A review of laboratory techniques and their use in the diagnosis of Leptospira interrogans serovar hardjo infection in cattle

CR SMITH*, PJ KETTERER?, MX McGOWANS and BG CORNEYt

SUMMARY: This paper reviews the laboratory diagnosis of Leptospira hardjo infection in cattle. Two genotypes of L hardjo, Hardjoprajitno and Hardjobovis, have been identi- fied in cattle, but only Hardjobovis has been isolated in Australia. There are problems with diagnosis and control of bovine leptospirosis. Infection is usually subclinical and the serological titres vary greatly in peak and duration. Leptospires may be excreted in urine for up to 18 months. Low microscopic agglutination test titres may be significant in unvaccinated herds as indicators of endemic infection. Vaccines differ in their compo- sition, and their efficacy is difficult to evaluate. The serological response after vaccination is difficult to differentiate from the response after infection. Pregnant cows that become infected may abort, but this is usually after the serological response has peaked. There- fore, paired serum samples are of little use in diagnosing abortion caused by L hardjo. Fluorescent antibody techniques are more sensitive than dark field microscopy for detection of leptospires in urine and tissue samples. Techniques for culture have im- proved but are still difficult to perform and take 3 months or longer for results to be known. DNA probes and polymerase chain reaction tests are very sensitive and specific, quick to perform, and can be used on fluid and tissue samples. Aust Vet J 71 : 290 - 294

Introduction Leptospirosis caused by infection with Leptospira interrogans

serovar harajo (L hurdjo) is responsible for considerable financial losses in cattle in the UK (Ellis 1986a), but its economic effects in NZ and Australia are uncertain (Dixon 1983; Chappel et a1 1989). However, it is a serious zoonosis in these two countries. Two genotypes have been described: Hardjobovis and Hardjoprajitno, but so far, only Hardjobovis has been identified in Australia (Robinson et nl1982; Skilbeck and Davies 1989; Ramadass and Marshall 1990). L hardjo vaccines sold in Australia differ in composition; most are derived from Hardjobovis strains, but some are derived from Hard- joprajitno. Because of difficulty in diagnosing current L hardjo infections, veterinarians have a problem in determining the efficacies of these vaccines (Hancock et a1 1984; Bolin et a1 1989~; Hjerpe 1990). The purpose of this article is to review current and recently proposed methods for laboratory diagnosis of L hardjo infection and to assist practitioners interpret serological and other laboratory results when they are investigating suspected cases of L hardjo infection.

Pathogenesis Differences have been observed in the pathogenicity of the two

genotypes of L hardjo. Ellis et af (1988) found Hardjoprajitno to be more commonly associated with clinical disease and Hardjobovis was associated with renal infection suggesting that Hardjoprajitno is more pathogenic. However, in a recent Australian study of strains of L hardjo isolated from cases of bovine agalactia and abortion, Hardjobovis was the only genotype identified (Djordevic et a1 1993). It is only recently that differences in genotype have been recognised and in many earlier reports of L har4o infection the genotype has not been characterised.

* Dayboro Veterinary Surgery, Department of Farm Animal Medidne and Production, University of Queensland. PO Box 3, Dayboro, Queensland 4521

t Animal Research Institute, Queensland Department of Primary Industries, Fairfield Road, Yeerongpilly. Queensland 4105

1 Department of Farm Animal Medicine and Production, Universlty of Queensland, PO Box 125, Kenmore, Queensland 4069

Infection with L hurdjo can occur directly via mucous membranes and through abraded or water-softened skin (Ellis 1984). Leptospires can be found in the blood 4 to 10 days after infection. This transient leptospiraemia may last from a few hours to 7 days (Ellis 1986b). Clinical signs are usually not observed but pyrexia, anorexia and agalactia associated with a ‘flaccid’ mastitis have been described (Ellis et a1 1976; Higgins et a1 1980). Organisms invade the body tissues including the kidney, mammary gland, liver, brain, and genital tract. The serological response initiated results in the produc- tion of specific antibodies that opsonise leptospires, facilitating elimination of the organism from most parts of the body (Thiermann 1984b). However, leptospires that reach the proximal renal tubules, genital tract and mammary gland appear to be protected from circu- lating antibodies (Marshall 1985). They persist and multiply in these sites, and may be excreted and transmitted to susceptible, in-contact animals. Urine is the major source of infection for lateral transmission.

