kvÄllssymposium 2008 vaccinering av hund och katt

Intervet AB Box 6103 102 33 StockholmTel 08–522 216 60www.intervet.se

Kennelhosta – vad är det?Jessica Ingman, Bitr. Statsvet., Sekt. f. häst, hund och katt, SVA

Nobivac KC® vet. – nytt vaccin frånIntervet/Schering-Plough Animal HealthAgneta Gustafsson, Teknisk chef, I/SPAH

Svenska erfarenheter avNobivac KC® vet. Anette Johansson, Distriktsvet., Kiruna

Presentation av SVAs rabiestiter-studie Louise Treiberg-Berndtsson, Bitr. Stats-vet., Sekt. f. virologisk diagnostik, SVA

Kort bensträckare

Nobivac® Tricat Novum vet. – nytt vaccin från Intervet/Schering-Plough Animal HealthAnna-Karin Lieber, Produktchef, I/SPAH

Vaccination av hund och katt 2008Ulrika Windahl, Bitr. Statsvet., Sekt. f. häst, hund och katt, SVA

Paneldiskussion med föredrags-hållarna

KVÄLLSSYMPOSIUM 2008Vaccinering av hund och kattStockholm 25 november – Göteborg 26 november – Lund 27 november

Kennelhosta – vad är det? Bitr. statsveterinär Jessica Ingman Enheten för djurhälsa och antibiotikafrågor, Statens veterinärmedicinska anstalt

Kennelhosta • Är ett samlingsbegrepp för kikhosteliknande symtom hos hund. • Akut, mycket smittsam luftvägsinfektion som karakteriseras av akut insättande

hostattacker (kväljningar/slem/nosflöde). • Multifaktoriell etiologi. • Graden av symtom kan variera beroende på vilket eller vilka agens som är

inblandade. • Infektion med enbart ett agens ger oftast lindrigare symtom. • Prognosen god vid okomplicerade infektioner, men immuniteten kan vara

kortvarig.

Smittspridning • Infektion sprids snabbt och effektivt bland hundar som hålls tillsammans i

större grupper. • Smitta direkt nos-nos-kontakt, hosta/nysningar, indirekt via händer/föremål. • Kliniska symtom uppträder vanligtvis inom 3-5 dagar efter infektion, även om

inkubationstid för kennelhosta anges till 3-10 dagar. • För de vanligaste virala agens kan utsöndring av virus ske upptill två veckor

efter infektion. • För B. bronchiseptica kan dock utsöndring pågå betydligt längre tid.

Etiologi • Virus

- Hundens parainfluensavirus typ 2 (CPiV-2) - Hundens adenovirus typ 2 (CAV-2) - Hundens herpesvirus typ 1 (CHV-1) - Canine respiratory corona virus (CRCoV) - (valpsjuka…)

• Bakteriella agens

- Bordetella bronchiseptica - Mykoplasma-arter, streptokocker, pasteurella-arter, koliforma bakterier och

pseudomonas-bakterier har också påvisats hos hostande hundar, men då främst som sekundärinfektion eller eventuellt bifynd.

• Hundens parainfluensavirus typ 2 och Bordetella bronchiseptica är de agens som

enligt litteraturen oftast isoleras från hundar med infektiös trakeobronkit, men flera andra virus och bakterier kan påverka symtombild och sjukdomsförlopp.

Hundens parainfluensavirus typ 2 • CPiV-2, canine parainfluenza virus • RNA-virus, familjen Paramyxoviridae • Det vanligaste virus som isoleras från luftvägarna hos hund i samband med

kennelhostesymtom. • Orsakar vid ensam infektion vanligen en relativt lindrig infektion i övre

luftvägarna med kortvarig, övergående hosta och minimal allmänpåverkan. • (Katter kan bli subkliniskt infekterade och utsöndra virus – okänt dock om

betydelse för smittspridning hos hund.)

Hundens adenovirus typ 2 • Virus som är nära släkt med det virus (CAV-1) som orsakar HCC, men CAV-2

angriper huvudsakligen luftvägarna. • Infektion orsakar vanligen inga påtagliga kliniska symtom. • De flesta hundar är idag vaccinerade mot CAV-2 (ger skydd mot CAV-1 som

orsakar HCC).

Hundens herpesvirus typ 1 • Är ett virus som också angriper de övre luftvägarna hos hund, men som

vanligtvis inte orsakar några tydliga kliniska symtom. Anses inte längre vara någon väsentlig orsak till kennelhosta.

• (Har betydelse vid dödlig infektion hos nyfödda valpar.)

CRCoV • Canine respiratory corona virus • Relativt nyupptäckt virus som isolerats från hundar med kennelhostesymtom. • Är olikt från CCoV – den enteriska formen • Har oftast associerats med lindriga kliniska symtom.

Valpsjukevirus • CDV, canine distemper virus • Kan ge respiratoriska symtom och har därför tidigare beskrivits i samband med

kennelhostekomplexet, men har idag mindre betydelse pga. utbredd vaccination.

B. bronchiseptica • Gramnegativ, aerob bakterie. • Kan förekomma i luftvägarna även hos friska individer. • Hundratals olika isolat påvisade med olika virulens och patogenicitet. • I experimentella studier orsakar infektion med enbart B. bronchiseptica typiska

symtom på kennelhosta framför allt hos unga hundar. • Bakterien kan utsöndras från övre luftvägarna i 3-4 månader efter

genomgången infektion.

Sammanfattningsvis om kennelhosta • Kennelhosta är ett komplext komplex - flera möjliga agens, multifaktoriell

etiologi. • Patogena agens kan vara olika vid olika utbrott. • En hund som haft kennelhosta är inte skyddad mot att få sjukdomen igen vid

ett senare tillfälle.

Vet. Res. 38 (2007) 355–373 355c© INRA, EDP Sciences, 2007DOI: 10.1051/vetres:2006058

Review article

Canine respiratory viruses

Canio B*, Vito M

Department of Animal Health and Wellbeing, Faculty of Veterinary Medicine of Bari, Italy

(Received 27 April 2006; accepted 28 August 2006)

Abstract – Acute contagious respiratory disease (kennel cough) is commonly described in dogsworldwide. The disease appears to be multifactorial and a number of viral and bacterial pathogenshave been reported as potential aetiological agents, including canine parainfluenza virus, canineadenovirus and Bordetella bronchiseptica, as well as mycoplasmas, Streptococcus equi subsp.zooepidemicus, canine herpesvirus and reovirus-1,-2 and -3. Enhancement of pathogenicity by mul-tiple infections can result in more severe clinical forms. In addition, acute respiratory diseasesassociated with infection by influenza A virus, and group I and II coronaviruses, have been de-scribed recently in dogs. Host species shifts and tropism changes are likely responsible for theonset of these new pathogens. The importance of the viral agents in the kennel cough complex isdiscussed.

kennel cough / respiratory disease / dogs / viruses

Table of contents

1. Introduction ...................................................................................................... 3552. Canine adenovirus type 2 ..................................................................................... 3563. Canine herpesvirus ............................................................................................. 3574. Canine influenzavirus .......................................................................................... 3595. Canine parainfluenzavirus .................................................................................... 3606. Canine reovirus .................................................................................................. 3627. Canine respiratory coronavirus .............................................................................. 3638. Pantropic canine coronavirus................................................................................. 3649. Conclusions ...................................................................................................... 365

1. INTRODUCTION

Infectious tracheobronchitis (ITB) orkennel cough is the term used by veterinar-ians to describe an acute, highly contagiousrespiratory disease in dogs affecting thelarynx, trachea, bronchi, and occasionallythe nasal mucosa and the lower respiratorytract [6].

* Corresponding author:[email protected]

Mild to severe episodes of cough andrespiratory distress are characteristic clin-ical features recognized in affected dogs.ITB has worldwide distribution and is rec-ognized as one of the most prevalent infec-tious diseases of dogs. The disease is fre-quently described in dogs housed in groupsin rehoming centers and boarding or train-ing kennels.

Two clinical forms of ITB have been de-scribed. The uncomplicated form is most

Article available at http://www.edpsciences.org/vetres or http://dx.doi.org/10.1051/vetres:2006058

http://www.edpsciences.org/vetres

http://dx.doi.org/10.1051/vetres:2006058

356 C. Buonavoglia, V. Martella

common and is characterized as a dry,hacking cough, often in association withgagging and retching behavior. The dogsare affected by a self-limiting, primarilyviral infection of the trachea and bronchi.A complicated form of ITB is describedin puppies or immuno-compromised dogs.In the complicated forms, secondary bac-terial infections and involvement of pul-monary tissue overlaps the viral process.The cough is associated with mucoid dis-charges. The condition may progress tobronchopneumonia and, in the most severeinstances, to death [6].

Multiple agents, bacterial and viral, areimplicated in the aetiology of ITB. Co-isolation of viral and bacterial pathogensis frequent, while experimental infectionswith single pathogens may result in sub-clinical or mild forms of disease, suggest-ing a multi-factorial pathogenesis. Manyagents likely play a role in ITB, suchas canine parainfluenza virus [2], canineadenovirus [55], Bordetella bronchisep-tica [18], and mycoplasmas [36, 128].Streptococcus equi subsp. zooepidemicus,has been associated with severe to fa-tal respiratory forms in dogs alone or inmixed infections [36, 61, 173]. Recently,outbreaks of influenza A virus, initiallymisdiagnosed as ITB, have been reportedin the USA [45,173]. In addition, novel ca-nine coronaviruses, a pantropic variant ofCCoV type II [29] and the canine respira-tory coronavirus virus, CRCoV [60], havebeen detected from the respiratory tractof either symptomatic or asymptomaticdogs. Canine herpesvirus and reovirus-1,-2,-3 have rarely been reported from dogswith kennel cough but are not thoughtto play a major role in the disease com-plex [86,97]. Vaccines are available againstsome of these infectious agents but regularvaccination in kennels often fails to preventITB.

An overview of the viral agents thathave been associated with ITB in dogs,

with particular regards to newly describedviruses, is reported.

2. CANINE ADENOVIRUS TYPE 2

Canine adenovirus type 2 (CAV-2) de-termines unapparent to mild infection ofthe respiratory tract and is regarded as oneof the causes of the common widespreadITB [154]. CAV-2 has also been implicatedin episodes of enteritis [76, 98] and hasbeen detected in the brain of dogs withneurological signs [19].

The virus was first detected in 1961,in Canada, from dogs affected by laryngo-tracheitis [55]. The isolate, strain TorontoA26/61, was characterized as an aden-ovirus, and was initially considered to bean attenuated strain of canine adenovirustype 1 (CAV-1). Subsequently, structuraland antigenic differences were observedand strain A26/61 was proposed as theprototype of a distinct canine adenovirus,designated as type 2 (CAV-2) [65, 75, 104,107, 152, 172]. CAV-1 and CAV-2 werefound to be genetically different by restric-tion endonuclease analysis [10, 75] and byDNA hybridization [106]. The completesequence analysis of both the CAV-1 andCAV-2 genome has revealed about 75%nucleotide identity [49, 114]. AlthoughCAV-1 and CAV-2 are related geneticallyand antigenically [109,168], they have dif-ferent tissue tropism. Vascular endothelialcells and hepatic and renal parenchymalcells are the main targets of CAV-1, whilethe respiratory tract epithelium and, to alimited degree, the intestinal epithelium,are the targets of CAV-2 [3, 140, 153]. Inaddition, the two types display differenthemagglutination patterns [105].

