WIMMERA RYE GRASS TOXICITY IN WESTERN AUSTRALIA
P. H. BERRY,^^ B.V.S., and J. L. WISE, B.Ag.Sc. Department of Agriculture, Katunning, Western Australia, 631 7 Introduction
Two syndromes characterised by staggering and convulsions in livestock grazing rye grass have been reported. Rye grass staggers, a disease of sheep and cattle grazing young perennial rye grass, Lolium perenne, has been described in the United States of America (Shaw and Muth 1949), New Zealand (Cunningham and Hartley 1959), Great Britain (Davies and Farmer 1961, Clegg and Watson 1961) and Australia (Healey 1970). Morbidity rates vary from 5 to 75% but mortality rates are low (Cunningham and Hartley 1959), the signs disappearing soon after the removal of stock from affected pastures. The aetiological involvement of the high alkaloid content of young perennial rye grass has been examined (Aasen et a1 1969). Wimmera rye grass toxicity (WRGT), a disease with similar clinical signs but high mortality rates, occurs on mature annual Wimmera rye grass, L. rigidum, with seeds parasitised by a nematode and a bacterium. In South Australia, McIntosh et a1 (1967) described the seasonal occurrence of WRGT from spring until the following opening winter rains, the limited farm distribution and the stock morbidity and mortality rates, signs and pathology. They experimentally reproduced the disease by feeding mature parasitised Wimmera rye grass to sheep and parasitised seed to guinea pigs. Later, Price (1973) identified the parasitic nematode in South Australia as a new species, Anguina lolii, and gave a detailed account of its life cycle. A. lolii, which reproduces in seed galls in mature Wimmera rye grass, acts as a vector for a Corynebacterium sp which grows as a yellow slime over the surface of parasitised plants and in some galls. Such parasitism often causes distorted growth of Wimmera rye grass. To eradicate WRGT from farms Price (1973) suggested the use of fallowing, desiccant herbicides, and hard grazing in early spring to reduce the Wimmera rye grass content of pastures and thus interrupt the parasitic nematode’s life cycle. Early summer burning of Wimmera rye grass pastures was also suggested as a means of con11
Present address: C/-Animal Health-Laboratory, DeoGment of Agriculture, Jarrah Rd, South Perth, Western Australia 6151.
Australian Veterinary Journal, Vol. 5 1, November, 1975
trolling the disease by destroying the nematode within galls. In Western Australia, Gwynn and Hadlow ( 197 1) reported the occurrence of a clinically identical syndrome in sheep grazing Wimmera rye grass parasitised by a nematode, Anguinu sp, and a Corynebacterium sp. The syndrome was experimentally reproduced in sheep and guinea pigs fed Wimmera rye grass harvested from paddocks in which losses from WRGT had occurred (M. E. Nairn, personal communication). Since the 1971 report from Western Australia, further sheep losses and the first confirmed cattle deaths have occurred in WRGT outbreaks over a much wider area. The objects of this paper are to report information gathered from the outbreaks recorded between 1970 and 1974 and to describe measures aimed at assessing the distribution of parasitised Wimmera rye grass in the affected district. Materials and Methods Study of Outbreaks Between November 1970 and March 1974, 58 outbreaks of WRGT in sheep and cattle were investigated. Diagnosis was based on signs and autopsy findings and the presence of parasitised Wimmera rye grass in feed grazed by affected stock. At each outbreak details of the geographical location, grazing history, age and type of stock involved, clinical signs, morbidity and mortality rates and autopsy findings were recorded. Haematoxylin and eosin stained sections of formalin fixed lung, liver, kidney, spleen, lymph node, thymus, skeletal and cardiac muscle, brain and spinal cord from 12 sheep and 1 cow from 4 WRGT outbreaks were submitted to histopathological examination. Estimations of serum levels of inorganic phosphorus, calcium and magnesium were made on samples collected from 10 sheep exhibiting signs of WRGT during 1 outbreak. Treatment of 25 affected sheep with 6 to 10 ml of 50% chloral hydrate solution was attempted during 1 outbreak. Treatment of 4 adult cattle, 1 each with chloral hydrate, pentobarbitone sodium*, acetyl promazinet, and xylazinet, respectively, was also applied during one outbreak in cattle. Farmers instituted 4 different managerial practices (other than cropping only) to gain use from the affected paddocks. Immediately after an outbreak, interrupted grazing, where stock were moved in and out of affected * “Sagatal”, May and Baker Ltd, West Footscray, Victoria.
t
“Acepromazine”, Apex Laboratories Pty Ltd, Enfield, New South Wales. Bayer Pharmaceutical Company, Botany, New South WalLs.