Leptospires are excreted, often intermittently, in urine for a variable period. Shedding times ranging up to 241 days (Hellstrom and Blackmore 1979) or 542 days (Thiermann 1982) have been recorded. Higher rates of renal carriage have been observed in younger cattle than older cows (Ellis et a1 1981a). L hardjo infections persist for up to 142 days in the pregnant bovine uterus, and 97 days in the non-pregnant uterus (Thiermann 1982). Infection of the pregnant uterus may cause infection and death of the foetus. Leptospires may be detected in the uterine discharge for up to 8 days after calving, and persist in the oviduct for up to 22 days after calving (Ellis 1984). In bulls, L hardjo has been isolated from the testis, epididymis, and seminal vesicles (Ellis et a1 1986), but the period of persistence of infection is unknown. L hardjo can be recovered from the mammary gland for up to 91 days after infection (Thiermann 1981).

When foetal infection is a sequel to invasion of the uterus, abortion may occur 4 to 12 weeks after the acute phase of infection (Ellis 1986b). Aborted foetuses are usually expelled in an autolysed state. Premature weak calves and full-term weak or stillborn calves may also be produced.

Serological Responses The first serological response in animals infected with L hardjo is

the production of immunoglobulin M (IgM) antibodies. These rise

290 Auslrdian Veterinary Journal Vol. 71, No. 9, September 1994

rapidly but may fall to an undetectable concentration by 4 weeks after infection (Adler et a1 1982). Within 1 to 2 weeks of infection, IgG, antibodies appear, and at 3 months they make up 80% of antibodies detected in the microscopic agglutination test (MAT) (Marshall 1985).

The MAT titre peaks 11 to 21 days after infection (Ellis and Michna 1977) but may vary from the order of 3200 to a concentration that is not detectable (Stringfellow et a1 1983). The MAT titre declines gradually over 11 months (Hodges and Ris 1974) but the period of persistence is variable. Titres of 50 to>400 may persist for 12 months (Mackintosh et a1 1980), or for 2 years (Worthington 1982). How- ever, in one study ofabortion caused by L hardjo, 22.8% of aborting cows had no detectable antibodies at the time of abortion (Ellis et a1 1982b). In another study, 19.6% of L hardjo renal carriers had no detectable agglutinating antibodies (Ellis et a1 198 1 b).

Vaccination induces the production of antibodies that are mainly of the IgG class (Bey and Johnson 1986). This may be one reason that agglutinating antibody titres after vaccination are usually lower than titres after natural infections (Kingscote and Proulx 1986). Generally, titres reach their peak 2 weeks after a two-dose vaccina- tion course (Stringfellow et a1 1983). Individual peak titres vary greatly from 0 to a maximum of about 800. Hardjobovis vaccines appear to induce higher agglutination titres than Hardjoprajitno vaccines (Bolin et a1 1989~) despite the fact that Hardjoprajitno is routinely used in the MAT test (JK Elder, personal communication). Antibodies after vaccination decrease rapidly (Marshall et a1 1979; Hodges and Day 1987). Allen et a/ (1982) showed that 95% of vaccinated heifers did not have MAT antibodies 20 weeks after the second of 2 vaccinations given 4 weeks apart. The absence of MAT antibodies is not necessarily an indication that protection has waned (Schollum and Marshall 1985). Vaccinated animals were protected from natural L hardjo challenge for many months after their MAT titres became undetectable (Mackintosh et a1 1980; Hancock et a1 1984). Vaccination of animals that have been previously sensitised by either vaccination or infection, may result in a higher antibody response than that achieved after initial vaccination (Mackintosh and Marshall 1980; Hjerpe 1990).