Infection with CAV-2 appears to bewidespread in dogs that are not immune toCAV-1 or CAV-2. CAV-2 was isolated from34 out of 221 throat swabs of pups with andwithout respiratory signs that were takento a veterinarian for vaccination [4]. Like-wise, pups in pet shops and in laboratory

Canine respiratory viruses 357

animal colonies were found to carry CAV-2in the respiratory tract [6,20]. By converse,CAV-2 was not detectable in dogs vacci-nated in a rehoming center [61].

The host range of CAV-2 includesa broad number of mammalian species.Wild-life animals may be a source of in-fection for domestic dogs. The overallprevalence of antibodies to canine aden-oviruses in European red foxes (Vulpesvulpes) in Australia was 23.2% withmarked geographical, seasonal and age dif-ferences [134], while the prevalence of an-tibodies was 97% in Island foxes (Urocyonlittoralis) in the Channel Islands, Califor-nia [70]. Antibodies to CAV-2 were alsodetected in free-ranging terrestrial carni-vores and in marine mammals in Alaskaand Canada, including black bears (Ursusamericanus), fishers (Martes pennanti),polar bears (Ursus maritimus), wolves(Canis lupus), walruses (Odobenus ros-marus) and Steller sea lions (Eumetopiasjubatus) [30, 120, 147].

The route of infection of CAV-2 is oro-nasal. The virus replicates in non-ciliatedbronchiolar epithelial cells, in surface cellsof the nasal mucosa, pharynx, tonsillarcrypts, mucous cells in the trachea andbronchi in peribronchial glands and type2 alveolar epithelial cells. In addition tothese tissues, the virus can be isolatedfrom retropharyngeal and bronchial lymphnodes as well as from the stomach andthe intestine. The peak of replication isreached by 3–6 days post infection. Sub-sequently, virus loads rapidly decline, inrelation to the production of antibodies,and CAV-2 usually can not be isolated by9 days post infection. Respiratory signsare consistent with damage of bronchialepithelial cells. There may be evidenceof narcotising bronchitis or bronchiolitisand of bronchiolitis obliterans. Infection oftype 2 alveolar cells is associated with in-terstitial pneumonia [5, 6, 9, 35, 47, 90].

Dogs exposed to CAV-2 alone rarelyshow spontaneous disease signs, although

lung lesions can be extensive. When addi-tional bacterial or viral agents are involved,the ITB complex can be observed [5, 6].

Antibodies to CAV-2 antigens havebeen demonstrated by hemagglutination-inhibition, agar gel diffusion, virus precipi-tation, complement fixation and by neutral-ization [90]. Protection appears to correlatewith the neutralizing antibody levels [3,8].

Nasal or throat swabs appear to be suit-able for virus isolation. Primary dog kid-ney cells have been used successfully forisolation and cultivation of CAV-2 [55].However, a variety of cell lines are simi-larly susceptible to CAV-2 and to CAV-1[171]. Demonstration of CAV-2 antigen byimmunofluorescence in acetone-fixed lungsections or tissue imprints is used for di-agnosis of CAV-2. A polymerase chainreaction (PCR) assay has been developedto detect canine adenoviruses and to dis-tinguish between CAV-1 and CAV-2 [82].

Modified live CAV-2 vaccines provedto be highly effective in reducing the cir-culation of CAV-2 in canine populations.Dogs vaccinated with CAV-2 develop im-munity to both CAV-1 and CAV-2 [3, 8].In a similar fashion, dogs vaccinated withCAV-1 develop immunity to both CAV-1and CAV-2 [43]. However, the use ofCAV-2 for immunization of pups againstboth canine adenovirus types has elimi-nated safety side-effects encountered withCAV-1 vaccines, i.e. the occurrence of oc-ular lesions [24,48,90]. Maternally-derivedantibodies in pups may prevent active im-munization after vaccine administration upto the age of 12-16 weeks [8]. Vaccineadministration by the intranasal route hasbeen proposed to overcome the interfer-ence of maternal antibodies [8], but prod-ucts for intranasal vaccination are not mar-keted.

3. CANINE HERPESVIRUS

Canine herpesvirus (CHV) is a mem-ber of the Alphaherpesvirinae subfamily


of the Herpesviridae [159]. CHV was firstdescribed in the mid 1960s from a fatalsepticemic disease of puppies [32]. Infec-tion of susceptible puppies of less thantwo weeks of age may result in fatal gen-eralized necrotizing and hemorrhagic dis-ease, while pups older than two weeks andadult dogs often do not show any clinicalsigns [32]. Infection in older dogs appearsto be restricted to the upper respiratorytract [7]. CHV is also transmitted transpla-centally, resulting in fetal death [77].

Serological surveys have shown a rela-tively high prevalence of CHV in house-hold and colony-bred dogs. The prevalenceof antibodies in dogs was 88% in Eng-land, 45.8% in Belgium, and 39.3% inthe Netherlands [129, 133, 135]. Serologi-cal studies in Italy have revealed a simi-lar prevalence in kennelled dogs (27.9%),while the prevalence was lower in pets(3.1%) [142].

The host range of CHV is restricted todogs [91]. However, antibodies to CHVhave been detected in the sera of Europeanred foxes (Vulpes vulpes) in Australia [134]and Germany [156] and in sera of NorthAmerican river otters (Lontra canadensis)from New York State [88], while a CHV-like virus has been isolated from captivecoyote pups [64].

CHV appears to be a monotypic virus,as defined by antigenic comparison of var-ious isolates [32, 122]. The gene structureof CHV has yet to be determined, sinceno CHV strain has been completely se-quenced and only a few genes have beenidentified [73, 96, 132]. Restriction map-ping, southern blot hybridization and se-quence analysis have shown that the over-all structure of CHV resembles those ofother alphaherpesviruses and that CHV isgenetically related to feline herpesvirus(FHV-1), phocid herpesvirus 1 and to theequid herpesviruses 1 and 4 [103,130,138,169].

Like other herpesviruses, attachment ofCHV to permissive cells (MDCK) ap-

pears to be mediated by heparan sulfate,as observed for FHV and for other her-pesviruses [99, 116].

After both symptomatic and asymp-tomatic infections, dogs remain latently in-fected and virus may be excreted at unpre-dictable intervals over periods of severalmonths, or years. Reactivation of latentvirus may be provoked by stress (move-ment to new quarters, introduction of newdogs) or, experimentally, by immunosup-pressive drugs (corticosteroids) or antilym-phocyte serum. Latent virus, demonstratedby the polymerase chain reaction, persistsin the trigeminal ganglia, but other sitessuch as the lumbo-sacral ganglia, tonsils,and parotid salivary gland have been iden-tified [31, 33, 112, 119].

Canine herpesvirus has been detected indogs with ITB [22] but its role remainscontroversial. Experimental infection hasbeen shown to cause mild clinical symp-toms of rhinitis and pharyngitis [7] or toresult in ITB-related disease [85]. Experi-mental infection by the intravenous routein adult foxes results in fever, lethargy andrespiratory signs, while peroral infectiondoes not [131].

A long-term survey in a populationof dogs in a rehoming center has evi-denced CHV in 9.6% of lung and 12.8%of tracheal samples. CHV infections oc-curred later than other viral infections. Incontrast to CRCoV and CPIV, that were de-tected more frequently within the first andsecond week, respectively, CHV was de-tected more frequently at weeks 3 and 4after dog introduction in the kennel. Inter-estingly, CHV infection was apparently re-lated to more-severe respiratory signs [61].Whether the presence of CHV is responsi-ble for increased disease severity or vice-versa is not clear. In a 1-year study intraining centers for working dogs, sero-conversion to CHV appeared to be morefrequent in dogs infected by CRCoV [62],a finding that is more consistent with virusreactivation after disease-induced stress.


An inactivated, subunit vaccine hasbeen available in Europe since 2003. Thevaccine is specifically indicated for bitchesduring pregnancy. The vaccine was shownto provide good immunity to newborn pupsafter two injections had been administeredto their dams.

4. CANINE INFLUENZAVIRUS

Influenza is globally the most eco-nomically important respiratory disease inhumans, pigs, horses, and in the avianspecies. Influenza A viruses have en-veloped virions of 80 to 120 nm in di-ameter, with about 500 spikes of 10 to14 nm in length radiating outward from thelipid envelope [170]. The genome is com-posed of eight segments of single-strandedRNA that segregate independently. Thespike proteins, HA (hemagglutinin) andNA (neuraminidase), elicit neutralizing an-tibody response and provide the basis for adual classification system by H (1 to 16)and N (1 to 9) subtypes [67, 170].

Distribution of the various subtypes isspecies-restricted but interspecies trans-missions may occur, notably betweenavians and mammalians [170]. H3N2,H2N2 and H1N1 strains have been re-sponsible for influenza-associated diseaseand mortality in the last decades in hu-mans, while fatal infections by the highlypathogenic H7N7 and H5N1 avian strainshave occurred sporadically [41, 66, 151].In pigs, influenza A infection is caused byH1N1 and H3N2 subtypes [27]. Two dif-ferent subtypes of equine influenza virus,H7N7 and H3N8, have been associatedwith disease in horses [149, 162, 163]. Vir-tually all the H and N subtypes have beensignaled in the avian species that act as avast, continual reservoir for mammalians[164, 170].

Until recently, dogs were regardedunanimously as non-susceptible hosts toinfluenza virus A. In the 1980s, antibod-ies to human influenza A viruses were

detected in the sera of dogs by hemaggluti-nation inhibition and serum neutralizationassays, suggesting a possible exposure ofdogs to human influenza viruses [28].

The first documented evidence of in-fluenza A in dogs was obtained in 2004 inthe USA, where outbreaks of severe res-piratory disease were reported in Floridaracing greyhounds [45]. Additional out-breaks of respiratory disease were reportedin 2004 in 6 states and 2005 in 11 statesthroughout the USA and the infection wasalso confirmed in pet dogs. These casesoccurred in animal shelters, humane soci-eties, rescue groups, pet stores, boardingkennels, and veterinary clinics [45, 173].

The viruses were found to agglu-tinate chicken erythrocytes and twostrains were isolated in Madin-Darbycanine kidney cells (MDCK) fromlung and bronchioalveolar lavagefluid [45, 173], A/ca/Florida/43/2004and A/ca/Iowa/13628/2005. Retrospectiveserological investigation demonstratedthat the virus was present before 2004,but not before 1998, and a strain,A/eq/Florida/242/03, was isolated fromarchival tissues of a greyhound that haddied from hemorrhagic bronchopneumoniain 2003 [45].

