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Studies on the Pathogenesis of Infection with Hyostrongylus Rubidus(Nematoda). The Effects of Levels of Infection of up to 150, 000 Infective Stage Larvae on the Growing Pig
Br. vet.
J. (1972), 128, 138
STUDIES ON THE PATHOGENESIS OF INFECTION WITH HYOSTRONGYLUS RUB/DUS (NEMATODA). THE EFFECTS OF LEVELS OF INFECTION OF UP TO 150,000 INFECTIVE STAGE LARVAE ON THE GROWING PIG. 1. NUTRITIONAL STUDIES By I. J.
LEAN*,
I. V.
HERBERT AND
J. B.
CASTELINO*
Department of Agricultural and Forest Zoology, University College of North Wales, Bangor
SUMMARY
Colostrum deprived specific pathogen-free (S .P.F. ) and conventionally reared minimal disease growing pigs have been experimentally infected with H. rubidus infective larvae. Infection doses have ranged from 3000-150,000 larvae administered as one dose; previously infected animals have also been reinfected. Animals have been maintained in metabolism crates on diets containing 16 per cent and 13 per cent crude protein, and the following indices recorded: liveweight gains, nitrogen retention, food conversion efficiencies and apparent digestibility. Statistical analysis of these data shows no consistent differences between infected and control animals in each trial. INTRODUCTION
Hyostrongylus rubidus, the small red stomach worm of swine, is a trichostrongylid nematode which has received a remarkable amount of publicity in recent years. The commercial interest in pig helminthoses has seemingly changed from a pronounced emphasis on Ascaris suum infection, a parasite of considerable importance but one which has been relatively well controlled for a number of years, to hyostrongylosis and oesophagostomiasis. Early workers (e.g. Hassall & Stiles, 1892) considered H. rubidus to be of little importance, and that clinical symptoms following infection only appear when the animal is concurrently infected with some other pathogen or under some form of "stress" such as poor housing, undernutrition, malnutrition, pregnancy or stocking density effects. It is not surprising perhaps that field reports attribute a wide range of pathogenic effects to the worm, some conflicting. These include ulceration and gastritis, haemorrhagic anaemia, capricious appetite, increased heartbeat, emaciation, and death. Infertility and agalactia are two conditions mentioned in association with breeding stock. Stomach changes are associated particularly with the larval stages in the gastric mucosa. Little work has been done on the effects of the worm on the growing pig. * Present addresses: 1. J. Lean, Department of Agriculture, Wye College, Nr. Ashford, Kent ;
J. B. Castelino, Department of Zoology, University College, Nairobi, Kenya.
H. RUBIDUS INFECTION IN THE PIG
139
Field data has been more generally concerned with prevalence in sows at autopsy with no precise measurement of the specific effects of hyostrongylosis in the absence of other factors. Unsubstantiated claims for the pathogenicity of the worm are a characteristic feature of the literature. The trials reported here have attempted to measure the effects of single infective doses of H. rubidus upon the growing pig in the absence of concurrent infection, and under standard conditions. MATERIALS AND METHODS
Colostrum deprived (specific pathogen free, S.P.F.) and conventionally reared minimal disease animals from seven litters of either Large White or Large White cross Landrace growing pigs were used in the five trials recorded. H. rubidus eggs were obtained from experimental donor animals and the larvae cultured to the infective stage as detailed by Castelino, Herbert & Lean ( I97 0a ). TA BLE I THE PIGS U TILIZED IN THE NUTRITIONAL TRIALS, THE AGES AT WHICH THEY WERE INFECTED AND SLAUGHTERED, AND THE NUTRITIONAL REGIMES INVOLVED
Trial number Total number of pigs Sex ratio-male/female
2
3
4
5
7
8
12
6
4
4/3
4/4
6/6
6/0
2/2
Age at weaning
28 days
28 days
Birth
35 days
35 days
Age at infection
7 days
40 days
74 days
73 days
127 days
2/2
2/ 2
4/4
3/0
1/ 1
Number infected-male/female Level of infection
Diet while on trial
Metabolism crates Slaughter weight, kg
(a) Sow milk creep 16 % crude protein feed pellets ad lib. meal (b) 16% crude protein meal Yes 45
Yes 45
10 X 10 3 reinfected at 45 kg 20 X 10 3
20
16 % crude protein meal
13 % crude protein meal
16 % crude protein meal
Yes go
Yes 45
Yes 45
X
10 3
15
x
10'
Note: Pre-trial Treatment of Animals All litters had available 22 per cent crude protein pellets from one week after birth. At weaning animals were introduced to the diet on which they were fed during the trial period . Trial 3 animals were fed milk substitute for the first 3 weeks of life.