$ ”Rompun”
525
paddocks every 2 or 3 days, and mechanical dislodgement of seed from Wimmera rye heads to reduce the availability of parasitised seed to grazing stock, were measures attempted. In seasons subsequent to an outbreak, mowing or heavy grazing of parasitised pastures in late winter and early spring, and autumn pasture burning followed by cropping, were attempted. Distribution of Parasitised Wimmera Rye Grass Collection of rye grass samples - The distribution of parasitised Wimmera rye grass was investigated by the collection of 750 pasture samples in 100 paddocks on the 26 farms where outbreaks had occurred, 38 samples from 15 paddocks on 9 neighbouring farms, and 20 samples from 20 paddocks on 12 other farms throughout the region. In one 72 ha Wimmera rye dominant paddock, where 45 of 99 cattle died from WRGT in November 1972, the distribution of Anguina sp and Corynebacterium sp was investigated in detail in February 1973. When assessing the presence or absence of Anguina sp and Corynebacterium sp in a paddock the number of samples collected for examination was in proportion to the paddock area and variability of soil types. At 4 to 20 different locations, where macroscopic evidence of parasitism was evident or where Wimmera rye was most dominant on different soil types, 50 t o 200 Wimmera rye heads were collected for microscopic examination. In the paddock examined in detail, 50 to 100 g Wimmera rye grass samples were collected from 179 pegged sample points on a 6 0 m grid over the entire paddock. At each point the soil type was also noted. Examination of rye grass samples - When assessing the presence of Anguina sp and Corynebacterium sp samples were hand threshed and the seed so obtained soaked in water for 12 hours. Using a stereomicroscope each seed sample was scanned for the presence of nematode galls and bacterially infected seeds as described by McIntosh et a1 (1967) and Price (1973). After soaking normal seeds the caryopses have an ovular, evenly-pigmented appearance, whereas galls and the caryopses of bacterially infected seeds are pointed, usually without pigment near the pointed extremity. Nematode galls contain up t o 7 adult or 2,110 larval A . Zolii, while the endosperm and germ of bacterially infected seed is replaced by yellow slime. When an estimation of the level of parasitism was required, from each sample a 100-seed random subsample was examined and the number of nematode
galls and bacterially infected seed expressed as a percentage of the total. Investigution of means of spread of Wimmera rye grass parasites - The means by which the Anguina sp and Corynebacterium sp were spread were investigated by comparing the distribution of affected paddocks and farms with geographical and meteorological influences. The movement of farm machinery, produce and stock, both on and between affected farms was also examined by questioning property owners. Bacteriological Examination of Corynebacterium sp in Purusitised Lolium rigidu m Mature rye grass showing macroscopic evidence of parasitism was collected in December 1973 from a paddock in which a WRGT outbreak had recently occurred. Dried yellow slime was scraped from the seed heads, emulsified in a saline solution and Gram stained smears prepared. The saline slime suspension was inoculated onto Price’s medium (Kado and Heskett 1970) and a
526
nutrient agar* containing 1.5% crushed mature rye grass (NARG) medium and incubated at 30°C and at room temperature. Colonies grown after 5 days were Gram stained and Gram positive organisms cultured on the NARG medium at room temperature for 4 days. These small, discrete, yellow or cream colonies were again Gram stained, catalase tested and submitted to other biochemical tests listed in Table 2.
Results
Study of Outbreaks Occurrence and incidence -The 58 outbreaks of WRGT between November 1970 and March 1974 occurred on 26 different farms and resulted in the deaths of 3,366 sheep and 46 cattle. The number of outbreaks and total mortalities recorded in sheep in the years 1970 to 1974 are presented in Table 1. The paddock outbreaks of WRGT occurred each year between November and April on different types of feed containing parasitised Wimmera rye grass. The monthly frequency of outbreaks is shown in Figure 1. Seventy-nine per cent of outbreaks occurred on pasture. Other outbreaks took place on either cereal stubble ( l l % ) , pasture and stubble grazed concurrently (4% ) , cereal crop ( 2 % ) , cereal screenings left heaped in a paddock (2% ) or hay ( 2 % ) . The WRGT outbreak on hay occurred in housed calves in May 1973 and is not included in the outbreaks shown in Figure 1. In 33 of 46 outbreaks of WRGT in stock grazing pasture signs developed within 2 to 7 days of the introduction of stock to the affected paddock, or as Wimmera rye pastures matured and parasitised seed became available in late October or early November. In 13 other outbreaks on pasture livestock signs took longer than 7 days to appear. In stubble paddocks the grazing period taken for signs to develop was usually more than 4 weeks after the introduction of stock. In the single outbreak that occurred in an unharvested barley crop in which parasitised Wimmera rye grass also grew, signs appeared 2 days after the accidental introduction of stock. The disease was seen in all ages of stock: 70% of outbreaks involved adult sheep and cattle, 15% ewes and lambs to 4 months of age, 13% lambs 6 to 10 months old and 2% (1 outbreak) 3-month-old calves. Morbidity rates ranged between 2 % and 100%. Mortality rates recorded in 56 outbreaks in sheep were: under 5 % in 26 outbreaks, 5 % to 15% in 14, 15% to 25% in 5 , 25% to 50% in 6 and over 50% in 5 . In 2 outbreaks in cattle mortality rates of “Oxoid”, Abbott Laboratories Pty Ltd, Cronulla. New South Wales.
Australian Veterinary Journal, Vol. 51, November, 1975
TABLE 1 Mortalities of Sheep due to Wimmera Rye Grass Toxicity and Progressive Total of Number of Farms with Parasitised L. rigidum in the Years 1970-74 Total mortalities each year* Number of farms reporting first outbreak Number of reported outbreaks each year Progressive total of number of farms with Darasitised rve mass * Numerator indicates number of deaths, denominator
1970-7 1
1971-72
1972-73
1973-74
550/3540 5 9
1247/5670 4 13
889/4736 8 12
68119720 9 22
5
9
17
20% ( 1 of 5 calves) and 45% (45 of 99 mixed-
age cattle) were recorded. Clinical Signs - Signs of WRGT in sheep were characterised by staggering, collapse, convulsions, other nervous signs and death. When an affected flock was driven a few hundred metres a proportion of animals collapsed into ventral or lateral recumbency. Some animals remained down for only 5 to 10 seconds and showed no other signs. In other recumbent animals some or all of the following signs were seen: neck ventroflexion, opisthotonus, head nodding, forelimb, hind-limb and digital extension, tetanic and clonic convulsions, and posterior extension of the hind limbs of sheep in ventral recumbency. From 5 seconds to a few minutes after initiaI collapse affected stock usually regained their feet and staggered away with either a stiff-legged, jumping, or swaying gait. After a further 1 or 2 minutes these animals either apparently recovered or wandered aimlessly about, showing depression and ataxia. These and other apparently normal stock would continue to collapse, either spontaneously or upon being driven, showing further convulsions and other of the afore-
n
I
Outbreaks on pasture Outbreaks involving crops and stubbles
VI
Y
4
$20-
1
m I-
3
0 L
0 ct
-
15-
w
:fit-
P
2
.... ...... ..
-
5-
rrr
... ..-.._ ... ... ...-.. Z i i
n OCT
NOV
DEC
JAN
FEB
MAR
31
indicates number at risk.
APR
Figure 1. Month of occurrence of 57 field outbreaks of Wirnrnera rye grass toxicity in Wesern Australia in the period 1970-74.
