We report here the complete results of the 1984 survey (Table 1) and the results of FECRTs conducted by this laboratory from 1987 to 1991 (Table 2). The later FECRTs were conducted according to industry guidelines (Anon 1989) and differ fmm those of the 1977 and 1984 studies in that FECs were done 10 to 14 days after treatment and resistance was defmed as failure to reduce the FEC by 95%(with the lower 95%confiidence l i t of that estimate being c 90%) and the pre-treatment control group mean FEC for each genus was not less than 150 epg. The flocks included in the FECRTs from 1987 to 1991 were selected on the basis of their response to the recommendation, made as part of the WORMKILL* program,to check a n t h e w tic efficacy or because, for other reasons, anthelminticresistance was suspected. These FECRTs confirmed the finding of the 1984 survey that anthehhic resistance in the important sheep nematode species is very commonin the New England Region Most flocks have resistance in at least one of themore important speciesof internal parasites. The flocks on which these FECRTs were conducted are likely to be a biased sample of the New England population because of the basis of selection. Accordingly, it is difficult to draw conclusions about apparent differences in frequency of resistance between the Northern Tablelands and the North West Slopes and Plains (Table 2). Likewise, the results of these FECRTs cannot be closely compared with those of the 1984 and 1977 surveys. As well as differences in method of selection, the definition of resistance for the FECRTs was differentfrom that which applied to the earlier surveys. Furthermore, a 10 to 14 day post-treatment sampling was used in the 1987 to 1991 FECRTs (to eliminate the effects of a possible temporary depression of egg production) whereas a 4-day interval applied in the 1977 and 1984 surveys. However, opinion is divided on the matter of post-treatment depressionofegg laying. McKenna (199O)presentsevidencethat differences in results between the shorter and longer posttreatment intervals may be of little practical significance. Also, Beveridge et a1 (1990) remark that there is no fm published evidence of a temporag depression of egg production occurring in the field. Scott et al(1991), however, found a reduction in the fecundity of anthelmintic resistant adult H contortus after exposure to ivermectin or benzimidazoles in vivo and concluded that a post-treatment period of at least seven to 10 days should be used in FECRTs. The results of the 1987 to 1991 FECRTs support the finding of the 1984 survey, and subsequentfield experience,that anthelmintic resistance in the important sheep nematodes is very common in the New England region. Very few flocks tested were without resistance (detectable by a FECRT) in at least one of the more important species: H contortus, T colubriformis or 0 circumcincta. The prediction by Dash (1986) that multiple resistance in T cotubriformis and consequent control failures would emerge as a problem in the Northern Tablelands has been confiied by this study and by field experience in recent years. In addition, H contortrcs isolates recovered by this laboratory were recently confiied to be resistant to closantel (Rolfe e f ul 1990). Recognising the importance of anthelmintic resistance in the New England region, the WORMKILL program is undergoing continual revision and development. For example, because the possible emergence of closantel resistance was anticipated, the number of treatments each year with this drug was reduced a few years ago, with some farms using only one annual treatment in late Spring instead of 3 treatments a year as specified in the original program. B m d o n (1980) suggested that parasite control shouldinvolve more complex systems with such components as chemotherapy, grazing and animal management, and the induction of immunity being involved. The WORMKILL

WORMKILL. A strategic drenching program for sheep in summer rainfall areas; NSW Agriculture, Rural Lands Protection Boards and CSIRO

Australian VeterinaryJournal Vol69, No 8, August 1992

program, ernphasising as it does not only strategicdrenching but also grazing management, nutrition and management factors such as time of weaning and periodic monitoring of FECs, has clearly followed this line of development. The technical assistance of Peter Choice and Pennie Glock in our parasitology section, as well as the cooperation of the laboratory's veterinary clients, Rural Lands Protection Board Rangers (particularly H R Officer), and Dr Douglas Gray of the University of New England, is gratefully acknowledged. For their involvement in the 1984 survey, we acknowledge Stephen Sinclair and Kerry Speak, as well as Drs P Andrew, B Chick, R Everett, P Greentree, E Hall and R Maher.

