on liver and pancreatic metabolism were demonstrated. The antioxidant formulation used in this study was based on grape seed extract, quercetin, blueberry, resveratrol, and strawberry and blackberry dried extracts (Vitaberry Plus). All these compounds contain anthocyanins and polyphenols, which have antioxidant effects. The total proanthocyanidin content of grape seed extract also included catechins and epicatechins and possesses antioxidant properties and protects the vascular system (28). The present study clearly showed a decrease in dROM species after the administration of a specific diet enriched with antioxidants. This scavenger activity Figure 2. Graphical representation of oxidative stress parameters concen- was in agreement with other studies about the antioxidant effects tration of dogs. A significant decrease in dROMs and BAP concentration of single active ingredients included in antioxidant supplements. was observed after 18 weeks of supplementation with SD and CD diet, respectively (*P , 0.05). A — dROMs and B — BAP values before (T0) For example, it has been demonstrated that supplementing with and after 18 weeks (T1) of diet supplementation. grape seed extract decreased the activity of the oxidative stress- responsive transcription factors NF-kB and Nrf2 in the duodenal mucosa of pigs (29). metabolic and degenerative diseases. Among the mechanisms that In a similar study, grape seed extract significantly reduced can influence health status, the balance of oxidative stress plays a (by 60%) the formation of azoxymethane-induced aberrant crypt relevant role. Metabolic oxidative reactions constantly take place foci, probably due to its activity in reducing the levels of cyclin D1, in animals in order to balance the production of free radicals with COX-2, and iNOS involved in the foci genesis (30). While grape antioxidant molecules. seed and blueberry extract are endowed with antioxidant activity, The significant reduction (P , 0.05) of dROMs and BAP values quercetin supplementation is known to prevent age-related dis- observed in dogs fed the SD diet during the first experimental period eases in dogs (31). Moreover, the anthocyanins and polyphenols in clearly demonstrated the ability of the diet to reduce oxidative stress, blueberry extract have been shown to exert antioxidant effects in

2000;64:0–00 The Canadian Journal of Veterinary Research 211 Figure 3. Graphical representation of metabolic profile of dogs. A significant decrease in GPT concentration was observed after SD diet supplementa- tion (3L, *P , 0.05). (A) albumin, (B) alkaline phosphatase, (E) calcium, (G) creatine phosphokinase, (I) Gamma-glutamyl transpeptidase, (J) glucose, (K) Glutamic-oxaloacetic transaminase, (L) Glutamic — pyruvic transaminase, (O) total protein (continues on next page).

212 The Canadian Journal of Veterinary Research 2000;64:0–00 Figure 3. Graphical representation of metabolic profile of dogs. A significant decrease in GPT concentration was observed after SD diet supplementa- tion (3L, *P , 0.05). (A) albumin, (B) alkaline phosphatase, (E) calcium, (G) creatine phosphokinase, (I) Gamma-glutamyl transpeptidase, (J) glucose, (K) Glutamic-oxaloacetic transaminase, (L) Glutamic — pyruvic transaminase, (O) total protein (continues on next page).

2000;64:0–00 The Canadian Journal of Veterinary Research 213 Figure 3. Graphical representation of metabolic profile of dogs. A significant decrease in GPT concentration was observed after SD diet supplementa- tion (3L, *P , 0.05). (A) albumin, (B) alkaline phosphatase, (E) calcium, (G) creatine phosphokinase, (I) Gamma-glutamyl transpeptidase, (J) glucose, (K) Glutamic-oxaloacetic transaminase, (L) Glutamic — pyruvic transaminase, (O) total protein.

blueberry extract has also demonstrated genoprotective and behav- ioral effects (35). Specifically, 30 mice received a supplementation of blueberry extract for 30 d. After that period, all treated animals had a higher significant memory retention in all test sessions after 7 and 30 d of supplementation, while in-vitro examination revealed significantly reduced DNA damage in hippocampal tissues than in the control group. Resveratrol (3,5,49-trihydroxystilbene), a polyphenol naturally occurring in grapes, berries, peanuts, and other vegetables, has also been shown to have antioxidant, anti-inflammatory, and anti-­ proliferative properties (36,37). Although there is a lack of scientific studies on resveratrol supplementation in pets, it has been demon- strated that resveratrol was able to protect from oxidative stress in hepatic steatosis conditions, significantly increasing GSH and GPx levels in liver tissues and significantly decreasing ROS levels in 30 mice (38). Antioxidant and anti-inflammatory activity have been ascribed to strawberry extract due to its high content of flavonoids (39). For example, a daily intake of 500 g of strawberries for 9 d increased non- urate plasma 2,2-diphenyl-1-picrylhydrazyl radical decomposition, thus possibly decreasing the risk of systemic oxidants over activity (excessive activity) (40). Antioxidant and anti-inflammatory activities were also investi- gated for a polyphenolic blackberry extract by means of the oxygen radical absorbance capacity assay (ORAC) (41). The ORAC value of the blackberry extract was higher than the ORAC of quercetin and ellagic acid. Moreover, the blackberry extract inhibited superoxide Figure 4. Graphical representation of oxidative stress parameters con- production by NADPH oxidase (nicotinamide adenine dinucleo- centration of dogs. A — dROMs values and B — BAP before (T0) and tide phosphate-oxidase) in THP-1 cells and nitrite production in after 18 weeks (T1) of diets supplementation. J774A.1 cells after 4 h of incubation, showing a scavenger activity, while a reduction of nitrites was observed after 24 h of incubation. A dog’s diet may be qualitatively and quantitatively appropriate some pathological conditions, such as hypobaric hypoxia, due to to the type and intensity of the activity as well as to its age, breed, the inhibition of lipid peroxidation by interfering with glutathione and gender. Balanced nutrition could therefore be crucial to restor- activity (32,33). The antioxidant levels of sled dogs supplemented ing good overall health status. This study confirms that long-term with blueberries were studied and blood creatine kinase levels and supplementation with a specific diet enriched with antioxidants oxidative status were evaluated 48 h after exercise (34). Although the is able to regulate the antioxidant status and general health status exercise protocol did not influence concentrations of creatine kinase, of working dogs involved in animal-assisted intervention (AAI) supplementation with blueberries significantly increased the total programs and prevent damage due to oxidative stress before and antioxidant status after exercise. In addition to being an antioxidant, after their work.

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216 The Canadian Journal of Veterinary Research 2000;64:0–00 Article

Evaluation of transmission infrared spectroscopy and digital and optical refractometers to identify low immunoglobulin G concentrations in alpaca serum Ibrahim Elsohaby, Jennifer J. Burns, Christopher B. Riley, J. Trenton McClure

Abstract This study aimed to evaluate the digital Brix and optical serum total protein (STP) refractometers for measuring concentrations of serum immunoglobulin G (IgG) in alpacas and compare them to IgG concentrations measured by the reference method of radial immunodiffusion (RID) assay. The appropriate cutoff point for Brix and STP refractometers and the transmission infrared (TIR) spectroscopy method was determined for low IgG concentrations (, 10 g/L). Serum samples were collected from alpacas (N = 169) and tested by both refractometers. The correlation between Brix % and STP was high [correlation coefficient (r) = 0.99]. However, the correlation coefficients between Brix % and STP with serum RID-IgG concentrations were only 0.56 and 0.55, respectively. Twenty-one (12.4%) of 169 alpaca serum samples had IgG concentrations of , 10 g/L. Using receiver operator characteristic curve (ROC) analysis, the optimal cutoff points for the TIR assay, digital Brix, and optical STP refractometers for assessing low IgG (RID , 10 g/L) were 13 g/L, 8.8%, and 50 g/L, respectively. The TIR assay showed higher sensitivity (Se = 95.2%) and specificity (Sp = 96.8%) than either the digital Brix (Se = 90.5% and Sp = 65.5%) or optical STP (Se = 81% and Sp = 73.7%) refractometers for assessing alpacas with low IgG. In conclusion, the Brix and STP refractometers lack accuracy in measuring alpaca IgG concentrations, but may be useful for screening animals for low serum IgG. However, the TIR assay with a cutoff point of 13 g/L was more appropriate for identifying low IgG than either refractometer. Another study that focuses on neonatal crias is recommended in order to evaluate the usefulness of these assays for field diagnosing of failure of transfer of passive immunity (FTPI).

Résumé Cette étude visait à évaluer un réfractomètre Brix digital et un réfractomètre optique pour protéines sériques totales (PST) pour mesurer les concentrations sériques d’immunoglobulines G (IgG) chez les alpagas et de les comparer aux concentrations d’IgG mesurées par la méthode de référence d’immunodiffusion radiale (IDR). La valeur seuil appropriée pour les réfractomètres Brix et PST ainsi que pour la méthode de spectroscopie infrarouge à transmission (SIT) fut déterminée pour des faibles concentrations d’IgG (, 10 g/L). Des échantillons de sérum ont été prélevés d’alpagas (N = 169) et testés avec les deux réfractomètres. La corrélation entre le % Brix et PST était élevée [coefficient de corrélation (r) = 0,99]. Toutefois, les coefficients de corrélation entre % Brix et le PST et les concentrations d’IgG déterminées par IDR étaient seulement de 0,56 et 0,55, respectivement. Vingt-et-un (12,4 %) des 169 échantillons de sérum d’alpaga avaient des concentrations d’IgG , 10 g/L. En utilisant l’analyse de la courbe de la fonction d’efficacité du récepteur, les valeurs seuils optimales pour l’épreuve SIT, et les réfractomètres Brix digital, et PST optique pour évaluer un faible niveau d’IgG (IDR , 10 g/L) étaient de 13 g/L, 8,8 % et 50 g/L, respectivement. L’épreuve SIT a montré une sensibilité (Se = 95,2 %) et une spécificité (Sp = 96,8 %) plus élevée que le réfractomètre Brix digital (Se = 90,5 % et Sp = 65,5 %) ou le réfractomètre PST optique (Se = 81 % et Sp = 73,7) pour évaluer les alpagas avec de faibles concentrations d’IgG. En conclusion, les réfractomètres Brix et PST manquent de précision pour mesurer les concentrations d’IgG d’alpagas, mais peuvent être utiles pour vérifier les animaux pour des faibles concentrations d’IgG sériques. Toutefois, l’épreuve SIT avec une valeur seul de 13 g/L était plus appropriée que les deux réfractomètres pour identifier des valeurs faibles d’IgG. Une autre étude qui s’attardait aux crias nouveau-nés est recommandée afin d’évaluer l’utilité de ces épreuves pour le diagnostic des déficiences de transfert d’immunité passive. (Traduit par Docteur Serge Messier)

Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island C1A 4P3 (Elsohaby, Burns, Riley, McClure); Infectious Diseases, Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig City 44511, Sharkia Province, Egypt (Elsohaby); Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North 4442, New Zealand (Riley). Address all correspondence to Dr. Ibrahim Elsohaby; telephone: (902) 566-6063; fax: (902) 566-0823; e-mail: [email protected] Received November 24, 2016. Accepted January 11, 2017.

2017;81:217–222 The Canadian Journal of Veterinary Research 217 Introduction Table I. Descriptive statistics of immunoglobulin G (IgG) concentrations, Brix percentage (%) and serum total protein Camelids are born hypogammaglobulinemic as the diffuse epi- (STP) measurements in 169 alpaca serum samples theliochorial structure of their placentation does not allow immuno- Parameters Method Median Minimum Maximum globulins to pass from dam to fetus (1). Newborn camelids therefore IgG (g/L) RID 24.8 3.9 52.9 rely on the transfer of maternal immunoglobulins through intake IgG (g/L) TIR 26.7 22.3 51.8 of the dam’s colostrum in order to acquire passive immunity (2). Brix (%) Digital 9 7.2 12.5 Failure of transfer of passive immunity (FTPI) occurs when newborn STP (g/L) Optical 54 38 84 camelids fail to ingest or absorb a sufficient amount (, 10 g/L) of RID — radial immunodiffusion assay; TIR — transmission infrared colostral IgG within 48 h of birth (1,3,4). There is a recognized asso- spectroscopy; Digital — Brix refractometry; Optical — serum total ciation between FTPI and increased infectious disease, including protein (STP) refractometry. septicemia, diarrhea, pneumonia, and septic arthritis, in neonatal camelids (5–7). Early diagnosis of FTPI is therefore a vitally impor- tant part of neonatal camelid husbandry and is critical in decreasing 6 farms in Ontario and New Brunswick, Canada (n = 82) and 3 farms neonatal morbidity and mortality rates (8). in the Adelaide Hills region of South Australia (n = 93). All alpacas Several assays are available to measure IgG concentrations directly appeared clinically healthy and were not visibly dehydrated. Age or indirectly and to assist in assessing conditions characterized and gender data were only available for 129 of the 175 alpacas. Of by low IgG, such as FTPI in camelids (1,9). Direct methods of these 129 alpacas, 81 were female and 48 were male. Four of these assessment include the radial immunodiffusion (RID) assay (10), alpacas were , 2 mo old, 5 were 2 to 3 mo old, 15 were 3 to 6 mo immunoturbidimetric­ (IT) assay (11), and transmission infrared old, 12 were 6 mo to 1 y old, and 93 were . 1 y old. (TIR) spectroscopy (12). Indirect methods include zinc sulfate In Canada, blood samples were collected from alpacas by jugular turbidity (13), glutaraldehyde coagulation assay, serum gamma- venipuncture into a sterile, plastic vacutainer tube without anti­ glutamyl transferase (GGT) activity (14), and total serum protein coagulant, then centrifuged at 1500 3 g for 10 min at room tempera- and globulin concentration measurements (4,13). The RID assay is ture. Serum was collected into a cryovial and frozen at −20°C until the historical gold standard method for direct quantification of IgG transport to the Atlantic Veterinary College (AVC). Blood samples level in camelids (15). Radial immunodiffusion (RID) has significant collected from alpacas in Australia were taken for hematologic and disadvantages, however, including the time it takes to obtain results biochemical analysis as part of a separate study. The remaining (18 to 24 h), higher costs than other methods, and a lack of agree- serum was stored at −80°C before being shipped frozen to AVC, ment about which commercially available RID kits should be used UPEI, Canada where it was stored at −80°C for later batch analysis. to measure IgG concentration in camelids (10,11). While infrared spectroscopy has recently been used to measure IgG direct measurement methods alpaca serum IgG (12), refractometry is the most common indirect Results of serum RID and TIR IgG (g/L) measurement for the method for estimating serum IgG and assessing FTPI on-farm (13). same samples from a related previous study were used (12). In the Previous studies have evaluated optical serum total protein (STP) earlier study, a commercial alpaca RID IgG assay (Triple J Farms, refractometry for assessing FTPI in neonatal camelids (13,16). To Bellingham, Washington, USA) and transmission infrared (TIR) the authors’ knowledge, however, no study has been published spectroscopy (Tensor 37; Bruker Optics, Coventry, UK) were used. that describes the use of digital Brix refractometry for measuring Radial immunodiffusion (RID) IgG assay was used as a reference serum IgG concentrations and assessing IgG of , 10 g/L, which is method to determine each alpaca’s serum IgG concentration. Each the cutoff point for FTPI in alpacas. serum sample and assay standard was tested in replicates of 5 to The objectives of this study were to i) evaluate the digital Brix and generate an average IgG concentration value. For TIR spectroscopy, optical STP refractometers for measuring serum IgG concentrations each sample was tested in replicates of 6 as described in a previous in alpacas and compare values with those measured by the reference study (12). Sample spectra were collected over the wavenumber method of radial immunodiffusion (RID) assay; and ii) determine range of 4000 to 400 cm21, with a nominal resolution of 4 cm21, with the optimal cutoff points for assessing low IgG concentrations 512 scans collected for data acquisition. Data for the measured TIR (, 10 g/L) in alpacas using digital Brix and optical STP refractom- IgG values were used to calculate an optimal cutoff point to identify eters and the transmisssion infrared (TIR) spectroscopy method. low IgG (, 10 g/L) for this study. IgG indirect measurement methods Materials and methods Only 169 of the original 175 serum samples were available for testing by refractometers, as there was not enough volume in the Study population and sampling remaining 6 samples to allow for further testing. Samples were vor- Banked sera from a previous study stored at −80°C were used texed for 10 s and serum Brix (%) and STP (g/L) levels were deter- for this study (12). The study was approved by the University of mined using a digital Brix refractometer (PAL-1; Atago, Bellevue, Prince Edward Island (UPEI) Animal Care Committee and the Washington, USA) and an optical handheld refractometer (Westover Animal Ethics Committee of the University of Adelaide. Alpacas RHC 200ATC handheld refractometer; Woodinville, Washington, (N = 175) enrolled in this study between 2009 and 2011 were from USA), respectively. The same person conducted both refractometer

