AHL Newsletter. Animal Health Laboratory. May 1, 2005, AHL User s Guide and Fee Schedule. In this issue:

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1 Animal Health Labratry AHL Newsletter Vlume 9, Number 2, page 9 ISSN In this issue: May 2005 User s Guide & Fee Schedule 9 Tny van Dreumel retries 9 Bisafety & submissins 10 Bacterial cunts f clstrum & bedding 10 Ruminants - BVDV in alpaca 11 Milk Mycplasma bvis culture - r-t PCR 12 Scrapie resistance PrP gentyping 12 Bvine haptglbin 12 Avian - Pet bird pathlgy 13 Swine PRRSV utbreak 14 Hrses - Neurlgic disease surveillance 14 Cmpanin animals - Canine leptspirsis 15 Ehrlichia canis case 16 May 1, 2005, AHL User s Guide and Fee Schedule We hpe that this cmprehensive surce f infrmatin will save yu time and help yu make the mst f ur services. The Guide/Schedule is als available n the Web at We have adjusted sme f ur fees t reflect increasing csts f materials. We have added sme new tests t the test menu and have drpped sme tests that have been used infrequently r that have becme utmded. This is part f ur nging effrt t make the best use f testing resurces t bring yu services that are current and meet tday's needs. Significant changes in the May 1, 2005, Fee Schedule include: Update f ur prfessinal staff cntact list t include: Dr. Jim Fairles, Client Services Veterinarian Dr. Durda Slavic, Bacterilgist ANIMAL Added: HEALTH infrmatin abut wnership f specimens and islates LABORATORY new AHL cntact: inf@ahl.uguelph.ca nte abut discuraging return f carcasses reference intervals fr cavity fluids teststerne and prgesterne interpretatin infrmatin index f tests by species, as well as tests by discipline new mlecular-based (PCR) tests canine and feline heartwrm prfiles t include CBC and bichemistry prfile May 1, 2005 User s Guide and Fee Schedule Fr clients in specialty practice, we wuld be happy t prvide the Fee Schedule fr yu srted by test name, methd, etc., within yur species f primary interest. Please cntact Helen Oliver at (519) , ext We will cntinue t strive t prvide the mst cst-effective testing strategies t help yu deal with animal health challenges, and will keep yu psted n changes in test availability and fees thrugh this quarterly AHL Newsletter - als available n the Web at AHL Tny van Dreumel retires After 38 years f service t the University f Guelph and the Ontari Ministry f Agriculture and Fd, Dr. Tny van Dreumel retired April 30, A 1963 graduate f the Ontari Veterinary Cllege, Tny wrked in the prvincial diagnstic lab in Winnipeg fr 3 years, earned an MSc at the University f Manitba in 1967, and became bard certified in anatmic pathlgy by the American Cllege f Veterinary Pathlgists in Frm 1970 t 1997, he was Scientific Crdinatr f the Veterinary Labratry Services Branch in OMAFRA and a part-time mammalian pathlgist. He served as the President f the Wrld Assciatin f Veterinary Labratry Diagnsticians, and as a site visit auditr fr the American AVLD. Since 1997, he has served as a mammalian pathlgist in the AHL-Guelph lab, and until recently supervised the Mycplasmlgy lab as well. Tny has had a lng and distinguished career in veterinary pathlgy, has numerus publicatins t his credit, and has been an invited speaker at natinal and internatinal meetings. Yur clleagues wish yu a lng and happy retirement Tny. AHL