All pigs were weighed weekly and rations adj usted to the mean live weight of the group. Trial I animals were weighed on alternate days from birth to a weaning age of 28 days. Meal was then fed at a basic rate of 830 gJheadJday at 18 kg increasing by a further 180 gJheadJd ay for each 5-kg liveweight gain to 55 kg, then by a further 90 gJhead/day for each 5-kg liveweight gain to 90 kg. Water was added to the meal at a rate of 500 ml/500 g meal fed. Groups of 6 animals were penned in metabolism crates for seven-day
140
BRITISH VETERINARY JOURNAL, 128, 3
periods on alternate weeks, except for Trial 4 where the animals were crated continuously for 30 days. Animals were introduced to the crates by penning them in for a period of at least seven days before beginning collection of faeces and urine for experimental purposes. ''''hen not in the crates animals were penned singly to r educe the possibility of cross-infection and in order that infected animals could be observed more fully. Faecal and urine samples were collected daily and weighed. The faeces were then stirred for two minutes in a Hobart Mixer and one-fifth of the material was retained for a five-day collection pool. The urine was similarly mixed and an aliquot retained. Sulphuric acid (10 per cent) was added to the urine collection vessels beneath the metabolism crates, and the daily sub-samples were stored in a freezer at - 20 °C until analysed. Samples from trial 4 were analysed on an alternate day basis to provide a more detailed picture of nitrogen retention during the larval development period and during the peak of egg production. Nitrogen analysis of faeces, urine, and feed samples was carried out on a Technicon autoanalyser system (Siriwardene, 1966). Pooled samples were blended in the Hobart mixer, and the sub-samples taken for analysis predigested in order to speed up the autoanalyser system. Samples were periodically checked by repeating the analysis using a standard Markham apparatus (Markham, 1942). Daily examination of faeces for helminth eggs commenced 15 days after infection and continued until the peak of egg production passed; from then on counts were made at weekly intervals until the pigs were slaughtered. Saturated magnesium sulphate solution was used as the flotation agent in the Clayton Lane system of analysis. This method was used because H. rubidus has a low fecundity and consequently faecal egg counts are seldom high. All animals were taken to a commercially acceptable weight for slaughter, at which time the stomachs of infected and control animals were examined for H. rubidus. An estimate of the degree of ulceratien was made, and the worms were extracted using Reinecke's (1967) method. RESULTS
Animals remained in good condition throughout the trials, and at no time was a depression in appetite seen. Some animals scoured slightly during the second week of the infection but examination of the faeces of infected animals failed to show any voided larvae, or traces of mucus or blood. Occasionally infected animals coughed up a clear mucus just prior to feeding; examination of this material did not reveal worms. No vomited feed was ever seen. H. rubidus eggs were first detected in the faeces 19 days after infection; the numbers reached a peak by the 24th day and then declined (see Castelino, Herbert & Lean, 1970). In some instances a slight rise in egg counts of short duration was noted up to 60 days after infection. Reinfection with 20,000 larvae at 45 kg liveweight in Trial 3 animals showed no rise in egg production after the second infection.
H. RUBIDUS INFECTION IN THE PIG
The mean values recorded for nitrogen retention, apparent digestibility, and feed conversion efficiency in trial 4 animals are plotted in Fig. I. The records are typical of those for all the trials (see Lean, 1969). The data on liveweight gain of all the trials over varying time periods are summarized in Table II. Statistical analysis of these data (by variance ratio
18
2 c co
g"
16 14 12
Z 10
• ___ e
A.
•
Point of Infection
~. ./
8
%
95 90
B.
~.--
85
2
4
6
8
10
12
14
16
18
20
22
24
Days
Fig.
1.