Molecular and antigenic analyses ofinfluenza viruses isolated from the var-ious influenza outbreaks in racing grey-hounds revealed that the canine strains areclosely related to H3N8 equine influenzaviruses [45, 173]. The HA and NA genesof the canine isolates are genetically close(96%–98% nucleotide identity) to the HAand NA genes of recent H3N8 equineinfluenza viruses. Sequence and phyloge-netic analysis of all the 8 genome segmentsindicated that the canine influenza virusesform a monophyletic group, a finding thatis consistent with a single interspeciesvirus transfer, and that the virus likely gotadapted to the canine host by accumulationof point mutations rather than by exchangeof genome segments via reassortment with


other influenza virus A strains, since all thegenome segments are of equine origin [45].

Two distinct clinical forms have beendescribed in dogs infected with influenzavirus with illness rates being nearly 100%.A milder illness is described in most dogs,which is characterized by initial fever andthen cough for 10 to 14 days, followedby recovery. The cough is usually moist,but in some dogs can be dry and resemblethe ITB complex. A thick nasal dischargemay be described, which is usually causedby a secondary bacterial infection. A per-acute death associated with haemorrhagein the respiratory tract has been observedin about 5% of the dogs. The severe formis accompanied by rapid respiration andhigh fever (40–41 ◦C). Post-mortem ex-amination of dogs dead after the peracuteform revealed extensive haemorrhage inthe lungs, mediastinum and pleural cav-ity. The lungs exhibited extensive red tored-black discoloration with moderate tomarked palpable firmness. Mild fibrinouspleuritis was also noted. Histological ex-amination revealed tracheitis, bronchitis,bronchiolitis and suppurative bronchop-neumonia. Lung sections were character-ized by severe hemorrhagic interstitial orbronchointerstitial pneumonia. Patchy in-terstitial change with alveolar septal thick-ening, coagula of debris in the alveoli, andassociated atelectasis were evident, alongwith foci of pyogranulomatous bronchoin-terstitial pneumonia and dilatation of air-ways by degenerate cells and debris. Scat-tered vasculitis and vascular thrombi werealso observed [45, 173]. The disease hasbeen reproduced by experimental inocula-tion of the virus [45].

Therapeutic administration of broad-spectrum antimicrobial drugs reduces theseverity but can not control the dis-ease [173]. In the milder forms, a thickgreen nasal discharge, which most likelyrepresents a secondary bacterial infection,usually resolves quickly after treatmentwith a broad-spectrum bactericidal antimi-

crobial. In the more severe forms, pneu-monia is likely caused by bacterial super-infection, and responds best to hydrationand broad-spectrum bactericidal antimi-crobials.

No vaccine is available to protectdogs against canine influenza. Vaccina-tion against other pathogens causing res-piratory disease, however, may help pre-vent more common respiratory pathogensfrom becoming secondary infections ina respiratory tract already compromisedby influenza infection. The canine in-fluenza virus appears to be easily inacti-vated by common disinfectants (e.g., qua-ternary ammonium compounds and bleachsolutions). Protocols should be establishedfor thoroughly cleaning and disinfectingcages, bowls, and other surfaces betweenuse, as well as for disinfections of person-nel before and after handling of animals.

There is no rapid test for direct diag-nosis of acute canine influenza virus in-fection. Serological assays may detect an-tibodies to canine influenza virus as earlyas 7 days after onset of clinical signs. Inequipped laboratories, viral isolation ontissue cultures and reverse transcription(RT)-PCR or real time PCR analysis maybe applied to fresh lung and tracheal tissuesof dogs that have died from pneumonia andto respiratory secretion specimens from illanimals.

5. CANINE PARAINFLUENZAVIRUS

Canine parainfluenzavirus (CPIV) wasfirst reported in the late 1960s from labora-tory dogs with respiratory disease [2] andfrom a sentry dog with respiratory diseaseof the upper tract [44]. Subsequent studiesrevealed that the virus was frequent in dogswith respiratory disease [11, 22, 42, 110,137, 158], suggesting a key role, alongwith Bordetella bronchiseptica, in the ae-tiology of ITB.

Parainfluenza viruses include importantpathogens of the respiratory tract of mam-mals and birds. The term parainfluenza


was originally adopted after the influenza-like symptoms observed in infected pa-tients and after the influenza-like hemag-glutination and neuraminidase activitiesexhibited by the virus particles. Parain-fluenza viruses are classified in the fam-ily Paramyxoviridae, subfamily Paramyx-ovirinae. CPIV is antigenically similar tothe simian virus 5 (SV5) and to porcine,bovine, ovine and feline parainfluenzaviruses [1, 127]. Sequence analysis of thefusion protein-encoding gene has revealedthat CPIV has 99.3% nucleotide similar-ity to porcine parainfluenza virus, 98.5%to SV5 and 59.5% nt to human parain-fluenza virus 2 [111]. Accordingly, CPIVis regarded as a host variant of SV5, withinthe genus Rubulavirus and has been ten-tatively proposed as parainfluenza virus 5(PI-5) [39]. Viruses genetically similar toSV5 have been detected in humans in moreoccasions although the relationship to anyhuman disease remains contentious [39].

The virus is composed of a singlestranded RNA genome of negative po-larity and is surrounded by a lipid en-velope of host cell origin. The genomeof SV5 contains seven genes that en-code eight proteins: the nucleoprotein(NP), V/phosphoprotein (V/P), matrix(M), fusion (F), small hydrophobic (SH),hemagglutinin–neuraminidase (HN), andlarge (L) genes [92]. The HN proteinis involved in cell attachment to initiatevirus infection and mediates hemaggluti-nation [100]. In addition, HN has neu-raminidase activity. The F protein mediatesfusion of the viral envelope with the cellmembrane [143]. The V protein blocks in-terferon (IFN) signaling and inhibits IFNsynthesis. Interaction of the virus with theIFN system is regarded as a critical factorin the outcome of the infection [23,54,121,146].

Parainfluenza is highly contagious andthe prevalence of infection appears to berelated to the density of the dog population.CPIV is excreted from the respiratory tract

of infected animals for 8-10 days after in-fection and is usually transmitted by directcontact with infected aerosol [6]. The virusmay spread rapidly in kennels or shelterswhere a large number of dogs are kept to-gether. The virus was detected in 19.4%of tracheal and 9.6% of lung samples ofdogs in a rehoming centre where ITB wasendemic and persisted, in spite of regu-lar vaccination against canine adenovirustype-2, distemper and parainfluenza [61].

There is evidence that cats, hamstersand guinea pigs may naturally be in-fected with CPIV/SV5 or a very closelyrelated virus [81, 144]. In addition, aCPIV/SV5-like strain, termed SER, wasrecently isolated from the lung of a fetusof a breeding sow with porcine respira-tory and reproductive syndrome [79, 155].Antibodies to CPVI have been demon-strated in 20 of 44 wildlife species in eightAfrican countries [74]. Also, antibodies toCPVI have been detected in non-captiveblack bears (Ursus americanus) and fishers(Martes pennanti) in Canada [120], sug-gesting circulation of CPIV-like viruses inwildlife animals. Even more interestingly,CPIV/SV5-like viruses may infect humansand non-human primates [39].

CPIV infection is usually restricted tothe upper respiratory tract in dogs oftwo weeks of age or older [6]. Althoughviremia is considered an uncommon event,CPIV has been recovered from the lungs,spleen, kidneys and liver of laboratorydogs with mixed infections [22]. After ex-perimental infection of dogs, CPIV repli-cates in cells of the nasal mucosa, phar-ynx, trachea and bronchi. Small amountsof virus can be recovered from the locallymph nodes, but not from other lymphatictissues. In naturally infected dogs, simulta-neous infections with other viral and bac-terial agents are quite common and clinicalsigns may be more severe [2, 22, 137].

Symptoms generally occur 2–8 days af-ter infection. CPIV produces mild symp-toms lasting less than six days, but


infection is usually complicated by otherpathogens. In the non-complicated forms,clinical signs include low-grade rise intemperature, deep sounding dry cough, wa-tery nasal discharge, pharyngitis and ton-sillitis [6]. Most dogs appear healthy andactive. In the complicated forms, describedmostly in immunocompromised animalsor young unvaccinated puppies, the symp-toms may progress and include lethargy,fever, inappetance, and pneumonia.

CPIV has also been isolated from a dogwith temporary posterior paralysis [63]and this isolate, termed CPI+, causedacute encephalitis when injected intracra-nially into gnotobiotic dogs [13]. From onesuch experimentally infected dog, a vari-ant, termed CPI2, was isolated that hadphenotypic and genotypic differences fromCPI+. CPI2 is attenuated in ferrets and itmore readily establishes persistent infec-tions in tissue culture cells. The biologi-cal changes and the ability to block IFNsignaling have been mapped to the P/V-N-terminal common domain of the V pro-tein [14–17, 38, 148].

In experimentally infected dogs pe-techial hemorrhages have been describedin lung lobes between 3 and 8 dayspost infection [2, 26]. Histological ex-amination has revealed catarrhal rhinitisand tracheitis with mono- and poly-morphonuclear cell infiltrates in the mu-cosa and submucosa. Bronchi and bron-chioli may contain leukocytes and cellulardebris.

Laboratory diagnosis may rely on viralisolation from nasopharyngeal or laryngealswabs, using primary cells or cell linesderived from dog kidneys. A wide rangeof canine, feline, bovine, simian, and hu-man cells are permissive for CIPV andmonkey kidney cells have also been usedsuccessfully [6]. In the first passage, thevirus usually does not induce cytopathiceffects and virus antigens may be demon-strated by hemadsorption or immunoflu-orescence [2, 44]. RT-PCR may also be

applied to respiratory secretions, nasopha-ryngeal/laryngeal swabs and tracheal/lungtissues [61]. Serological investigations byhemagglutination inhibition and the virusneutralization test may be useful to screenanimals for the presence of specific anti-bodies.

Attenuated vaccines have been devel-oped against CPIV. A parenteral CPIVvaccine is available in combination withother antigens. These vaccines alone rarelyprovide protection against contracting theinfection, although they help to reduce theseverity of the disease. Vaccination of allanimals, notably of puppies, is indicatedin kennels or in pet shops. Strict hygienewith thorough cleaning and disinfectionof cages and food and water containers,good ventilation and adequate populationdensity are essential for controlling virusspread.

6. CANINE REOVIRUS

Mammalian orthoreoviruses (MRV) arenon-enveloped, double-stranded (ds) RNAviruses included in the genus Orthore-ovirus within the family Reoviridae. MRVare responsible for either symptomatic orasymptomatic infections in mammals andpossess a broad host range [157].

The reovirus genome contains tendsRNA segments, which are designed aslarge (L, three segments), medium (M,three segments), or small (S , four seg-ments) on the basis of the electrophoreticmobility [118]. Three MRV serotypes havebeen recognized by cross-evaluation withspecific sera in neutralization and inhi-bition of haemagglutination assays [136,141]. Neutralization and HA activities arerestricted to a single reovirus gene seg-ment, S 1 [165], that encodes for the pro-teins σ1 and σ1s. The σ1 protein, a fi-brous trimer located on the outer capsidof the virion [68, 69], is responsible forviral attachment on cellular receptors [95,167], serotype-specific neutralization [12],and hemagglutination [166]. Analysis of


the S 1 gene of MRV belonging to dif-ferent serotypes has shown a strict cor-relation between sequence similarity andviral serotype [34, 56, 117]. Conversely,the other genome segments do not displayany correlation to viral serotype, suggest-ing that MRV have evolved independentlyof serotype in the various species [25, 37,71, 87, 94].