2 AHL Newsletter, Vlume 9, Number 2 10 Bisafety and sample submissin Jim Fairles, Linda McCaig The packaging f sample submissins sent t the Animal Health Labratry is, fr the mst part, excellent. We d ccasinally receive samples that have been brken, damaged r leaking. It is imperative that we realize the bisafety aspects f such submissins. With the wide array f zntic agents (e.g., Salmnella, Cxiella, Chlamydphila) that practitiners as well as the labratry must deal with, prtectin frm expsure t these agents is very imprtant. By the time the sample gets t the testing areas in ur labratry, many peple have handled it, including yur staff, the transprtatin cmpany, and ur staff. Until an agent is identified r a diagnsis is made, we d nt knw the agent invlved, s all samples must be treated with respect. Occasinally samples arrive that are cntaminated with fecal material, have fecal material in the packing material, leaking jars, leaking plastic bags, etc. Our standard perating prcedures indicate that we must cntinue the unpacking f these submissins in a bisafety cabinet using prtective gear. Besides expsing staff t the agents, this delays the sample arriving at the lab area fr testing. As well, it raises cncerns abut cntaminatin f ther samples in the package, and the pssibility f false-psitive results. If yu suspect a zntic agent, please indicate this n the submissin frm and place the frm in a plastic bag where it is easily accessible n pening the uter packaging. The staff in Specimen Receptin can then be alerted f any ptential cncerns. Fr example, any submissins frm vine abrtins are autmatically pened in a bisafety cabinet due t the ptential presence f Cxiella r Chlamydphila. Thanks in advance fr yur cntinued attentin t packaging f submissins. T serve yu better, we will be cntacting yu if samples are received that raise bisafety cncerns. AHL Bacterial cunts f clstrum and bedding Jim Fairles, Durda Slavic Several articles and presentatins lately have discussed the crrelatin f the cntaminatin f the envirnment and the ptential fr disease. Fr example, calf scurs and clstral bacterial lad, and stall bedding and mastitis. These crrelatins are difficult t make and will require much further research. Intuitively, a clean envirnment wuld lgically seem t reduce the spread f disease. Prtcls have been develped fr sample selectin and quantifying bacterial lad. The AHL is nw ffering clstrum and bedding bacterial cunts. Please cntact us fr the prtcls, sample submissin requirements, and price. AHL AHL Newsletter June Vlume 9, Number 2 Editr: Grant Maxie, DVM, PhD, Diplmate ACVP Editrial Assistant: Ms. Helen Oliver The AHL Newsletter is published quarterly (March, June, September, December) by the Animal Health Labratry, Labratry Services Divisin, University f Guelph. Its missin is t infrm AHL clients and partners abut AHL current activities, and labratry-based animal disease events and disease trends. All material is cpyright Ideas and pinins expressed herein d nt necessarily reflect the pinins f the University r the editr. Articles may be reprinted with the permissin f the editr and with apprpriate credit given t the AHL Newsletter. Mailing address & cntact infrmatin: (please return all undeliverable Canadian addresses t:) Animal Health Labratry Labratry Services Divisin, University f Guelph Bx 3612, Guelph, Ontari, Canada N1H 6R8 Phne: (519) ext ; fax: (519) hliver@lsd.uguelph.ca ISSN Canada Pst Publicatins number Cntributrs t this issue: Frm the Animal Health Labratry: Patricia Bell-Rgers, BSc, MSc Brian Binningtn, DVM, Dip Path, Diplmate ACVP Hugh Cai, DVM, MSc Susy Carman, DVM, Dip SA Med, PhD Jsepha DeLay, DVM, DVSc, Diplmate ACVP Jim Fairles, DVM, MBA Murray Hazlett, DVM, DVSc, Diplmate ACVP Brent Hff, DVM, DVSc Gaylan Jsephsn, DVM, Dip Path Emily Martin, DVM, MSc, Diplmate ACPV Linda McCaig, MLT Beverly McEwen, DVM, MSc, PhD, Diplmate ACVP Davr Ojkic, DVM, MSc, PhD Lis Parker, BSc Janet Shapir, DVM, Dip Path, Dip Eq Surg Durda Slavic, DVM, MSc, PhD Other cntributrs: Nancy Carr, MD, Elginburg, ON Hilary Burgess DVM, Jhn Presctt VetMB PhD, Kris Rutsal DVM DVSc Diplmate ACVP, Pathbilgy, OVC Beth Hanselman DVM, VTH, OVC Our cntinued thanks t all f the nn-authr AHL clerical, technical, and prfessinal staff wh cntribute t the generatin f results reprted in the AHL Newsletter.