Trial 4 animals. Nitrogen retention (A), apparent digestibility (B) and feed conversion efficiency (C). Each point is the mean of three values. •
• Infected groups, 0 Non-infected groups,
o
TABLE 11 MEAN LIVEWEIGHT GAIN (KG/DAY) OF INFECTED AND NON-INFECTED ANIMALS USED IN TRIALS 1 -5 (SEE TABLE 1 FOR EXPERIMENTAL REGIME)
Trial number and weight range
Infected animals ±S,D, kg/day
Non-infected animals ±S,D, kg/day
I,
From infection for (18-45 kg)
24
days
0'20 0'40
0'10 0'20
0'20 0'40
0'12 0'23
2,
From infection for (18-45 kg)
35
days
0'10 0'40
0'05 0'20
0'10 0'40
0'05 0'20
3,
From infection for ~18-45 kg) 45-90 kg)
35
days
0'20 0'30 0'50
0'07 0'11 0'18
0'20 0'40 0'50
0'10 0'20 0'25
4, From infection for (18-45 kg)
35
days
0'30 0'40
0'17 0'23
0 '3 0 0'40
0'17 0'23
5, From infection for (18-45 kg)
35
days
0'50 0 '40
0'35 0'28
0'50 0'40
0'35 0'28
BRITISH VETERINARY JOURNAL, 128, 3
analyses) shows no consistent differences between infected and control animals in each trial. Analysis of haematological data for each trial also fails to show differences between infected and control animals (see Lean, Herbert & Castelino, 1972). Variation in the nitrogen retention data tends to be high, some of this being attributed to losses of ammonia through evaporation, and difficulties in separating faeces and urine when female animals were used. Examination of the stomachs of all the animals at slaughter revealed a very small residual population of worms, with relatively few immature stages. The stomach surface, especially in the fundic to pyloric region, was usually covered with healed crater type ulcers of a few mm diameter. Table III demonstrates the type of recovery from a single infection of 150,000 larvae (Trial 5). TABLE III WORM RECOVERY DATA
Number of animals
4
ON ANIMALS INFECTED
Days postinfection
21 35 56
WITH
Adults
282 3 53 20 54 2
150,000
LARVAE
(TRIAL
5)
Larvae
Percentage recovery
3461 190- 18 52 196
20'9 3,6-6,8 0'5
These results were collected from Trial 5 animals with others of the same litter which were infected at the same age and slaughtered at varying times from patency onwards. The results show that approximately 2 I per cent of the larvae fed to the pig developed to patency but their numbers subsequently declined rapidly, so that by 56 days post-infection the residual population was no higher than that recorded from groups which had been infected at a much lower level. The egg counts in this group infected with 150,000 larvae, expressed as eggs/g faeces (Castelino et at., 1970), reached a higher peak than that recorded for infective doses of 20,000 larvae given to conventionally reared, as opposed to colostrum deprived, animals. The stomach of the animal slaughtered 2 I days post-infection was inflamed but not haemorrhagic, worms were thickly aggregated in the characteristic clear mucus on the stomach surface. The stomach of the animal slaughtered 56 days post-infection had, as Table III shows, very few worms, the presence of crater shaped ulcers being the only indication of gross pathological change. In no infection recorded here were worms detached in the ingesta, nor were they ever found as Larsen (1967) described in the intestine as far down as the colon. DISCUSSION
The effects of nematodes upon liveweight gain, feed conversion efficiency, etc., in the growing pig have tended to take second place to studies involving pathological changes in parasitized tissues, and to the efficiency of anthelmintics on
H. RUBIDUS INFECTION IN THE PIG
143
worm removal. Field reports of prevalence are numerous-Crocker & Biester (1920); Skrjabin & Bekensky (1925); Drezenic (1964); and Gerber (1968) have all found H. rubidus in growing pigs- but experimental work on infections of H. rubidus in the absence of other parasites and its effect on the growing pig seem to be confined to the work of Porter (1940) and Davenport (1967). Porter (1940) failed to demonstrate a difference in the liveweight gains of infected and non-infected animals; Davenport (1967) did, however, show a significant different in the liveweight gains of the animals he used. The results of our trials agree closely with those of Porter (1940) who recorded liveweight gains over periods varying between 34 and 44 days, and who gave small doses of larvae over much of this period. Porter's highest single infection was 4800 larvae to a 35-day-old pig; our single dose to animals of a similar age has been 20,000. Davenport (1968, personal communication) has given a maximum dose of 15,000 larvae to an IS·5-lb animal. He analysed liveweight gain on a percentage increase basis, and demonstrated a significant difference between infected and control animals during the larval development period. This method of analysis was not adopted in the present study. Differences in interpretation of results could be due to this factor and also possibly to the feeding regimes used. Davenport obtained his animals from differing sources and they were housed together. Numerous descriptions have been made of the changes