However, the serological response of calves vaccinated at 3 months of age was lower than those vaccinated at 6 months of age (Schollum and Marshall 1985) because of the presence of maternal antibody. In calves aged 4 to 18 weeks, Palit et a1 (1991) showed that the titre before vaccination determines the serological response to vaccina- tion, with the rise in titre after vaccination being inversely propor- tional to the titre before vaccination.

Similarly, cattle that were experimentally infected with L hurdjo after vaccination, failed to show a serological response despite becoming leptospiruric (Marshall eta/ 1979; Mackintosh et a1 1980; Flint and Liardet 1980; Allen el a1 1982; Bolin et a1 1989~). Broughton et a1 (1984) postulated that antibodies circulating at the time of experimental infection have the capability to remove lepto- spires rapidly from the blood, resulting in insufficient stimulus for a serological response. However, Bolin et a1 (1989c), suggested that lipopolysaccharides on the surface of the leptospires are poor activa- tors of B lymphocytes so that a secondary serological response may not occur.

The maturity of the foetal immune system at the time of infection determines the serological iesponse of the foetus. By 4 to 5 months of gestation, the foetus can produce circulating IgM and IgG anti- bodies (Sawyer et a1 1973) and since maternal antibodies cannot cross the placental barrier (Brambell 1970), the presence of lepto- spiral antibodies in the foetus is indicative of infection of the foetus. Ellis et a1 (1982a) in the UK, demonstrated that 30 (17%) of 173 aborted foetuses infected with L hardjo had antibodies to L hardjo, but no leptospiral antibodies were detected in 130 foetuses from slaughter houses, despite L hardjo being isolated from 6 of these foetuses (Ellis et a1 1982~).

Microscopic Agglutination Test The MAT is the most widely used laboratory test for diagnosis of

leptospirosis (Ellis 1986b). Most workers have adopted a procedure similar to that described by Chappel (1993).

Because agglutinating antibodies wane, the sensitivity of the MAT in detecting animals infected for more than two years is low, probably less than 50% (Blackmore 1985a). To increase the sensitivity for detecting such infections in epidemiological studies, Blackmore (1985b) suggested the use of a lower initial serum dilution. Sullivan (1 970; 1972) could not correlate MAT responses with the occurrence of leptospiruria. Few false positive reactions occur in cattle, as the surface antigens of leptospires are not shared with other organisms. However, cross-reactions caused by exposure to leptospires of the same serogroup can occur, for example, infection by L bulcunicu (Mackintosh et ul 198 1) and L medanensis can produce false positive L hardjo reactions. In general terms, the specificity and sensitivity of the MAT test are high, but sensitivity declines when animals are tested a considerable time after infection (Blackmore I985a). When evaluating their sensitivity, laboratory tests are normally compared against the presence or absence of L hardjo in the urine or tissues of the animal being sampled. The definitive test is culture of the organism (Thiermann 1984b).

A major concern is the failure of the MAT to differentiate between titres after vaccination and those after natural infection since the titres may be of similar magnitude (Adler et a1 1982; Hodges and Day 1987). However, as already mentioned, titres after infection are, in general, higher and persist longer than vaccination titres; therefore the height of the titre and interval since vaccination, can, in some instances support the diagnosis of natural infection. Infection is more readily diagnosed by serological tests in unvaccinated herds or animals. Vaccinated cattle that subsequently become infected, may not mount an agglutinating antibody response (Marshall et a1 1979; Allen et a1 1982; Bolin et a1 1989~).

In the late 1960s and early 1970s, MAT titres as high as 300 000 were reported with natural and experimental L hardjo infections (Sullivan 1972) but much lower peak titres (3200 is rare) have been seen in veterinary diagnostic laboratories in recent years in Australia.