218 The Canadian Journal of Veterinary Research 2000;64:0–00 r = 0.56 r = 0.56 12 80

70 10

60 Brix (%) 8 50 Serum total protein (g/L) Serum total protein

40 6 0 10 20 30 40 50 0 10 20 30 40 50 IgG concentration from RID assay (g/L) IgG concentration from RID assay (g/L)

Figure 1. Correlation plot between Brix percentage (%) determined by Figure 2. Correlation plot between serum total protein (STP) determined digital Brix refractometry and immunoglobulin G (IgG) determined by by optical handheld refractometry and immunoglobulin G (IgG) deter- radial immunodiffusion (RID) assay for 169 alpaca serum samples. mined by radial immunodiffusion (RID) assay for 169 alpaca serum samples. measurements at room temperature. However, the optical refrac- from TIR were normally distributed (P = 0.321). Descriptive statistics tometry to determine STP was conducted first to avoid observer (median, minimum, and maximum) of IgG concentrations in alpaca bias. Both refractometers were cleaned and calibrated with distilled serum obtained by Brix and STP measurement are shown in Table I. water before each reading. The median IgG concentration measured by RID assay (24.8 g/L) in the present study was marginally higher than that previously Statistical analysis published, which ranged from 17.2 g/L to 23.4 g/L (3,4,11,14). The Statistical analyses were carried out using statistical software range of IgG concentrations in these studies was more likely to be (Stata Version 14.0; Stata Corporation, College Station, Texas, USA higher, however, because they were not limited to healthy alpacas. and MedCalc for Windows Version 15.8.0; MedCalc Software, The RID and TIR assays were conducted at an earlier time than the Mariakerke, Belgium). A value of P , 0.05 was considered statisti- refractometers, with the serum stored in a freezer at 280°C between cally significant. The data were evaluated for normality by applying testing. Degradation of IgG between testing periods is unlikely as the Shapiro-Wilk test. Descriptive statistics for IgG measurements it has been previously reported that freezing at this temperature were calculated by RID, TIR spectroscopy, digital Brix, and optical over a period of years has no significant effect on IgG concentra- STP refractometers. Spearman rank coefficient of correlation was tion (17,18). Only 21 of the 169 alpacas had an IgG concentration of calculated to determine the relationship among measurements by , 10 g/L, the most common cutoff point used to determine FTPI (1), RID, Brix, and STP refractometers. which resulted in a true prevalence of 12.4%. This value falls within Alpacas with serum RID IgG concentrations of , 10 g/L were the range of FTPI prevalence (9% to 20.5%) previously reported in considered to have low IgG, which is consistent with the definition the literature (4,15). The differences in the prevalence of low IgG of FTPI in camelid neonates (1,3,4). A receiver operating charac- concentrations (, 10 g/L) in the present and previous studies may teristic (ROC) analysis was conducted on data obtained with TIR be related to the age of alpacas enrolled in these earlier studies, as spectroscopy and Brix and STP refractometers to determine the well as to management practices on the farms, including biosecurity optimal cutoff point based on the Youden index. Sensitivity (Se), measures, nutrition, vaccination programs, geographic location, and specificity (Sp), accuracy, Youden index (J), and area under the climate (19). Since alpacas in this study population were of various curve (AUC) were used to compare the diagnostic performance of ages and included crias and adults, we are limited in assessing the each method. Accuracy, which was defined as the percentage of clinical usefulness of these assays for diagnosing FTPI. A future correctly classified samples, was calculated as follows: [Se 3 p] 1 study focusing on neonatal crias is therefore recommended in order [(Sp) 3 (1 2 p)], where p represents the prevalence of samples with to better evaluate the usefulness of TIR spectroscopy (12), Brix, and low IgG (, 10 g/L) observed in the studied population. Finally, STP refractometers for diagnosing FTPI. kappa statistic was used to assess the level of agreement among the results of TIR spectroscopy, Brix, and STP refractometers versus the Correlations reference RID assay, as well as between results of digital Brix and The Spearman rank correlation coefficient between IgG levels optical STP refractometers. was 0.56, as estimated by digital Brix refractometry and RID assay (Figure 1). The STP-based serum IgG measurements were correlated Results and discussion with those assessed by RID assay (r = 0.55; Figure 2). These findings While the IgG values from RID, Brix %, and STP assays were not were lower than correlations reported between immunoturbidimet- normally distributed (P , 0.001, respectively), the values for IgG ric (IT) (11) or infrared spectroscopy methods (12) and RID-based

2000;64:0–00 The Canadian Journal of Veterinary Research 219 Table II. Test characteristics for detecting low serum IgG r = 0.99 80 (, 10 g/L) in 169 alpaca serum samples by transmission infrared (TIR) spectroscopy and Brix and optical serum total 70 protein (STP) refractometers as calculated using the radial immunodiffusion (RID) assay as the reference

60 Refractometers Test characteristics TIR Brix STP Cutoff point # 13 g/L # 8.8% # 50 g/L 50 Sensitivity (Se) 95.2 90.5 81

Serum total protein (g/L) Serum total protein Specificity (Sp) 96.8 65.5 73.7 40 Accuracy 96.6 68.5 74.6 Youden index (J) 0.92 0.56 0.55 7 8 9 10 11 12 13 Area under the curve (AUC) 0.99 0.81 0.81 Brix (%) Kappa 0.82 0.30 0.33 Figure 3. Correlation plot between serum total protein (STP) determined Cutoff point — optimal criteria determined by receiver operating by optical handheld refractometry and Brix % determined by digital Brix characteristic curves (ROCs). refractometry for 169 alpaca serum samples.

100 (95.2%; 95% CI: 90.9 to 98.2) and Sp (96.8%; 95% CI: 92.3 to 98.9) for the TIR spectroscopic diagnosis of low IgG was achieved at # 13 g/L Se: 95.2 (Figure 4). The calculated Se and Sp for the digital Brix refractometer 80 Sp: 96.8 using # 8.8% as the optimal cutoff point were 90.5% (95% CI: 69.6 to Criterion: # 13 98.8) and 65.5% (95% CI: 57.3 to 73.2), respectively (Figure 5). This compares to the best combination of Se (81%; 95% CI: 58.1 to 94.6) 60 and Sp (73.7%; 95% CI: 65.8 to 80.5) for the optical STP refractometer at a cutoff point of # 50 g/L (Figure 6). The optimal Se and Sp values for the TIR spectroscopic approach were higher than those of the Brix and STP refractometers. Also,

Sensitivity 40 the Se (95.2%) of TIR spectroscopy at a cutoff point of # 13 g/L was higher than the Se (81%) from the original study when a cutoff point of 10 g/L was used for the same samples (12). The objectives of the 20 original study (12), however, were to develop an algorithm that best predicted serum IgG concentration values over the TIR spectrum AUC: 0.99 and looked at how well the TIR spectroscopic method performed 0 against the RID using the same cutoff value of , 10 g/L for each 0 20 40 60 80 100 assay. In this study, we used ROC analysis to determine the TIR IgG 100-Specificity value that best correlates with serum IgG of , 10 g/L as determined by the reference RID assay. The Se and Sp of the TIR spectroscopy Figure 4. Receiver operating characteristic (ROC) curve, the best combina- tion of sensitivity and specificity for transmission infrared (TIR) spectros- using a cutoff IgG of # 13 g/L value was similar or higher when copy obtained at a cutoff point of # 13 g/L in 169 alpaca serum samples. compared with the reported Se and Sp for other available methods to assess low serum IgG in alpacas (11,13,25). The digital Brix refrac- measurements of IgG concentrations. Furthermore, the correlations tometer showed higher Se and slightly lower Sp than the optical STP between the digital Brix or the optical STP refractometers and RID refractometer at a cutoff point of # 8.8%, which indicates that digital assay were lower than those reported for bovine (20–22) and lamb Brix may be preferred as a screening test compared to optical STP sera (23). There was a high correlation between digital Brix and opti- refractometer. The Se and Sp of optical STP refractometer at a cutoff cal STP refractometers values (r = 0.99; Figure 3), which indicates that point of # 50 g/L were close to the Se (71%) and Sp (80%) previ- there was no advantage of one method over the other. Although a ously reported with the same cutoff point (13). When a cutoff point similarly high level of correlation between Brix and STP refractom- of 51.5 g/L was used, however, a higher Se (87.5%) and Sp (87.9%) eters has previously been reported (24), correlation coefficients from for assessment of low serum IgG were reported (25). The difference 0.73 to 0.91 have been reported for other species (20–22). in the optimal cutoff points between studies may be due to varia- tions between instruments or differences in animal populations (22). Test characteristics for detection of low IgG Test characteristics of the TIR spectroscopy, digital Brix, and Agreement among tests optical STP refractometers for assessing alpacas with low IgG con- The agreement among results of TIR spectroscopy and Brix and centration (, 10 g/L) were calculated using optimal cutoff points STP refractometers for assessing alpaca serum with low IgG concen- determined by ROCs (Table II). The optimal combination of Se trations (, 10 g/L) compared to the reference RID assay is presented

220 The Canadian Journal of Veterinary Research 2000;64:0–00 100 100

Se: 90.5 80 Sp: 65.5 80 Criterion: # 8.8 Se: 81 Sp: 73.7 Criterion: # 50 60 60

Sensitivity 40 Sensitivity 40

20 20

AUC: 0.81 AUC: 0.81 0 0 0 20 40 60 80 100 0 20 40 60 80 100 100-Specificity 100-Specificity

Figure 5. Receiver operating characteristic (ROC) curve, the best combi- Figure 6. Receiver operating characteristic (ROC) curve, the best combi- nation of sensitivity and specificity for digital Brix refractometry obtained nation of sensitivity and specificity for optical serum total protein (STP) at a cutoff point of # 8.8% in 169 alpaca serum samples. refractometery obtained at a cutoff point of # 50 g/L in 169 alpaca serum samples.

in Table II. The overall percentages of agreement among results and Dr. Nicole Jewett (New Brunswick, Canada) for providing the of TIR spectroscopy, Brix, and STP refractometers to the reference samples used in this study. This study was partially supported by RID assay, i.e., accuracy, with a corresponding kappa value are also the Atlantic Canada Opportunities Agency. listed in Table II. The level of agreement between the results of the 2 refractometers was 85.2%, however, with a kappa value of 0.66. References This finding shows that TIR spectroscopy had a substantial agree- ment with the RID assay that was better than that observed for both 1. Wernery U. Camelid immunoglobulins and their importance refractometers. The analytic advantage of TIR is that the serum IgG for the new-born — A review. J Vet Med B Infect Dis Vet Public content creates a unique molecular absorption (fingerprint) in the Health 2001;48:561–568. infrared spectrum, which can be used for qualitative and quantitative 2. Walker P, Tibary A. Neonatal care of camelids: A review and case measurement (26). However, refractometers measure STP or Brix %, reports. J Camel Pract Res 1999;6:255–263. which indirectly related to serum IgG concentration as a portion of 3. Bravo P, Garnica J, Fowler M. Immunoglobulin G concentrations total solids (13). The level of agreement found between the 2 refrac- in periparturient llamas, alpacas and their crias. Small Rumin tometers (85.2%) was similar to the level of agreement (85%) reported Res 1997;26:145–149. between 2 refractometers for assessing FTPI in dairy calves (20). 4. Weaver DM, Tyler JW, Scott MA, Wallace LM, Marion RS, In conclusion, this study found that the TIR spectroscopic method Holle JM. Passive transfer of colostral immunoglobulin G in using the calculated optimal cutoff point of 13 g/L provided bet- neonatal llamas and alpacas. Am J Vet Res 2000;61:738–741. ter sensitivity, specificity, and accuracy for diagnosing low IgG 5. Adams R, Garry F. Llama neonatology. Vet Clin North Am Food (, 10 g/L) than digital Brix or optical STP refractometers at their Anim Pract 1994;10:209–227. optimal cutoff points of 8.8% and 50 g/L, respectively. The digital 6. Dolente BA, Lindborg S, Palmer JE, Wilkins PA. Culture- Brix and optical STP refractometers were poor predictors of serum positive sepsis in neonatal camelids: 21 cases. J Vet Intern Med IgG concentrations, and although they may be useful for screening 2007;21:519–525. animals for low IgG (, 10 g/L), further confirmation testing would 7. Whitehead CE. Management of neonatal llamas and alpacas. Vet be required due to the limited specificity of the assays. Another study Clin North Am Food Anim Pract 2009;25:353–366. focusing on neonatal crias is recommended to further evaluate the 8. Fowler ME, Bravo PW. Neonatology. In: Fowler ME, ed. Medicine specific usefulness of TIR spectroscopy for diagnosing FTPI. and Surgery of Camelids, 2nd ed. Ames: Iowa State University Press, 1998:507–524. Acknowledgments 9. Berne BH. Differing methodology and equations used in quan- titating immunoglobulins by radial immunodiffusion — A The authors thank Cynthia Mitchell for technical assistance, comparative evaluation of reported and commercial techniques. Professor Michael Reichel (University of Adelaide, Australia), Clin Chem 1974;20:61–69.

