SHORT CONTRIBUTIONS TABLE 1 Number of horses with local reaction to strangles vaccination

Reactions to strangles vaccination CSL Limited, 45 Poplar Road, Parkville, Victoria 3052

H SMITH

Strangles is an acute bacterial disease of the upper respiratory tract caused by Streptococcus equi. It is characterised by abscesses of the lymph nodes of the head and neck. In severe cases infection may spread to internal organs and lymph nodes. This form of the disease is callcd bastard strangles.

An aluminium adjuvanted vaccine* based on the surface (M) protein of thc organism is available. This cell-free vaccine replaced an early bacterin administered in dose volumes increasing from 2 to 20 ml,. Adverse reactions were frequent after administration of the bacterin. The M protein vaccine, when used as recommended, in- creases the resistance of the vaccinated horse to infection. Within herds vaccination limits the severity of outbreaks (Hoffman et a1 1991). Dcspite this a recent survey found that the recommended vaccination programme was rarely followed (Jorm 1990).

Primary vaccination against strangles generally takes place in foals. It is recommended that the first dose of I mL of vaccine be given at 3 months of age with two further I mL doses at intervals of at least two weeks between each dose. Vaccination at an earlier age is carried out in some areas, but has not been universally recommended because of anecdotal reports of an increase in susceptibility to strangles in foals vaccinated when less than 3 months of age.

The intramuscular route is recommended to minimise the visible site reaction and for ease of administration. Annual revaccination is recommended. Where the risk of infection is high, such as breeding complexes, training stables and situations where young horses are assembled from a wide area, six-monthly revaccination may give increased protection.

This report describes adverse reactions reported to the manufacturer after vaccination of Australian and New Zealand horses. Probable causes are discussed. Purpura haemorrhagica was not observed.

Records have been maintained since the M protein vaccine was first sold in 1976. Seventy-one separate reports on a total of 3557 animals have been received in which 235 individual horses reacted adversely after vaccination. Of these 40 were 1 year or less in age (Tables1 and 2). During this period over 2 million doses of the vaccine have been sold. This represents three reports per 100 000 doses or about one reaction per 10 000 doses sold.

Local reactions at the vaccination site account for 72% of reports (Table I). The swellings, which are firm or oedematous, may be hot or painful to touch, raised 1 to 2 cm above the surrounding skin and up to 15 cm by 6 to 7 cm in size. They usually appear the day after vaccination and persist for about 5 to 7 days.

Site reactions may be due to an acute inflammatory reaction to the aluminium adjuvant or to an allergic immediate or delayed type hypersensitivity to the vaccine. In horses with high levels of precipi- tating antibody to S equi antigen-antibody-complexes may form resulting in an Arthus or local Type III hypersensitivity reaction (Tizard 1992). Gentle massage of the site after vaccination may reduce the occurrence of reactions to the adjuvant. Nevertheless, small granulomatous nodules may persist at the site of administration of aluminium adjuvanted vaccine for many months (Fawcett 1984). Hypersensitivity reactions are usually reported in horses previously vaccinated against, or recently exposed to, strangles. Where such a reaction is suspected the horse may respond to treatment with anti- histamines. Increasingly severe reactions are reported when sub- sequent doses are given within a 2- to 3-month interval.

Rarely skin organisms are carried in with the vaccine by the vaccination needle resulting in infection of the site and a discharging

* Equivac-S@ CSL Limited,Parkville, Vic

Type of swelling Oedematous Abscess* Pain Firm

+ 26 2

- 117+ 4t 21 ~ ~~

All horses were adults except: * 20 foals, 1 yearling, Streptococcus zooepidemicus and

7 7 foals, 1 yearling $ 1 foal, 1 yearling

Streptococcus equisimilis isolated

TABLE 2 Numbers of horses with systemic reactions to strangles

vaccination

Stiffness 23' Arthritis 14 Fever 11 Oedema -Ventral abdominal 5

Lower leg 4 Founder 6 Lameness 6 Lethargy 5* Respiratory signs 3+ Colic 2: Ataxia 2 Urticaria 2 Failure to protect 244

All horses were adults except: * 2 foals t 1 yearling, 2 foals $ 1 foal 6 1 yearling, 1 foal

abscess. This is an undesirable complication, but does not appear to interfere with the immune response. Its occurrence can be minimised by careful preparation of the vaccination site, as recommended by the manufacturer.