Australian Veterinary Journal, Vol. 51, November, 1975
mentioned nervous signs. Animals recumbent for long periods remained in lateral recumbency and exhibited periods of violent convulsions, opisthotonus, tachycardia, dyspnoea, frothing at the mouth and nystagmus, and took up to 2 days to die. In 2 outbreaks of WRGT affecting flocks of ewes in late pregnancy and from which 30 of 700 and 20 of 350 had died, abortions occurred in an estimated 10% of the surviving ewes. No infectious causes of abortion were found during these outbreaks. The signs seen in cattle were essentially the same as those observed in sheep. After the removal of affected stock from parasitised Wimmera rye grass, signs and mortalities continued for up to 10 days, but more commonly they had ceased after 4 days. Some sheep or cattle did not manifest initial signs of WRGT until 2 or 3 days after removal of the affected flock or mob from parasitised rye grass. Clinical pathology - Biochemical estimations of serum inorganic phosphorus, calcium and magnesium levels showed that all were within the normal ranges. Autopsy findings - A pale, friable liver, haemorrhages in several organs, and lung congestion were the most constant gross autopsy findings. Histological changes observed in tissue sections were non-specific and variable. Treatment and control practices -Treatments attempted produced no apparent beneficial effects. The managerial control practices instituted by farmers were also unsuccessful in preventing recurrences of WRGT in stock grazing "outbreak" paddocks, in both the season of the initial outbreak and in subsequent seasons. Distribution of Parasitised Wimmera Rye Grass Parasitised Wimmera rye grass was found on the 26 properties on which WRGT occurred and on 5 other farms. The totaI paddock area of parasitised Wimmera rye on the 26 outbreak properties was 2,712 ha or 12% of their total arable area. On individual outbreak farms the affected paddock area was between 21% and 527
TABLE 2 Biochemical and Pigment Characteristics of 6 Isolates of Corynebacterium sp from L. rigidum Parasitised by Anguina sp in Western Australia, Compared with the Corynebacterium s p Isofated in South Australia bv Price (19731 Isolate Western Australia
Observation
1
Pigment Catalase Carbohydrate Fermentation glucose* sucrose maltrose glycerol lactose xylol starch Indole production Gelatin liquefaction Nitrate reduction
T + + + + + C
A
A A A d -
y = Bright Yellow. c = Cream. d = Doubtful.
76% of the total farm area. Three farms had more than 35% of their total grazing area affected. In the 750 pasture samples collected from farms on which WRGT had occurred infection levels from zero to approximately 60% for both Anguirra sp and Corynebacterium sp were found, although the highest levels in some paddocks where WRGT had occurred were less than 2%. In one stubble paddock where stock grazed without loss, levels were 12%. In the survey of properties adjacent to farms on which WRGT had occurred, macroscopic evidence of Anguina sp and Corynebacterium sp parasitised Wimmera rye grass was found on 3 properties. Anguina sp and the associated Corynebacterium sp were found in samples from 1 paddock each on 2 of the other 12 farms investigated. In the detailed paddock survey, parasitised Wimmera rye grass was found in 128 of the 179 samples collected. Levels of parasitism for nematode galls and bacterially infected seed in the samples collected ranged from zero to 44% and 55% respectively. Parasitised rye grass was found throughout the paddock on all soil types but the highest levels of parasitism were found where the rye grass pasture was densest on clay soil. Means of spread of rye grass parasites - On affected farms, as was the general practice throughout the region, paddocks were cropped in a 3- to 5-year rotation and usually more than 1 paddock on each farm was cropped each year. 528
2
A A A A
A
-
3 C
4 Y
A d A d d - A A A d d
5 C
6 Y
South Australia Price ( 1973)
+Y
A A A A A A d A A A d
-
+
-
- - - -
A A A A
-
A = Acid. = Gas was not produced from glucose; the other carbohydrates were not tested for gas production. = Positive. - = Negative.
+
Consequently, movement of machinery between paddocks often took place. Hay and grain harvested in one paddock on affected farms was commonly fed on the ground in various other paddocks. Occasional machinery and stock movements had occurred between farms under investigation, prior to the confirmation of the presence of parasitised rye in their paddocks. A farm on which the presence of parasitised rye grass was found in 1968 was worked in partnership with another property 40 km away. All cultivating, seeding, haymaking and harvesting machinery was shared and moved annually between the two farms, where they were used in turn. The second farm had its first confirmed WRGT outbreak in 1973. Commercial seed graders and contract haycarters commonly moved their machinery from farm to farm through the district. On some adjacent farms and paddocks with parasitised Wimmera rye grass, the location of affected paddocks followed common water courses and creek systems. Bacteriological Examination of Corynebacterium sp in Paraitised Lolium rigidum Examination of the smears of yellow slime from parasitised rye grass showed predominantly small, Gram positive bacilli. Small Gram positive, catalase positive bacilli with 6 slightly different ranges of biochemical activity were isolated. A summary of these biochemical findings compared with those of the Corynebacterium sp isolated by Price (1973) are presented in Table 2. Australian Veterinary Journal, Vol. 51, November, 1975
Disrussion