References Anon (1989) Anthelmintic Resistance. Report of the Working Party for the AnimalHealthCommittee ofthe StandingCommittee ondgricultwe,SCA Technical Report Series, No. 28 BeveridgeI, EIIisNJS.RileyMJandBrown TH (1990)Awt Vet J67: 413 Bmsdon RV (1980) Vet Parasitol6: 185 Dash KM (1986)Awt Vet J63: 45 Hotson IK,Campbell NJ and SmealMG (1970)Awt Vet J 4 6 : 356 Le Jambre LF, Southcott WH and Dash KM (1976) Inf J Pararitol 6:217 Le Jambre LF, Southcott WH and Dash KM (1 977) Inf J Parasitol7: 473 Le Jambre LF. Southcott WH and Dash KM (1978)Awt Vet J54:570 McKenna PB (1990)NZ Vet J38: 142 Rolfe PF, Boray JC. Fitzgibbon C. Parsons G,Kemsley P and Sangster N (1990) Aurt Vet 167: 29

ScottEW,BaxterPandArmourJ(1991)ResVetSci500:247 SmealMG, Gough PA, Jackson AR, and Hotson M (1968) Awl Vet J & 108 Webb RF, McCuUy CH, Clarke FL,Greentree P and Honey P (1979) Awt Vet J 55: 422 (Accepted for publication 13 May 1992)

Pasteurella multocida septicaemia in two calves Queensland Department of Primary Industries, Rockhampton Veterinary Laboratories, Rockhampton, Queensland4700 Department of Food and Agriculture, Benalla Agriculture and Veterinary Centre, Benalla, Victoria 3672

BD HILL

RB JOHNSON

Haemorrhagic septicaemia is a specific form of acute pasteurellosis of cattle and water buffaloes, which is caused by Pasteurella multocida capsular antigen types Carter B or E and somatic antigen Heddleston types 2 and 5 (Brogden and Packer 1979; Carter and Chengappa 1981). The disease has a high mortality rate and is one of the most economically important diseases of livestock in South East Asia. It differs from pneumonic pasteurellosis that is associated with infection by P multocida (Carter A serotype) and P haemolytica. Bovine pneumonic pasteurellosis appears as a fibrinous bronchopneumonia (Gourlay et a1 1989) and in contrast to haemonhagic septicaemia, has a longer clinical course and a lower mortality rate (Blood and Radostits 1989). This paper describes cases of P multocida septicaemia in two calves in central Queensland where the pathological findings were suggestive of haemorrhagic septicaemia. In Decembex 1989, two Brahman calves, 3 to 5 days old, died on a farm near Rockhampton. Clinical signs had included depression, excessive salivation, laboured breathing and lameness associated with swollen limbs. One of these calves (calf 1) was submined, 8 h after death, for necropsy. These calva were on what was normally swampy pasturebut prevailing weather at the t h e was dry. 197

Figure 1. Coomassie blue-stained sodium dodecyl sulphatepolyauylamide gel ele&ophoresis profiles of lysates of 3 Pasreurela rnuMdastrains: laneA (0019 lnsein), hne B (0132Katha),laneC (0444 neonatal calf isolate). Molecular weight standards are listed to the right of the figure. The amow indites the position of the 32 kD band.

In February 1991, Brahman calves on another farm near Rockhamptonwereobservedwiththesameclinical signs as those described above. Affected calves were 3 to 5 days old and in good body condition. The farm was flood-bound, and it was not possibleto gain access to the entire herd to assess the total number of calves affected. At least 3 calves exhibiting the above signs had died. A fourth affected calf (calf 2) was submitted alive to the laboratory for euthanasia and necropsy. Serum biochemistry revealed elevated gamma glutamyltransferase (510 IU/l) and total b i l i b i n (3 1p o l / l ) concentrations. Haematology showed that the calf was leucopenic (2.4 x l@A). Similar changes were observed in both calves. There was extensive subcutaneous strawcoloured oedema especially over the lumbar backline and down the limbs. This reaction, together with haemorrhage, also involved peritendonous tissues on the caudal aspects of the metacarpals and metatarsals. There was an excess of strawcoloured joint fluid in all limb joints. The synovial membranes appeared roughened, but articular surfaces were normal. Skeletal muscles showed extensive haemonhage interspersed with areas of pallor. These changes were more severe in hip and thigh muscles. The visceral and parietal pleura in calf 1 were hyperaemic and fibrinousexudate waspresent over a small area (about 2 cm diameter) of the right anterior lobe. The thoracic viscera of calf 2 appeared grossly normal except that the lungs were a dull grey colour. Excessive quantities of bloodtingedfluid(about300ml)werepresent in theperitoneal cavities. Most peripheral lymph nodes were oedematous and swollen. Microscopic examination of the tissues revealed similar changesinbothca1ves.There wasadiffuseinterstitialpneumoniawith alveolar septa thickened by infiltrating neutrophils and mononuclear inflammatory cells. Numerous alveolar septa1 capillaries were occludedby bacterial emboli. Theseemboli were often at the centre of foci of parenchymal necrosis, which were 198