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222 The Canadian Journal of Veterinary Research 2000;64:0–00 Article

Cystic duct pressures after ligation with a novel absorbable device in an ex vivo caprine cholecystectomy model Andrea J. Sundholm Tepper, Odd V. Höglund, Bonnie G. Campbell, Chi-Ya Chen, Boel A. Fransson

Abstract Laparoscopic cholecystectomy is the standard of care in human medicine for gall bladder disease. Although infrequently reported in veterinary literature, laparoscopic cholecystectomy is an option for uncomplicated gall bladder disease in canine patients. Due to the risk of cystic duct ligature slippage or clip dislodgement, we wanted to explore the use of a LigaTie; a novel absorbable medical device modeled after a cable tie. Our object was to describe the use of the LigaTie in a caprine cadaveric study of cholecystectomies as a model for canine patients and demonstrate the leak pressure of the cystic duct compared with cholecystectomies performed with 2 large endoscopic hemoclips. Samples of caprine gall bladder, liver, and cystic duct were collected. The cystic duct was ligated with either 2 large endoscopic hemoclips or a LigaTie. Maximum cystic duct pressure was recorded. Results showed that there was no statistically significant difference in the maximum cystic duct pressure achieved for cystic ducts ligated with 2 large endoscopic hemoclips or the LigaTie (P = 0.865). No leakage was observed from the cystic duct, hemoclip, or LigaTie site in either group. Supraphysiologic pressures were achieved in both groups and high pressure occlusion of the infusion pump determined the maximum intraluminal pressure achieved. Based on these results, the LigaTie may provide advantages in minimally invasive surgery, especially when considering ligation of a friable or thickened cystic duct during laparoscopic cholecystectomy. Future in vivo studies are warranted to determine minimally invasive maneuverability, tissue interaction, complications, and outcomes.

Résumé La cholécystectomie par laparoscopie est le standard de soin en médecine humaine pour les maladies de la vésicule biliaire. Bien que rapportée peu fréquemment dans la littérature vétérinaire, la cholécystectomie par laparoscopie est une option pour les maladies non-compliquées de la vésicule biliaire chez les patients canins. Due au risque de glissement de la ligature du canal cholédoque ou au déplacement de l’agrafe, nous avons voulu explorer l’utilisation de LigaTie; un nouveau dispositif médical résorbable modélisé d’après une attache de câble. Notre objectif était de décrire l’utilisation de LigaTie dans une étude sur des cadavres de chèvres de cholécystectomies comme modèle pour des patients canins et de démontrer la pression de fuite du canal cholédoque comparativement à des cholécystectomies réalisées avec deux larges agrafes hémostatiques endoscopiques. Des échantillons de vésicules biliaires, de foies et de canal cholédoques caprins ont été prélevés. Le canal cholédoque était ligaturé avec soit deux larges agrafes hémostatiques endoscopiques ou du LigaTie. La pression maximale dans le canal cholédoque fut enregistrée. Les résultats ont montré qu’il n’y avait pas de différence statistiquement significative dans la pression maximale atteinte dans le canal cholédoque par les deux méthodes de ligature (P = 0,865). Aucune fuite ne fut observée des sites de ligature du canal cholédoque que ce soit du groupe avec agrafes ou celui avec LigaTie. Des pressions supra-physiologiques ont été atteintes dans les deux groupes et l’occlusion de la pompe à infusion due à la haute pression a déterminé la pression intraluminale maximale atteinte. En fonction de ces résultats, le LigaTie est avantageux comme méthode chirurgicale minimalement invasive, surtout si l’on considère la ligature d’un canal cholédoque friable ou épaissi durant une cholécystectomie par laparoscopie. Des études in vivo ultérieures sont nécessaires afin de déterminer la manœuvrabilité invasive minimale, l’interaction tissulaire, les complications et les résultats. (Traduit par Docteur Serge Messier)

Department of Veterinary Clinical Sciences (Sundholm Tepper, Campbell, Chen, Fransson), College of Veterinary Medicine, Washington State University, Pullman, Washington, USA; Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden (Höglund). Address all correspondence to Dr. Boel A. Fransson; telephone: (509) 335-5669; e-mail: [email protected] Dr. Fransson’s current address is Washington State University, College of Veterinary Medicine, 205 Ott Road, Pullman, Washington, 99164, USA. Dr. Chen’s current address is the Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Atlantic Veterinary College, 550 University Avenue, Charlottetown, Prince Edward Island, C1A 4P3, Canada. Disclaimer. The LigaTies were provided by Dr. Odd Höglund, founder and partner of Resorbable Devices AB, which is the legal manufacturer of the device. The devices were donated for research purposes. Received December 1, 2016. Accepted February 6, 2017.

2017;81:223–227 The Canadian Journal of Veterinary Research 223 Introduction strength of the LigaTie was retained during healing time of the blood vessels. In contrast, non-absorbable cable ties, previously The laparoscopic revolution started in the late 1980s and early described in veterinary medicine for ovariohysterectomy, have 1990s, with minimally invasive surgery (MIS) for cholecystectomy resulted in adverse complications such as fistulous draining tracts, and adrenalectomy in humans (1). By the early 2000s, cholecys- retroperitoneal abscesses, and granuloma formation (18–20); their tectomy via laparoscopic approach had become the procedure of use in surgery is generally considered not appropriate. choice, with more than 75% of cholecystectomies in North America Due to the movement towards minimally invasive surgery and being performed laparoscopically (2), a number rising to exceed 90% need for a secure and reliable device for cystic duct ligation, we within the last decade (3). The procedure is typically conducted on wanted to explore the use of the LigaTie. The objective of this study an outpatient basis with conversion to open surgery occurring in was to describe the use of the LigaTie in an ex vivo caprine cadav- only 5% to 10% of cases (2). Advantages of laparoscopic procedures eric cholecystectomy model and demonstrate the leak pressure of are well-documented in human medicine and it is known that lapa- the cystic duct compared with cholecystectomies performed with roscopic cholecystectomy results in decreased post-operative pain, 2 large endoscopic hemoclips. Our null hypothesis was that the more rapid return to normal activity, and improved cosmesis (4). leak pressure of the cystic duct occluded with a LigaTie and 2 large Likewise, studies in small animal patients have shown reduced pain endoscopic hemoclips in an ex vivo cadaveric model would not be (5,6) and more rapid return to normal activity (7,8). Laparoscopic different. cholecystectomy in canine patients is infrequently reported in vet- erinary literature but is gaining popularity as a minimally invasive Materials and methods option. Mayhew et al (9) described laparoscopic cholecystectomy for uncomplicated mucoceles in 6 dogs. All dogs had successful lapa- This study was approved by the Washington State Institutional roscopic cholecystectomy without conversion to an open approach Animal Care and Use Committee. Twelve goats were used in the and clinical signs improved or resolved in all cases. More recently, study. A caprine model was chosen due to anatomical similarities short-term outcome of 16 dogs with laparoscopic cholecystectomy to dogs, and the limited availability of fresh canine cadavers. There was determined to be good, with 6/16 (38%) being converted due to were 4 males, 2 females, and 6 wethers. Breeds represented were necrotic or ruptured gall bladder (10). In addition, possible compli- Lamancha (n = 3), Boer cross (n = 3), alpine cross (n = 2), Saanen cations of laparoscopic cholecystectomy in dogs include cystic duct (n = 2), and pygmy cross (n = 2). Goats were quarantined for a rupture and bile spillage from cystic duct ligation (11). minimum of 14 d prior to administration of general anesthesia, to Options for laparoscopic cystic duct ligation include extracorpore- ensure appropriate health status. A complete physical examination ally or intracorporeally tied sutures, and hemoclips (9). Cystic duct and packed-cell volume/total protein (PCV/TP) was carried out ligation with a vessel sealant device has also been described in a before induction of general anesthesia for a terminal surgery labo- cadaveric study with normal hepatobiliary systems (12). Concern ratory. Goats were euthanized for reasons unrelated to this study. for hemoclip slippage, especially in thickened or friable cystic duct Immediately after euthanasia, a right paracostal approach was tissue, and challenges encountered with intra/extracorporeally conducted to access the duodenum, common bile duct, gall bladder, suture ligation have encouraged research into alternative methods and right liver lobes. In the original study design, a duodenotomy for cystic duct ligation (13). was completed and the common bile duct was cannulated retro- The LigaTie (Resorbable Devices AB, Uppsala, Sweden) is an grade from the intestinal lumen with a 4 Fr double lumen catheter absorbable self-locking device that was manufactured based on the (Milacath; Mila International, Florence, Kentucky, USA) and secured principle of a cable tie. It is currently not commercially available to the common bile duct with a circumferential suture. A chole- in the USA. Initially, the device was made of polydioxanone (14); cystectomy was carried out through the right paracostal approach however, because of concerns of pliability, it is now a co-polymer with the cystic duct being ligated with either 2 large stainless steel of glycolide and trimethylene carbonate (15). The original intent of endoscopic hemoclips (Horizon Metal Ligation Systems; Teleflex, the LigaTie was vessel ligation due to the excellent tissue grip and Morrisville, North Carolina, USA) or the LigaTie (Resorbable Devices ability to close to a loop end diameter of zero (14). Höglund et al AB, Uppsala, Sweden). A pilot sample elucidated multiple com- (15) described its use for ligation of the spermatic cord in dogs, munications between the biliary system and the pancreatic duct in with specific interest in the feasibility as well as biocompatibility. the goat, and infusion of fluid through a retrograde catheter (which The study showed that no complications were found related to the resided in the common bile duct) resulted in marked pancreatic device and that the subcutaneous tissues were capable of resorbing edema. Therefore, we were unable to maintain and determine the the polyglycolic based co-polymer, indicating appropriate biocom- true pressure within the cystic duct as infused fluid exuded into the patibility. The same device was also used for ligation of the canine pancreas. In the final study design, after the paracostal approach ovarian pedicle and its performance was compared to traditional was performed, the gall bladder, and approximately 4 cm of the suture ligation (16). This feasibility study showed that the ligation cystic duct was removed with the surrounding parenchyma of the time of the mesovarium was significantly shortened by using the right medial and quadrate liver lobe by sharp transection. The gall self-locking implant versus a single traditional ligature. A study by bladder was examined for gross pathology and gently expressed to Aminlashgari et al (17) tested the mechanical performance of the ensure a patent cystic duct. The cystic duct was dissected from the device and its degradation over time in vitro as well as short-term surrounding liver parenchyma with blunt dissection until a stalk tests in vivo in pigs. Results of this study suggested that sufficient large enough to fit 2 endoscopic hemoclips (Figure 1) or a LigaTie

224 The Canadian Journal of Veterinary Research 2000;64:0–00 Figure 1. Gall bladder catheterized with double lumen catheter (black Figure 3. Gall bladder catheterized with double lumen catheter (large arrow). Cystic duct ligated with 2 large endoscopic hemoclips (white black arrow). One lumen is connected to a pressure transducer (small arrow). black arrow) and the other is connected to the fluid ingress.

site of the clips or LigaTie. If no leakage was observed, the maximum pressure achieved with the fluid pump was recorded. Data analysis Statistical analysis was performed with a commercially available software program (GraphPad Prism 5; GraphPad Software, La Jolla, Californai, USA). Leak pressure between the 2 groups was normally distributed as determined with a D’Agostino-Pearson omnibus K2 test and the means were analyzed using a Student t-test. Mean weight of the goats was likewise normally distributed and the means were analyzed using a Student t-test. A P-value , 0.05 was considered significant.