MRV have a wide geographic distri-bution and can virtually infect all mam-mals, including humans [157]. In carni-vores, MRV infections have been reportedsporadically, although all three serotypeshave been isolated from dogs and cats [21,46, 51, 57, 89, 97, 102, 108, 113, 145].

As in other mammalians, the aetiologi-cal role of MRV in respiratory diseases ofdogs is still unclear. MRV-1 strains havebeen recovered from dogs with pneumo-nia [97] or enteritis [4], in association witheither canine distemper virus or canine par-vovirus type 2. MRV-2 and MRV-3 havebeen isolated from dogs with disease of theupper respiratory tract [21] and with diar-rhea [51, 89], respectively. Only an MRV-3enteric strain has been characterized at themolecular level in the S1 and L1 segments.The highest nucleotide identity was foundto a murine strain in the S 1 segment (93%)and to human and bovine strains in theL1 segments (90%), revealing the lack ofspecies-specific patterns [51]. By PCR, re-ovirus RNA was detected in 50/192 rectalswabs from dogs with diarrhea. Also, re-ovirus RNA was detected in 9/12 ocularswabs, in 10/19 nasal swabs of dogs withocular/nasal discharge, whereas it was notdetected in the oro-pharynx [57]. Thesedata suggest that reoviruses are commonin dogs, both in the enteric or in the respi-ratory tract, although the viruses are shedin low amounts. Experimental infectionswith MRV in germ- and disease-free dogsfailed to give conclusive results [4, 80].Accordingly, it appears that MRV do notexert direct pathogenic activity and, morelikely, act in synergism with other respira-

tory pathogens, aggravating the course ofconcomitant infections [4].

Diagnosis of reovirus infection is usu-ally based on virus isolation on cellcultures, electron microscopy and poly-acrilamide gel electrophoresis (PAGE).These methods proved to be poorly sen-sitive [115] and likely underestimate thepresence of MRV in animals and humans.RT PCR protocols have been developed fordetection of MRV and for prediction of theMRV serotype [51, 94, 115].

7. CANINE RESPIRATORYCORONAVIRUS

Members of the Coronaviridae fam-ily are enveloped viruses, 80–160 nmin diameter, containing a linear positive-stranded RNA genome. Coronavirusesare currently classified into four distinctgroups based on sequence analysis andgenome structure and on the antigenicrelationships [101, 139, 150]. The coro-navirus structural proteins include thespike glycoprotein, the membrane gly-coprotein and the nucleocapsid protein.The hemagglutinin-esterase glycoproteinis found only on the surface of group 2coronaviruses [60].

Three different coronaviruses have beenidentified in dogs thus far [60, 125]. Theenteric canine coronaviruses (CCoV) aredistinguished into two genotypes, I and II,and are included in group 1 coronavirusesalong with feline coronaviruses (FCoV)type I and type II, transmissible gastroen-teritis virus of swine (TGEV), porcinerespiratory coronavirus (PRCoV), porcineepidemic diarrhea virus (PEDV) and hu-man coronavirus 229E [59]. The evolutionof CCoV is tightly intermingled with thatof FCoV I and II [125]. Canine respiratorycoronavirus (CRCoV) was first detectedin the United Kingdom in 2003 from tra-chea and lung tissues of dogs [60]. Byphylogenetic analysis of the polymerase,CRCoV was found to segregate with group


2 coronaviruses, along with bovine coro-naviruses (BCoV) and human coronavirusstrain OC43 (HCV-OC43) [60]. Sequenceanalysis of the S protein-encoding generevealed a high genetic similarity to thebovine strain BCoV and to the humanstrain OC43 (96.9 and 97.1% at the nu-cleotide level and 96.0 and 95.2% atthe aa level, respectively) [60], suggest-ing a recent common ancestor for thethree viruses and demonstrating the occur-rence of repeated host-species shifts [161].Conversely, CRCoV was found to be ge-netically and antigenically different fromthe enteric canine coronaviruses (less than21.2% aa in the S gene).

By RT-PCR, CRCoV RNA has been de-tected both in asymptomatic and in symp-tomatic dogs, that suffered from mild ormoderate respiratory disease [60]. Analy-sis of archival samples has identified CR-CoV in 2 out of 126 dogs affected byrespiratory diseases in Canada [58].

Taking advantage of the close geneticrelatedness between CCRoV and BCoV,ELISA assays have been set up usingBCoV antigen and have been used toscreen canine sera, revealing the presenceof specific antibodies in 30.1% of dogs atthe time of entry in a rehoming kennel [60].In a further study, antibodies were de-tected in 22.2% and 54.2% of dogs on theday of entry into working kennels in Lon-don and Warwickshire, respectively [62].In larger sero-epidemiological surveys, theprevalence of CRCoV was demonstratedto be 54.7% in North America, 36.6%in the United Kingdom [126], 17.8% inJapan [84] and 32% in Italy [53] whilethere was no evidence for CRCoV-specificantibodies in cats [84]. By examiningthe relationship between the age of dogsand the presence of CRCoV antibodies, asteady increase in the seropositivity rateswas observed, with the highest prevalenceamong dogs of 7–8 years (68.4%) [126].

Attempts to isolate CRCoV from tis-sue of the respiratory tract, using canine

lung fibroblasts, MDCK, HRT-18G andfewf-4 cells were unsuccessful [60,84] andthis has hampered, thus far, the evaluationof CRCoV patho-biological properties andthe CRCoV role in canine respiratory dis-eases.

The role of CRCoV in ITB is notclear. Sero-conversion was observed inimmunologically-naïve dogs after intro-duction in a kennel where infected dogswere housed, revealing a highly contagiousnature [60]. Dogs sero-negative to CRCoVwere statistically more prone to developrespiratory disease than dogs with antibod-ies to CRCoV, providing indirect evidencefor a pathogenic role of CRCoV [60]. Itis likely that CRCoV alone may induceonly subclinical or mild respiratory symp-toms. However, coronavirus replicationcan damage the respiratory epitheliumand lead to bacterial superinfections. Thehuman respiratory coronavirus 229E candisrupt the respiratory epithelium andcause ciliary dyskinesia [40]. Accordingly,virus-induced alterations of the respiratoryepithelium would trigger the replication ofother pathogens, causing respiratory dis-eases resembling the ITB complex.

8. PANTROPIC CANINECORONAVIRUS

Enteric CCoV usually cause mild tosevere diarrhea in pups, whereas fatalinfections have been associated mainlywith concurrent infections by canine par-vovirus, canine adenovirus type 1 or caninedistemper virus [50, 123, 124].

Thus far, two genotypes of entericCCoV have been described, namely CCoVtype I and CCoV type II [125]. Molecu-lar methods able to distinguish between thetwo genotypes have revealed that mixed in-fections by both genotypes occur at highfrequency in dogs [52].

Recently, a fatal, systemic diseasecaused by a highly virulent CCoV strain


was reported, which was characterizedby severe gastrointestinal and respiratorysymptoms [29]. The disease occurred inseven dogs housed in a pet shop in theApulia region, Italy. The dogs displayedfever (39.5–40 ◦C), lethargy, inappetance,respiratory distress, vomiting, hemorrhagicdiarrhea, and neurological signs (ataxia,seizures) followed by death within 2 daysafter the onset of the symptoms. A markedleukopenia, with total WBC counts be-low 50% of the baseline values, was alsoreported. Necropsy examination revealedsevere gross lesions in the tonsils, lungs,liver, spleen and kidneys. Extensive lo-bar subacute bronchopneumonia was ev-idenced both in the cranial and caudallobes, along with effusions in the thoraciccavity.

By genotype-specific real-time RT-PCRassays, CCoV type II RNA was detected inthe intestinal content and parenchymatousorgans, including the lungs, and a coron-avirus strain was successfully isolated oncell cultures from lungs and other tissues.

Sequence analysis of the 3’ end of theviral genome showed a point mutation inthe S protein and a truncated form of thenonstructural protein 3b, due to the pres-ence of a 38-nt deletion and to a frame shiftin the sequence downstream of the dele-tion that introduced an early stop codonin ORF3b. Either point mutations or dele-tions in the structural spike glycoproteinand in the nonstructural proteins have beenassociated to changes in tropism and vir-ulence of coronaviruses [72, 78, 83, 93,160]. The porcine respiratory coronavirus(PRCoV), a spike (S) gene deletion mu-tant of transmissible gastroenteritis virus(TGEV), causes mild or subclinical respi-ratory infections in pigs [93].

Experimental infection of dogs with thevirus isolate resulted in a severe systemicdisease that mimicked the clinical signsobserved in the outbreak. However, olderpuppies were able to recover from theinfection. The pathogenic CCoV variants

should be suspected when unexplainableepisodes of severe to fatal disease occur inpups. Epidemiological studies are requiredto determine whether the pantropic CCoVstrain is a new coronavirus variant emerg-ing in the canine population or if it is awide-spread infectious agent of dogs thatusually goes undetected. Vaccination tri-als are necessary to determine whether theCCoV vaccines currently available are ef-fective against the highly virulent CCoVstrain.

9. CONCLUSIONS

The development of new diagnostictechniques and the extensive use of molec-ular analysis are quickly providing anincreasing amount of information on theepidemiology of respiratory viruses, on themolecular basis of pathogenicity and onthe mechanisms that drive virus evolution.In the last decades, evidence has been col-lected for the emergence of novel virusesby host species shift or by change of tissuetropism due to genome mutations. Prophy-laxis of the ITB complex relies on the useof vaccines based on selected pathogens(CAV-2, CPIV and Bordetella bonchisep-tica) and those vaccines are not alwayseffective in preventing ITB, suggesting thatother pathogens may also play a role inrespiratory diseases of dogs. Whether thedetection of new respiratory pathogens re-quires the development of novel prophy-laxis tools is an issue that surely deservesmore attention. At the same time, intensifi-cation of surveillance activity is paramountto monitor the emergence and spread ofnovel pathogens, to investigate their epi-demiology and plan adequate measures ofcontrol.