heavily infiltrated by neutrophils. In calf 1 there was a fibriiopurulent pleurisy with the exudate containing numerous bacteria. Alveolar lumens in this case were flooded with a fibriious exudate. Both calves had a severe suppurativemyositis withnumerous bacteria present throughout the lesions in skeletal muscle. Splenic red pulp had numerous foci of necrosis, which contained bacterial colonies, but there was only minimal neutrophil response. Heart, liver and kidney appeared normal in calf 2, but in calf 1 scattered capillaries in these organs were occluded by bacterial emboli but without an apparent inflammatory reaction. Stainingtissues by the Brown and Brenn method (Humason 1%2) showed that all the bacteria described above were small gram-negative rods. Using standard techniques, swabs from the heart, lung, liver, carpal joints and skeletal muscle from both calves were cultured for aerobic and anaerobic bacteria. In addition, mesenteric lymph node, elbow joint and spleen were cultured from calf 2. P multocida was isolated in pure culture from all the above sites. No virus culture was attempted. P multocida isolated from calf 2 was examined for similarities to reference strains that cause haemorrhagic septicaemia. In the absence of a single universally recognised definitive test for haemorrhagic septicaemia,three different procedures were used. The electrophoreticprofile of the isolate (Figure 1) was consistent with those of other P multocida strains (Johnson et a1 1991) but differed from those of 2 haemorrhagic septicaemiareference isolates, Katha and lnsein (Dawkins et a1 1990), in that it lacked a major protein band at 32 kD. This band is characteristic of Carter group B, the serotype responsible for haemorrhagic septicaemia in Asia and North America (Johnson et al 1991). The serological properties of the organism were examined using both an antigen detection ELISA, specific for haemorrhagic septicaemia strains, and a conventional typing method. The former assay, which has a sensitivity of at least 86% and a specificityof 99%, is based on the reactivity of a rabbit antiserum raised against a crude LPS extract of a haemorrhagic septicaemia strain of P multocida (Dawkins et a1 1990). The P multocida isolate from calf 2 was negative in this test. The conventional typing of the isolate revealed that it was of the Heddleston type 3,4 (somatic antigen type). In contrast, organisms causing haemorrhagic septicaemia are Heddleston types 2 and 5 (Brogden and Packer 1979). These results showed that the isolate differed in protein profile and serological type from strains causing haemorrhagic septicaemia. Histopathological and bacteriological studies of the disease observed in the 2 outbreaks indicated that it was septicaemic in nature and not typical of pneumonic pasteurellosis.The reason that the calves developed septicaemic pasteurellosis remains uncertain, although other potential factors such as viral infection or toxic injury were not investigated. The authors thank Dr C Morrow, Victorian Institute of Animal Science, Attwood, for performing Heddleston typing on the organisms, and Mrs M Houge, Miss J Sleep andMrs H Wakeham for technical assistance. This work was funded by the Australian Centre for InternationalAgriculturalResearch (ACIAR), project number 8907.

References Blood I X and Radostits OM (1989)VeterinaryMedicine, 7th edn, Bailliere Tindall. London, p 658 Brogden KA and Packer RA (1979)Am J Vet Res 4 0 1332 Carter GR and Chengappa MM (1981) Recommendations for a Standard System of Designating S e r o t p s of Pastewella multocida. In: 24th Ann Proc Am Assoc Vet Lab Diagnost. p 37 Dawkins HIS. Johnson RB, Spencer TL and Patten BE (1990)Res VefSci 49261 Gourlay RN, 'Ihomas LH and Wyld SG (1989)Res Vet Sci 47:185 Humason GL (1%2) Animal Tissue Techniques, Freeman, San Francisco JohnsonRB, Dawkins HJSand Spencer TLS (1991)AmJVetRes52:1644 (Acceptedfor publication 2 9 April 1992)

Australian VeferinatyJournal V o l 6 9 , No 8, August 1992