Figure 2. Gall bladder with cystic duct ligated with the LigaTie (white Results arrow). Twelve goats of various breeds were included in this study. There were no statistically significant differences between the weight of goats between groups (P = 0.564). Abnormal physical examination (Figure 2) was exposed. The LigaTie was placed in a similar fashion findings included mucoid nasal discharge in 3, lice in 1, and stimu- as a cable tie and tension was placed on the LigaTie “tail” until no lus induced myotonia which was clinically irrelevant in 1. Results further closure of the device could be achieved. Ligation method was of preoperative PCV, and total protein were within normal limits in designated for the first sample based on a coin toss, and thereafter all goats. Goats were treated by the attending clinician for abnormal the method was alternating in the subsequent samples. Once either preoperative findings and were considered healthy before anesthesia the hemoclips or LigaTie had been placed, the apex of the gall blad- and euthanasia. Subjectively, all gall bladders and livers were free der was cannulated using a 4 Fr double lumen catheter (Milacath; of gross disease, and when expressed, normal bile easily passed Mila International). One port of the double lumen catheter was con- through the cystic duct, demonstrating patency. All cystic ducts were nected to a 1-liter bag of sterile saline infused with fluorescein dye , 5 mm in diameter and visibly free of pathology. in order to augment identification of leakage within the system. The There were 12 samples included in the study: 6 ligated by hemo- other port was connected to an extension set and pressure transducer clips and 6 ligated by the LigaTie. The mean cystic duct pressure (Hewlett Packard CMS 24 Omicare; HP, Palo Alto, California, USA) achieved in the hemoclip group was 310.5 mmHg [6 75.4 mmHg (Figure 3). The transducer was calibrated to atmospheric pressure standard deviation (SD)]. The mean cystic duct pressure of the before testing each gall bladder. No reinforcement or ligation was LigaTie group was 317.2 mmHg [6 55.9 mmHg]. There were no needed at the catheter-gall bladder interface due to the apparent statistically significant differences between the maximum cys- water tight seal created when fluid infusion was initiated. Fluid was tic duct pressures between groups (P = 0.865). No leakage was infused through the catheter at a rate of 999 mL/h while the system observed from the cystic duct, hemoclip site, or LigaTie site in was observed for leakage. Leak pressure was recorded when fluid either group. However, very small amounts of fluid were observed was seen extruding from the gall bladder wall, cystic duct, or at the diffusely exuding from the gall bladder wall in 4 samples at

2000;64:0–00 The Canadian Journal of Veterinary Research 225 pressures . 330 mmHg. Also, in 1 sample, a small amount of leak- One main advantage with using the LigaTie on the cystic duct is age was observed around the catheter insertion site at the apex of the its ability to occlude and compress tissue without major limitation gall bladder, at a pressure of . 230 mmHg. Gentle digital pressure on duct size or tissue pliability. During cholecystectomies, the cystic at the catheter entrance site was enough to allow continued filling duct may be friable and not amendable to extensive dissection or of the gall bladder without loss of pressure. The final pressure mea- manipulation. Additionally, if the tissue is fibrous or dilated, large sures were obtained when the infusion pump stopped due to the hemoclips may not adequately occlude the duct or provide adequate high pressure within the system. hemostasis of the cystic artery; risking slippage and biliary leakage (9). The LigaTie is flexible and could be used on any size cystic duct with a single, secure ligation with no collateral thermal damage. Discussion Laparoscopic cholecystectomy is performed in other species, includ- The results of this study indicate that there was no difference ing wild animals (25), in which the LigaTie may be an alternative for between the maximum cystic duct pressure occluded with 2 large wide cystic ducts with fibrous walls. endoscopic hemoclips or a LigaTie in an ex vivo caprine cadaveric The ideal use for the LigaTie is implementing it in minimally inva- model for cholecystectomies. sive surgery, specifically laparoscopic cholecystectomy. However, Both the large hemoclip and LigaTie group reached supraphysi- there is hesitation for complete laparoscopic cholecystectomies in ologic pressures within the gall bladder and cystic duct. Small canine patients which is because of the inability to flush the com- amounts of fluid that diffusely seeped through the gall bladder wall mon bile duct to confirm patency. Recent reports have described at pressures . 330 mmHg were observed in 4 gall bladder samples. open cholecystectomies without catheterization of the common bile This did not affect the overall pressure achieved within the gall duct (26). Malek et al (26) also found that 53.5% of common bile bladder or cystic duct because the transducer would continue to ducts were not catheterized before cholecystectomy. No significant show increases in pressure. This equivocal leakage was assumed association was found in patient outcome between the methods of to be secondary to the marked intraluminal increases in hydrostatic catheterizing the common bile duct during cholecystectomy and pressure and the infusion pump eventually stopped when the pres- whether or not the common bile duct was catheterized. However, sure became too high. Therefore, it is possible that higher pressures recommendations on the necessity or technique for flushing the could have been achieved with manual pressure and leakage from common bile duct during surgery could not be made based on their the hemoclips or LigaTie site could have been observed. However, findings (26). A recent study by Scott et al (27) described periopera- the pressures obtained are already supraphysiologic and testing tive complications and outcomes of laparoscopic cholecystectomy to failure would not be clinically relevant. The normal common in 20 dogs. Six dogs (30%) required conversion to an open cholecys- bile duct pressure in a dog is 12 mmHg (21). Although the normal tectomy; however, all dogs were discharged from the hospital and caprine cystic duct or common bile duct pressure is unknown, it is no significant difference was found between hospitalization time in unlikely to reach pressures close to those noted in this study, indicat- laparoscopic versus conversion to an open procedure (27). Patients ing that both large hemoclips and the LigaTie may be appropriate with evidence of extra-hepatic biliary obstruction were not included for cystic duct ligation in dogs. Additionally, there are no known in the study; therefore, patency of the common bile duct was not studies that measure cystic or common bile duct pressures in dogs confirmed intraoperatively. Based on that study, laparoscopic cho- with hepatobiliary disease. In humans, common bile duct pressures lecystectomy can be performed successfully for uncomplicated can increase up to 29.4 mmHg with gallstones and suppurative gall bladder disease with careful case selection. The authors of the cholangitis (22). current study are not necessarily advocating for laparoscopic cho- The LigaTie was manufactured to display characteristics of hemo- lecystectomy without catheterizing of the common bile duct, rather, stasis and excellent tissue grip making possible applications of the understanding that it may be an option and prospective studies are device quite diverse. Initial studies found that a single device placed required to help further define the ideal patient. on the renal artery of pigs showed complete hemostasis in 12 arteries Limitations of this study include small sample size, using caprine for 5 min (14). To further document resistance to slippage, the ability cadavers instead of live or cadaver canine models, and the inability of the device to withstand a ligature slip-off was tested by applying a to model a standard cholecystectomy. However, given that this study force of 10 N (14). The LigaTie has successfully been used in vivo on was intended to be a proof of concept, future studies can be more ovarian pedicles (15) and the spermatic cord (23) with histological targeted in both the species and study design to better analyze the evidence of vascular occlusion and biocompatibility. Future applica- clinical uses of LigaTie in traditional open and laparoscopic chole- tions such as lung lobectomies are currently being investigated at cystectomy. An analysis of the histologic differences of a caprine the authors’ institutions. In humans, the use of a self-locking non- gall bladder compared to a canine gall bladder was not conducted. absorbable inert polymer clip (Hem-O-Lok clip) has been shown to Given there were no differences between the groups in this study be a secure option during basic laparoscopic procedures including and the supraphysiologic pressures that were obtained, the differ- cholecystectomy, appendectomy, splenectomy, and nephrectomy ence in histologic appearance would likely not be clinically signifi- (24). These clips can ligate up to 10-mm of tissue through a 5-mm cant. It would have been ideal to be able to model a clinical canine portal or up to 15-mm of tissue through a 10-mm portal (24). Due cholecystectomy in this study’s caprine model. However given the to the concern of migration or dislodgement of metallic clips, these gross anatomic differences from a canine cadaver, this was not pos- locking clips have been preferred for various procedures and share sible. Instead, it was decided that the focus would be on mechanical common advantages to the LigaTie. testing, in a consistent and repeatable manner, although the design

226 The Canadian Journal of Veterinary Research 2000;64:0–00 set-up is not applicable to in vivo conditions. Additionally, no attempt and Thoracoscopy. 1st ed., Ames, Iowa: Wiley-Blackwell, 2015: was made to place the LigaTie in a laparoscopic model; therefore, 156–166. we cannot comment on the ability to maneuver the device in that 14. Höglund OV, Hagman R, Olsson K, Mindemark J, Borg N, setting. Instrument design is underway at our institution to use the Lagerstedt AS. A new resorbable device for ligation of blood LigaTie in a minimally invasive fashion. vessels — A pilot study. Acta Vet Scand 2011;53:47. In conclusion, no statistically significant differences were found 15. Höglund OV, Hagman R, Olsson K, Carlsson C, Södersten F, in the maximum cystic duct pressures between 2 large endoscopic Lagerstedt AS. Ligation of the ovarian pedicles in dogs with a hemoclips and a LigaTie in a caprine cholecystectomy model. 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2000;64:0–00 The Canadian Journal of Veterinary Research 227 Short Communication Communication brève

Evaluation of serum symmetric dimethylarginine in dogs with heartworm infection Bom-Sul Choi, Hyeongsun Moon, Sang-IL Suh, Changbaig Hyun

Abstract This study evaluated the circulating levels of serum symmetric dimethylarginine (SDMA) in 12 dogs with different severities of heartworm disease (HWD) and assessed the biochemical renal markers (blood urea nitrogen, creatinine). Dogs were classified into 2 groups based on the severity of clinical signs. Group A — asymptomatic to mild clinical signs, group B — moderate to severe clinical signs. The serum SDMA levels were higher in dogs in group B. Although the serum SDMA levels in dogs in group A were also higher than those of the control dogs, the difference was not statistically significant. There was a good correlation between renal markers and severity of clinical signs. This study demonstrated that the glomerular filtration rate was significantly decreased in dogs in group A; therefore, earlier detection of renal impairment is required for successful management of dogs with HWD.

Résumé La présente étude visait à évaluer les niveaux sériques de diméthylarginine symétrique (DMAS) chez 12 chiens atteints de maladie du vers du cœur (MVC) de différentes sévérités et d’examiner les marqueurs biochimiques rénaux (azote uréique sanguin, créatinine). Les chiens ont été classés en deux groupes sur la base de la sévérité des signes cliniques. Le Groupe A — asymptomatique à signes cliniques légers, groupe B — signes cliniques modérés à sévères. Les niveaux de DMAS sériques étaient plus élevés chez les chiens du groupe B. Chez les chiens du groupe A, bien que les niveaux sériques de DMAS étaient également plus élevés que ceux des chiens témoins, la différence n’était pas statistiquement significative. Il y avait une bonne corrélation entre les marqueurs rénaux et la sévérité des signes cliniques. Cette étude a permis de démontrer que le taux de filtration glomérulaire était diminué de manière significative chez les chiens du groupe A; ainsi, une détection précoce de déficience rénale est nécessaire pour la gestion réussie des chiens avec MVC. (Traduit par Docteur Serge Messier)

Heartworm disease (HWD) is one of the most life-threatening from a private animal shelter. The owner of the shelter gave consent infectious heart diseases in dogs, and typically causes cardiac and for the dogs to participate in this study. The ages of dogs enrolled in pulmonary disorders including right-side congestive heart failure, this study could not be determined, although all dogs were mature. endarteritis, and pulmonary hypertension with abnormal immune Heartworm preventive drugs were not given to the dogs in this reactions (1). Clinical signs are generally related to worm burden (1) study population. The affected dogs received no medical therapy and azotemia is often seen in dogs with HWD (1,2). for HWD prior to renal testing. The dogs had no significant medical Symmetric dimethylarginine (SDMA) has recently been evaluated history, but had undergone deworming and vaccination. Heartworm as a renal biomarker that can efficiently detect early renal dysfunc- infection in this study population was diagnosed using a commercial tion in dogs (3). The level of serum SDMA has shown a significant enzyme-linked immunosorbent assay (ELISA) test kit (SNAP 4Dx relationship with the glomerular filtration rate (GFR) because it is Plus Test, IDEXX Laboratory, Westbrook, Maine, USA) used accord- only excreted through renal clearance in dogs (3). Unlike serum ing to the manufacturer’s directions. Dogs with HWD were further blood urea and creatinine, SDMA concentration is not significantly divided into 2 groups based on clinical signs using physical, radio- affected by age, body mass, and systemic diseases (4). Furthermore, graphic, or echocardiographic evaluation. Briefly, dogs in group A SDMA, compared to creatinine, tends to start increasing at an earlier [n = 6, 7.2 6 3.4 kg of body weight (BW)] had either no or mild clini- stage of renal failure in dogs (3). Therefore, SDMA may have good cal signs with/without abnormal findings on physical, radiographic, potential for early diagnosis of renal impairment in dogs with HWD. or echocardiographic examinations (i.e., occasional cough with or The aim of this study was to evaluate the diagnostic value of serum without exercise intolerance, mild pulmonary infiltration, and pul- SDMA in dogs with HWD-mediated renal disorders. monary arterial dilation, few visible worms in pulmonary arteries, The study population consisted of 12 dogs infected with HWD and no evidence of pulmonary hypertension). While dogs in group B (6.6 6 4.5 kg, mixed breed) and 6 HWD-negative clinically normal (n = 6, 5.4 6 3.1 kg BW) had moderate to severe clinical signs with control dogs (8.7 6 6.5 kg, mixed breed). All dogs were acquired abnormal findings on physical, radiographic, or echocardiographic

Department of Small Animal Internal Medicine, College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea (Choi, Suh, Hyun); Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA (Moon). Address all correspondence to Dr. Changbaig Hyun; fax: 82 33 244 2367; e-mail: [email protected] Received August 3, 2016. Accepted September 23, 2016.