In reports of MAT results from veterinary laboratories, titres of > 100 for leptospiral serovars are interpreted as positive, 100 as suspect, and 50 as negative. These arbitrary values are meant to give an indication of clinical significance (evidence of recent infection) but they apply poorly to L hurdjo infection. Bey and Johnson (1986) suggested that a titre of 40 and more should be considered positive for recent infection with L hardjo. However, titres of this order can persist for two years after infection. Blackmore (1985b) believes a low serum dilution, in the order of 25, is needed to detect past infections for epidemiological studies. Ellis (1 986b) stated that if foetal agglutinating antibodies are to be detected, the initial dilution should be 10.

Because of the frequent low or possibly negative MAT titres in animals recently infected with L hurdjo, making a diagnosis on the basis of a serological result from one animal is extremely difficult (Elder et a1 1985). Ellis et al(1982b) reported that there was no value in examining paired serum samples from individual cows after abortion because titres are either falling or static at the time of abortion (Ellis 1986b). Ellis et a1 (1982b) found from limited data that if serum from an aborting cow had a titre of > 1000, then there was an 80% probability that the foetus was infected. Up to 17% of infected foetuses may have MAT antibodies (Ellis et a1 1982a) and titres as low as 10 may be significant (Ellis 1986b).

The MAT is best used as a screening test when investigating the possibility of L hardjo infection in groups or herds of cattle. At least 30 animals (or 10% of large groups) should be bled and animals of various ages should be included (Stoenner 1972; Hathaway et al 1986).

Australian Veterinary JoumalVol. 71, No. 9, September 1994 291

Disadvantages of the MAT are well known. The test is laborious and time-consuming. Live leptospires must be propagated to carry out the test with the risk of infection of laboratory staff(Cousins et a1 1985).

Enzyme-linked Immunosobent Assay Deficiencies of the MAT led some workers to develop enzyme-

linked immunosorbent assays (ELISA) (Adleretull982). The tnajor benefit of the ELISA is that it can be specific for IgM antibodies or IgG antibodies. A positive IgM-specific ELISA result can therefore indicate that infection has occurred within the previous month (Adler et al1982). Using an ELISA, Cousins et a1 (1985) detected IgM from 1 to 5 weeks after experimental infection of 6 animals. It has been suggested that the ELISA and MAT measure different antibodies (Adler et a1 1982). Goddard et a1 (1991) reported better correlation of MAT results with their IgM-specific ELISA than with their IgG-specific ELISA. However, Bercovich et a1 (1 990) found a 90% correlation between their IgG,-ELISA and the MAT. Their test also had extremely good serogroup specificity. Differences in the performance of respective E L S A may be due to differences in components, particularly variations in method of antigen preparation.

Various ELISA are reported to be more sensitive than the MAT. Thiermann and Garrett (1 983) found their ELISA detected antibodies in 39 (64%) of 6 1 sera from vaccinated cattle while the MAT detected only 1 1 (18%). There were no false positive results from known negative controls. Cousins et a1 (1985) found no relationship be- tween ELISA results and the occurrence of leptospiruria. Chappel et a1 (1989) detected antibodies in 6 of 1 1 infected foetuses using an IgM-ELISA; the other 5 foetuses were seropositive to MAT and one of the 1 1 was seropositive to both. Presumably foetuses that were positive to the MAT and negative to the ELISA had been infected for more than 4 weeks and only IgG antibodies were present. In the same study, results of IgM-ELISA from the sera of cows with the infected foetuses gave better correlation with foetal infection than the MAT.

Like the MAT, ELISA fail to differentiate between antibodies resulting from vaccination or infection (Thiermann 1984a). How- ever, the increased sensitivity of ELISA, which also enhances its ability to identify recent infections, may one day see it in common use. The fact that live antigens are not required also gives the ELISA advantages over the MAT.