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N o b i v a c® KC

KC – bredare och snabbare skydd

N o b i v a c® KC

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N o b i v a c ® K C

Nobivac® KC – produktprofil

• Försvagat levande vaccin för intranasaltbruk:

• >108.3 cfu B. bronchiseptica stam B-C2 • 103.8 TCID50 CPi

• Dos: 0.4 ml• Ges i ena näsborren• Immunitet efter 72 tim


Intranasala vacciner – fördelar

• Inducerar lokal och systemiskimmunitet

• Snabb immunitetsstart• Fullt skydd efter en enda vaccination • Kan ges till MDA-positiva valpar*

* Jacobs et al. WSAVA 2006


Systemisk Immunitet - IgG

Efter injektions- / intranasal vaccination:

IgGIgG

Vid smitta - Inhalerade Bb• Fäster till cilierna

• Orsakar infektion

• Inflammationen frisätter IgGtill ytan och cilierna

• Infektion begränsas men förhindras inte


Lokal immunitet - IgA

Celler

Plasma-cell

Lumen Bb

Vaccin Bb

IgAIgA

Bb Intranasalt vaccinadministreras på mukosansom vid naturlig infektion:

• IgA frisätts i mukosan

• Submukösa plasmacellerstimuleras till IgA-produktion

• Vid smitta - IgA binder inhalerade Bordetellabronchiseptica


Intranasala vacciner – nackdelar

• Ibland svårt att administrera• Kan förekomma nysningar efter

vaccination • Vaccinerade hundar kan utskilja Bb

(apatogen)


Effektivietsstudie1 års immunitet

• 12 hundar vacc vid 3 v ålder

• + 6 kontroller samma ålder

• Challenge med Bb och CPi efter 56 veckor

• + 6 10v valpkontroller

• Provtagna och observerade i 3 veckor

Jacobs et al. 2005 Vet Rec, 157, 19-23.


Effektivietsstudie1 års immunitet – resultat

Efter vacc - signifikant lägre:

- virusisolering (p=0,05)

- mv kliniska poäng (61%, p=0,009)

- mv kliniska poäng valpar (90%, p=0,001)

Jacobs et al. 2005 Vet Rec, 157, 19-23.


Effektivitetsstudie skydd på 72 tim

• Valpar vacc. vid 8 v– 20 vacc. (10 + 10) + 10 kontroller

• Challenge med Bb 108,4 CFU/ml i 2ml aerosol efter 48 eller 72 tim

• Bedömning av kliniska symptom

Gore et al. (2005) Vet Rec 156, 482-483


4

18

0

5

10

15

20

25

Vaccinates (n=10) Controls (n=10)

Effektivitetsstudie skydd på 72 tim – resultat

Kliniska poäng efter Bb Challenge - mv

Kliniska symptom minskade med 73%, signifikant (p=0.002)


Excellent säkerhetsprofil• Experimentella säkerhetsstudier:

2-veckors valpar x 1



Dräktiga i alla tre trimestrar

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• Fältstudier:- 1289 hundar varierande ålder, ras och kön(varav 104 st dräktiga)

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• Resultat:0,1 % visade

milda, övergående

symptom

Excellent säkerhetsprofil


Nobivac® KC – sammanfattning

• Bivalent skydd mot kennelhosta

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• Sgs ingen virusutsöndring efter CPi-challenge

• Endast EN dos

• Mycket snabb immunitetsstart Bb – 72h!

• Duration minst 12 mån

• Kan användas ihop med andra Nobivac-vacc*

• Excellent säkerhetsprofil – 2v valpar, dräktiga* Jacobs et al. Vet Rec (2007) Jan 13, 160, 41-45.


Vilka hundar ska vaccineras mot kennelhosta?

• Både SVS/SVAs och WSAVAs expertgrupper rek. vaccination till hundar som vistas i hundrika miljöer

• SKK rek. Vaccination inför utställningar, prov och tävlingar


Nobivac® KC – vaccinationsteknik

• Låt ägaren stå framför hunden• Stå själv bakom hunden • Håll om nosen med ena handen• Droppa i dropparna, inte spraya


Förpackningar och priser

Apotek IntervetDirekt

5 doser 310:- -25 doser 1385:- 1299:-


AnvAnvAnvääändndnd NobivacNobivacNobivac® KC till KC till KC till hundarhundarhundar i i i allaallaalla åååldrarldrarldrar nnnääärrr ettettettbredarebredarebredare ochochoch snabbaresnabbaresnabbare skyddskyddskyddmot mot mot kennelhostakennelhostakennelhosta ööönskasnskasnskas!!!

KC KC –– bredare och snabbare skyddbredare och snabbare skydd

Svenska erfarenheter av vaccination med intranasalt kennelhostevaccin Anette Johansson, distriktsveterinär i Kiruna

Det är femtonde året i rad som vi vaccinerar våra hundar samt en del andra slädhundar med intranasalt kennelhostevaccin. Första gången var 1994 och då fick hundarna två vaccinationer med en månads intervall. Efter varje vaccination fick de vila en vecka. Under de första åren vaccinerade vi två gånger. De fick en första vaccination tidigt på hösten och en andra i december i god tid innan tävlingssäsongen drog igång på allvar. Under de följande tio åren har de vaccinerats bara en gång per säsong och det har gett ett tillräckligt skydd. Vi vaccinerar under perioden oktober till december. Eftersom hundarna får vila några dagar (oftast 5 dagar) har vi vaccinerat när det har blivit isigt före och omöjligt att träna eller om vi har varit bortresta. Från början var det ett vaccin av annat fabrikat men nu har vi använt Nobivac KC i flera år. Det intranasala vaccinet ger ett mycket bra skydd. Sedan vi började använda intranasalt vaccin har våra vaccinerade hundar inte drabbats av kennelhosta trots att smittan har grasserat i omgivningarna. Vi har oftast inte vaccinerat valpar och så sent som i vintras smittades valparna när grannarnas hundar hade hosta. Men ingen av de vuxna hundarna fick hosta. Risken för biverkningar lär vara högre med intranasalt vaccin och det är därför vi bara har vaccinerat de vuxna hundarna. Det är också anledningen till att hundarna får vila från träningen veckan efter vaccination. Det är nog också viktigt att hundarna är i god kondition och friska för övrigt. Genom åren har någon enstaka hund hostat efter vaccinationen. Det har varit hundar som inte tidigare har varit vaccinerade med intranasalt vaccin. Det är enda biverkningarna vi har sett på våra hundar. Däremot har jag ett par fall av allvarligare biverkningar på två andra kennlar. Det första var ett spann som skulle tävla på medeldistanstävlingen Alpirod som gick i Alperna. Det var redan 1992 och första gången jag sökte licens på intranasalt vaccin. Hundarna vaccinerades en tid innan de skulle åka på tävlingen. De blev riktigt sjuka med hosta och andra symtom från luftvägarna. En del fick pneumoni. Förmodligen var inte hundarna i tillräckligt god kondition eller kanske rentav nerkörda av för mycket träning. Det var ingen mönsterkennel vad gäller utfodring och kennelmiljö. En annan kennel med ca 20 hundar vaccinerades i januari månad för ca 10 år sedan. Hundhållningen var bra och träningen seriös. Ändå blev flertalet av hundarna sjuka med hosta och luftvägssymtom. Även då fick någon hund lunginflammation. Kennelhosta grasserade redan när hundarna vaccinerades. Sannolikt var de redan smittade och vaccinationen gjorde utbrottet allvarligare. Jag har mycket goda erfarenheter av det intranasala vaccinet. Jag vet inte om risken för biverkningar är mindre idag än tidigare men jag rekommenderar att man vaccinerar i god tid innan säsongen börjar på allvar, att hundarna är i god kondition och att man erbjuder dem en viloperiod efter vaccinationen.

Jämförande studie mellan vaccinationsantikroppar mot rabies. Louise Treiberg Berndtsson, leg.vet, enheten för virologi, immunbiologi och parasitologi, SVA. Bakgrund. I och med Sveriges inträde i EU fick hundar och katter möjlighet att utan karantän föras in i Sverige. Djuren måste dock vara vaccinerade mot rabies och dessutom kontrollerade att de erhållit en skyddande titer på minst 0,5 IE/ml serum 120 dagar efter senaste vaccination. SVA har sedan detta blev möjligt tagit emot blodprov/serum för analys. Analysen görs i enlighet med OIEs och EUs community reference laboratory, CRL instruktioner, med deltagande i årliga ringtester. SVA testar årligen flera tusen prover för rabies antikroppar. De första åren fanns i Sverige bara ett godkänt vaccin, Rabisin Vet, men sedan 1998 finns 2 st godkända vaccin då även Nobivac Rabies Vet är inregistrerat i Sverige. Vi tyckte oss märka efter en tid att det var fler hundar som inte blev godkända om de var vaccinerade med Nobivac Rabies Vet än de som var vaccinerade med Rabisin vet. Vi fick också indikationer från veterinär och kliniker att fler hundar än tidigare inte blivit godkända. Vi visste sedan tidigare att det skilde sig om djuret var vaccinerat en eller två gånger samt om vaccinet även innehöll komponenten Leptospiros. Med hjälp av medel från Intervet och Merial beslöts att en undersökning av prov tagna under ett år skulle undersökas huruvida det var någon skillnad mellan vaccinerna. Vi beslöt också att samtidigt undersöka om ålder, kön, ras samt om djuret är vaccinerat en eller två gånger har betydelse. Material Serum från 6.881 hundar, provtagna i Sverige analyserade år 2005 på SVA, avdelningen för virologi. Prov har skickats in från hela landet av olika veterinärer och kliniker. Av de 6.881 hundarna som ingår i studien var 3.584 st (52,08%) vaccinerade med Nobivac Rabies Vet och 3.297 st (47,91%) vaccinerade med Rabisin vet. Analysmetod Serumproverna analyserades med FAVN-testen, en modifierad serumneutralisations test utarbetad av AFSSA, Nancy, OIEs referenslab och EUs CRL-lab. (Cliquet et al, 1998). Resultat Vid studien kunde några faktorer påvisas som har stor betydelse för om hunden ska klara gränsen på 0,5IE/ml eller ej.

1. En eller två immuniseringar. Dubbla vaccineringar har stor betydelse för om hunden ska klara gränsen eller ej om hunden är vaccinerad med Nobivac ® Rabies Vet.

2. Hundens ålder Hundar under ett års ålder klarade sig något sämre än äldre hundar om de vaccineras med Nobivac Rabies Vet.

3. Skillnader mellan de 2 olika vaccinerna Av de 3.584 st hundarna vaccinerade med Nobivac Rabies Vet klarade 87,28% gränsen 0,5 IE/ml Av de 3.297 st hundarna vaccinerade med Rabisin Vet klarade 97,15% gränsen 0,5 IE/ml

Kön på hunden spelade ingen roll.

Varför skillnad i resultat? Vaccinerna är framställda av olika virusstammar. Nobivac ® Rabies vet innehåller RIV från Pasteur Institutet och Rabisin® Vet innehåller Wistar G 57 också från Pasteur Institutet. I analysmetoden används CVS-11 virus som kan vara närmare besläktat med Wistar G 57 (likheter i G-proteinet) och därmed lättare fångar upp antikroppar riktade mot detta virus.