Systemic signs of stiffness, arthritis, lameness, founder and leth- argy described in 24% of reports may occur alone or in association with a reaction at the site. Fever, respiratory signs, colic and ataxia are less frequent (Table 2). These signs may be due to cytokines or complement fragments released during Type 111 or delayed-type hypersensitivity reactions to Sequi antigens or as a result of inflam- mation (Morris 1987; Tizard 1992). As with local reactions systemic signs disappear by 5 to 7 days after vaccination in most cases.

Swelling of the lower legs was reported in 4 horses. Despite the absence of petechial haemorrhages in skin or mucous membranes, this syndrome was reported as purpura. These cases are believed to be due to allergic reaction of the Type 111 class. In Type 111 hyper- sensitivity, mediators activated by circulating antigen-antibody- complexes damage the endothelium of small blood vessels resulting in localised escape of intravascular fluid and oedema of the surrounding tissues (Tizard 1992). Immune complexes containing IgA and S equi antigens have been found in horses with post- strangles purpura haemorrhagica (Galen and Timoney 1985). Urticaria, which was reported in two horses, is usually associated with immediate or Type I hypersensitivity but may also be a manifestation of immune complex mediated or toxic vasculitis (Morris 1987). Horses that react systemically may benefit from treatment with antihistamines and anti-inflammatory agents.

No reports have been received of classical purpura haemorrhagica after vaccination with the M protein vaccine. Nor has it been reported

Australian Veterinaty Journal Vol. 7 1, No 8, August 1994 257

in increased incidence in vaccinated horses on subsequent exposure to strangles infection, In this series two of 24 vaccinated horses were reported to have developed post-strangles lower leg oedema. Never- theless, purpura haemorrhagica is reported after use ofother strangles vaccines, and M antigen has been identified in immune complexes recovered from horses with post-strangles purpura (Galen and Timoney 1985; Sonea 1987). Therefore it must be considered as a potential adverse reaction to use of any strangles vaccine.

Revaccination is not recommended for 12 months where a severe local or systemic reaction has occurred. This contraindication is historically based. Flowever. the probable aetiological role of im- mune complexes in many of these events indicates a long interval before revaccination is warranted.

J'urpura haemorrhagica is a recognised sequel ofclassical strangles. Recently a correlation has been found between purpura haemor- rhagica, high blood concentrations of IgA and a persistent focus of S equi infection (Galen and Timoney 1985). Although details of incidence are difficult to obtain. a study \\as carried out at the Swedish remount depot ti-om 1969 to 1971 (Hafer et 01 1973). Twenty-sevcn cases of purpura haemorrhagica occurred over a 3- month period in 1438 horses being acclitnatised. Eleven cases fol- lowed typical strangles and the remainder non-abscess forming. pharyngitis or bronchitis. The number of strangles cases was not given. but by extrapolation from graphs. one in seven cases of strangles might have been complicated b!, purpura haemorrhagica (Hafer et a / 1973). A similar ratio is reported by others (Sweeney ef a/ 1987). l h e stress in the remount depot was severe with up to four viruses recovered from nasal swabs that were collected preced- ing or during the strangles cpisodes. The incidence of purpura haemorrhagica coincided with the incidence of strangles, but the mechanism leading to purpura (vascular damage caused by antigen- antibody-complexes) suggests that it should follow the precipitating condition by several days (Reef 1987). Ifthis is the case. purpura haemorrhagica in the Slvedish remount depot may be related not to strangles but rather to all cascs of respiratory disease. This would gibe an incidence of one cast of purpura to eve5 forty cases of phavngitis. bronchitis or typical strangles.

Thc absence or very Ion incidence of infectious diseases known to be associated with purpura haemorhagica. such as equine influenza and equine arteritis. may account for the apparent low incidence of purpura haemorhagica in clinical cases of strangles in Australia.

In our experience purpura haemorrhagica is not a complication of vaccination with vaccine prepared from M protein although some evidence of immune-mediated vasculitis. such as oedema of the lower legs and body \\all. may follow vaccination in rare cases. Where hypersensitivity or antigen-antibody-complex disease are suspected treatment \vith antihistamine and anti-inflammatory agents or corticosteroids is indicated. Vaccine should not be rcadministered within 12 months.

References Fawcett HA (1984)Arch Dermaiol l20:1318 Galen JE and Timoney JF (1985)Jlmmunol135: 3134 Hafer B, Slcck F. Gerber 11, Loher J,Nicolet J and Paccaud MF (1973) froc

3rd In1 CunfEquine Inf Dis. edited by Bryans JT and Gerber H. Karger. Basel, p 527

Hoffman AM. Slaemptli HR. Prcscott JFandViel L (1991).4mJ I Z I ResS2: 5 89

Jorrn IR (1990) .41cZi I et J 67 436 Morris DD (1987) JAni IGt .\ltdAssoc 191:460 ReefV (19x7) Cirrrent 73ierupy rn Eqtrine .\ledicine. edited by Robinson NE.