228 The Canadian Journal of Veterinary Research 2017;81:000–0002017;81:228–230 examinations (i.e., persistent coughing, severe exercise intoler- Table I. Correlation of clinical severity of heartworm disease ance, signs related to right-sided congestive heart failure, marked (HWD) and symmetric dimethylarginine (SDMA) concentrations pulmonary infiltration with pulmonary arterial dilation and right with other variables ventricular enlargement, echocardiographic evidence of pulmonary Clinical severity SDMA hypertension with . 2.8 m/s peak velocity of tricuspid regurgitation R P-value R P-value or . 2.2 m/s peak velocity of pulmonary regurgitation. BW 0.03 0.751 0.116 0.355 To minimize the influence of feeding, all dogs were fasted for 12 h Clinical severity — — 0.732 0.001 before the collection of blood samples. Whole blood was withdrawn BUN 0.434 0.002 0.743 0.001 from either the cephalic or jugular veins for determination of serum CRE 0.555 0.01 0.648 0.01 level of SDMA. Blood samples were drawn directly into sterile tubes SDMA 0.738 0.001 — — (Vacutainer tubes; BD, Franklin Lakes, New Jersey, USA) and then BW — bodyweight; BUN — blood urea nitrogen; CRE — creatinine: centrifuged at 1.500 3 g for 10 min at 4°C. The SDMA concentrations R-correlation coefficient. were determined by a reference laboratory (IDEXX Laboratories). Levels of blood urea nitrogen (BUN) and creatinine were determined using an automated biochemistry analyzer (VetScan VS2, Abaxis, Union City, California, USA). renal impairment are limited to dogs with apparent renal disease or Statistical analyses were done using commercially available dysfunction, although direct measurement of GFR with creatinine or statistical software (SPSS 15.0 for Windows; IBM, New York, USA). iohexol can detect earlier stages of renal impairment (5). However, Descriptive statistics for quantitative variables between study groups indirect methods using BUN and creatinine are less sensitive, and were calculated using analysis of variance (ANOVA) with Tukey’s are influenced by age, body mass, and other systemic diseases (5). multiple comparison test. The correlation between renal markers Recent veterinary studies found that the level of circulating SDMA (e.g., BUN, creatinine, and SDMA) was determined by Pearson’s was more accurate and useful for early detection of renal dysfunc- coefficient of bivariate correlation analysis. A probability value less tion in dogs, and is not significantly affected by lean body mass than 0.05 was considered statistically significant in our analysis. and age (3,4). Our study also demonstrated gradual but significant Serum SDMA concentrations were 8 6 2 mg/dL in controls, elevation of serum SDMA with progression of HWD, implying 13 6 5 mg/dL in group A, and 21 6 4 mg/dL in group B. Serum that renal impairment might be more obvious with progression of BUN was 18 6 7 mg/dL in controls, 26 6 13 mg/dL in group A, and clinical signs in HWD. Unlike creatinine, the serum level of SDMA 41 6 25 mg/dL in group B. Serum creatinine was 0.7 6 0.2 mg/dL started to increase in dogs with ~40% loss of nephrons (3). In this in controls, 1.0 6 0.4 mg/dL in group A, and 1.5 6 0.5 mg/dL in study, the serum level of SDMA was higher than the reference range group B. Pair-wise comparisons showed significant differences in in 2/6 dogs in group A and 6/6 dogs in group B, while the serum serum BUN, creatinine, and SDMA concentrations in control and level of creatinine was higher in 1/6 dogs in group A and 2/6 dogs disease groups (P , 0.05), as well as in HWD groups with different in group B. This finding suggests that, in dogs, circulating SDMA severities of clinical signs (P , 0.05). can detect renal impairment earlier than does creatinine. Further, Based on previous veterinary references (3,5), upper limits were one recent canine study of chronic mitral valve disease found that set as 25 mg/dL for BUN, 1.4 mg/dL for creatinine, and 14 mg/dL SDMA increased with the severity of mitral insufficiency and indi- for SDMA. The number of dogs with BUN higher than the upper cated renal impairment earlier than did creatinine in dogs (8). The limit was 0/6 in controls, 2/6 in group A, and 3/6 in group B; the increased serum levels of SDMA in dogs with advanced stages of number with creatinine higher than the upper limit was 0/6 in HWD (i.e., group B) in this study suggested a higher risk of renal controls, 1/6 in group A, and 2/6 in group B. The number of dogs impairment in dogs with higher worm burden and severe clini- with SDMA above upper limit values was 0/6 in controls, 2/6 in cal signs. Interestingly, 2/6 dogs in group A in this study showed group A, and 6/6 in group B. serum SDMA levels higher than the reference range, suggesting Bivariate analysis revealed close correlations among renal mark- that many dogs without clinical signs related to HWD could have ers (i.e., SDMA, creatinine, and BUN) were found. Serum BUN, reduced GFR via impaired renal perfusion or renal injury due to creatinine, and SDMA concentrations were not correlated with body immunological causes, although they generally appeared clinically weight but were closely correlated with the severity of clinical signs normal. Therefore, earlier intervention to prevent renal injuries (Table I). Serum SDMA concentration was correlated with serum might be beneficial for successful management of HWD in dogs. BUN and creatinine concentration (Table I). Routine SDMA testing in dogs with HWD will help clinicians to Azotemia is one of the most common complications of advanced more properly decide the choice of HW therapeutic protocols (e.g., heart failure caused by HWD and other heart diseases (6,7). Unlike slow kill versus melarsomine), the timing of initiation of adulticidal other heart diseases, HWD in dogs can induce azotemia through therapy and management guideline during adulticidal treatment, immune-mediated glomerulonephritis caused by microfilaria and based on the renal status of dogs. debris of dead adult worms, while lower cardiac output-induced There are several limitations in this study. First, the study popula- ischemic renal injury in decompensated heart failure is a major risk tion was small, which may not provide sufficient data to adequately factor for azotemia (1). Therefore, early detection of renal impair- correlate SDMA with the severity of HWD in dogs. Second, the ment in HWD would enable clinicians to establish a safe but efficient SDMA levels of all affected dogs in this study population could management plan in dogs with HWD. Current diagnostic tests for not be monitored over time owing to refusal by the owners. We

2000;64:0–00 The Canadian Journal of Veterinary Research 229 obtained SDMA test results over time for only a few affected dogs 2. Atkins CE, Vaden SL, Arther RG, et al. Renal effects of Dirofilaria (2/6 dogs in group B) after melarsomine therapy and found that the immitis in experimentally and naturally infected cats. Vet Parasitol SDMA levels returned to the reference range 2 mo after treatment 2011;176:317–323. (unpublished data). Since melarsomine is eliminated by the kidney 3. Nabity MB, Lees GE, Boggess MM, et al. SDMA assay valida- to an extent, further studies should be aimed at investigating the tion, stability, and evaluation as a marker for early detection influence of melarsomine on renal function and SDMA levels in a of chronic kidney disease in dogs. J Vet Intern Med 2015;29: larger study population. In addition, further study should also be 1036–1044. aimed at identifying which therapeutic protocol (e.g., slow kill or 4. Hall JA, Yerramilli M, Obare M, Yerramilli M, Melendez LD, melarsomine-based protocol) is more suitable for HWD infected dogs Jewel DE. Relationship between lean body mass and serum with prolonged kidney damage. Nevertheless, our study demon- renal biomarkers in healthy dogs. J Vet Intern Med 2015;29: strated that the GFR was significantly decreased in dogs with lower 808–814. worm burden and milder clinical signs; therefore, earlier detection 5. Braun JP, Lefebvre HP. Kidney function and damage. In: of renal impairment is required for successful management of dogs Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical Biochemistry with HWD. Our study also implied that earlier recognition and stabi- of Domestic Animals. 6th ed. London, UK: Elsevier, 2008: lization of renal impairment prior to and during adulticidal therapy 485–528. might be more important for clinicians to achieve therapeutic goals 6. Ronco C, Haapio M, House AA, Anavekar N, Bellomo R. Cardio­ by slow kill or melarsomine. renal syndrome. J Am Coll Cardiol 2008;52:1527–1539. 7. Tang WH, Mullens W. Cardiorenal syndrome in decompensated References heart failure. Heart 2008;96:255–260. 8. Choi B-S, Moon H-S, Seo S-I, Hyun C. Evaluation of serum 1. Calvert CA, Rawlings CA. Canine heartworm disease. In: cystatin-C and symmetric dimethylarginine concentrations in Fox PR, ed. Canine and Feline Cardiology. New York, New York: dogs with heart failure from chronic mitral valvular insufficiency. Churchill Livingstone, 1988:519–549. J Vet Med Sci 2017;79:41–46.

230 The Canadian Journal of Veterinary Research 2000;64:0–00 Short Communication Communication brève

Maternal and fetal arterial blood gas data in normotensive, singleton, isoflurane anesthetized sheep at 124–126 days of gestation Claire M. Loughran, Matthew W. Kemp, Gabrielle C. Musk

Abstract The aim of this case series was to describe the differences between maternal and fetal blood-gas results during anesthesia. Sixteen singleton adult merino ewes weighing 60.1 6 5.1 kg at 125.7 d (124 to 126 d) gestation were anesthetized. Maternal (radial) and fetal (umbilical) arterial blood gas samples were collected 79 6 6 min after the start of anesthesia if maternal mean arterial pressure

(MAP) was stable and . 65 mmHg. Fetal pH, partial arterial pressure of oxygen (PaO2), glucose, arterial hemoglobin oxygen saturation (SaO2), sodium, and chloride were significantly lower and fetal partial arterial pressure of carbon dioxide (PaCO2), lactate, hematocrit, total hemoglobin, potassium, and calcium were significantly higher than maternal blood-gas values. Fetal pH, PaO2, and BE were lower and fetal lactate was higher than fetal umbilical arterial samples previously reported, which may indicate a non-reassuring fetal status. Further refinement of the ovine experimental model is warranted with fetal monitoring during maternal anesthesia.

Résumé L’objectif de l’étude était de décrire les différences dans les résultats d’analyse des gaz sanguins maternel et fœtal durant l’anesthésie. Seize brebis mérinos primipares pesant 60,1 6 5,1 kg à 125,7 j (124 à 126 j) de gestation ont été anesthésiées. Des échantillons de sang artériel maternel (radiale) et fœtal (ombilicale) ont été prélevés 79 6 6 min après le début de l’anesthésie si la pression artérielle moyenne (PAM) maternelle était stable et . 65 mmHg. Pour le sang fœtal, les valeurs de pH, de la pression artérielle partielle en oxygène (PaO2), du glucose, de la saturation en oxygène de l’hémoglobine artérielle (SaO2), du sodium, et du chlorure étaient significativement inférieures, et les valeurs de la pression artérielle partielle en dioxyde de carbone (PaCO2), du lactate, de l’hématocrite, de l’hémoglobine totale, du potassium, et du calcium étaient significativement supérieures que celles du sang maternel. Les valeurs fœtales du pH, de la PaO2, et de BE étaient plus basses et le lactate fœtal étaient plus élevées que les valeurs d’échantillons provenant du sang artériel ombilical fœtal rapportées précédemment, ce qui pourrait indiquer un statut fœtal non-rassurant. Des améliorations de ce modèle expérimental ovin sont souhaitées avec un suivi fœtal durant une anesthésie maternelle. (Traduit par Docteur Serge Messier)

Pregnant sheep are frequently used as an experimental model for housed in a purpose built biomedical research facility and cared for the human maternal-fetal unit (1–4). During experimental research, as previously described (7,8). maternal health is routinely assessed while fetal health is more Sixteen singleton adult merino ewes weighing 60.1 6 5.1 kg at difficult to assess and is not routinely performed. This approach 125 d (124 to 126 d) gestation (term is 150 d) were anesthetized as has its limitations as fetal health is not always synonymous with previously described (7,8). In brief, the ewes were pre-medicated maternal health (5). There is limited information in the literature with a combination of acepromazine [0.03 mg/kg body weight (BW)] regarding fetal blood gas results and their relationship with mater- and buprenorphine (0.01 mg/kg BW) by intramuscular injection. nal blood gas results during maternal anesthesia in sheep. The aim Forty-five minutes later, anesthesia was induced with a combina- of this study was to describe the differences between maternal and tion of midazolam (0.25 mg/kg BW) and ketamine (5 mg/kg BW) fetal blood gases in normotensive and normocapnic third trimester by intravenous injection. Anesthesia was maintained with isoflu- singleton ewes during general anesthesia. These data were collected rane (vaporizer settings were 1% to 2%) in 100% oxygen delivered opportunistically as the animals were part of a larger project requir- through a circle breathing system. Ewes were placed in dorsal ing fetal intervention during maternal anesthesia, laparotomy, and recumbency in the Trendelenburg position with the table tilted at hysterotomy. 25°C and were mechanically ventilated (Aestiva 5 with Smartvent The use of sheep in this study was approved by the Animal Ethics 7900; Datex-Ohmeda, GE Healthcare, Bromma, Sweden), with a tidal Committees of the University of Western Australia and Murdoch volume of 7 to 10 mL/kg BW, I:E ratio 1:2 and a peak inspiratory

University in accordance with the Code of Practice for the Care pressure of 10 to 25 cmH2O. Intravenous fluid therapy was adminis- and Use of Animals for Research and Teaching (6). All sheep were tered during anesthesia with a balanced isotonic electrolyte solution

College of Veterinary Medicine, School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia (Loughran); School of Women’s and Infants’ Health, The University of Western Australia, Perth, Western Australia 6009, Australia (Kemp); Animal Care Services, The University of Western Australia, Perth, Western Australia 6009, Australia (Musk). Address all correspondence to Gabrielle Musk; e-mail: [email protected] Received September 7, 2016. Accepted November 14, 2016.