Although 58 outbreaks of WRGT on 26 farms were reported to the authors between 1970 and 1974, approximately 5 more farms are believed to have incurred stock losses during 1973-74. These outbreaks were not reported because stock owners thought this would reduce the value of their farm and returns from stock sales. Between 1970 and 1974 the occurrence of 57 paddock outbreaks between late October and April coincided with the seasonal pattern of WRGT observed in South Australia by McIntosh et a1 (1967), and the toxic phase in the life cycle of Anguinn sp with the associated Corynebacterium sp defined by Price ( 1973). In Western Australia the different frequency of outbreaks on pasture and stubble in months between October and April reflected the grazing management systems commonly used in the region. Stubbles are only available after harvest in December and January. The greater number of outbreaks recorded in sheep, compared with cattle, was related to the population of each in the region. The availability of feed other than parasitised rye grass in toxic paddocks influenced the occurrence of WRGT. Observations showed that stock took a longer time to develop signs of WRGT after their introduction to cereal stubbles containing a large bulk of crop residue amongst which parasitised rye grass grew. This may have been due to a reduced daily intake of toxic Wimmera rye grass material by grazing stock. Results of a pen-feeding trial (unpublished data) showed that sheep could consume reduced amounts of parasitised rye grass daily for up to 75 days before signs of WRGT eventually developed. Alte-natively, the extended period taken for stock to develop WRGT signs in certain paddocks containing parasitised rye grass may have been due to their grazing alternative feed only, before intake of toxic material commenced. Clinical signs, including abortions, together with macroscopic and microscopic autopsy findings observed in the outbreaks in Western Australia, were similar to those described in South Australia (McIntosh et al 1967), although aimless wandering of semi-recovered stock was a feature of some Western Australian outbreaks. Mortality rates recorded during some outbreaks in Western Australia were higher than those reported from South Australia. Livestock morbidity and mortality rates, and the time taken for signs to abate after removal of stock from affected pastures were related to intake of parasitised Wimmera rye grass by affected stock. In a number of outbreaks during Australian Veterinary Journal, Vol. 51, November, 1975
1974, when stock owners were aware of the disease, immediately recognised the first signs, and removed stock from affected paddocks, mortality rates were usually low. However, in other outbreaks losses were. frequently high because stock had been left unobserved on toxic pastures for 2 or 3 weeks. Shortcomings of control measures attempted by farmers were obvious. Interrupted grazing failed because of a poor understanding of levels of toxicity and varying stock grazing behaviour in different paddocks. Measures used to dislodge rye grass seed from erect Wimmera rye grass pasture in early summer offered some initial success, but the longer stock grazed such pastures the more likely they were to eventually consume enough parasitised seed from the ground to elicit WRGT signs. Mowing in early spring failed because of the variation in stage of maturation of Wimmera rye grass plants at the time of mowing and subsequent regrowth of rye grass. Heavy grazing was usually an impractical management procedure. On farms where a significant paddock area was affected there simply were not enough stock to maintain sufficient grazing pressure during the critical spring period to prevent most rye grass plants flowering. Control procedures involving autumn burning of pasture were unsuccessful for reasons discussed by Price ( 1973 ) . Investigation of the distribution of parasitised Wimmera rye grass and WRGT outbreaks showed that since the initial report of WRGT in Western Australia (Gwynn and Hadlow 1971) the syndrome has emerged as a threat to livestock production in a significant proportion of the agricultural area of Western Australia. Whether the problem has spread since 1971 to all the farms presently involved is doubtful. The finding of parasitised Wimmera rye in paddocks and on farms where stock losses had not been observed suggests the nematode and bacterial parasites might exist in a paddock for at least one season before its presence is realised. It is likely that spread is a slow continuing process, but observations made in spring and early summer of 1974-75 and not included in the results reported in this paper suggest that the rate of spread is increasing. Although the level of parasitism of Wimmera rye grass was higher in areas of denser rye grass in the paddock surveyed in detail, the presence cf parasitised rye throughout the paddock was not surprising. Cultivating and harvesting in preceding cropping years would have encouraged spread. 529
The bacteriological findings indicate that the Corynebacterium sp associated with nematode infested L. rigidwm in Western Australia, although showing slightly different biochemical activities, is similar to the bacterium isolated by Price (1973). No attempt has yet been made to compare the Anguina sp nematode associated with WRGT in this state with A . lolii (Price 1973) found in South Australia. summary
Outbreaks of Wimmera rye grass toxicity in the south-west of Western Australia, and the associated distribution of Wimmera rye grass parasitised by Anguina sp and Corynebacterium sp were investigated. Between 1970 and 1974, 58 outbreaks occurred on 26 farms in sheep and cattle grazing pasture, cereal crop, stubble, screenings, or hay containing parasitised Wimmera rye grass. Morbidity rates up to 100% and mortality rates up to 77% were observed. Clinical signs were characterised by staggering, collapse, periods of convulsions and other nervous signs, often followed by death. A pale, friable liver and haemorrhages in different organs were the most constant gross autopsy findings, but no specific histopathological changes were observed. Symptomatic treatment of affected animals and agronomic control measures attempted by farmers were unsuccessful in controlling the disease. The distribution of parasitised Wimmera rye grass was studied on farms in the region, and in detail in one paddock where heavy losses from Wimmera rye grass toxicity had recently occurred. Parasitised Wimmera rye grass was also found
530