3 AHL Newsletter, Vlume 9, Number 2 11 RUMINANTS Bvine viral diarrhea virus (BVDV) in alpaca clinical illness, early pregnancy lss, abrtin and persistent infectin Susy Carman, Nancy Carr, Jsepha DeLay, Murray Hazlett A chrnically ill 3-mnth-ld, lw-birth-weight alpaca cria, with intermittent pneumnia and nasal discharge, and his dam were added t a herd f 52 alpaca in eastern Ontari. The cria died 5 mnths later. Necrpsy examinatin was nt perfrmed. The dam had been n 3 different farms during her pregnancy, tw in Alberta and a different farm in Ontari, where the cria was brn. In the same year, bth Alberta farms experienced reprductive lsses, including abrtin and stillbirth, and all 3 farms fund animals with antibdy t BVDV. Nne f the 4 farms had knwn cntact with sheep r cattle. Three mnths after arrival f the cria at the farm in eastern Ontari, 9 alpacas experienced clinical illness ver a 6-wk perid, with sre muth, anrexia and lethargy. Nne had diarrhea. Subsequently, ne animal died f hepatic lipidsis fllwing a perid f anrexia, 2 alpaca had early pregnancy lss, and 2 abrted. Seventeen f 19 alpaca with varius histries f clinical illness, early pregnancy lss, abrtin, delivery f live brn cria, r clinically unaffected were fund t have antibdy t BVDV type 1 and 2, with titers highest t BVDV type 1. One alpaca fetus was abrted at 5.5 m gestatin, 3 m after the time f clinical illness. Immunhistchemistry (IHC) fr BVDV was psitive fr fetal tissues. Nncytpathic (ncp) BVDV was islated frm the skin f this fetus, but nt frm ther fetal tissues. The ther abrted fetus was nt tested. Twelve cria were later brn uninfected. A single BVDV-infected male cria was brn 9 m after the clinical illness in the herd. His dam had been at 2 m gestatin at the time f clinical illness in the herd, and she was later fund t be BVDV antibdy psitive. The cria had a lw birth weight fr the Ontari farm. Althugh he had chrnic diarrhea and episdic lacrimatin, he cntinued t gain weight well until euthanasia. His fleece lked slightly less dense than that f ther cria f the same age (Figure 1), with lnger straight hair-like fibers frm primary fllicles extending past wllike fibers frm secndary fllicles. Ncp BVDV was islated frm the placenta at birth and at 3 and 26 d f age frm buffy cat cells. Nested reverse transcriptin PCR (nrt- PCR) was psitive fr BVDV type 1 fr EDTA bld and plasma fr bth 3 and 26 d. The cria cntinued t be psitive fr BVDV at euthanasia at 46 d by virus islatin n plasma, serum and multiple tissues, nrt-pcr n plasma and serum, antigen detectin ELISA n serum and ear ntch, and IHC n multiple tissues, including the cerebral crtex, retina, and pinna. The cria remained BVDV antibdy negative t BVDV type 1 and type 2. All ncp BVDV islates frm the abrted fetus and the PI cria were typed as BVDV type 1 using nrt-pcr. A diagnsis f persistent infectin (PI) with BVDV was made. This is the first cnfirmed reprt f a BVDV PI alpaca cria. Hwever, it can be speculated that the chrnically ill cria that came t the farm prir t the utbreak f clinical illness was an unrecgnized PI cria. Figure 1. The persistently infected alpaca cria at 4 wk f age, with lnger straight hair-like fibers frm primary hair fllicles extending abve the wl-like fibers frm secndary hair fllicles. This reprt cnfirms that diagnstic tests designed fr the identificatin f BVDV infectin in cattle can be used effectively t identify BVDV in alpaca. Abrtins due t BVDV have been previusly reprted in llama and in alpaca. Belknap et al. recmmend that BVDV infectin shuld be cnsidered in llamas with abrtin, diarrhea, unthriftiness and weight lss. This recmmendatin shuld nw be extended t alpaca. Persistently infected alpaca cria are an imprtant surce f BVDV infectin fr the alpaca industry, where dams and their cria are cmmnly sent t different farms fr breeding. These PI cria have great ptential t infect naïve alpaca herds. AHL References 1. Belknap EB, et al. Bvine viral diarrhea in New Wrld camelids. J Vet Diagn Invest 2000;12: Gyal SM, et al. Islatin f bvine viral diarrhea virus frm an alpaca. J Vet Diagn Invest 2002;14:

4 AHL Newsletter, Vlume 9, Number 2 12 Milk Mycplasma bvis culture and real-time PCR Hugh Cai, Lis Parker, Patricia Bell-Rgers The AHL nw ffers a real-time PCR assay fr the detectin f Mycplasma bvis in bvine milk and lung tissue samples. We validated the assay by testing 165 individual mastitic milk samples and 53 pneumnic lung samples in parallel with islatin in culture. The sensitivity and specificity were 100% and 99.3% fr milk, and 96.6% and 100% fr lung tissue samples. It has detectin limits similar t culture islatin, but results are available in 1-3 days instead f 5-10 days. The fee fr the real-time PCR is $24/ sample. The fee fr individual milk Mycplasma culture islatin is $10/sample, as f May T achieve the best culture islatin results, please submit fresh milk samples - freezing and thawing can reduce the viable numbers f Mycplasma in milk. Fr further infrmatin please cntact Hugh Cai (ext ) r the Mycplasmlgy lab (ext ). AHL Reference Biddle MK, et al. Effects f strage time and thawing methds n the recvery f Mycplasma species in milk samples frm cws AHL scrapie resistance PrP gentyping Hugh Cai As f May 2005, the fee fr real-time PCR gentyping will be $28/sample fr three cdns (136, 154 and 171). It is wrthwhile t have all three cdns analyzed. A survey n 14,000 British sheep (1) shwed that all three cdns, especially 136 and 171 are related t the susceptibility t scrapie (Table 1). Please cllect and submit a 3-5 ml EDTA bld sample frm each sheep t be tested. Fr further infrmatin please cntact Dr. Hugh Cai at ext r the Mlecular Bilgy/Mycplasmlgy lab at ext AHL Reference 1. Baylis M. Risk f scrapie in British sheep f different prin prtein gentype. J Gen Virl 2004;85(Pt 9): Table 1. Gentype resistance t scrapie Gentype (136, 154, 171 /136, 154, 171) Cases f scrapie per year per millin sheep ARR/ARR 0.0 ARR/ARH 0.0 AHQ/ARH 0.0 ARR/AHQ 0.3 ARR/ARQ 0.4 ARH/ARH 2.0 AGQ/AHQ 5.0 ARQ/ARH 5.2 ARR/VRQ 6.3 ARQ/AHQ 8.7 ARQ/ARQ 36.9 ARQ/VRQ ARH/VRQ VRQ/VRQ Haptglbin in rutine analysis f bvine serum Brent Hff Haptglbin (Hp) has prven t be a very valuable aid in the diagnsis f many bvine diseases. Hp serum levels are increased pstpartum, especially in cws that develp retained placenta, mastitis, r displaced abmasum (DA). Increased haptglbin is part f the acute phase reactin t inflammatin, resulting frm the release f cytkines frm leukcytes at the site f injury, especially in diseases with acute inflammatin, such as pneumnia, mastitis, enteritis, and endmetritis. With very high levels f Hp, it is likely that these specific rgans are invlved. Hp levels crrelate well with ttal serum prtein levels and negatively with albumin and AST activity. Fuerll et al. fund the highest Hp cncentratins in cws and calves with pneumnia (range g/l), fllwed by cws with DA (range g/l), as well as cws with mastitis and endmetritis (range g/l) (reference interval g/l). Cattle ill with DA plus an additinal prblem had the fllwing Hp cncentratins (g/l median): enteritis 1.10, brnchitis 0.95, nephritis 0.90, and laminitis Interestingly, the leukcyte cunt did nt crrelate with the Hp cncentratin. These wrkers fund that Hp was useful acutely, but had limited prgnstic value, as the animals with infectin recvered with treatment. In rutine testing f cattle, elevated serum Hp indicates serius inflammatin (pneumnia, mastitis, enteritis). Nte that Hp increases during acute parturient stress, and shuld nt be used as an indicatr f inflammatin in the first week after calving. Remember that Hp is affected by hemlysis, and samples must be handled carefully. AHL Reference Fuerll M, et al. 5 th Internatinal Cllquium n Animal Acute Phase Prteins. Dublin, 2005.