2017;81:231–234 The Canadian Journal of Veterinary Research 231 Table I. Mean 6 SD of the measured indices from ewes (N = 16) to maternal hypotension (MAP , 65 mmHg) at the time of arterial over the duration of anesthesia prior to collection of arterial blood gas collection. Sixteen sets of arterial blood gas results were blood subsequently analyzed. Maternal physiologic parameters, ETCO2, peripheral capillary oxygen saturation measured by the pulse oxim- Parameter Values eter (SpO ), and MAP at the time of blood gas collection were within Heart rate (beats/min) 86.7 6 17.9 2 normal limits (Table I). Ten of the sheep had a mean arterial blood Respiratory rate (breaths/min) 10.1 6 1.9 pressure between 55 and 65 mmHg for 5 to 15 min at the beginning MAP (mmHg) 70.8 6 5.5 of anaesthesia. This hypotension was managed with fluid therapy ETCO (mmHg) 37.6 6 3.4 2 (temporary increase in the rate of infusion up to the high end of the Temperature (°C) 38.2 6 0.9 aforementioned range for both the crystalloid and the gelofusin) and Isoflurane vaporizer setting (%) 1.5 6 0.5 alteration of the vaporizer setting. Fetal pH, PaO , glucose, SaO , Time of blood gas collection after onset of 78.8 6 5.9 2 2 Na1, and Cl2 were significantly lower than maternal values while anesthesia (min) 1 21 fetal PaCO2, lactate, Hct, tHb, K , and Ca were significantly higher MAP — mean arterial pressure; ETCO2 — end tidal carbon dioxide. than maternal values (Table II). Maternal CaO2 was significantly

higher than fetal CaO2. (Compound Sodium Lactate solution; Baxter Healthcare, Toongabbie, This study compared paired maternal and fetal arterial blood New South Wales, Australia) at 10 to 20 mL/kg per hour and succi- gas data at a single time point from dorsally recumbent, normo- nylated gelatin solution (Gelofusine 4%; Braun Australia, Bella Vista, tensive isoflurane-anesthetized singleton ewes and their fetuses at New South Wales, Australia) at 2 to 10 mL/kg per hour as required a gestational age of 125 d (124 to 126 d). Comparison of these data to maintain normotension (MAP . 65 mmHg). The physiological demonstrated a significantly higher fetal PaCO2, lactate, Hct, tHb, changes associated with anesthesia were monitored continuously K1, and Ca21 relative to maternal results and a significantly lower 1 2 and displayed on a multivariable monitor (Advisor Vital Signs fetal umbilical PaO2, glucose, SaO2, Na , Cl , and CaO2 relative to Surgivet Monitor V9212AR; Smiths Medical, Lincoln Park, New maternal arterial values. Jersey, USA). These parameters included heart rate, respiratory rate, Simultaneous fetal and maternal arterial blood gas samples electrocardiography, pulse oximetry, esophageal temperature, inva- in this study revealed maternal PaO2 within the expected range, sive blood pressure, and inspiratory and ETCO2. The experimental but fetal PaO2 lower than values recorded in previous studies (8). procedure involved laparotomy and hysterotomy to access the fetus. Anesthetized pregnant sheep breathing up to 98% O2, almost 5 times

While remaining in utero the fetal trachea was intubated and the the oxygen concentration of room air, are expected to have a PaO2 of fetal lung was ventilated. At the end of the procedure the ewe and approximately 484 mmHg 6 21 mmHg according to the alveolar fetus were euthanized with intravenous pentobarbitone 160 mg/kg gas equation (9). This value is similar to the maternal PaO2 reported (Valabarb, Pentobarbitone 320 mg/mL; Jurox, Rutherford, New in this study. Maternal circulating oxygen is transported across South Wales, Australia). the placenta and into the fetal circulation by passive diffusion in

The paired maternal and fetal arterial blood gas samples were response to a pressure gradient. Consequently, fetal PaO2 should collected 70 to 90 min after the onset of anesthesia and analyzed be significantly lower than maternal values, as reported in this provided maternal MAP had been stable for 15 min and was greater study. The fetal PaO2 from the singleton animals in this study was than 65 mmHg at the time of blood sample collection. Maternal arte- unexpectedly lower than those reported for twin fetuses from anes- rial blood samples were collected from the radial arterial catheter thetized ewes (8). This discrepancy may indicate a greater degree and fetal arterial samples were collected from the umbilical artery. of maternal-fetal placental perfusion mismatch in the current study The samples were collected into heparinized syringes, any air bub- and a reduction in the oxygen transfer between the maternal and bles were expelled and a cap was placed on the syringe immediately. fetal blood. Furthermore, and perhaps more importantly, the fetal

Samples were analyzed within 15 min of collection using a benchtop PaO2 values in this study were taken at 79 min 6 6 min, whereas arterial blood gas analyzer (RapidLab 1265; Siemens Healthcare the fetal twin arterial samples were collected at 28 min 6 6 min and Diagnostics). The data collected from these analyses included pH, 56 6 15 min after the onset of anesthesia for the first and second

PaO2, PaCO2, BE, lactate, glucose, hematocrit (Hct), tHb, SaO2, twin, respectively, indicating that the duration of anesthesia may 1 1 21 2 sodium (Na ), potassium (K ), calcium (Ca ), and chloride (Cl ). negatively impact fetal PaO2.

Maternal and fetal arterial oxygen content (CaO2; mL/L) was cal- Despite the low PaO2 of fetal blood, fetal tissue oxygen require- 21 culated as follows: CaO2 (mL/L) = [([Hb] (gdL ) 3 1.36 3 SaO2 (%)) ments may exceed those of the mother. Consequently, the fetus has

1 (0.0031 3 PaO2 (mmHg))] 3 10. Normally distributed data were adapted to lower PaO2 levels by having a higher Hct and hemoglo- compared with a Student’s t-test. Otherwise a Mann–Whitney bin concentration with a greater affinity for oxygen than maternal Rank Sum test was used. All tests were performed with Prism 6, hemoglobin. Maternal ovine Hct decreases during anesthesia (10). 2015 (Graphpad Software; La Jolla, California, USA); P , 0.05 was The maternal Hct reported in the current study, however, was lower considered to be statistically significant. Normally distributed data than values reported previously in anesthetized non-pregnant sheep. are presented as mean 6 SD, otherwise data are expressed as median This finding is consistent with a previous report that pregnant sheep (interquartile range). are anemic during anesthesia (8). The fetal Hct in this study was

Nineteen pairs of maternal and fetal blood gas results were significantly higher than maternal values and when the CaO2 in obtained, but 3 pairs of results were excluded from analysis due fetal and in maternal blood is compared, it is apparent that although

232 The Canadian Journal of Veterinary Research 2000;64:0–00 Table II. Mean 6 SD or median (interquartile range) of measured and calculated indices of arterial blood gas data from the ewe and fetus during isoflurane anesthesia in singleton sheep at 124 to 126 d of gestation Difference between maternal and Maternal Fetal fetal samples P-value pH 7.4 6 0.05 7.2 6 0.07 0.2 6 0.09 , 0.0001 BE (mmol/L) 1.4 6 2.4 20.9 6 3.0 2.3 6 4.3 0.0231

PaCO2 (mmHg) 42.1 6 5.1 73.0 6 9.1 230.9 6 8.5 , 0.0001

PaO2 (mmHg) 486.3 6 51.0 20.1 6 4.2 461.2 6 51.0 , 0.0001 Hct (%) 19.6 6 2.6 35.6 6 2.7 215.9 6 3.5 , 0.0001 tHb (g/dL) 6.6 (6.1–7.2) 12.1 (11.3–12.5) 25.4 [26.5–(24.2)] , 0.0001

SaO2 (%) 99.9 (99.8–99.9) 54.7 (44.7–60.7) 45.2 (39.2–55.2) , 0.0001 Glucose 3.0 6 0.5 1.0 6 0.2 2.2 6 0.4 , 0.0001 Lactate (mmol/L) 1.4 6 0.5 3.63 6 0.9 22.3 6 1.0 , 0.0001 Ca21 (mmol/L) 1.1 (1.0–1.1) 1.7 (1.6–1.8) 20.6 [20.7–(20.5)] , 0.0001 Na1 (mmol/L) 144.1 6 1.5 137.6 6 1.7 6.4 6 1.5 , 0.0001 K1 (mmol/L) 3.0 (2.8–3.2) 4.0 (3.7–4.3) 21.0 [21.1–(20.7)] , 0.0001 Cl2 (mmol/L) 111.2 6 3.7 100.1 6 2.0 11.06 6 3.9 , 0.0001

CaO2 (mL/L) 104.9 6 12.2 83.2 6 18.8 20.6 6 19.6 0.0006

BE — base excess; PaCO2 — partial arterial pressure of carbon dioxide; PaO2 — partial arterial pressure of oxygen; 21 Hct — hematocrit; tHb — total hemoglobin; SaO2 — percentage hemoglobin saturation with oxygen; Ca — 1 1 2 calcium; Na — sodium; K — potassium; Cl — chloride; CaO2 — arterial oxygen content of the blood.

maternal CaO2 is significantly higher, the difference is smaller than this physiological state occurs when maternal PaCO2 values are might be expected. It is unknown whether anemia during anesthesia considerably lower than those being recommended here (14). of pregnant sheep is acute or chronic and this finding warrants fur- Maternal blood glucose concentrations in this study were within ther investigation. Pre-operative blood samples were not collected the expected range, but fetal glucose concentrations were lower in this study. than values recorded in previous studies (8). This discrepancy may Simultaneous fetal and maternal arterial blood gas samples in this relate to the timepoint at which the sample was collected, which study revealed that maternal PaCO2 was within the target range, but was 79 min 6 6 min after anesthesia. The fetus is dependent on fetal PaCO2 was higher than values recorded in the fetus of conscious maternal circulation to supply glucose so low fetal blood glucose pregnant sheep (73 mmHg versus 52 mmHg) (11). Ventilation in this could indicate a failure of placental transfer due to the development study was controlled with the aim of maintaining an ETCO2 of 35 to of maternal-fetal placental perfusion mismatch over time. Maternal

45 mmHg. Consequently the maternal PaCO2 was similar to values lactate values in this study were within the expected range, but reported in conscious pregnant ewes breathing room air at 103 to fetal lactate was higher than values recorded in previous studies (8). 104 d of gestation and confirms findings of previous studies which Lactate production by the fetus is a direct end product of anerobic failed to demonstrate a significant difference between the PaCO2 of metabolism. conscious pregnant and non-pregnant sheep (12). Fetal PaCO2 in this Consistent with previous reports, the maternal pH in this study study was significantly higher than maternal PaCO2. These figures was similar to values recorded in conscious sheep and fetal pH was are consistent with those in other studies; however, the gradient for significantly lower than maternal levels (8,10). However, the fetal the exchange of CO2 between maternal and fetal blood was higher pH values in this study were lower than those recorded in fetal than the gradient reported in twin pregnant anesthetized ewes (8). samples taken from singleton conscious ewes (11). A specific defini-

The increased CO2 gradient could indicate a greater fetal-maternal tion of acidemia in the fetus is difficult to describe as the fetus has perfusion mismatch in this study. The fetal PaCO2 in this study adapted to withstand short periods of acidosis. Acute fetal acidosis was 40% higher than fetal values recorded in singleton conscious is almost always respiratory in origin, but quickly progresses to a ewes, indicating fetal hypercapnia (11). Based on the fetal-maternal mixed respiratory metabolic acidosis as seen in this study by the

PaCO2 gradient observed in this and other studies it may be war- increase in fetal BE compared to fetal samples from anesthetized ranted to target mild maternal hypocapnia (PaCO2: 30 to 35 mmHg) and conscious sheep (8,10). during anesthesia to reduce fetal PaCO2 to a level comparable to The electrolyte concentrations in this study varied between fetal measurements in conscious ewes (11). Maternal hypocapnia is maternal and fetal circulations. Ewes were slightly hypocalcemic, normally tolerated for short periods, with few apparent effects (13). hypokalemic, normonatremic, and normochloremic (15). Fetal elec- Extreme hypocapnia can reduce placental blood flow in sheep, but trolyte levels were significantly lower (CL2 and Na1) and higher

2000;64:0–00 The Canadian Journal of Veterinary Research 233 (Ca21 and K1) than maternal levels and lower than ranges found in 4. Uemura K, Shimazutsu K, McClaine RJ, et al. Maternal and 1- to 3-month-old sheep (16). Reference ranges are not available for preterm fetal sheep responses to dexmedetomidine. Int J Obstet fetal lambs and there is no information in the literature regarding Anesth 2012;21:339–347. electrolyte levels during gestation. The fetal electrolyte levels mea- 5. Künzel W, Misselwitz B. Unexpected fetal death during preg- sured in this study were similar to values in twin fetuses, with the nancy — A problem of unrecognized fetal disorders during ante- exception of K1, which was lower in this study (8). natal care? Eur J of Obstet Gynecol Reprod Biol 2003;110:S86–S92. Although the fetuses in this study underwent ventilation, the 6. National Health and Medical Research Council. Australian code placental circulation was intact. This intervention does not alter for the care and use of animals for scientific purposes, 8th ed fetal blood gas results (17) so based on the results of this study, [guidelines on the Internet]. Canberra, Australia: National ovine maternal health as assessed by arterial blood gas analysis and Health and Medical Research Council c2013. Available from: routine physiological monitoring during anesthesia, does not neces- https://www.nhmrc.gov.au/_files_nhmrc/publications/ sarily constitute fetal health. While this study highlights poorer fetal attachments/ea28_code_care_use_animals_131209.pdf Last blood gas parameters in anesthetized sheep compared to fetal blood accessed February 21, 2017. gas parameters from conscious sheep, this finding is not unexpected 7. Davis J, Musk GC. Pressure and volume controlled mechani- given the negative impact of anesthesia on the fetal and maternal cal ventilation in anaesthetized pregnant sheep. Lab Anim cardiovascular system, and by extension, placental blood flow, and 2014;48:321–327. nutrient and waste product transfer. Improvements in fetal blood 8. Musk GC, Kemp MW. Maternal and fetal arterial blood gas data gas results (lower PaCO2 and lactate; higher pH, PaO2, and BE) in during general anaesthesia for caesarean delivery of preterm future studies may be possible with refinements of the anesthetic twin lambs. Lab Anim 2016;50:198–203. protocol: determination of the optimal approach to the management 9. Dugdale A. Blood gas analysis. In: Veterinary Anaesthesia: of hypotension (fluid therapy versus vasopressors or inotropes); Principles to Practice. Oxford, England: Wiley-Blackwell, 2010: sophisticated regional anesthesia; neuromuscular blockade; intra- 182–197. operative analgesic drug infusion (e.g., fentanyl), and propofol based 10. Onmaz AC, Gunes V, Atalan G, Gelfert CC, Atalan G. Compari­ total intravenous anesthesia (18). son of arterial and venous blood gas values in sheep before and This case series presents the differences between maternal and during isoflurane anaesthesia. Revue Méd Vét 2009;160:356–361. fetal arterial blood gas samples in dorsally recumbent isoflurane 11. Fleming JA, Lambert TF, Walker AM. The effects of bupivacaine anesthetized singleton pregnant ewes. Despite the ewes being on the umbilical circulation and placental gas exchange in the normotensive and normocapnic, the fetus showed signs of acidosis fetal lamb. Anesth Analg 1987;66:1121–1126. signaling non-reassuring fetal status. Further work is required to 12. Mathai S, Booth LC, Davidson JO, et al. Acute on chronic expo- determine both the significance of these findings on fetal health sure to endotoxin in preterm fetal sheep. Am J Physiol Regula beyond delivery, and to optimize the anesthetic regime for pregnant Integr Comp Physiol 2013;304:R189–R197. ewes at different gestational ages in a biomedical research setting. 13. Laffey JG, Kavanagh BP. Hypocapnia. N Engl J Med 2002;347: 43–53. 14. Levinson G, Shnider SM, deLorimier AA, Steffenson JL. Effects of Acknowledgments maternal hyperventilation on uterine blood flow and fetal oxy- Thanks is also extended to Astrid Armitage, Andrew Wilson, and genation and acid-base status. Anesthesiology 1974;40:340–347. the Animal Care Services staff at the Large Animal Facility, for their 15. Seeler DC. Fluid, electrolyte, and blood component therapy. In: expert care and husbandry of the pregnant ewes. Tranquilli WJ, Thurmon JC, Grimm KA, eds. Lumb and Jones’ Veterinary Anaesthesia and Analgesia. 4th ed. Ames, Iowa: Blackwell Publishing, 2007:183–201. References 16. Eshratkhah B, Sadaghian M, Nezhad MS, Sabri V, Geyglou BF. 1. Collins JJP, Kuypers E, Nitsos I, et al. LPS-induced chorioam- Evaluation of electrolytes normal values in blood of Moghani nionitis and antenatal corticosteroids modulate Shh signaling sheep breed. J Anim Vet Adv 2008;7:437–440. in the ovine fetal lung. Am J Physiol Lung Cell Mol Physiol 17. Hillman NH, Moss TJ, Nitsos I, Jobe AH. Moderate tidal volumes 2012;303:L778–L787. and oxygen exposure during initiation of ventilation in preterm 2. Gisslen T, Hillman NH, Musk GC, et al. Repeated exposure to fetal sheep. Pediatr Res 2012;72:593–599. intra-amniotic LPS partially protects against adverse effects of 18. Seehase M, Houthuizen P, Collins JJ, Zimmermann LJ, intravenous LPS in preterm lambs. Innate Immunity 2014;20: Kramer BW. Propofol administration to the fetal-maternal unit 214–224. reduces cardiac oxidative stress in preterm lambs subjected 3. Kuypers E, Collins JJP, Kramer BW, et al. Intra-amniotic LPS to prenatal asphyxia and cardiac arrest. Pediatr Res 2016;79: and antenatal betamethasone: Inflammation and maturation 748–753. in preterm lamb lungs. Am J Physiol Lung Cell Mol Physiol 2012;302:L380–L389.