in paddocks and on farms where losses due to Wimmera rye grass toxicity had not been diagnosed. Acknowledgments
The authors wish to acknowledge Dr R. Gwynn and Dr A. Hadlow for their initial field records. Also we express grateful acknowledgment to Miss 0. Goss and Mr P. McR. Wood for the examination of Wimmera rye grass material, also Dr R. B. Richards, Dr J. Dickson and Dr M. Bond for assistance with histopathology and Mrs S . Ball for bacteriology. We also wish to thank Mr J. Burdass, Mr T. Marfleet, Mr S . Bull and other officers of the Department of Agriculture, Katanning, for their assistance in the field. References Aasen, A. L., Culvenor, C. C. J., Finnie, E. P., Kellock, A. W. and Smith, L. W. (1969)-Aust. 3. agric. Res. 20: 71. Clegg, F. G. and Watson, W. A. (1961)--Vet. Rec. 73: 731. Cunningham, I. J. and Hartley, W. J. (1959)-N.Z. Vet. J . 7: 1. Davies, E. T. and Farmer, P. E. (1961)-Vet. Rec. 73: 130. Gwynn, R. and Hadlow, A. J. (1971)-Aust. vet. J . 47: 408. Healey, J. S. (1970)-Agric. Gaz. N.S.W. 81: 92. McIntosh, G. H., Rac, R. and Thomas, M. R. (1967) -Aust. vet. J . 43: 349. Price, P. C. (1973)-"1nvestigation of a nematodebacterium disease complex affecting Wimmera rye grass". Ph.D, Thesis, University of Adelaide. Shaw, J. N. and Muth, 0. H. (1949)--/. Am. vet. med. Ass. 114: 315. (Received for publication 3 September 1974)
Australian Veterinary Journal, Vol. 51, November, 1975
P. H. BERRY,^^ B.V.S., and J. L. WISE, B.Ag.Sc. Department of Agriculture, Katunning, Western Australia, 631 7 Introduction
Two syndromes characterised by staggering and convulsions in livestock grazing rye grass have been reported. Rye grass staggers, a disease of sheep and cattle grazing young perennial rye grass, Lolium perenne, has been described in the United States of America (Shaw and Muth 1949), New Zealand (Cunningham and Hartley 1959), Great Britain (Davies and Farmer 1961, Clegg and Watson 1961) and Australia (Healey 1970). Morbidity rates vary from 5 to 75% but mortality rates are low (Cunningham and Hartley 1959), the signs disappearing soon after the removal of stock from affected pastures. The aetiological involvement of the high alkaloid content of young perennial rye grass has been examined (Aasen et a1 1969). Wimmera rye grass toxicity (WRGT), a disease with similar clinical signs but high mortality rates, occurs on mature annual Wimmera rye grass, L. rigidum, with seeds parasitised by a nematode and a bacterium. In South Australia, McIntosh et a1 (1967) described the seasonal occurrence of WRGT from spring until the following opening winter rains, the limited farm distribution and the stock morbidity and mortality rates, signs and pathology. They experimentally reproduced the disease by feeding mature parasitised Wimmera rye grass to sheep and parasitised seed to guinea pigs. Later, Price (1973) identified the parasitic nematode in South Australia as a new species, Anguina lolii, and gave a detailed account of its life cycle. A. lolii, which reproduces in seed galls in mature Wimmera rye grass, acts as a vector for a Corynebacterium sp which grows as a yellow slime over the surface of parasitised plants and in some galls. Such parasitism often causes distorted growth of Wimmera rye grass. To eradicate WRGT from farms Price (1973) suggested the use of fallowing, desiccant herbicides, and hard grazing in early spring to reduce the Wimmera rye grass content of pastures and thus interrupt the parasitic nematode’s life cycle. Early summer burning of Wimmera rye grass pastures was also suggested as a means of con11
Present address: C/-Animal Health-Laboratory, DeoGment of Agriculture, Jarrah Rd, South Perth, Western Australia 6151.
Australian Veterinary Journal, Vol. 5 1, November, 1975
trolling the disease by destroying the nematode within galls. In Western Australia, Gwynn and Hadlow ( 197 1) reported the occurrence of a clinically identical syndrome in sheep grazing Wimmera rye grass parasitised by a nematode, Anguinu sp, and a Corynebacterium sp. The syndrome was experimentally reproduced in sheep and guinea pigs fed Wimmera rye grass harvested from paddocks in which losses from WRGT had occurred (M. E. Nairn, personal communication). Since the 1971 report from Western Australia, further sheep losses and the first confirmed cattle deaths have occurred in WRGT outbreaks over a much wider area. The objects of this paper are to report information gathered from the outbreaks recorded between 1970 and 1974 and to describe measures aimed at assessing the distribution of parasitised Wimmera rye grass in the affected district. Materials and Methods Study of Outbreaks Between November 1970 and March 1974, 58 outbreaks of WRGT in sheep and cattle were investigated. Diagnosis was based on signs and autopsy findings and the presence of parasitised Wimmera rye grass in feed grazed by affected stock. At each outbreak details of the geographical location, grazing history, age and type of stock involved, clinical signs, morbidity and mortality rates and autopsy findings were recorded. Haematoxylin and eosin stained sections of formalin fixed lung, liver, kidney, spleen, lymph node, thymus, skeletal and cardiac muscle, brain and spinal cord from 12 sheep and 1 cow from 4 WRGT outbreaks were submitted to histopathological examination. Estimations of serum levels of inorganic phosphorus, calcium and magnesium were made on samples collected from 10 sheep exhibiting signs of WRGT during 1 outbreak. Treatment of 25 affected sheep with 6 to 10 ml of 50% chloral hydrate solution was attempted during 1 outbreak. Treatment of 4 adult cattle, 1 each with chloral hydrate, pentobarbitone sodium*, acetyl promazinet, and xylazinet, respectively, was also applied during one outbreak in cattle. Farmers instituted 4 different managerial practices (other than cropping only) to gain use from the affected paddocks. Immediately after an outbreak, interrupted grazing, where stock were moved in and out of affected * “Sagatal”, May and Baker Ltd, West Footscray, Victoria.
t
“Acepromazine”, Apex Laboratories Pty Ltd, Enfield, New South Wales. Bayer Pharmaceutical Company, Botany, New South WalLs.