brought about in the stomach wall by the action of larval and adult H. rubidus, one of the most recent being that of Kendall, Thurley & Peirce (1969). Most do not deal with experimentally produced infections but with an examination of material collected after emergency slaughter or in the course of routine veterinary work. Some authors (Connan, 1967; Jacobs, 1967) have suggested that the gastritis due to H. rubidus might cause the same sort of protein digestive upset as occurs in bovine ostertagiasis. Larsen (1967) points out that although gross morphological similarities between gastric and abomasal hyperplasia are seen to exist, important differences in pathogenicity do occur. A primary symptom of ostertagiasis is chronic diarrhoea. Very few authors have found scouring in hyostrongylosis, although Dodd (1960) does mention it as a symptom of infection in very young animals. Shanks (1965) considers the occurrence of this symptom to be due to some other agent. Nielsen (1966) failed to demonstrate a gastro-intestinal protein loss in sows with a history of H. rubidus infection although he has shown this to occur in cattle with ostertagiasis and other diarrhoeic conditions. Larsen (1967) suggests that the emaciation seen in adult swine with H. rubidus infection under field conditions is mainly due to a deficient gastric digestion though he presented no evidence to show that this was so. Although the post-mortem results recorded in the infections noted in this study (Lean, 1969) indicate periods of intense worm activity in the early period of infection, the data in liveweight gain, nitrogen retention and apparent digestibility have failed to show a significant difference between animal groups or individuals on each trial. The results graphed for the infection period of Trial 4 are typical of those for any of the levels of infection so far considered.
BRITISH VETERINARY JOURNAL, 128, 3
The values showed no significant deviation from those found in uninfected controls. None of these values were in any way altered when animals received larval doses of 20,000 or up to 150,000. This suggests that neither the larvae nor the adult worms were in sufficiently great numbers in the stomach mucosa, nor present for a sufficiently long time to produce a level of irritation which would disturb gastric activity to an extent which affected the digestive system of the host. It may be postulated that a ration normally given to the growing animal contains an excess of protein such that any effect of the parasite would be masked, whereas a lower crude protein level might increase the effect and duration of H. rubidus infection, since competition for available material would be greater and the animal would be less capable of withstanding the demands of the parasite. Trial 4 animals were therefore fed a diet containing 13 per cent crude protein. The protein level in this diet, although not particularly low, was below that normally fed to growing pigs. Although these animals had a reduced weight gain when compared to other trial groups, there was no significant differences between the infected and non-infected animals on the diet. Animals from each trial were usually slaughtered together, although those in Trial 3 were slaughtered over a three-week period which was a reflection of the differing starting weights of the animals. Crocker & Biester (1920), Castle (1932) and Clay (1938) all considered that H. rubidus was only of importance on those occasions when the vitality of the host animal was lowered as a result of other disease processes or stresses. In the present study, although the animals were exposed to considerably higher infections than they would normally meet on any single occasion in the field, they were not, so far as we are aware, subjected to any stress factors other than those caused by experimentation. There is reason to believe from reinfection experiments not recorded here that the intake of small numbers of larvae over a longer period produces no measureable effect upon the stomach. Nutrition was adequate, and housing conditions good. Animals were not exposed to draughts or adverse temperature conditions. It is consequently suggested that the worms did not reach a position of dominance in the host parasite relationship, and were unable to produce changes of a demonstrable magnitude in our experiments. It is pointed out that the statistical validity of such experiments as those recorded here would be improved by the use of more animals, when marginal differences between infected and non-infected animals might be detectable at the infection levels used. However, such high rates of infection rarely appear to occur naturally in the United Kingdom, and it seems reason able to conclude that H. rubidus is not likely to be commercially important in growing animals under good husbandry conditions and in the absence of other disease-causing agents. ACKNOWLEDGEMENTS
This work was supported by a grant from the Meat and Livestock Commission to whom we offer our thanks.