Complement Fixation Tests Complement-fixing antibodies are detectable for a shorter time

after infection than MAT antibodies (Hodges and Ris 1974). The CFT titres after vaccination are highly variable, ranging from 20 to 640 and persisting for I to'14 weeks, and generally they agree with MAT titres (Hodges and Day 1987). The CFT has been claimed to be as reliable, for the detection of cows with leptospiruria, as the MAT (Hodges et a1 1979). The CFT can be semi-automated and is therefore less labour intensive than the MAT and it also has the advantage of using a killed antigen (Hodges and Weddell 1977). CFT usually does not provide for the differentiation of serovar of leptospire, which restricts its usefulness.

Microscopic Examination Dark field microscopy (DFM) has been used for more than 40 years

to demonstrate leptospires in bovine fluids (for example, urine) and tissues. Simmons (1950) and Doherty (1 966) examined urine by DFM to demonstrate L pornonu infection in cattle, and Doherty (1966) reported that DFM was superior to guinea pig inoculation techniques. Urine is collected from the suspect animal and formalin is added to a final concentration of 1% to prevent bacterial over- growth. The sample is centrifuged at 3450 g for 20 to 30 minutes, then a drop of the deposit under a coverslip is examined by DFM

(Hodges et a1 I979). A positive result is dependent on the observation of intact leptospires, which are often confused with proteinaceous filaments known as pseudo-leptospires (Rahman and Macis 1979; Waitkins and Zochowski 1990). Therefore a skilled microscopist is required (Palmer 1988). DFM can not determine the serovar of any leptospires seen.

Examination of tissues and fluids for leptospires by microscopy can be improved by fluorescent antibody tests (FAT). Direct or indirect techniques using fluorescein-labelled antibodies may be used (Bolin et a1 1989b). The technique is rapid, and may be used with frozen (Ellis et a1 1982a) as well as fresh tissues or urine (Bolin et a1 1989a). The FAT is more sensitive than DFM, detects degenerated as well as intact leptospires and, unlike DFM, it may be serovar-specific.

Histological Staining Techniques Examination of fixed foetal tissues for leptospires has been used

for survey and experimental studies of reproductive loss in cattle. Leptospires stain poorly with aniline dyes (Ellis 1978), and silver impregnation techniques are used to demonstrate organisms (Drury and Washington 1967; Ellis and Michna 1976). Warthin-Starry, modified Dieterle (Van Orden and Greer 1977; Bolin et ul 1989% c) and other silver stains have been used. Silver impregnation stains can detect only intact organisms. Immunochemical techniques include an immunogold-silver labelling (Skilbeck and Chappel 1987) and an immunofluorescent histological stains (Skilbeck 1986). In general, histological and immunochemical techniques lack serovar specificity.

Microbiological Culture Definitive diagnosis of leptospirosis is usually achieved by culture

and serological identification of the infecting organism (Thiermann 1984b). Isolation may be attempted from foetal kidneys (Ellis et a1 1982a), liver (Thiermann 1984b), lung (Thiermann 1984a) and aqueous humour (Ellis 1978). When sampling autolysed foetuses, Thiermann (1984a) obtained higher recovery rates from aqueous humour than from kidney or liver. Small tissue samples may be homogenised by expelling 1 mL oftissue from a sterile 10 mL plastic syringe (without needle) into a tube of Ellinghausen McCullough Johnson Harris culture medium (EMJH) (Thiermann 1984a). Smith et a1 (1967) and Ellis and Michna (1976) reported that post-mortem autolysis of the foetus rapidly killed the leptospires, but Ellis et a1 (1982a) found that attention to components of the culture medium and use of a dilution culture technique gave greater success with foetal tissues.