Biologicals 33 (2005) 269e276

www.elsevier.com/locate/biologicals

The influence of homologous vs. heterologous challengevirus strains on the serological test results of rabies

virus neutralizing assays

Susan M. Moore*, Teri A. Ricke, Rolan D. Davis, Deborah J. Briggs

Department of Diagnostic Medicine, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA

Received 19 May 2005; accepted 12 June 2005

Abstract

The effect that the relatedness of the viral seed strain used to produce rabies vaccines has to the strain of challenge virus used to

measure rabies virus neutralizing antibodies after vaccination was evaluated. Serum samples from 173 subjects vaccinated witheither purified Vero cell rabies vaccine (PVRV), produced from the Pittman Moore (PM) seed strain of rabies virus, or purified chickembryo cell rabies vaccine (PCECV), produced from the Flury low egg passage (Flury-LEP) seed strain of rabies virus, were tested

in parallel assays by RFFIT using a homologous and a heterologous testing system. In the homologous system, CVS-11 was used asthe challenge virus in the assay to evaluate the humoral immune response in subjects vaccinated with PVRV and Flury-LEP wasused for subjects vaccinated with PCECV. In the heterologous system, CVS-11 was used as the challenge virus in the assay toevaluate subjects vaccinated with PCECV and Flury-LEP was used for subjects vaccinated with PVRV. Although the difference in G

protein homology between the CVS-11 and Flury-LEP rabies virus strains has been reported to be only 5.8%, the use ofa homologous testing system resulted in approximately 30% higher titers for nearly two-thirds of the samples from both vaccinegroups compared to a heterologous testing system. The evaluation of equivalence of the immune response after vaccination with the

two different vaccines was dependent upon the type of testing system, homologous or heterologous, used to evaluate the level ofrabies virus neutralizing antibodies. Equivalence between the vaccines was achieved when a homologous testing system was used butnot when a heterologous testing system was used. The results of this study indicate that the strain of virus used in the biological

assays to measure the level of rabies virus neutralizing antibodies after vaccination could profoundly influence the evaluation ofrabies vaccines.� 2005 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Keywords: CVS-11; Flury-LEP; RFFIT; Serological testing; Rabies vaccine

1. Introduction

The immune response to rabies vaccination involvesactivation of rabies virus-specific B cells which differen-tiate into plasma cells producing antibody and memoryB cells. Although antibodies specific for the rabies virusglycoprotein (G) and nucleoprotein (N) (as well as other

* Corresponding author. Tel.:C1 785 532 5650; fax:C1 785 532 4474.

E-mail address: [email protected] (S.M. Moore).

1045-1056/05/$30.00 � 2005 The International Association for Biologicals.

doi:10.1016/j.biologicals.2005.06.005

rabies viral proteins) are produced after vaccination,published reports indicate that it is the antibodiesspecifically directed against antigenic components ofthe G protein that neutralize the rabies virus [1]. Rabiesvirus-specific CD4C T cells, primarily induced by therabies virus N protein, assist in B cell immunoglobulinclass switching and immunoglobulin production. Due tothe lack of a well-established practical method tomeasure the cellular immune response against rabiesvirus and because rabies virus neutralizing antibodies

Published by Elsevier Ltd. All rights reserved.

mailto:[email protected]

http://www.elsevier.com/locate/biologicals

270 S.M. Moore et al. / Biologicals 33 (2005) 269e276

(RVNA) are critical for protection against rabiesinfection, the standard method to verify that an immuneresponse has occurred after rabies vaccination is toevaluate the level of RVNA in sera. The World HealthOrganization (WHO) recognizes two RVNA assays toassess the humoral immune response after rabiesvaccination: the Rapid Fluorescent Focus InhibitionTest (RFFIT) and the Fluorescent Antibody VirusNeutralization Test (FAVN). Both assays utilize theChallenge Virus Standard (CVS-11) strain of rabiesvirus as the challenge virus to quantitate the neutrali-zation activity of RVNA produced in response toa rabies vaccine [2,3]. Previous studies have demon-strated the significant influence that the strain ofchallenge virus used in an assay has on the measurementof vaccine potency [4,5]. Indeed, published reportsindicate that higher vaccine potency values are achievedwhen a homologous challenge virus is used for potencytesting as compared to when a heterologous challengevirus strain is used. A similar effect has been demon-strated in the serological test results from serum samplesassayed for the presence of specific antibody againstdifferent genotypes of lyssaviruses including rabies virus.For example, higher RVNA titer values were obtainedagainst rabies virus, (lyssavirus genotype 1) as opposedto lyssavirus genotypes 2e7 when the source of theantibody that was evaluated was pooled sera frompersons vaccinated against rabies virus (lyssavirusgenotype 1) [6]. Additionally, another study reportedvariations in RVNA titer values when two different CVSstrains were used as the challenge virus [7].

There are several cell culture rabies vaccines licensedfor use throughout the world. Many of these vaccinesare produced from different rabies virus seed strainsincluding: Pittman Moore (PM) rabies virus strain usedto produce human diploid cell rabies vaccine (HDCV),purified Vero cell rabies vaccine (PVRV) and purifiedduck embryo cell rabies vaccine (PDEV); Flury high eggpassage (Flury-HEP) or Flury low egg passage (Flury-LEP) rabies virus strain used to produce two differenttypes of purified chick embryo cell rabies vaccine(PCECV); and Kissling rabies virus strain of ChallengeVirus Standard used to produce rabies vaccine adsorbed(RVA). The PM and Kissling rabies virus strainsoriginated from the brain of a rabid cow in France in1882 and the Flury-LEP strain originated from a hu-man patient in the USA who died of rabies in 1939.Investigation of the phylogenetic trees of the G and Nrabies virus proteins originating from different vaccineseed strains indicates a much closer relationship existsbetween the PM and CVS strains of rabies virus thanbetween the Flury-LEP and the CVS strains (Fig. 1).Published reports also indicate areas of differences existbetween the amino acid sequence of the G protein ofCVS and PM and the G protein of CVS and Flury-LEPrabies virus strains (Fig. 2). It is important to note that

there are no amino acid sequence differences in theknown, mapped antigenic sites [8]. However, six of theeight known antigenic sites (epitopes) of the G proteinare conformational and any amino acid changes in closeproximity to these epitopes could potentially affect thefolding of the protein [8]. Additionally, the transmem-brane region has been reported to affect folding of theectodomain resulting in subtle conformational changesof the antigenic sites [9]. The production of RVNAinvolves a process of fine-tuning of specificity resulting inthe selection of B cell clones with the highest avidity toa specific antigen. The potential differences in theG protein antigenic sites of the original seed virus strainsused in the production of the different rabies vaccinescould result in the preferential production of antibodieswith the highest affinity for antigenic sites resembling thevaccine seed virus strain. Thus, the strain of challengevirus used in an RVNA assay and the type of vaccine thata person was vaccinated with could profoundly influencethe serological test results after vaccination. If this iscorrect, RVNA assays using homologous testing systems(wherein the strain of challenge virus used in the testingassay is very closely related to the seed virus strain usedto produce the vaccine that a subject received) wouldreport higher titer values than heterologous testingsystems (wherein the strain of challenge virus used inthe testing system is less closely related to the seed virusstrain used to produce the vaccine that a subjectreceived). The following study was conducted to de-termine the influence that the strain of rabies virus(homologous vs. heterologous) used as the challengevirus in a serological assay and the strain of seed virusused in the production of the rabies vaccine that a subjectreceived has on the quantitative evaluation of RVNA.

2. Materials and methods

2.1. Challenge virus

Two strains of rabies virus were evaluated as thechallenge virus in the RFFIT assays used to quantitatethe amount of RVNA present in serum samples. TheCVS-11 strain was obtained from the Centers forDisease Control and Prevention (Atlanta, GA). Seedvirus of the CVS-11 was grown on BHK cells to producestock virus. The Flury-LEP strain was obtained fromChiron Vaccines (Marburg, Germany); stock virus wasgrown in primary chicken fibroblasts. Stock viruspreparations were titered to obtain a working dilutionof 50 TCID50.

2.2. Serum samples

Serum samples used in the analyses were obtainedfrom subjects who had received the same simulated

271S.M. Moore et al. / Biologicals 33 (2005) 269e276

Fig. 1. Phylogenetic relationship of rabies virus strains (courtesy of Dr Iris Stalkamp, Institut fur Virologie, Giessen, Germany).

post-exposure vaccination regimen with either PCECV(nZ 86) or PVRV (nZ 87). Subjects did not receiverabies immune globulin (RIG). Serum samples that werecollected on day 14 and day 90 after initial vaccinationwere included in the study. Serum samples wererandomly placed into five testing groups (Groups 1through 5). Each group contained from 60 to 120 serumsamples including samples from subjects vaccinated withPCECV as well as subjects vaccinated with PVRV. Allserum samples were coded to ensure that testing wasconducted blindly and unsorted by vaccine group.

2.3. Equilibration of working dilutionof challenge virus

The working dilution of the challenge virus wasequilibrated to 50 TCID50 for both the CVS-11 and theFlury-LEP rabies virus strains. The titer of the challengevirus was calculated for each test set of serologicalsamples in order to assure equivalence in testing criteria.For all test runs, the titer of the challenge virus wasmaintained within one standard deviation of the average

calculation (41.1 TCID50) for virus titer throughout theentire evaluation.

2.4. Serological testing

The RFFIT, using CVS-11 and Flury-LEP as thechallenge virus strains in parallel, was used to assay allserum samples, as previously described [10]. Briefly,100 mL of each serum sample, in duplicate, was dilutedin serial fivefold dilutions and loaded into 8-well Lab-tek chamber slides after which 100 mL of the challengevirus, at a concentration of 50 TCID50, was added.Slides were incubated at 37 �C for 90 min after which200 mL of a suspension of 5! 105 BHK cells was addedto each well. Slides were placed in a 5% CO2 incubatorat 37 �C for 24 h. After incubation the slides werewashed and fixed in 80% cold acetone, dried and stainedwith FITC conjugated anti-rabies antibody (Chemicon,Temecula, CA). Twenty fields/well were examined under160! magnification using a fluorescence microscope forthe presence of rabies virus and RVNA titers werecalculated using the Reed and Muench method. Re-ciprocal titers were used in the evaluations in order to


Fig. 2. Amino acid alignment of the rabies glycoprotein from Flury, CVS and PM strains (courtesy of Dr Iris Stalkamp, Institut fur Virologie,

Giessen, Germany). There are fewer amino acid sequence changes from CVS to PM (filled arrows) than CVS to Flury (open arrows). The changes are

not in areas of the mapped antigenic sites of the rabies glycoprotein (shaded triangles). The transmembrane sequence is indicated by the boxed area.

eliminate the need to calculate international units usingtiter results obtained from an international rabiesreference serum that originated from subjects onlyvaccinated with a rabies vaccine produced from a PMseed strain of rabies virus.

2.5. Statistical analyses

After all serum samples were tested separately withboth the CVS-11 and the Flury-LEP rabies challengevirus strains, the identification of the two vaccinationgroups (PVRV and PCECV) was unblinded and theRVNA titers were statistically analyzed to determine theeffect of serological testing by means of a homologousvs. heterologous test. To determine whether any strain-dependent difference in neutralizing antibody wasmagnified at higher titers, the titer results (both day 14and day 90) were sorted into response groups, thegeometric mean titer (GMT) of the groups was

calculated, and the GMT by challenge virus wascompared. Additionally, to determine whether matura-tion of the antibody response amplified the differences inGMT, the titer responses by day of serum drawn weresorted and the GMT of the groups was calculated andcompared by challenge virus.