Sonea I (1987) Current Therap In Equrne .lledicine. edited by Robinson NE.

Swecney CK, Whitlock RH. hleirs DA, Whitehead SC and Barningham SO

'Tizard IK (1992) l'eierinar,~ Immzmolog.: An Introdttctron, 4th edn,

(Accepted.for pirhlrcation 23 March 1994)

Saunders. Philadelphia, vol 2. p 312

Saunders, Philadelphia vol 2, p 590

(1987) , J , 4 m Itt.Ifec.d.4s.r~~ 191:1446

Saunders. Philadelphia p 270

Strain variation in the killing of staphylococci by avian heterophils

School of Veterinary Studies, Murdoch University, Murdoeh. Western Australia 6 I50

JN MILLS

A bactericidal assay using chicken heterophils was dcvelopcd to determine whether or not there was a correlation betwecn the intracellular survival of strains of staphylococci and their known virulence. The virulence of the four strains of staphylococci used in the study had been assessed previously by Kibenge et n / ( 1983). Two pathogenic strains ( I 78 I and 1036) and one non-pathogenic strain of Staplidococcus nitreiw ( 1333) and one non-pathogenic strain of S lyicrts ( 1609) were used.

Log-phase bacteria cultured for 4 h in tryptonc soy broth ('lX13) were washed twice in TSB. adjusted to a concentration of9 x IO"/ml, and opsonised by the addition of0.5Yn pooled normal chicken serum (NCS) at 37°C for 30 min. No precipitating antibody to the organisms was detccted in thc NCS from specitic pathogen-tree (SW) chickens by agar gel immunoprccipitation tests. For each comparativc bacter- icidal assay. heparinised blood was collected from 4 normal mature SPF chickens. IHeterophils were separated by I'crcoll* discontinuous density gradient centrifugation as described previously (Mills and Wilcox 1993). pooled. washed twice in antibiotic-free RPMI 1640 mediumt and adjusted to aconccntration o f 9 x IO'hL. Counts were performed using a haeniocytomcter chamber and phase-contrast microscopy .

For each assay. 0. I ml> ofopsoniscd bacteria was added to 0.9 mL of pre-warmed heterophils providing a ratio of 10 hetcrophils to 1 bacterium. in sterile, siliconised glass tubes (1 20 m m x I5 mm) with screwcap lips. and the samples were incubated at 37°C with constant agitation (100 shakes per min). The assay conditions used contain higher proportions of heterophils than in a previous report (Andrea- son et a/ 1991). but were similar to assay conditions reported by Finlay-Jones et a/ (1984) using mammalian leukocytes. 'I'hesc con- ditions were found necessary to overcome the unavoidable presence of nucleated erythrocytes in the heterophil preparations and the apparent resistance of one of the strains of bacteria used. Medium RPMI 1640 was substituted for the hcterophil preparation in control assays for each bacterial strain. To assess the cffect of the addition of NCS on the bactericidal activity of the heterophils, 0.1 mL NCS (final concentration 10%) was added to the mixtures in scparate assays.

After 0. 90 and 120 min, 0.5 ml, samples were removed and immediately diluted in 5 mL of 0. I % Triton X- 100: in phosphate buffered saline to lyse the heterophils. The number ofcolony forming units (CFU)/mL was determined in duplicate after serial dilutions in TSB and incubating inoculated nutrient agar plates at 37°C for 18 h. Changes in concentrations of viable staphylococci were calculated by subtracting the number of CFU/mL at 90 and 120 min from the initial concentration using values converted to logarithms to the base 10. The viability of heterophils was assessed at the end of each experiment by the exclusion of 0.5% trypan blue and found to be

Analysis of variance tests of the results showed a significant (P 0.05) decrease in survival of the non-pathogenic S aureiis (1333) compared with the other strains, in the bactericidal assays in which NCS was excluded, whereas the non-pathogenic S hvicus showed no

97% to 100%.

* Pharmacia Fine Chemicals. Uppsala, Sweden t Flow Laboratories, Stanmore, NSW $ BDH Ltd, Poole, UK

258 Australian I.Zferinary./ournal Vol. 71, No. 8, August 1994