$ ”Rompun”
525
paddocks every 2 or 3 days, and mechanical dislodgement of seed from Wimmera rye heads to reduce the availability of parasitised seed to grazing stock, were measures attempted. In seasons subsequent to an outbreak, mowing or heavy grazing of parasitised pastures in late winter and early spring, and autumn pasture burning followed by cropping, were attempted. Distribution of Parasitised Wimmera Rye Grass Collection of rye grass samples - The distribution of parasitised Wimmera rye grass was investigated by the collection of 750 pasture samples in 100 paddocks on the 26 farms where outbreaks had occurred, 38 samples from 15 paddocks on 9 neighbouring farms, and 20 samples from 20 paddocks on 12 other farms throughout the region. In one 72 ha Wimmera rye dominant paddock, where 45 of 99 cattle died from WRGT in November 1972, the distribution of Anguina sp and Corynebacterium sp was investigated in detail in February 1973. When assessing the presence or absence of Anguina sp and Corynebacterium sp in a paddock the number of samples collected for examination was in proportion to the paddock area and variability of soil types. At 4 to 20 different locations, where macroscopic evidence of parasitism was evident or where Wimmera rye was most dominant on different soil types, 50 t o 200 Wimmera rye heads were collected for microscopic examination. In the paddock examined in detail, 50 to 100 g Wimmera rye grass samples were collected from 179 pegged sample points on a 6 0 m grid over the entire paddock. At each point the soil type was also noted. Examination of rye grass samples - When assessing the presence of Anguina sp and Corynebacterium sp samples were hand threshed and the seed so obtained soaked in water for 12 hours. Using a stereomicroscope each seed sample was scanned for the presence of nematode galls and bacterially infected seeds as described by McIntosh et a1 (1967) and Price (1973). After soaking normal seeds the caryopses have an ovular, evenly-pigmented appearance, whereas galls and the caryopses of bacterially infected seeds are pointed, usually without pigment near the pointed extremity. Nematode galls contain up t o 7 adult or 2,110 larval A . Zolii, while the endosperm and germ of bacterially infected seed is replaced by yellow slime. When an estimation of the level of parasitism was required, from each sample a 100-seed random subsample was examined and the number of nematode
galls and bacterially infected seed expressed as a percentage of the total. Investigution of means of spread of Wimmera rye grass parasites - The means by which the Anguina sp and Corynebacterium sp were spread were investigated by comparing the distribution of affected paddocks and farms with geographical and meteorological influences. The movement of farm machinery, produce and stock, both on and between affected farms was also examined by questioning property owners. Bacteriological Examination of Corynebacterium sp in Purusitised Lolium rigidu m Mature rye grass showing macroscopic evidence of parasitism was collected in December 1973 from a paddock in which a WRGT outbreak had recently occurred. Dried yellow slime was scraped from the seed heads, emulsified in a saline solution and Gram stained smears prepared. The saline slime suspension was inoculated onto Price’s medium (Kado and Heskett 1970) and a
526
nutrient agar* containing 1.5% crushed mature rye grass (NARG) medium and incubated at 30°C and at room temperature. Colonies grown after 5 days were Gram stained and Gram positive organisms cultured on the NARG medium at room temperature for 4 days. These small, discrete, yellow or cream colonies were again Gram stained, catalase tested and submitted to other biochemical tests listed in Table 2.
Results
Study of Outbreaks Occurrence and incidence -The 58 outbreaks of WRGT between November 1970 and March 1974 occurred on 26 different farms and resulted in the deaths of 3,366 sheep and 46 cattle. The number of outbreaks and total mortalities recorded in sheep in the years 1970 to 1974 are presented in Table 1. The paddock outbreaks of WRGT occurred each year between November and April on different types of feed containing parasitised Wimmera rye grass. The monthly frequency of outbreaks is shown in Figure 1. Seventy-nine per cent of outbreaks occurred on pasture. Other outbreaks took place on either cereal stubble ( l l % ) , pasture and stubble grazed concurrently (4% ) , cereal crop ( 2 % ) , cereal screenings left heaped in a paddock (2% ) or hay ( 2 % ) . The WRGT outbreak on hay occurred in housed calves in May 1973 and is not included in the outbreaks shown in Figure 1. In 33 of 46 outbreaks of WRGT in stock grazing pasture signs developed within 2 to 7 days of the introduction of stock to the affected paddock, or as Wimmera rye pastures matured and parasitised seed became available in late October or early November. In 13 other outbreaks on pasture livestock signs took longer than 7 days to appear. In stubble paddocks the grazing period taken for signs to develop was usually more than 4 weeks after the introduction of stock. In the single outbreak that occurred in an unharvested barley crop in which parasitised Wimmera rye grass also grew, signs appeared 2 days after the accidental introduction of stock. The disease was seen in all ages of stock: 70% of outbreaks involved adult sheep and cattle, 15% ewes and lambs to 4 months of age, 13% lambs 6 to 10 months old and 2% (1 outbreak) 3-month-old calves. Morbidity rates ranged between 2 % and 100%. Mortality rates recorded in 56 outbreaks in sheep were: under 5 % in 26 outbreaks, 5 % to 15% in 14, 15% to 25% in 5 , 25% to 50% in 6 and over 50% in 5 . In 2 outbreaks in cattle mortality rates of “Oxoid”, Abbott Laboratories Pty Ltd, Cronulla. New South Wales.
Australian Veterinary Journal, Vol. 51, November, 1975
TABLE 1 Mortalities of Sheep due to Wimmera Rye Grass Toxicity and Progressive Total of Number of Farms with Parasitised L. rigidum in the Years 1970-74 Total mortalities each year* Number of farms reporting first outbreak Number of reported outbreaks each year Progressive total of number of farms with Darasitised rve mass * Numerator indicates number of deaths, denominator
1970-7 1
1971-72
1972-73
1973-74
550/3540 5 9
1247/5670 4 13
889/4736 8 12
68119720 9 22
5
9
17
20% ( 1 of 5 calves) and 45% (45 of 99 mixed-
age cattle) were recorded. Clinical Signs - Signs of WRGT in sheep were characterised by staggering, collapse, convulsions, other nervous signs and death. When an affected flock was driven a few hundred metres a proportion of animals collapsed into ventral or lateral recumbency. Some animals remained down for only 5 to 10 seconds and showed no other signs. In other recumbent animals some or all of the following signs were seen: neck ventroflexion, opisthotonus, head nodding, forelimb, hind-limb and digital extension, tetanic and clonic convulsions, and posterior extension of the hind limbs of sheep in ventral recumbency. From 5 seconds to a few minutes after initiaI collapse affected stock usually regained their feet and staggered away with either a stiff-legged, jumping, or swaying gait. After a further 1 or 2 minutes these animals either apparently recovered or wandered aimlessly about, showing depression and ataxia. These and other apparently normal stock would continue to collapse, either spontaneously or upon being driven, showing further convulsions and other of the afore-
n
I
Outbreaks on pasture Outbreaks involving crops and stubbles
VI
Y
4
$20-
1
m I-
3
0 L
0 ct
-
15-
w
:fit-
P
2
.... ...... ..
-
5-
rrr
... ..-.._ ... ... ...-.. Z i i
n OCT
NOV
DEC
JAN
FEB
MAR
31
indicates number at risk.
APR
Figure 1. Month of occurrence of 57 field outbreaks of Wirnrnera rye grass toxicity in Wesern Australia in the period 1970-74.