H. RUBIDUS INFECTION IN THE PIG
145
I. J. Lean was a Sir William Roberts scholar and J. B. Castelino a Commonwealth Scholar. REFER ENCES
CAsTELINO, J . B., HERBERT,!. V. & LEAN, I.J. (1970). R es. Vet. Sci. U,7. CAsTLE, A. (1932) . Vet. J. 88, 474. CLAY, A. L. (1938). Aust. vet. J. 14. 194. CONNAN, R. M. (1967). Vet. Rec. 80, 424. CROCKER, W. T. & BIESTER, H. E. (1920). J. Aust. vet. med. Ass. 57, 427. DAVENPORT, P. G. (1967). Vet. Rec. 81, 390. DODD, D. C. (1960) . N.;:;. vet. J. 8, 100. DREZENIC, 1. (1964). Veter. Arhiv. 34. 283. GERBER, H. C . (1968) . Thesis, Free University, Berlin. HASSALL, A. & STILES, C. W. (1892) . J. camp. Med. vet. Arch. 13,207. JACOBS, D. E. (1967) . Vet. Rec. 81,198. KENDALL, S. B., THURLEY, D. C. & PEIRCE, M. A. (1969). J. camp. Path. 79, 87. LARSEN, S. (1967). Acta vet. scand. 8, 347. LEAN,!. J. (1969). Ph.D. Thesis, University of Wales, Bangor. LEAN, I.J., HERBERT,!. V. & CASTEUNO,J. B. (1972). Br. vet. ]. 128,000. MAIurnAM, R. (1942). Biochem. J. 36, 790. NIELSEN, K. (1966). Acta vet. scand. 7, 321. PORTER, D. A. (1940). Proc. helminth. Soc. Wash. 7, 20. REINICKE, R. K. (1967). Onderstepoort J. vet. Res. 34. 547. SHANKS, P. L. (1965). N;:;. vet.], 13,38. SIRIWARDENE, J. A. DA S. (1966). J. Sci. Fd Agric. 17,456. SKR]ABIN, K . I. & BEKENSKY, P. W. (1925). B erl. tieriir;;tl. Wschr. 41, 52. (Receivedfor publication 20 October 1971)
Etudes sur la pathogenicite de I'infection per Hyostrongylus rubidus (neDlatode). Effets des tall" d'infection allant jusqu':ii 150.000 larves en phase infectieuse chez Ie cochon en voie de croissance I. Etudes de nutrition (Lean et al.) Resulne. On a infecte experimentalement avec des larves infectieuses de H . rubidus des cochons en voie de croissance prives de colostrum, sans pathogene specifique (S.P.F.) et eleves conventionellement avec Ie minimum de maladies. L es doses d'infection s'etalient de 3.000 11 150.000 larves administrees en une seule dose; des animaux infectes auparavant ont egalement ete reinfectes. On a maintenu les animaux dans des cages metaboliques avec des regimes contenant 16 pour cent et 13 pour cent de proteine brute et on a enregistre les indices suivants; augmentation du poids du foie, retention d'azote, efficacites de conversion de la nourriture et digestibilite apparente. Les analyses statistiques de ces donnees ne montrent aucune difference consistante entre les animaux infectes et les temoins au cours d e chaque essai. Studien iiber die Pathogenese der InIektion Dlit Hyostrongylus ruhidus (NeIDatoda) Der Effekt von Infektionen Dlit bis zu 150000 Larven iDl infektiosen StadiUln auf heranwachsende Schweine I. Erniihrungsstudien (Lean et al.) ZusaD1Dleruassung. Schweine, die kein Colostrum (S.P.F.) erhalten hat ten und in der ublichen Weise als "minimal disease" Schweine aufgezogen worden waren, wurden experimentell mit H. rubUlus infektioser Larven infiziert. Die Dosis erstreckte sich von 3000 bis zu 150000 Larven, die als Einzeldosis gegeben wurden. Auch vorher infizierte Tiere wurden erneut infiziert. Sie wurden in StoffWechselkafigen mit einer Diat, die 16 Prozent und 13 Prozent Protein enthielt, gehalten, und die folgenden Daten wurden registriert : Gewichts-
BRITISH VETERINARY JOURNAL, 128, ::5 anstieg, Stickstoffretention, Leistungsfahigkeit hinsichtlich der Futterverwertung und augenscheinliche Verdaulichkeit der Nahrung. Estudios sobre la patogenesis de infecci6n con Hyostrongylus rubidus (nelDatodes). Efectos de los niveles de infecci6n de hasta 150.000 larvas en fase infectiva sobre el cerdo en crecimiento I. Estudios nutritivos
(Lean et al.) Res1UDen. Cerdos en crecimiento privados de calostrum, libres de pat6genos especificos y criados con metodos convencionales fueron infectados experimentalmente con larvas infecciosas de H. Tubidus. Las dosis de infecci6n oscilaron entre 3000 y 150.000 larvas adrninistradas en una sola dosis; los animales infectados previamente tam bien fueron reinfectados. Los animales se mantuvieron cajas de metabolismo en dietas que contenian 16 por ciento y 13 por ciento de proteinas crudas, registnindose a continuaci6n los siguientes indices; ganancia en peso en vivo, retenci6n de nitr6geno, eficiencias de conversi6n de los alimentos y digestibilidad aparente. Los analisis estadisticos de estos datos no muestran diferencias importantes entre los ani males infectados y los con troles en cad a una de las pruebas.