Isolation of leptospires from urine is not as difficult as isolation from foetuses, but still requires patience and persistence. Culture of urine obtained with the aid of diuretics gives the best results (Thier- mann 1984a). Commonly, frusemide is injected intravenously at a rate of 0.5 to 0.8 m a g , and the second voiding of urine is collected (Nervig and Garrett 1979), being careful to minimise contamination. Urine must be added to a suitable culture medium, for example EMJH, immediately after collection and despatched as quickly as possible to the diagnostic laboratory. Chappel (1993) describes detailed techniques for culture of leptospires.

Leptospires are difficult to isolate from vaccinated animals even though their presence may be detected using other means (Bolin et af 1989b). Possible reasons for this include: the presence of antibodies (Stuart 1956) or other substances that inhibit the growth of L hardjo in vitro; presence of nonviable leptospires (Bolin et a1 1989~); or insufficient numbers of leptospires in the samples.

Isolation of L hardjo can be a prolonged exercise with only half of the isolations being made within 10 weeks (WA Ellis, personal communication). Culture media are examined by DFM at least fortnightly for three months after sampling. The procedure is labour- intensive, expensive, and requires skill and experience. Identification of isolates to the serovar level is usually carried out at reference

292 Austmiian Vereriruoy Journal Vol. 71, No. 9, September 1994

laboratories and uses time-consuming, cross-absorption agglutina- tion procedures. For these reasons leptospiral culture is not used routinely in diagnostic laboratories. A rapid, sensitive, and serovar- specific technique that can detect leptospires in fresh, frozen or fixed samples is required (Bolin et a1 1989b).

DNA Probes and Polymerase Chain Reactions Detection of leptospires in tissues using a DNA genomic probe was

first described by Terpstra et a1 (1986). Millar et a1 (1987) used a DNA probe prepared from L pomona to demonstrate leptospires in pig urine and foetal calf serum. The probe detected heterologous serovars L hardjo or L turussovi with only slightly reduced sensitivity and did not react with DNA of porcine isolates of Stuphylococcus aureus, Escherichiu coli or Streptococcus sp. McCormick et a1 (1989) found their DNA probes to be less sensitive than culture, but had advantages of being rapid, less laborious, and applicable to frozen tissues. Le Febvre (1 987) described a DNA probe that could identify L hardjo genotype Hardjobovis in cattle. Zuemer and Bolin (1988) cloned a repetitive DNA sequence from Hardjobovis, and found it to be a sensitive and specific probe for diagnosis of Hard- jobovis leptospirosis. This probe was more sensitive than either fluorescent antibody or cultural techniques for diagnosis and was also highly specific (Bolin et ul 1989b).

Van Eys et a1 (1989) developed a polymerase chain reaction (PCR) for L hardjo. They could detect less than 10 leptospires in urine and media seeded with Hardjobovis using the PCR followed by Southern blotting (Southern 1975). Woodward et a1 (1991) also reported a PCR for Hardjobovis that was based on a repetitive sequence. Their PCR was specific, but no data on sensitivity were reported. In time, DNA-based techniques will probably provide rapid and sensitive diagnostic techniques that are serovar- and genotype-specific.

Discussion Over the last forty years, many laboratory techniques have been

used to aid in the diagnosis of infection with L hardjo. Serological and microbiological detection of chronically infected animals is difficult, as is the confirmation of leptospirosis as a direct cause of reproductive losses in a herd. Serological tests fail to differentiate between antibodies produced after vaccination and after infection but higher titres are indicative of infection. Microscopic examination lacks serovar specificity and is not sensitive enough to detect degen- erated leptospires. Fluorescent antibody microscopy has improved sensitivity and may be serovar specific. Immunochemical staining techniques lack serovar specificity and have not been accepted by diagnostic laboratories for routine use. Culture techniques are labo- rious, expensive, may lack sensitivity and results are often not available for 3 to 6 months.

There is hope that DNA technology will provide diagnostic labo- ratories with a rapid, sensitive, and specific test for L hurGo, which can be performed on fresh or frozen tissues or fluids, and possibly on fixed material. Veterinary clinicians would benefit greatly from the availability of such a test, while epidemiological studies and vaccine trials would also be greatly enhanced.