5 AHL Newsletter, Vlume 9, Number 2 13 AVIAN Pet bird pathlgy submissins - tips fr veterinarians Emily Martin, Brian Binningtn, Jan Shapir The Animal Health Labratry has necrpsy and histpathlgy services available fr pet birds. These prcedures are valuable in supprting the veterinarian s clinical evaluatin. Samples submitted can include tissue bipsies, and tissues r the whle carcass f a deceased bird. In cases where a necrpsy is required, yu can submit the entire carcass fr a full pst-mrtem examinatin, r yu can perfrm the necrpsy yurself and submit tissue samples t the AHL. Bipsies If yu are investigating a feather abnrmality, it is imprtant t submit a sample f skin that includes a pulp feather. Sending a feather alne des nt prvide enugh tissue t evaluate histlgically. If yu have a suspect case f prventricular dilatin disease (PDD) yu can submit a crp bipsy. Please be aware that nly abut 50% f crp bipsies are cnsidered diagnstic fr PDD. Pst-mrtem examinatins If yu decide t submit a bird fr pst-mrtem examinatin, we recmmend that, as sn as the bird dies, yu dampen it with cld water and place it in a refrigeratr t prmptly chill the carcass. This will ffset the nrmal high bdy temperature in these species that cntributes t early pst mrtem change. If yu cannt submit the carcass shrtly after death, r if there is a prlnged wait fr the wner t decide if they want further testing dne, then the carcass can be frzen. Hwever, please nte that this is nt the ideal situatin as this will cause freeze/thaw artifacts in the tissues and make histlgic evaluatin mre difficult. If the bird is very small (e.g., finch), yu can retract the keel, bserve fr grss lesins, take samples f a few fresh tissues t hld frzen at yur clinic (see list belw), pen the skull t expse the brain, and then place the entire bird in the frmalin jar. (If necessary, the tail feathers and wings can be remved.) This allws us access t all f the tissue we may need fr histlgic evaluatin. When shipping whle carcasses t the AHL, please ship them n frzen ice packs sn after death. Ontari practitiners can ship t the AHL at n charge by Purlatr (use Purlatr accunt n ). If yu decide t d the necrpsy yurself, please submit a wide selectin f tissues fr histlgic evaluatin. As histlgic results may indicate the need fr additinal testing (e.g., bacterilgy, virlgy, txiclgy, Chlamydphila assays) we suggest that yu als save tissues frzen at yur clinic (freeze in a chest freezer). Depending n what yu see grssly, here are a few suggestins fr tissues t submit fr histlgy: frm all birds (minimum) - brain, heart, trachea, lung, liver, spleen, kidney, crp, prventriculus, ventriculus, pancreas, and intestines, plus any ther affected tissues detected at necrpsy. In additin t the minimum: frm juvenile birds - bursa, r claca with bursa attached. frm birds that are suspicius fr PDD - crp, prventriculus/gizzard (whle, cmbined), 3-4 pieces f small intestine, adrenal glands (cranial prtin f the kidney), and nervus system tissues as listed belw. frm neurlgic cases - brain (in situ), spinal crd (in situ), and peripheral nerves (brachial plexus, sciatic). Tissues t hld frzen at yur clinic can include liver, kidney, intestines and lung. The cst f a case is based n the number f birds submitted (if a full necrpsy case) and/r the number f slides cut. Yu can submit mre than ne histlgy jar t ensure the prper rati f 10 parts frmalin t 1 part tissue. Fees are as fllws: pst mrtem examinatin - $60 per pet bird; histlgy - $45 n a necrpsy case fr 1-5 slides, then $5/additinal slide; histlgy - $45 n a mail-in case fr 1-3 slides, then $5/ additinal slide. If yu have any questins regarding sample submissin, please call the labratry and ask t speak t ne f ur avian pathlgists. AHL Samples submitted can include tissue bipsies, and tissues r the whle carcass f a deceased bird Need submissin frms? Revised AHL submissin frms were distributed in January. The new frms are available n the Web in pdf frmat at and may be printed. Alternatively, fax yur request t us at (519) , and we will custmize the frms that yu require with yur clinic default infrmatin.