234 The Canadian Journal of Veterinary Research 2000;64:0–00 Short Communication Communication brève

Towards an improved estimate of antimicrobial use in animals: Adjusting the “population correction unit” calculation Brian R. Radke

Abstract International comparisons of animal antimicrobial use (AMU) have typically been based on total national estimates of antimicrobials sales standardized by the national animal biomass calculated as the population correction unit (PCU). The objective of this paper was to compare the currently accepted PCU calculation with that of the adjusted population correction unit (APCU), which re-evaluates the standard animal weights used in the calculation and accounts for animal lifespan. The APCU calculation resulted in substantial changes to the 2009 national biomass estimates for cattle, pigs, and poultry in 8 European countries and Canada. The estimated national biomass for cattle increased 35% to 43%, while the estimated national biomass of pigs and poultry typically decreased by approximately 51% and 87%, respectively. Among the 9 countries, the total national APCU ranged from an increase of 1% to a decrease of 40% relative to PCU, and these differences were statistically significant. Adjusted population correction unit is preferred over PCU in comparing and contrasting AMU among animals with different lifespans because it is more transparently derived and is a reasonable approximation of the animal biomass at risk of antimicrobial treatment.

Résumé Les comparaisons internationales de l’utilisation d’antimicrobiens chez les animaux (UMA) ont typiquement été basées sur les totaux nationaux estimés de ventes d’antimicrobiens standardisés pour la biomasse animale nationale calculée comme l’unité de correction pour la population (UCP). Les objectifs de cet article étaient de comparer les calculs d’UCP présentement acceptés à ceux de l’unité de correction pour la population ajustée (UCPA), qui réévalue les poids animaux standards utilisés dans les calculs et tient compte de la durée de vie des animaux. Les calculs de l’UCPA ont entrainé des changements substantiels aux estimés nationaux de 2009 de la biomasse pour les bovins, porcs et volailles dans 8 pays européens et le Canada. La biomasse nationale estimée pour les bovins a diminué de 35 % à 43 %, alors que les biomasses nationales estimées pour les porcs et les volailles ont typiquement diminué d’environ 51 % et 87 %, respectivement. Parmi les neuf pays, l’UCPA nationale totale variait d’une augmentation de 1 % à une diminution de 40 % relativement à l’UCP, et ces différences étaient statistiquement significatives. L’UCPA est préférée par rapport à l’UCP pour la comparaison et la mise en contraste de l’UMA chez les animaux avec différentes durées de vie étant donné qu’elle est dérivée de manière plus transparente et qu’elle est une approximation raisonnable de la biomasse animale à risque d’un traitement antimicrobien. (Traduit par Docteur Serge Messier)

Due to global public health concerns, antimicrobial use (AMU) in In contrast, national estimates of antimicrobial usage standardized animals is of significant interest, including international comparisons using PCU are available for over 25 countries, including Canada, of AMU. These comparisons have typically been based on total national and encompass use in all food-producing species (4). Therefore, for estimates of antimicrobials sales standardized by the national animal the foreseeable future it is likely PCU will continue to be used in biomass calculated as the population correction unit (PCU). This international comparisons of animal AMU, as well as in comparison approach has been criticized in favor of daily defined dose animals of usage between species (5). metrics (DDDA) which account for drug potency and usage at a species The purpose of PCU is to control for animal demographics, which level, if not by animal age or weight (1). However, current and future can vary over time within a country, and between countries. The implementation of DDDA is hampered by its high resource demands PCU is calculated by totalling the number of livestock or poultry (2), including antimicrobial use by species, if not by animal age or in an animal category multiplied by a standardized theoretical weight, and the lack of a global DDDA standard. A European Union weight of an animal in that category at the age it would most likely standard that has been under development since 2012, has recently be treated with antimicrobials, which is called average weights at been released and addresses poultry, pigs, and cattle while recognizing treatment (AWT) (6). the need for DDDAs for all food producing species including other There are 2 potential problems with the PCU method for calcu- ruminants, horses, fish, rabbits, and companion animals (2,3). lating animal biomass. Firstly, it is not clear how AWT is estimated

Plant & Animal Health Branch, Ministry of Agriculture, British Columbia. Address all correspondence to Dr. Brian Radke; telephone: (604) 556-3066; fax: (604) 556-3015; e-mail: [email protected] Dr. Radke’s current address is 1767 Angus Campbell Road, Abbotsford, British Columbia V3G 2M3, Canada. Received June 15, 2016. Accepted November 14, 2016.

2017;81:235–240 The Canadian Journal of Veterinary Research 235 or how it is related to antimicrobial use. Instead, PCU calculations For the animal categories most commonly included in PCU calcu- typically reference Monforts (7) and the European Medicines Agency lations, established AWT values were obtained from the European (8), that simply define AWT as the mean body weight for animals Medicines Agency (6) (Table I). raised for slaughter, and the maximum body weight for other animal Adjusted weights (AW) were arrived at in several different ways groups (e.g., breeding animals). Even based on these definitions, depending on the data available. For the cattle categories, the slaugh- some of the currently used AWT values do not appear to accurately ter (ending) weights were calculated by dividing the average carcass reflect animal weights. For example, the average mature cow weighs weight by a live-to-carcass weight conversion factor (13) to deter- approximately 600 kg (9–12), while a weight of 425 kg is currently mine the average weight of live animals. Average carcass weights for used for PCU calculations. 28 EU countries were calculated by dividing the total animal weight Antimicrobial use in an animal population is affected by the at slaughter for a given animal category, by the number of animals weight of the animals, and their length of life. The opportunity for slaughtered for that category (14). Subsequently, using Monforts’ antimicrobial use increases with increased length of life, and length definition, the mean body weight of the cattle slaughter categories of life of PCU’s livestock and poultry categories vary considerably. was calculated by averaging a birth weight of 45 kg and the final The PCU doesn’t take into account length of life (4) and this is the weight at slaughter. The AW of imported and exported cattle for second concern with the PCU method of estimating animal biomass. slaughter is the mean weight of slaughter heifers, bullocks, and bulls. The PCU’s failure to incorporate the variable lifespans of the animal For the remaining animal categories, Eurostat data regarding car- categories has potential implications not only for AMU comparisons cass weights and number of animals slaughtered were not available. between species, but also for comparisons of total national usage. For this reason, the international literature was reviewed to provide This failure potentially results in underestimation of AMU in coun- a contemporary estimate of animal weights in each category. It was tries with a preponderance of short-lived animal categories, such determined that these AWT are generally consistent with the (aver- as poultry, and overestimation in countries with disproportionately age) body weights defined elsewhere (9–12), including Canadian more longer-lived categories such as cattle. PCU calculations (Canadian Integrated Program for Antimicrobial It is important that PCU calculations accurately reflect animal Resistance personal communication, 2016). The AWT was therefore biomass for the animal categories of interest because inaccurate PCU used as AW for the non-cattle categories with the exception of pigs values may lead to erroneous conclusions when comparing and con- imported or exported for fattening. The AWT of these pigs is 25 kg. trasting AMU data. The objective of this paper is to compare the cur- However, Monforts (7) and the European Medicines Agency (8) rently accepted PCU biomass calculation with one that re-evaluates include weight of piglets up to 25 kg in the sow weight so the AW AWT (based on current data regarding production animal weights) for exported fattening pigs was set to zero (Table I). Similarly, the and accounts for the lifespan of the animal categories in question, 25 kg AWT for imported fattening pigs was set to zero as this weight using data from 8 European countries and Canada. is already recognized as the beginning weight in the slaughter pig’s Currently, a country’s PCU is calculated as follows: category (Table I). An animal category’s length of life (LL) was calculated using the 2 1 PCU = S ncAW Tc S niAW Ti S neAW Te (Equation 1) inverse of its number of cycles per year on an average farm. For c i e example, if the typical broiler farm has 9 cycles per year, then the where nj is the total number of animals in category j (i.e., for breed- average LL for broiler chickens is 0.11 y (1/9). Data regarding the ing animals, nj is the number of animals present in a year; if j are number of cycles per year for each animal category were obtained slaughter animals, nj is the total number of animals slaughtered from Monforts (7) and the European Medicines Agency (8). Neither annually); AWTj is the average weight at treatment of an animal reference included the number of cycles for slaughter heifers, bull- in j (kg); c is the animal categories raised and slaughtered within ocks or bulls. A LL of 1.5 y is assigned to slaughter heifers, bullocks, the country in question; i is the animal categories imported to the and bulls based on knowledge of these industries. country; and e is the animal categories exported from the country. Using the most recently published values of n for the 8 European The proposed equation, adjusted PCU (APCU), is as follows: countries (6) and Canadian data (Canadian Integrated Program for Antimicrobial Resistance personal communication, 2016), and 2 1 APCU = S ncLAWc S niLAWi S neLAWe (Equation 2) AWT (Table I), the 2009 PCU for the 9 countries was reproduced c i e using Equation 1. The APCU for each country was calculated using where LAWj is the life adjusted weight of an animal in category j. Equation 2, the same values of n, and LAW from Table I. A 2-tailed Life adjusted weight (LAW) is calculated as: paired t-test was used to determine whether total PCU and total APCU were significantly different among countries using Stata v.13.1

LAWj = AWjLLj (Equation 3) (StatCorp, College Station, Texas, USA) and APCU as a percentage change from PCU [i.e., (APCU-PCU)/PCU 3 100%] was calculated.