3. Results

The virus titer of CVS-11 and Flury-LEP, used as thechallenge virus in each of the five serological testinggroups, remained consistently equivalent throughout thetesting period (Table 1). There was a similar wide rangeof RVNA titers obtained for each vaccination group,independent of whether CVS-11 or Flury-LEP was usedas the challenge virus strain (Table 2). There were twooutlier reciprocal titer values in the PVRV vaccinationgroup, 9500 and 19 700, exhibited by the same subject


on days 14 and 90, respectively. The GMT for eachgroup indicates higher RVNA titers were reported whena homologous challenge virus strain was used in theserological assay. The RVNA test results of individualserum samples indicated that there was a clear trend toreport higher titers when a homologous testing system(CVS-11 used as the challenge virus in the RFFIT fortesting sera from subjects vaccinated with PVRV andFlury-LEP used as the challenge virus in the RFFIT fortesting sera from subjects vaccinated with PCECV)rather than a heterologous testing system (Flury-LEPused as the challenge virus in the RFFIT for testing serafrom subjects vaccinated with PVRV and CVS-11 usedas the challenge virus in the RFFIT for testing sera fromsubjects vaccinated with PCECV) was used (Fig. 3).

The RVNA values in both the PCECV and thePVRV vaccine groups included titers in the low, mediumand high range, regardless of which challenge virus wasused in the assay. Low, medium, and high ranges weredesignated for this set of results to determine possibletrends associated with the strength of the antibodyresponse. Nearly two-thirds of the samples from each

Table 2

Rabies virus neutralizing antibody (RVNA) titers from the Rapid

Fluorescent Focus Inhibition Test (RFFIT) using a homologous

challenge virus testing system and a heterologous challenge virus

testing system

Vaccine

administered

RFFIT testing

system

GMT (range)

Challenge virus Day 14 Day 90

PCECV Homologous 1855 265

Flury-LEP (320e6300) (45e1500)

Heterologous 1275 183

CVS-11 (145e5400) (45e1100)

PVRV Homologous 2364 274

CVS-11 (360e8500) (45e9500)

Heterologous 1448 188

Flury-LEP (70e8500) (45e19700)

Serum samples were obtained from subjects vaccinated with purified

chick embryo cell rabies vaccine (PCECV) or purified Vero cell rabies

vaccine (PVRV) and were assayed using CVS-11 and Flury-LEP as the

challenge viruses in the RFFIT.

Table 1

Titer of Challenge Virus Standard (CVS-11) and Flury low egg passage

(Flury-LEP), the two rabies virus strains used as the challenge viruses

for each serological testing group

Serological testing group Titer of CVS-11 Titer of Flury-LEP

1 42.1 41.1

2 40.0 41.2

3 41.0 40.0

4 41.4 42.3

5 41.1 40.9

Geometric mean 41.1 41.1

Virus titer is expressed in TCID50.

vaccine group reported higher titers when a homologouschallenge virus strain was used for the RFFIT assay,63% for PCECV and 65% for PVRV. Approximately30% of the serum samples tested in each vaccine groupreported titers that were the same value or similar(within one standard deviation) regardless of whetherthey were assayed using a homologous or a heterologouschallenge virus strain.

The percent reduction of reported RVNA titers,when switching from a homologous testing system toa heterologous testing system was 23%, 47%, and 33%,respectively, for the low, medium, and high responsegroups in the PVRV vaccination group and 27%, 25%,and 40% in the PCECV vaccination group (Fig. 4).Thus, there was no clear trend of higher or lower RVNAtiters related to the type of testing system, and whetherthe serum tested belonged to the low, medium, or highresponse group. The overall reduction in RVNA titervalues when switching from a homologous challengevirus assay to a heterologous challenge virus assay was33% for the PVRV vaccination group, and a 31%reduction for the PCECV vaccination group.

On both day 14 (data not shown) and day 90 theGMTs were higher when a homologous challenge virussystem was used for the PVRV and PCECV vaccinationgroups (Fig. 5).

4. Discussion

Neutralizing antibodies play a critical role in immuneprotection against rabies infection. Therefore, it is

1.50

2.00

2.50

3.00

3.50

4.00

1.50 2.00 2.50 3.00 3.50 4.00

titers (CVS) log dil

tite

rs

(F

lu

ry

) lo

g d

il

PCECV Subjects PVRV Subjects

Fig. 3. Serum samples from day 90 after administration of purified

chick embryo cell rabies vaccine (PCECV) or purified Vero cell rabies

vaccine (PVRV) given in a post-exposure prophylaxis regimen were

analyzed twice by RFFIT. In one assay CVS-11 was utilized as the

challenge virus in the RFFIT and in the second RFFIT, Flury-LEP

was utilized as the challenge virus. The rabies virus neutralization titer

(RVNA) result obtained for each serum sample was plotted according

to the challenge virus used in the RFFIT. The line of unity represents

expected RFFIT values that would be equivalent regardless of whether

CVS-11 or Flury-LEP rabies virus was used as the challenge virus for

patients vaccinated with PCECV or PVRV. Similar results were seen

with the day 14 results (data not shown).


Low

GM

T

60

80

100

120

140

160180200220240260

Medium200

300

400

500

600

700800900

100011001200

High1000

1500

2000

2500

3000

350040004500500055006000

PCECV homologousPCECV heterologous



PVRV homologousPVRV heterologous



Fig. 4. Depicted are the geometric mean titers (GMT) of serum samples analyzed by the Rapid Fluorescent Focus Inhibition Test (RFFIT) and

separated into high, medium and low titer results. Serum samples were collected from subjects vaccinated with purified Vero cell rabies vaccine

(PVRV) or purified chick embryo cell rabies vaccine (PCECV), and tested with RFFIT in either a homologous or heterologous testing system. A

homologous testing system included sera from subjects vaccinated with PCECV and analyzed by RFFIT using the Flury-LEP as the challenge and

sera from subjects vaccinated with PVRV and analyzed by RFFIT using the CVS-11 as the challenge virus. A heterologous testing system included

sera from subjects vaccinated with PCECV and analyzed by RFFIT using the CVS-11 challenge virus and sera from subjects vaccinated with PVRV

and analyzed by RFFIT using the Flury-LEP as the challenge virus.

appropriate to utilize RVNA assays to measure theimmune response after rabies vaccination rather thanrelying on antigen-binding assays, which do not measurethe function of the antibodies produced. Indeed,currently the most accepted method for measuring theimmune response to rabies antigen is to measurethe amount of RVNA in serum. In the United States,the Advisory Committee on Immunization Practices(ACIP) recommends that RVNA testing should beperformed using a virus neutralization assay; thosepersons at risk of contracting rabies should have theirRVNA levels measured periodically; and a boostershould be administered to persons at risk of contractingrabies when their RVNA titer falls below completeneutralization of a specific quality of rabies virus at a 1:5

serum dilution by the RFFIT (the World HealthOrganization recognizes this level to be 0.5 IU/mL)[11,12]. The evaluation of serological levels of RVNA isalso appropriate for patients who may have a question-able immune response after post-exposure prophylaxis,i.e. when vaccine was administered or stored inappro-priately, when a patient may be immunosuppressed, orwhen a patient may have had a severe adverse reactionto the vaccine. Finally, new rabies vaccines areevaluated, licensed and approved for use partly byassessing the level of RVNA produced after vaccinationin human subjects enrolled in clinical trials.

Asmentioned earlier, theCVS-11 strain of rabies virus,generally used as the challenge virus in the RFFIT assaysthat are used tomeasureRVNA, differs in how closely it is

CVS-11 Flury LEP CVS-11 Flury LEP

GM

T (

reci

proc

al ti

ters

)

10

100

1000PCECV heterologous

PCECV homologous

PVRV heterologous

PVRV homologous

Challenge Virus Strain:

0.69 (0.58 - 0.84)GMR(PCECV/PVRV) 1.48 (1.23 - 1.80)

1.00 (0.83 - 1.21)

Fig. 5. Serum samples from day 90 after administration of purified chick embryo cell rabies vaccine (PCECV) or purified Vero cell rabies vaccine

(PVRV) given in a post-exposure prophylaxis regimen were analyzed by the Rapid Fluorescent Focus Inhibition Test (RFFIT) for rabies virus

neutralizing antibodies, using different challenge virus strains. Depicted are geometric means of reciprocal titers (GMT), error bars represent 90%

confidence intervals. Geometric mean ratios (PCECV/PVRV) of the different challenge strain comparison groups were calculated (90% confidence

intervals in parentheses), resulting in equivalent titers when using the homologous challenge strain Flury-LEP for PCECV and CVS-11 for PVRV.


related to the PM and Flury strains of rabies viruses thatare used in the production of human rabies vaccines(Figs. 1 and 2). These differences are in some instanceslocated in areas that are in close enough proximity to theantigenic sites (and also in the transmembrane region) topotentially affect the conformation of the antigenic sites.It is possible that the differences between the strains ofseed virus used in the production of rabies vaccines areenough to cause slight conformational changes in theantigen-binding site of the antibodies that are inducedafter vaccination. These slight differences in the antigen-binding site could cause the antibody to have a higheraffinity for a challenge virus used in an in vitro assaythat more closely resembles the antigen that caused itsproduction in the first place. The results of our studyindicate that the degree of homology between the strain ofchallenge virus used in the RFFIT to measure the im-mune response after vaccination and the strain of seedvirus used to produce the vaccine that subjects receivedprofoundly affects the reported RVNA values. Indeed,the use of challenge virus strains with equivalent titers inRFFIT assays resulted in approximately 30% higherRVNA values in two-thirds of the serum samples weanalyzed when a homologous testing system was used. Inaddition, the level of the RVNA titer (high, medium orlow) had no obvious or consistent effect on the percentageof titer difference reported between the testing systems.