6 AHL Newsletter, Vlume 9, Number 2 14 SWINE PRRSV utbreak cntinues in suthwestern Ontari Susy Carman, Beverly McEwen, Gaylan Jsephsn, Jim Fairles In the Dec/04 AHL Newsletter, we dcumented the fall 2004 PRRSV utbreak in swine herds in suthwestern Ontari. Figure 1 is an updated epidemic curve, shwing the utbreak cntinuing int the winter f Numbers f cases submitted t the AHL fr PRRSV diagnsis using PCR increased frm 201 in the Oct-Dec 2004 interval t 323 fr the Jan-March 2005 interval, with the ttal number f cases fund t be psitive increasing frm 108 t 172. Even thugh the verall number f psitive cases increased, there was a very small decrease in the 3- mnth average percent PRRSV-psitive cases frm 55% t 53%. These data d nt include mnitring cases, in which semen was the sample submitted. Gene sequence analysis f the ORF5 envelpe gene fr PRRSV fr 100 strains identified frm 2003 t 2005 shws the utbreak nt t be frm a single surce (except fr integrated lps), with viruses distributed ver 19 phylgenetic branches, including 2 vaccine-like grups. AHL Number f cases Ja Apr- Jul-Sep Oct- 2002Ja Apr- Jul-Sep Oct- 2003Ja Apr- Jul-Sep Oct- 2004Ja Apr- Jul-Sep Oct % 50% 40% 30% 20% 10% 0% Figure 1. PRRSV PCR submissins tested by the AHL, Jan 1, Mar 31, 2005, with semen mnitring remved. % psitive cases Psitive cases Negative cases % psitive cases HORSES Equine viral neurlgic disease surveillance 2004 Jsepha DeLay, Beverly McEwen A surveillance prject t identify Ontari hrses infected with eastern equine encephalitis virus (EEEV) r West Nile virus (WNV) was carried ut frm Aug-Dec 2004, funded jintly by the Ontari Ministry f Agriculture and Fd (OMAF) and the Ontari Ministry f Health and Lng- Term Care, with the cperatin f CFIA. The 87 equine neurlgic cases submitted t the AHL cnsisted f 64 live hrses fr which serlgy was perfrmed fr WNV and EEEV, and 23 dead hrses frm which either carcasses r tissues were submitted fr necrpsy, histpathlgy, and EEEV and WNV PCR testing n brain. Immunhistchemistry (IHC) fr viral antigen was perfrmed n selected cases if fresh r frzen tissue was nt available fr PCR testing. Our interim reprt in the Dec 2004 AHL Newsletter described 10 Ontari hrses acutely infected with WNV between Aug-Oct, as identified by psitive WNV IgM ELISA and cmpatible clinical signs. The majrity f these hrses riginated frm the Nrth Bay / Sturgen Falls / Bracebridge area, with ther cases scattered thrughut suthwestern Ontari, and nne f the hrses had a knwn histry f recent travel. Of the 10 hrses diagnsed in 2004, 7 were nt vaccinated against WNV, 1 had an unknwn vaccinatin histry, 1 had received primary but nt bster vaccinatin, and 1 had been vaccinated less than 2 weeks prir t the nset f clinical signs. Three f the WNV-infected hrses died r were euthanized, and 6 survived the acute infectin. One case was lst t fllw-up. N additinal WNV cases were identified in Nv-Dec In 2003, the number f WNV-infected hrses in Ontari was similar, with 8 animals identified. The relatively lw numbers f cases in 2003 and 2004 are in sharp cntrast t the 70 WNVpsitive clinical cases diagnsed in Ontari in Fllwing predictins, the number f EEEVinfected hrses in Ontari this year was much lwer than the 10 cnfirmed and suspected cases identified in In 2004, 2 hrses were diagnsed with acute EEEV infectin, and bth animals were euthanized r died. One hrse, riginating frm eastern Ontari and with n histry f EEEV vaccinatin, was identified with acute EEEV infectin based n psitive EEEV IgM ELISA. In Nv 2004, an additinal hrse riginating frm suthwestern Ontari was identified with acute EEEV infectin based n IHC identificatin f EEEV antigen within suspicius histlgic lesins. This hrse als had histlgic lesins cnsistent with cncurrent wbbler syndrme, which cmplicated the clinical presentatin; the vaccinatin histry f this hrse was unknwn. AHL