Veterinarians investigating suspected L hardjo infections in cattle herds should collect at least 30 (or 10% from large groups) serum samples for serological testing. In endemically infected herds, ani- mals in the first lactation group are likely to have a higher prevalence of positive reactions, and titres are higher because of more recent infection in this group. In dairy cattle 10 animals from the first lactation group, 10 from the second lactation group and 10 older cows in the herd should be sampled for serological testing (Hathaway et a1 1986). A collection of urine from animals with MAT titres of 800 or greater may be useful for demonstration of organisms by fluores- cent antibody or cultural isolation (McClintock et a1 1993).

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Am Vet MedAssoc 182165

(Accepted for publication 13 April 1994)

Effects of xylazine on humans: a review JJ FYFFE

McIvor Road Veterinary Center, PO Box 991, Bendigo 3550

Xylazine [2-(2.6-dimethylphenylamino-4-H-5,6-dihydro-l.3] thiazine hydrochloride is an alpha2-adrenergic agonist whose chemical structure resembles the phenothiazines and the tricyclic antidepressants. It is also pharmacologically related to clonidine, which is used in human medicine to control arterial hypertension. Xylazine is an effective sedative in animals, in particular ruminants, and is widely used in the deer industry either alone as a sedative or in combination with other drugs for sedation, analgesia and general anaesthesia.

Xylazine acts on the central nervous system by activation or stimulation of alpha-adrenoceptors such as the alpha2-adreno- ceptors; this increases sympathetic discharge and reduces the release of norepinephrine. Through its central stimulation of alpha2- adrenergic receptors, xylazine has potent analgesic activity (Booth 1988).

Courses have been established recently in Australia and New Zealand to teach lay operators to remove velvet painlessly from their own deer after the administration of suitable analgesics.

These courses refer to the use of xylazine as one of the suitable analgesic agents. The medication recommended for use by lay operators is a 2% solution (20 mg/mL) but there are also products available that contain 50 mg/mL and 100 mg/mL, and a dry product that contains 500 mg of xylazine per vial. Concern has been expressed that the accidental self-administration of xylazine by lay operators may cause serious harm.

In view of these occupational health and safety issue concerns, published reports on the effects of xylazine on humans were reviewed.

The data bases of the Commonwealth Agriculture Bureau from 1973 to 1992 and Medline from 1966 to 1993 were searched for references. For the Commonwealth Agriculture Bureau survey the key word search was done on xylazine, human and toxicity while with the Medline database the search was done on ‘xylazine not animal’. All papers so discovered that were case reports have been included in this review.

Carruthers et a1 (1979) recorded the first case of toxicity of xylazine in a human. The male patient attempted to commit suicide by self-administration by intramuscular (IM) injection of about Ig of xylazine. The clinical signs of toxicity included hypotension, brady- cardia, respiratory and central nervous system depression, and hyperglycaemia. The patient recovered after 6 0 hours on a respirator.

Gallanosa et a1 (1981) described a case of deliberate self- administration ofxylazine in a woman. The patient attempted suicide by swallowing 400 mg of xylazine. The clinical signs included hypotension, bradycardia, miotic pupils and a slow pupillary light reflex. The patient was drowsy, disoriented, dozed off at intervals but was responsive to painful stimuli; she was placed on a ventilator and given intravenous fluid therapy. Frequent unifocal premature ventricular contractions were present and responded to treatment with intravenous lidocaine. Assisted ventilation was stopped after 24 hours and the patient was discharged from hospital after two days. Gas chromatographic analysis of the patient’s urine revealed xylazine as the only drug detected.

Spoerke et a1 (1986) reported a suicide attempt by a woman who injected 2400 mg of xylazine IM. This was an approximate dose rate of 22 mg/kg. On admission to hospital the patient was somnolent but

294 Austmlim Veterincqy Journal Vol. 71, No. 9, September 1994