ANNOUNCEMENT In October, 1971, a Society of Pharmacological and Environmental Pathologists was incorporated in the state of New Jersey, U.S.A. The objectives of this society are to foster the discipline of pharmacological and environmental p athology, to contribute to the development of criteria and standards for the evaluation of pathological changes induced by drugs, chemicals, or environmental agents, to promote the training of pharmacological and environmental pathologists, and to establish a registry of naturally occurring and induced diseases in laboratory animals. For additional information and application forms for membership please contact the Secretary-Treasurer (P.O. Box 276, Florham Park, New Jersey, 07932, U.S.A.).
J. (1972), 128, 138
STUDIES ON THE PATHOGENESIS OF INFECTION WITH HYOSTRONGYLUS RUB/DUS (NEMATODA). THE EFFECTS OF LEVELS OF INFECTION OF UP TO 150,000 INFECTIVE STAGE LARVAE ON THE GROWING PIG. 1. NUTRITIONAL STUDIES By I. J.
LEAN*,
I. V.
HERBERT AND
J. B.
CASTELINO*
Department of Agricultural and Forest Zoology, University College of North Wales, Bangor
SUMMARY
Colostrum deprived specific pathogen-free (S .P.F. ) and conventionally reared minimal disease growing pigs have been experimentally infected with H. rubidus infective larvae. Infection doses have ranged from 3000-150,000 larvae administered as one dose; previously infected animals have also been reinfected. Animals have been maintained in metabolism crates on diets containing 16 per cent and 13 per cent crude protein, and the following indices recorded: liveweight gains, nitrogen retention, food conversion efficiencies and apparent digestibility. Statistical analysis of these data shows no consistent differences between infected and control animals in each trial. INTRODUCTION
Hyostrongylus rubidus, the small red stomach worm of swine, is a trichostrongylid nematode which has received a remarkable amount of publicity in recent years. The commercial interest in pig helminthoses has seemingly changed from a pronounced emphasis on Ascaris suum infection, a parasite of considerable importance but one which has been relatively well controlled for a number of years, to hyostrongylosis and oesophagostomiasis. Early workers (e.g. Hassall & Stiles, 1892) considered H. rubidus to be of little importance, and that clinical symptoms following infection only appear when the animal is concurrently infected with some other pathogen or under some form of "stress" such as poor housing, undernutrition, malnutrition, pregnancy or stocking density effects. It is not surprising perhaps that field reports attribute a wide range of pathogenic effects to the worm, some conflicting. These include ulceration and gastritis, haemorrhagic anaemia, capricious appetite, increased heartbeat, emaciation, and death. Infertility and agalactia are two conditions mentioned in association with breeding stock. Stomach changes are associated particularly with the larval stages in the gastric mucosa. Little work has been done on the effects of the worm on the growing pig. * Present addresses: 1. J. Lean, Department of Agriculture, Wye College, Nr. Ashford, Kent ;