The AWj is the adjusted weight of an animal in j (kg) calculated using The 2009 PCU for the 9 countries is reported in Table II, as is the Monforts’ (7) and the European Medicines Agency’s (8) definitions APCU and the percentage change. For cattle, APCU was 35% to 43% of the animal weights (i.e., the mean weight for slaughter animal greater than PCU for each of the 9 countries, while for pigs, poultry, categories and the maximum weight for all other animal catego- sheep, and goats, the APCU was consistently less than the PCU. For ries). The LLj variable is the length of life for category j animals as example, using APCU, the national poultry biomass decreased by measured in years. 81% to 89%. The estimated national biomass of horses and fish were

236 The Canadian Journal of Veterinary Research 2000;64:0–00 Table I. Animal categories, population correction unit (PCU), average weight at treatment, and data used in calculating life adjusted weights PCU average weight at Adjusted Length Life adjusted treatment weight of life (LL) weight (LAW) Animal category (AWT) (kg) (AW) (kg) (year) (kg year) Cattle Slaughter cows 425a 627 1 627 Slaughter heifers 200b 269 (45, 493)e 1.5 404 Slaughter bullocks and bulls 425a 329 (45, 612)e 1.5 494 Slaughter calves and young cattle 140c 169 (45, 293)e 0.56 94 Imported/exported cattle for slaughter 425a 299j 1.5 449 Imported/exported cattle for fattening 140c 169 (45, 293)e 0.56 94 Livestock dairy cows 425a 627 1 627 Pigs Slaughter pigs 65d (25, 105)e 65 0.33 22 Imported/exported pigs for slaughter 65 65 0.33 22 Imported/exported pigs for fattening 25f 0 Livestock sows 240g 240 1 240 Poultry Slaughter broilers 1 1 0.11 0.11 Slaughter turkeys 6.5 6.5 0.37 2.4 Imported/exported broilers for slaughter 1 1 0.11 0.11 Sheep and goats Slaughter sheep and goats 20 (NAh, 40–45)e 20 0.5 10 Imported/exported sheep and goats for slaughter 20 20 0.5 10 Livestock sheep 75 75 1 75 Horses Living horses 400i 400 1 400 Fish Slaughter fishk

Rabbits Slaughter rabbits 1.4 1.4 0.15 0.21 a Adult cow weight. b 0-1-year-old bovine weight. c Veal calf weight. d Fattening pig (25 to 105 kg). e Beginning weight for animal category, ending weight for animal category. f Weaner pig (to 25 kg). g Weight for sow and piglets until 25 kg. h Not available. i Horses 600 kg, ponies 250 kg. j The mean of a slaughter heifer, bullock, and bull weight (i.e., 269 1 329/2). k Eurostat data available only as live-weight at slaughter; information on AWT is unavailable.

the same for APCU and PCU. The estimated national biomass for statistically significantly different (P = 0.02, t = 3.01, d.f. = 8) for the slaughtered rabbits decreased for France and Canada. 9 countries. For each country as a whole, the difference between APCU and Use of the 2 different animal biomass calculations (APCU versus PCU was variable. For Finland and Norway, APCU was respectively PCU) resulted in substantially different national values for most 11% and 1% greater than PCU. For the remaining 7 countries, APCU animal categories included in this analysis, as well as for most of was between 2% and 40% less than PCU. The APCU and PCU were the countries as a whole. These differences could have substantial

2000;64:0–00 The Canadian Journal of Veterinary Research 237 Table II. Calculation of population correction unit (PCU), adjusted population correction unit (APCU), and APCU as a percentage change (% D) from PCU for 8 European countries and Canada, 2009 (1000 tonnes) Czech Republic Denmark Finland France Netherlands Animal category PCU APCU % D PCU APCU % D PCU APCU % D PCU APCU % D PCU APCU % D Cattle 308 421 37% 403 566 41% 227 319 41% 3289 4437 35% 1009 1401 39% Slaughtered cows 52 76 80 119 36 53 756 1115 216 319 Slaughtered heifers 5 10 9 18 7 14 85 172 3 6 Slaughtered bullocks and bulls 47 54 47 55 62 72 500 581 26 30 Slaughtered calves and 2 1 19 13 0 0 220 148 208 139 young cattle Net exported cattle for slaughter 30 32 0 0 0 0 18 19 1 1 Net exported cattle for fattening 9 6 3 2 0 0 148 99 0 0 Net imported cattle for slaughter 0 0 0 0 0 0 0 0 0 0 Net imported cattle for fattening 0 0 0 0 0 0 0 0 109 73 Livestock dairy cows 163 241 244 360 122 179 1561 2303 664 979 Pigs 245 116 253% 1820 768 258% 190 88 254% 1941 836 257% 1484 668 255% Slaughtered pigs 211 70 1255 418 152 51 1619 540 898 299 Net imported pigs for slaughter 3 1 0 0 0 0 0 0 0 0 Net exported pigs for slaughter 0 0 80 27 0 0 37 12 315 105 Net exported pigs for fattening 0 0 162 0 0 0 0 0 7 0 Net imported pigs for fattening 9 0 0 0 0 0 0 0 0 0 Livestock sows 47 47 323 323 37 37 284 284 264 264 Poultry 154 17 289% 112 12 289% 57 8 286% 1179 229 281% 400 44 289% Slaughtered broilers 136 15 100 11 51 6 759 84 481 53 Slaughtered turkeys 0 0 0 0 6 2 377 140 0 0 Net exported broilers for 18 2 12 1 0 0 43 5 0 0 slaughter Net imported broilers for 0 0 0 0 0 0 0 0 81 9 slaughter Sheep and goats 15 15 21% 9 8 211% 8 8 25% 677 621 28% 103 93 210% Slaughtered sheep and goats 0 0 2 1 1 0 105 52 15 8 Net exported sheep for slaughter 0 0 0 0 0 0 8 4 6 3 Livestock sheep 15 15 7 7 7 7 565 565 82 82 Livestock horses 28 28 0% 70 70 0% 29 29 0% 168 168 0% 58 58 0% Live weight fish slaughtered 20 20 0% 34 34 0% 14 14 0% 234 234 0% 56 56 0% Slaughtered rabbits 0 0 0% 0 0 0% 0 0 0% 52 8 285% 0 0 0% Total 771 617 220% 2447 1458 240% 524 465 11% 7540 6533 213% 3109 2321 225%

effects on international comparisons of AMU as well as national For example, cattle AW estimates using Canadian data yielded comparisons among animal categories. For example, using PCU as results similar to the Eurostat data. In the future, AW values could the denominator, 2009 AMU in UK cattle is over 3-fold greater than be further improved by collecting international contemporary weight AMU in pigs and poultry (5). In contrast, 2009 AMU in UK cattle data for all animal categories as is currently collected for cattle. is less than the AMU in pigs and poultry when APCU is used as The European Medicines Agency has revised the weight for beef the denominator (calculations not shown). These differences are and dairy cows upwards to 500 kg in their DDDA calculations (2), primarily attributable to including length of life in the calculation, but these weights remain less than those suggested by the Eurostat although for cattle categories and traded fattening pigs, adjusting data (Table I). the weights used in the calculation also had an impact. Given that Second, although PCU is controlling for animal demographics analyses of AMU rely on accurate estimation of animal biomass to which vary among countries and includes standardizing for dif­ enable comparisons among animal categories and between countries, ferences in animal weights, it does not include controlling for differ- these results have significant implications on how AMU is calculated. ences in animals’ lifespans (4). Bondt et al (1) objected, in principal, Amending or replacing the conventional PCU biomass calcula- to the PCU approach of adding weights of breeding stock to those tions with the APCU calculation presented here should be considered of animals slaughtered during the year without accounting for for 2 reasons. First, the APCU uses weight values that are clearly length of life because this approach does not accurately reflect the defined and supported by current data regarding animal weights. population at risk for antimicrobial treatment. The DDDAs used in

238 The Canadian Journal of Veterinary Research 2000;64:0–00

Table II. (continued) Norway Sweden United Kingdom Canada Animal category PCU APCU % D PCU APCU % D PCU APCU % D PCU APCU % D Cattle 231 315 36% 331 456 38% 1678 2395 43% 3925 5490 40% Slaughtered cows 51 75 65 95 204 301 276 407 Slaughtered heifers 0 0 11 21 156 315 215 434 Slaughtered bullocks and bulls 76 89 96 111 537 623 703 816 Slaughtered calves and 2 1 10 6 6 4 41 28 young cattle Net exported cattle for slaughter 0 0 0 0 0 0 324 342 Net exported cattle for fattening 0 0 0 0 0 0 40 27 Net imported cattle for slaughter 0 0 0 0 14 15 0 0 Net imported cattle for fattening 0 0 0 0 3 2 26 24 Livestock dairy cows 102 150 150 222 792 1169 2332 3440 Pigs 32 27 216% 231 103 256% 674 306 255% 1874 793 258% Slaughtered pigs 8 3 192 64 587 196 1352 451 Net imported pigs for slaughter 0 0 0 0 32 11 0 0 Net exported pigs for slaughter 0 0 1 0 0 0 78 26 Net exported pigs for fattening 0 0 0 0 0 0 128 0 Net imported pigs for fattening 0 0 0 0 2 0 0 0 Livestock sows 24 24 38 38 121 121 316 316 Poultry 71 8 289% 76 9 288% 942 131 286% 718 116 284% Slaughtered broilers 71 8 73 8 839 93 620 69 Slaughtered turkeys 0 0 3 1 101 37 140 52 Net exported broilers for 0 0 0 0 2 0 12 1 slaughter Net imported broilers for 0 0 0 0 0 0 254 26 slaughter Sheep and goats 92 80 213% 46 43 26% 1915 1758 28% 56 49 213% Slaughtered sheep and goats 24 12 5 3 308 154 16 8 Net exported sheep for slaughter 0 0 0 0 6 3 21 0 Livestock sheep 68 68 41 41 1601 1601 41 41 Livestock horses 14 14 0% 141 141 0% 520 520 0% 417 417 0% Live weight fish slaughtered 0 0 0% 0 0 0% 197 197 0% 142 142 0% Slaughtered rabbits 0 0 0% 0 0 0% 0 0 0% 1 0 2100% Total 440 443 1% 825 752 29% 5925 5307 210% 7133 7007 22%

the Netherlands (15) and Denmark (16) account for animal lifespan. and contrasting AMU among animals with different lifespans. The The LL used in the APCU calculations are representative of Canadian methodology used to transparently derive APCU will increase the production practices. credibility of this measure of animal biomass, improve comparisons Population correction unit is a purely technical unit of measure- of AMU data among animal categories and countries, and foster ment and not a real value for the animal population biomass that increased acceptance and harmonization of AMU calculations. could potentially be treated with antimicrobial agents (6). By adjust- ing the animal weights and incorporating length of life, the APCU References approach is an improved approximation of the actual animal biomass at risk of antimicrobial treatment. 1. Bondt N, Jensen VF, Puister-Jansen LF, van Geijlswijk IM. Using a calculation that better reflects average weights and Comparing antimicrobial exposure based on sales data. Prev includes length of life for each animal category resulted in values Vet Med 2013;108:10–20. for total annual animal biomass that were significantly different 2. European Medicines Agency 2013. Revised ESVAC reflection than those obtained using a traditional PCU calculation. As a result, paper on collecting data on consumption of antimicrobial APCU provides a reasonable approximation of the actual animal agents per animal species, on technical units of measurement biomass at risk of antimicrobial treatment. Consideration should be and indicators for reporting consumption of antimicrobial given to replacing PCU with APCU in AMU calculations comparing agents in animals. EMA/286416/2012-Rev.1. Available from:

2000;64:0–00 The Canadian Journal of Veterinary Research 239 http://www.ema.europa.eu/docs/en_GB/document_library/ and Application in Antimicrobial Resistance in Bacteria of Scientific_guideline/2012/12/WC500136456.pdf Last accessed Animal Origin. Washington, DC: ASM Press, 2006:375–395. February 16, 2017. 11. DANMAP 2011 — Use of antimicrobial agents and occurrence 3. European Medicines Agency 2016. European surveillance of of antimicrobial resistance in bacteria from food animals, food veterinary antimicrobial consumption. Defined daily doses and humans in Denmark. ISSN 1600-2032. Available from: for animals (DDDvet) and defined course doses for animals http://www.danmap.org/Downloads/~/media/Projekt%20 (DCDvet). EMA/224954/2016. Available from: http://www.ema. sites/Danmap/DANMAP%20reports/Danmap_2011.ashx Last europa.eu/docs/en_GB/document_library/Other/2016/04/ accessed February 16, 2017. WC500205410.pdf Last accessed February 16, 2017. 12. Radke BR. Use of Over-the-Counter Antibiotics in BC Livestock 4. Government of Canada. Canadian Integrated Program for and Poultry, 2002–2012. 2014. Available from: http://www.agf. Antimicrobial Resistance Surveillance (CIPARS) 2014 Annual gov.bc.ca/lhmr/pubs/otcu_amu.pdf Last accessed February 16, Report. Guelph, Ontario: Public Health Agency of Canada, 2016. 2017. 5. UK-VARSS 2013. UK veterinary antibiotic resistance and sales 13. Agriculture and Agri-Food Canada 2013. Red meat sector con- surveillance report. Available from: https://www.gov.uk/govern version factors. Available from: http://www.agr.gc.ca/eng/ ment/uploads/system/uploads/attachment_data/file/440744/ industry-markets-and-trade/statistics-and-market-information/ VARSS.pdf Last accessed February 16, 2017. by-product-sector/red-meat-and-livestock/red-meat-market- 6. European Medicines Agency 2011. Trends in the sales of veteri- information-canadian-industry/carcass-weight/conversion- nary antimicrobial agents in nine European countries (2005–2009). factors/?id=1415860000020 Last accessed February 16, 2017. EMA/238630/2011. Available from: http://www.ema.europa.eu/ 14. Eurostat, 2014. Slaughtering in slaughterhouses — annual data. docs/en_GB/document_library/Report/2011/09/WC500112309. Available from: http://appsso.eurostat.ec.europa.eu/nui/show. pdf Last accessed February 16, 2017. do?dataset=apro_mt_pann&lang=en Last accessed February 16, 7. Montforts MHMM 1999. Environmental risk assessment for vet- 2017. erinary medicinal products. Part 1. Other than GMO-containing 15. Netherlands Veterinary Medicine Authority 2015. Usage of and immunological products. First update. RIVM report 601300 antibiotics in agricultural livestock in the Netherlands in 2014. 001. Available from: http://www.rivm.nl/bibliotheek/rap Trends and benchmarking of livestock farms and veterinarians. porten/601300001.pdf Last accessed February 16, 2017. Available from: http://www.autoriteitdiergeneesmiddelen.nl/ 8. European Medicines Agency 2008. Revised guideline on envi- Userfiles/pdf/SDa-rapporten/def-sda-rapport-ab-2014-engels- ronmental impact assessment for veterinary medicinal products v2-aangepast-102015-incl-erratum.pdf Last accessed February 16, in support of the VICH guidelines GL6 and GL 38. EMEA/ 2017. CVMP/ERA/418282/2005-Rev.1. Available from: http://www. 16. DANMAP 2014 — Use of antimicrobial agents and occurrence ema.europa.eu/docs/en_GB/document_library/Scientific_guide of antimicrobial resistance in bacteria from food animals, food line/2009/10/WC500004389.pdf Last accessed February 16, 2017. and humans in Denmark. Available from: http://www.danmap. 9. Jensen VF, Jacobsen E, Badger F. Veterinary antimicrobial-usage org/~/media/Projekt%20sites/Danmap/DANMAP%20 statistics based on standardized measures of dosage. Prev Vet reports/DANMAP%202014/Danmap_2014.ashx Last accessed Med 2004;64:201–215. February 16, 2017. 10. Grave K, Frokjer VF, McEwen S, Kruse H. Monitoring of antimi- crobial drug usage in animals. In: Aarestrup FM, ed. Methods

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