Australian Veterinary Journal, Vol. 51, November, 1975
mentioned nervous signs. Animals recumbent for long periods remained in lateral recumbency and exhibited periods of violent convulsions, opisthotonus, tachycardia, dyspnoea, frothing at the mouth and nystagmus, and took up to 2 days to die. In 2 outbreaks of WRGT affecting flocks of ewes in late pregnancy and from which 30 of 700 and 20 of 350 had died, abortions occurred in an estimated 10% of the surviving ewes. No infectious causes of abortion were found during these outbreaks. The signs seen in cattle were essentially the same as those observed in sheep. After the removal of affected stock from parasitised Wimmera rye grass, signs and mortalities continued for up to 10 days, but more commonly they had ceased after 4 days. Some sheep or cattle did not manifest initial signs of WRGT until 2 or 3 days after removal of the affected flock or mob from parasitised rye grass. Clinical pathology - Biochemical estimations of serum inorganic phosphorus, calcium and magnesium levels showed that all were within the normal ranges. Autopsy findings - A pale, friable liver, haemorrhages in several organs, and lung congestion were the most constant gross autopsy findings. Histological changes observed in tissue sections were non-specific and variable. Treatment and control practices -Treatments attempted produced no apparent beneficial effects. The managerial control practices instituted by farmers were also unsuccessful in preventing recurrences of WRGT in stock grazing "outbreak" paddocks, in both the season of the initial outbreak and in subsequent seasons. Distribution of Parasitised Wimmera Rye Grass Parasitised Wimmera rye grass was found on the 26 properties on which WRGT occurred and on 5 other farms. The totaI paddock area of parasitised Wimmera rye on the 26 outbreak properties was 2,712 ha or 12% of their total arable area. On individual outbreak farms the affected paddock area was between 21% and 527
TABLE 2 Biochemical and Pigment Characteristics of 6 Isolates of Corynebacterium sp from L. rigidum Parasitised by Anguina sp in Western Australia, Compared with the Corynebacterium s p Isofated in South Australia bv Price (19731 Isolate Western Australia
Observation
1
Pigment Catalase Carbohydrate Fermentation glucose* sucrose maltrose glycerol lactose xylol starch Indole production Gelatin liquefaction Nitrate reduction
T + + + + + C
A
A A A d -
y = Bright Yellow. c = Cream. d = Doubtful.
76% of the total farm area. Three farms had more than 35% of their total grazing area affected. In the 750 pasture samples collected from farms on which WRGT had occurred infection levels from zero to approximately 60% for both Anguirra sp and Corynebacterium sp were found, although the highest levels in some paddocks where WRGT had occurred were less than 2%. In one stubble paddock where stock grazed without loss, levels were 12%. In the survey of properties adjacent to farms on which WRGT had occurred, macroscopic evidence of Anguina sp and Corynebacterium sp parasitised Wimmera rye grass was found on 3 properties. Anguina sp and the associated Corynebacterium sp were found in samples from 1 paddock each on 2 of the other 12 farms investigated. In the detailed paddock survey, parasitised Wimmera rye grass was found in 128 of the 179 samples collected. Levels of parasitism for nematode galls and bacterially infected seed in the samples collected ranged from zero to 44% and 55% respectively. Parasitised rye grass was found throughout the paddock on all soil types but the highest levels of parasitism were found where the rye grass pasture was densest on clay soil. Means of spread of rye grass parasites - On affected farms, as was the general practice throughout the region, paddocks were cropped in a 3- to 5-year rotation and usually more than 1 paddock on each farm was cropped each year. 528
2
A A A A
A
-
3 C
4 Y
A d A d d - A A A d d
5 C
6 Y
South Australia Price ( 1973)
+Y
A A A A A A d A A A d
-
+
-
- - - -
A A A A
-
A = Acid. = Gas was not produced from glucose; the other carbohydrates were not tested for gas production. = Positive. - = Negative.
+
Consequently, movement of machinery between paddocks often took place. Hay and grain harvested in one paddock on affected farms was commonly fed on the ground in various other paddocks. Occasional machinery and stock movements had occurred between farms under investigation, prior to the confirmation of the presence of parasitised rye in their paddocks. A farm on which the presence of parasitised rye grass was found in 1968 was worked in partnership with another property 40 km away. All cultivating, seeding, haymaking and harvesting machinery was shared and moved annually between the two farms, where they were used in turn. The second farm had its first confirmed WRGT outbreak in 1973. Commercial seed graders and contract haycarters commonly moved their machinery from farm to farm through the district. On some adjacent farms and paddocks with parasitised Wimmera rye grass, the location of affected paddocks followed common water courses and creek systems. Bacteriological Examination of Corynebacterium sp in Paraitised Lolium rigidum Examination of the smears of yellow slime from parasitised rye grass showed predominantly small, Gram positive bacilli. Small Gram positive, catalase positive bacilli with 6 slightly different ranges of biochemical activity were isolated. A summary of these biochemical findings compared with those of the Corynebacterium sp isolated by Price (1973) are presented in Table 2. Australian Veterinary Journal, Vol. 51, November, 1975
Disrussion
Although 58 outbreaks of WRGT on 26 farms were reported to the authors between 1970 and 1974, approximately 5 more farms are believed to have incurred stock losses during 1973-74. These outbreaks were not reported because stock owners thought this would reduce the value of their farm and returns from stock sales. Between 1970 and 1974 the occurrence of 57 paddock outbreaks between late October and April coincided with the seasonal pattern of WRGT observed in South Australia by McIntosh et a1 (1967), and the toxic phase in the life cycle of Anguinn sp with the associated Corynebacterium sp defined by Price ( 1973). In Western Australia the different frequency of outbreaks on pasture and stubble in months between October and April reflected the grazing management systems commonly used in the region. Stubbles are only available after harvest in December and January. The greater number of outbreaks recorded in sheep, compared with cattle, was related to the population of each in the region. The availability of feed other than parasitised rye grass in toxic paddocks influenced the occurrence of WRGT. Observations showed that stock took a longer time to develop