In most cases the choice of the challenge virus strainused in a rabies virus neutralization assay does not playa critical role in the evaluation of RVNA titers; forexample, periodic titer evaluations and the determina-tion of an immune response after post-exposure pro-phylaxis where the exact titer level is less importantthan the actual detection of neutralizing antibody. Inaddition, the strain of rabies virus used in a rabies virusneutralization assay is unlikely to be a determiningfactor in the measurement of RVNA titers in personswhose pre-exposure series may be from one vaccinesource and subsequent booster(s) from another source.Similarly, persons who have had a rabies exposure willhave an immune response to the rabies antigens in theexposure strain and to the vaccine strain confoundingthe mix of antibodies produced. For all of the abovementioned reasons it would provide little benefit toroutinely measure the RVNA response using separaterabies virus challenge strains. In contrast, the measure-ment of the humoral immune response after vaccinationfor the specific purpose of evaluating a rabies vaccinemakes the choice of the challenge virus used in a rabiesvirus neutralizing assay extremely important. When theRVNA levels produced against vaccines made from twodifferent parent strains are compared using an assay thatemploys a particular challenge virus strain in the testingsystem, the combined effect of the quantity, functional-ity and specificity of the respective antibody responseis measured. As demonstrated by this study, if the

challenge virus used in the assay is more closely relatedto one parent virus strain than to the other, the titerresults obtained will be biased toward the homologousvaccine. Most importantly, the evaluation methods usedto confirm an absence of significant difference betweenthe immune response produced by two vaccines involvestatistical comparisons of the GMT by the geometricmean ratio (GMR). The Food and Drug Administration(FDA) defines bio-equivalence as ‘‘pharmaceuticalequivalents whose rate and extent of absorption arenot statistically different when administered to patientsor subjects at the same molar dose under similarexperimental conditions’’ [13]. In comparing the statis-tical evaluation of each vaccine, the confidence intervals(CI) of the GMR are examined. When the lower limit ofthe 95% CI is greater than 50% and the intervalincludes 100%, ‘‘non-inferiority’’ is achieved. To de-termine the stricter standard of ‘‘bio-equivalence’’, 90%CI of the GMR must lie within 80e125%. If thisequivalence test is applied for the day 90 results in ourstudy, the GMTs obtained for PCECV are inferior toPVRV when serum samples from subjects vaccinatedwith PCECV are tested in a heterologous testing systemusing the CVS-11 strain of challenge virus. Conversely,the GMTs obtained for PVRV are inferior to PCECVwhen serum samples from subjects vaccinated withPVRV are tested in a heterologous testing system usingthe Flury-LEP strain of challenge virus (Fig. 5). How-ever, when a homologous testing system is used to testthe serum samples for subjects in each vaccination group,not only are the two vaccines non-inferior, they areequivalent.

This report ascertains that the choice of challengevirus strain used in rabies virus neutralization assays toevaluate the production of RVNA titers after vaccina-tion should be taken into consideration when the titervalues will be used for the evaluation of new or existingvaccines. Clearly, if quantifying the immune response tothe vaccine is the objective, then using a homologousrabies virus strain in the testing would most appropri-ately reflect this goal. Finally, it is important toremember that modern cell culture rabies vaccines arehighly effective and cross-protection between strains hasbeen demonstrated [14,15]. The use of a heterologous orhomologous testing system to evaluate the level ofRVNA as a measure of complete ‘protection’ againstrabies infection is incorrect. To date, the level of RVNArequired to be ‘protective’ against infection in humansis not known for an obvious reason: it is unethical toconduct challenge experiments in humans to determinethe level of RVNA required for protection. On theother hand, the use of rabies virus neutralizing antibodytesting systems to measure the immune response tospecific rabies antigens and the response to rabiesvaccines should not only be accurate and precise, butalso meaningful.


Acknowledgments

The authors would like to thank the Centers forDisease Control and Prevention and Chiron Vaccinesfor donating the challenge viruses used in this study. Inaddition, our appreciation is extended to Dr IrisStalkamp for providing phylogenetic data and sequencecomparison information and to Dr Tony Stamp andMal Hoover for their valuable assistance in preparingsome of the figures used in this paper.

References

[1] Lafon M, Edelman L, Bouvet JP, Lafage M, Martchatre E.

Human monoclonal antibodies specific for the rabies virus

glycoprotein and N protein. J Gen Virol 1990;71:1689e96.[2] Smith JS, Yager PA, Baer GM. A rapid reproducible test for

determining rabies neutralizing antibody. Bull World Health

Organ 1973;48:535e41.[3] Cliquet F, Aubert M, Sagne L. Development of a fluorescent

antibody virus neutralization test (FAVN test) for the quantita-

tion of rabies-neutralising antibody. J Immunol Methods

1998;212:79e87.[4] Blancou J, Aubert MF, Cain E, Selve M, Thraenhart O,

Bruckner L. Effect of strain differences on the potency testing of

rabies vaccines in mice. J Biol Stand 1989;17(3):259e66.

[5] Ferguson M, Wachmann B, Needy C, Fitzgerald EA. The effect

of strain differences on the assay of rabies virus glycoprotein by

single radial immunodiffusion. J Biol Stand 1987;15:73e7.

[6] Smith JS. Molecular epidemiology. In: Jackson AC, Wunner WH,

editors. Rabies. 1st ed. San Diego, CA, USA: Academic Press;

2002. p. 79e111.

[7] Smith JS. Rabies serology. In: Baer GM, editor. The natural

history of rabies. 2nd ed. Boca Raton, FL, USA: CRC Press;

1991. p. 235e52.

[8] Tordo N. Characteristics and molecular biology of the rabies

virus. In: Meslin FX, Kaplan MM, Koproski H, editors.

Laboratory techniques in rabies. 4th ed. Geneva, Switzerland:

World Health Organization; 1996. p. 28e51.

[9] Maillard A, Gaudin Y. Rabies virus glycoprotein can fold in two

alternative, antigenically distinct conformations depending on

membrane-anchor type. J Gen Virol 2002;83:1465e76.

[10] Smith JS, Yager PA, Baer GM. A rapid fluorescent focus

inhibition test (RFFIT) for determining rabies virus-neutralizing

antibody. In: Meslin FX, Kaplan MM, Koproski H, editors.

Laboratory techniques in rabies. 4th ed. Geneva, Switzerland:

World Health Organization; 1996. p. 181e92.

[11] Centers for Disease Control and Prevention. Human rabies

prevention-United States, 1999: recommendations of the Advi-

sory Committee on Immunization Practices (ACIP). MMWR

Morb Mortal Wkly Rep 1999;RR-1:1e21.

[12] WHO Expert Committee on Rabies. 8th Report. Geneva,

Switzerland: World Health Organization; 1992 [WHO Technical

Report Series, No. 824].

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nitions. Food and Drug Administration, Health and Human

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Jackson AC, Wunner WH, editors. Rabies. 1st ed. San Diego,

CA, USA: Academic Press; 2002. p. 401e28.

[15] Lodmell DL, Smith JS, Esposito JJ, Ewalt LC. Cross-protection

of mice against a global spectrum of rabies virus variants. J Virol

1995;69:4957e62.

1

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Vaccination av hund och katt 2008 Bitr. stats veterinär Ulrika Windahl SVA, Enheten för djurhälsa och antibiotikafrågor Bra vacciner är när de används korrekt kostnadseffektiva läkemedel som förebygger sjukdom och lidande.

• Vaccinering ingår i en god djurhållning av hund och katt

• Syfte med vaccination: förbättra djurskyddet.

• Kan dock inte kompensera en i övrigt undermålig djurhållning

• En hög andel korrekt vaccinerade individer i en population: minskat generellt smittryck, dvs. bidrar till att skydda även

o individer som inte svarat på vaccinering

o ännu inte har vaccinerats

o vaccineringen misslyckats

o är dåligt skyddade mot sjukdom (t ex valpar, kattungar)

• ”Ett nej till vaccination mot allvarliga sjukdomar är detsamma som ett ja till kommande sjukdomsutbrott”

Risk: allvarliga infektioner ”glöms bort” tack vare effektiv vaccinering. Biverkningsriskerna överdrivs

• Ex: parvovirus, HCC tros vara utrotad i Sverige

• Grupper arbetar aktivt (internet) för att motverka vaccinering

• Misstroende mot veterinärer, humansjukvård

• Vetenskapligt underlag för rekommendationer

• Rapportera misstänkta fall av biverkningar! .

Vaccination av hund och katt 2008 Ulrika Windahl, SVA

Vaccinering skall liksom annan medicinsk behandling vara individbaserad, baseras på vetenskap och djurägaren skall vara informerad och involverad

• Vaccin skall användas till rätt djur vid rätt tillfälle = endast vid behov

• Onödiga vaccinationer: o Mot lindriga infektioner

o Som inte har någon positiv effekt mot infektionsrisk och utveckling av sjukdom

o Infektioner det vaccinerade djuret löper mycket liten risk att drabbas av

o Eller där risken för allvarlig biverkning av vaccinet är större än risken för allvarlig

sjukdom hos det ovaccinerade djuret.

Kunskap krävs om

• Förekomsten av ett smittämne i det land/område djuret vistas i

• Risken för klinisk sjukdom kopplad till det aktuella smittämnet.

• Hur djuret hålls

• Resor

• Krävs att djurägaren är delaktig och tillför nödvändig information.

Djurägaren har också behov av information från veterinär i samband med vaccinering.

• Djuret har normalt inte har ett gott skydd omedelbart efter förstagångsvaccinationen

• Vilka vacciner som inte skyddar (helt) mot infektion och smittspridning

• Vilken effekt vaccinet förväntas ge; varför det är indicerat ge vaccinet • Djur som vaccinerats intranasalt kan utsöndra vaccinstammen en kortare tid

• Korrekt information om biverkningar

• Normalt med viss reaktion, jämför barn (och häst)

• Tillägg till besöket: skriftlig information


Vaccinationsintervall = hur länge ger en vaccination ett immunologiskt tillfredställande skydd

• Texten i FassVet = en information från vaccintillverkaren. o De vaccinationsintervallen = de är de företaget dokumenterat effekten av vid registreringen

av vaccinet. o Företagen kan inte marknadsföra förhållanden som skiljer sig från de godkända

produktresuméerna.

• Andra studier kan visa på en annan (t.ex. längre varaktighet) av skyddet

• …..och annan effekt

• Veterinära Ansvarsnämnden 2003: ” ……VA i sin bedömning av anmälda fall beaktar så väl vetenskap som beprövad erfarenhet, varför vaccinationsintervall som bygger på sådan grund inte löper risk att ifrågasättas på grund av avvikelse från rekommenderade intervall i FassVet.”

• OBS: om en enskild veterinärs vaccinationsrutiner väsentligt avviker från tillverkarens rekommendationer ökar kravet på aktsamhet, och att veterinären håller sin vetenskapliga kunskap uppdaterad inom detta område. (Norska vaccinrapp -03)

Bristande möjlighet vaccinera kan leda till

- Dödsfall hos människa - Dödsfall vilda djur - Djurlidande, dödsfall husdjur - Svält…

Generell risk I-länder: för få djur vaccineras – men med onödigt många vacciner.

• Basvaccin (core vaccine) ”till alla” • Tilläggsvaccin (non-core vaccine) ”Till en del, i en del situationer” • ”Rekommenderas ej -vacciner” = i ett visst land/generellt • Vaccination enligt lagstiftning

www.sva.se Djurhälsa – hund, eller katt

SVS-SVA:s vaccinationsrapport 2003, arbete pågår med revidering = uppdatering

ABCD-vets.org

AAFP

AAHA

WSAVA Vaccination av hund och katt 2008 Ulrika Windahl, SVA

Basvaccin - katt

Tilläggs vaccin ‐ katt

Rekommenderas ej

Basvaccin - hund

Tilläggsvaccin - hund

Rekommenderas ej

Kattungar bas/core vaccine

Vuxna katter bas/core vaccine


Valpar bas/core vaccine

Vuxna hundar - bas/core vaccine

kvÄllssymposium 2008 vaccinering av hund och katt

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