7 AHL Newsletter, Vlume 9, Number 2 15 COMPANION ANIMALS Canine leptspirsis in Ontari mre cases in 2004 Beverly McEwen, Davr Ojkic, Jhn Presctt Increased serlgical submissins fr Leptspira spp. (Table 1) reflect the cncerns f ur clients abut canine leptspirsis. Table 1. Percent canine serlgical diagnses, Ontari dgs, by year, fr all Leptspira servars. Year Number f submissins Negative* n (%) Suspicius** n (%) Psitive*** n (%) (59) 6 (14) 11 (26) (66) 10 (18) 8 (14) (45) 20 (13) 63 (41) (47) 75 (35) 37 (17) (33) 91 (43) 49 (23) (29) 158 (37) 143 (34) (31) 282 (35) 267 (34) * titer < 1:40, ** titer 1:80 t 1:160, *** titer > 1:320 There were mre serpsitive dgs in 2004 than in previus years, which may in part be the result f submissins that dubled ver 2003, since the frequency f leptspirsis has remained at 34%. Hwever, the mild, wet fall f the last 2 years and 2000 may be assciated with the relative increase f leptspirsis and the data suggest that leptspirsis cntinues t increase in dgs in Ontari. Fr the past few years, leptspirsis has ccurred thrughut the year, althugh mst cases ccur in the fall. The largest number f psitive cases (n = 44, 16%) ccurred in Nvember Mst cases (63%) in 2004 ccurred between August and December, with fewer cases (21%) between April and July, and ccasinal cases (16%) between January t March. Serpsitive dgs were frm all regins in Ontari. The frequency f serpsitivity t L. autumnalis and L. bratislava have been cnsistently greater than ther servars since 2000 (Table 2). Serpsitivity t mre than ne servar ccurred in 180 f 267 (67%) psitive cases, cmpared t 61% in One dg was psitive t all servars, and 4 dgs were psitive t all servars except fr L. hardj. A cnsistent pattern f serpsitivity t individual servars was nt evident, s it is difficult t implicate Table 2. Percent serpsitivity f canine sera t varius Leptspira spp. servars, specific servars frm the data. The brad serpsitivity bserved in these sera is prbably a reflectin f the invlvement f several different servars in early canine leptspirsis as well as the brad crss-reactivity f IgM antibdies. IgM is the dminant immunglbulin in the early humral immune respnse. Nevertheless, the cnsensus is that resurging leptspirsis in dgs is the result in mst cases f servar gripptyphsa, acquired frm raccns, and t a lesser extent servar pmna acquired frm skunks, and servar bratislava acquired frm dgs (mstly), hrses, pigs and pssibly ther surces. It has still nt been reslved by islatin studies whether the high frequency f autumnalis serpsitives represents genuine infectin with this servar r is the result f crss-reacting antibdies. The high frequency f bth autumnalis and bratislava serpsitivity may be because these servars are nt currently included in the canine vaccines and therefre may be prprtinately increasing as causes f canine leptspirsis. Interpretatin f titers is als affected by immunizatin; dgs immunized with servars canicla and icterhaemrrhagiae may shw titers up t 320 r 640 in the first mnths after immunizatin (when f curse they wuld be prtected against these servars). Similar relatively lw serlgical respnses are likely als t ccur in dgs immunized with the newer vaccines cntaining servars L. gripptyphsa and L. pmna. Clients are reminded that a single serum sample, if negative, is inadequate t rule ut leptspirsis since serum btained early in the curse f disease may be negative fr antibdies, as is true f any acute infectin. Althugh nt as dramatic as the surge f canine leptspirsis in 2000, 2004 data shw that canine leptspirsis caused by several servars has nw becme well established thrughut Ontari and, perhaps in part because f increased awareness, has becme a mre cmmn diagnsis. Leptspirsis in animals is f public health cncern and is an infectin that can be cntrlled by vaccinatin. Certain vaccine manufacturers may be in prcess f including servar bratislava in the canine vaccine. AHL Servar % % % % % % % range * L. autumnalis ,480 L. bratislava ,480 L. gripptyphsa ,480 L. pmna ,480 L. icterhaemrrhagiae ,240 L. canicla ,240 L. hardj ** 320-1,280 - = serlgy nt dne, * values are the reciprcal f the titer, ** fewer cases tested fr L. hardj (n=559)