signs of WRGT after their introduction to cereal stubbles containing a large bulk of crop residue amongst which parasitised rye grass grew. This may have been due to a reduced daily intake of toxic Wimmera rye grass material by grazing stock. Results of a pen-feeding trial (unpublished data) showed that sheep could consume reduced amounts of parasitised rye grass daily for up to 75 days before signs of WRGT eventually developed. Alte-natively, the extended period taken for stock to develop WRGT signs in certain paddocks containing parasitised rye grass may have been due to their grazing alternative feed only, before intake of toxic material commenced. Clinical signs, including abortions, together with macroscopic and microscopic autopsy findings observed in the outbreaks in Western Australia, were similar to those described in South Australia (McIntosh et al 1967), although aimless wandering of semi-recovered stock was a feature of some Western Australian outbreaks. Mortality rates recorded during some outbreaks in Western Australia were higher than those reported from South Australia. Livestock morbidity and mortality rates, and the time taken for signs to abate after removal of stock from affected pastures were related to intake of parasitised Wimmera rye grass by affected stock. In a number of outbreaks during Australian Veterinary Journal, Vol. 51, November, 1975
1974, when stock owners were aware of the disease, immediately recognised the first signs, and removed stock from affected paddocks, mortality rates were usually low. However, in other outbreaks losses were. frequently high because stock had been left unobserved on toxic pastures for 2 or 3 weeks. Shortcomings of control measures attempted by farmers were obvious. Interrupted grazing failed because of a poor understanding of levels of toxicity and varying stock grazing behaviour in different paddocks. Measures used to dislodge rye grass seed from erect Wimmera rye grass pasture in early summer offered some initial success, but the longer stock grazed such pastures the more likely they were to eventually consume enough parasitised seed from the ground to elicit WRGT signs. Mowing in early spring failed because of the variation in stage of maturation of Wimmera rye grass plants at the time of mowing and subsequent regrowth of rye grass. Heavy grazing was usually an impractical management procedure. On farms where a significant paddock area was affected there simply were not enough stock to maintain sufficient grazing pressure during the critical spring period to prevent most rye grass plants flowering. Control procedures involving autumn burning of pasture were unsuccessful for reasons discussed by Price ( 1973 ) . Investigation of the distribution of parasitised Wimmera rye grass and WRGT outbreaks showed that since the initial report of WRGT in Western Australia (Gwynn and Hadlow 1971) the syndrome has emerged as a threat to livestock production in a significant proportion of the agricultural area of Western Australia. Whether the problem has spread since 1971 to all the farms presently involved is doubtful. The finding of parasitised Wimmera rye in paddocks and on farms where stock losses had not been observed suggests the nematode and bacterial parasites might exist in a paddock for at least one season before its presence is realised. It is likely that spread is a slow continuing process, but observations made in spring and early summer of 1974-75 and not included in the results reported in this paper suggest that the rate of spread is increasing. Although the level of parasitism of Wimmera rye grass was higher in areas of denser rye grass in the paddock surveyed in detail, the presence cf parasitised rye throughout the paddock was not surprising. Cultivating and harvesting in preceding cropping years would have encouraged spread. 529
The bacteriological findings indicate that the Corynebacterium sp associated with nematode infested L. rigidwm in Western Australia, although showing slightly different biochemical activities, is similar to the bacterium isolated by Price (1973). No attempt has yet been made to compare the Anguina sp nematode associated with WRGT in this state with A . lolii (Price 1973) found in South Australia. summary
Outbreaks of Wimmera rye grass toxicity in the south-west of Western Australia, and the associated distribution of Wimmera rye grass parasitised by Anguina sp and Corynebacterium sp were investigated. Between 1970 and 1974, 58 outbreaks occurred on 26 farms in sheep and cattle grazing pasture, cereal crop, stubble, screenings, or hay containing parasitised Wimmera rye grass. Morbidity rates up to 100% and mortality rates up to 77% were observed. Clinical signs were characterised by staggering, collapse, periods of convulsions and other nervous signs, often followed by death. A pale, friable liver and haemorrhages in different organs were the most constant gross autopsy findings, but no specific histopathological changes were observed. Symptomatic treatment of affected animals and agronomic control measures attempted by farmers were unsuccessful in controlling the disease. The distribution of parasitised Wimmera rye grass was studied on farms in the region, and in detail in one paddock where heavy losses from Wimmera rye grass toxicity had recently occurred. Parasitised Wimmera rye grass was also found
530
in paddocks and on farms where losses due to Wimmera rye grass toxicity had not been diagnosed. Acknowledgments
The authors wish to acknowledge Dr R. Gwynn and Dr A. Hadlow for their initial field records. Also we express grateful acknowledgment to Miss 0. Goss and Mr P. McR. Wood for the examination of Wimmera rye grass material, also Dr R. B. Richards, Dr J. Dickson and Dr M. Bond for assistance with histopathology and Mrs S . Ball for bacteriology. We also wish to thank Mr J. Burdass, Mr T. Marfleet, Mr S . Bull and other officers of the Department of Agriculture, Katanning, for their assistance in the field. References Aasen, A. L., Culvenor, C. C. J., Finnie, E. P., Kellock, A. W. and Smith, L. W. (1969)-Aust. 3. agric. Res. 20: 71. Clegg, F. G. and Watson, W. A. (1961)--Vet. Rec. 73: 731. Cunningham, I. J. and Hartley, W. J. (1959)-N.Z. Vet. J . 7: 1. Davies, E. T. and Farmer, P. E. (1961)-Vet. Rec. 73: 130. Gwynn, R. and Hadlow, A. J. (1971)-Aust. vet. J . 47: 408. Healey, J. S. (1970)-Agric. Gaz. N.S.W. 81: 92. McIntosh, G. H., Rac, R. and Thomas, M. R. (1967) -Aust. vet. J . 43: 349. Price, P. C. (1973)-"1nvestigation of a nematodebacterium disease complex affecting Wimmera rye grass". Ph.D, Thesis, University of Adelaide. Shaw, J. N. and Muth, 0. H. (1949)--/. Am. vet. med. Ass. 114: 315. (Received for publication 3 September 1974)
Australian Veterinary Journal, Vol. 51, November, 1975