8 AHL Newsletter, Vlume 9, Number 2 16 Ehrlichia canis identified in a canine neurlgy case Hilary Burgess, Brent Hff, Kris Rutsal, Beth Hanselman In February 2005, a yung adult, castrated male Pug was referred t the Ontari Veterinary Cllege because f ataxia. The wners had acquired the dg just ver a year prir t presentatin. He had a histry f intermittent ataxia with prgressin f clinical signs ver the previus 2 wk. The patient s travel histry was extensive, with the first 18 m f his life spent in Csta Rica, fllwed by a trip t Arizna 1 m prir t referral. On presentatin he had plyuria, plydipsia, decreased appetite, mild enlargement f submandibular lymph ndes, and bilateral vestibular ataxia. Hematlgy revealed mderate t marked nnregenerative anemia, marked thrmbcytpenia, mderate increase in band neutrphils, mild lymphpenia, and mild mncytsis. On serum bichemistry, there was mderate hyperglbulinemia, mderate hypalbuminemia, mildly increased bilirubins, a mderate increase in alkaline phsphatase, and a marked increase in alanine amintransferase. Bth pre- and pst-prandial bile acid levels were elevated (pre: 14 µml/l, reference interval 0-6; pst: 92 µml/l, RI 0-20). CSF analysis revealed markedly increased prtein cncentratin, and mderate predminantly nnsuppurative inflammatin. An Ehrlichia sp. mrula was nted in a single neutrphil during cytlgical evaluatin f the CSF (Fig. 1). Inclusins were als nted in neutrphils, mncytes and platelets n a buffy cat preparatin f a secnd bld sample. The patient s erythrid and megakarycytic lines were hypplastic n bne marrw evaluatin. An Ehrlichia canis ELISA (Snap 3Dx assay, IDEXX Labratries) was psitive. PCR was psitive fr E. canis and negative fr E. ewingii, Anaplasma phagcytphila and Anaplasma platys. While ccasinal platelets cntained inclusins resembling A. platys, this diagnsis was nt supprted by PCR analysis, and at this time the ability f E. canis t infect platelets has nt been dcumented. Ehrlichiae are gram-negative, plemrphic ccci that infect bld cells and replicate within membrane-bund vacules. Ehrlichia canis is transmitted by Rhipicephalus sanguineus (the brwn dg tick), requiring hr f tick attachment befre successful transmissin. E. canis has a wrldwide distributin, therefre it is difficult t identify the lcatin f riginal infectin in this case. Because ehrlichisis is a multisystemic disease, the clinical signs can vary greatly. Ehrlichiae can be present during the acute, subclinical r chrnic phases f disease. Mild, vague illness usually ccurs within 1-3 wk f infectin. At this pint, the mst cmmnly nted change in the clinical pathlgy is thrmbcytpenia resulting frm platelet cnsumptin in the face f vascular inflammatin, immune-mediated platelet destructin, and splenic sequestratin f platelets. After the acute phase f infectin, the infectin either reslves r enters a subclinical phase lasting frm weeks t years. Diagnsis f the disease is mst cmmnly made during the chrnic phase f infectin. As nted in ur case, clinical signs and clinical pathlgy changes tend t be nn-specific in the chrnic phase and include thrmbcytpenia, mild t mderate nn-regenerative anemia, hyperglbulinemia, hypalbuminemia, and increases in ALP and ALT. Bne marrw aspirates revealing hypplasia, as in ur case, generally suggest chrnic infectin. Clinical signs f ehrlichisis can include multiple neurlgic deficits including vestibular ataxia, as well as the nnspecific signs f lethargy, anrexia and weight lss. The neurlgical signs in this disease are secndary t vasculitis f the meningeal vessels and/r hemrrhage int the CNS. A definitive diagnsis can be achieved by visualizatin f intracellular Ehrlichia mrulae. This ccurs mre frequently in acute infectin than in the subclinical r chrnic phases. While the agent respnsible fr infectin can ften be presumed based n the cell type infected, this case is an excellent example f when cell trpism is nt abslute - mrulae were nted within bth mnnuclear cells and neutrphils. While E. canis generally infects mnnuclear cells, the PCR analysis ruled ut the cmmn agents that infect neutrphils, therefre the mrulae within the neutrphils were likely als E. canis. Se- Figure 1. Single Ehrlichia mrula in a neutrphil in a CSF sample. rlgy is an additinal test that is cmmnly used fr the diagnsis f ehrlichisis. Hwever, it shuld be nted that a psitive titer nly indicates expsure, nt necessarily active infectin. In additin, a negative titer shuld nt rule ut ehrlichisis as clinical signs can appear prir t a detectable antibdy respnse. Newsletter Figures are psted in full clr in the Web versin f the Newsletter -