J. B. Castelino, Department of Zoology, University College, Nairobi, Kenya.
H. RUBIDUS INFECTION IN THE PIG
139
Field data has been more generally concerned with prevalence in sows at autopsy with no precise measurement of the specific effects of hyostrongylosis in the absence of other factors. Unsubstantiated claims for the pathogenicity of the worm are a characteristic feature of the literature. The trials reported here have attempted to measure the effects of single infective doses of H. rubidus upon the growing pig in the absence of concurrent infection, and under standard conditions. MATERIALS AND METHODS
Colostrum deprived (specific pathogen free, S.P.F.) and conventionally reared minimal disease animals from seven litters of either Large White or Large White cross Landrace growing pigs were used in the five trials recorded. H. rubidus eggs were obtained from experimental donor animals and the larvae cultured to the infective stage as detailed by Castelino, Herbert & Lean ( I97 0a ). TA BLE I THE PIGS U TILIZED IN THE NUTRITIONAL TRIALS, THE AGES AT WHICH THEY WERE INFECTED AND SLAUGHTERED, AND THE NUTRITIONAL REGIMES INVOLVED
Trial number Total number of pigs Sex ratio-male/female
2
3
4
5
7
8
12
6
4
4/3
4/4
6/6
6/0
2/2
Age at weaning
28 days
28 days
Birth
35 days
35 days
Age at infection
7 days
40 days
74 days
73 days
127 days
2/2
2/ 2
4/4
3/0
1/ 1
Number infected-male/female Level of infection
Diet while on trial
Metabolism crates Slaughter weight, kg
(a) Sow milk creep 16 % crude protein feed pellets ad lib. meal (b) 16% crude protein meal Yes 45
Yes 45
10 X 10 3 reinfected at 45 kg 20 X 10 3
20
16 % crude protein meal
13 % crude protein meal
16 % crude protein meal
Yes go
Yes 45
Yes 45
X
10 3
15
x
10'
Note: Pre-trial Treatment of Animals All litters had available 22 per cent crude protein pellets from one week after birth. At weaning animals were introduced to the diet on which they were fed during the trial period . Trial 3 animals were fed milk substitute for the first 3 weeks of life.
All pigs were weighed weekly and rations adj usted to the mean live weight of the group. Trial I animals were weighed on alternate days from birth to a weaning age of 28 days. Meal was then fed at a basic rate of 830 gJheadJday at 18 kg increasing by a further 180 gJheadJd ay for each 5-kg liveweight gain to 55 kg, then by a further 90 gJhead/day for each 5-kg liveweight gain to 90 kg. Water was added to the meal at a rate of 500 ml/500 g meal fed. Groups of 6 animals were penned in metabolism crates for seven-day
140
BRITISH VETERINARY JOURNAL, 128, 3
periods on alternate weeks, except for Trial 4 where the animals were crated continuously for 30 days. Animals were introduced to the crates by penning them in for a period of at least seven days before beginning collection of faeces and urine for experimental purposes. ''''hen not in the crates animals were penned singly to r educe the possibility of cross-infection and in order that infected animals could be observed more fully. Faecal and urine samples were collected daily and weighed. The faeces were then stirred for two minutes in a Hobart Mixer and one-fifth of the material was retained for a five-day collection pool. The urine was similarly mixed and an aliquot retained. Sulphuric acid (10 per cent) was added to the urine collection vessels beneath the metabolism crates, and the daily sub-samples were stored in a freezer at - 20 °C until analysed. Samples from trial 4 were analysed on an alternate day basis to provide a more detailed picture of nitrogen retention during the larval development period and during the peak of egg production. Nitrogen analysis of faeces, urine, and feed samples was carried out on a Technicon autoanalyser system (Siriwardene, 1966). Pooled samples were blended in the Hobart mixer, and the sub-samples taken for analysis predigested in order to speed up the autoanalyser system. Samples were periodically checked by repeating the analysis using a standard Markham apparatus (Markham, 1942). Daily examination of faeces for helminth eggs commenced 15 days after infection and continued until the peak of egg production passed; from then on counts were made at weekly intervals until the pigs were slaughtered. Saturated magnesium sulphate solution was used as the flotation agent in the Clayton Lane system of analysis. This method was used because H. rubidus has a low fecundity and consequently faecal egg counts are seldom high. All animals were taken to a commercially acceptable weight for slaughter, at which time the stomachs of infected and control animals were examined for H. rubidus. An estimate of the degree of ulceratien was made, and the worms were extracted using Reinecke's (1967) method. RESULTS
Animals remained in good condition throughout the trials, and at no time was a depression in appetite seen. Some animals scoured slightly during the second week of the infection but examination of the faeces of infected animals failed to show any voided larvae, or traces of mucus or blood. Occasionally infected animals coughed up a clear mucus just prior to feeding; examination of this material did not reveal worms. No vomited feed was ever seen. H. rubidus eggs were first detected in the faeces 19 days after infection; the numbers reached a peak by the 24th day and then declined (see Castelino, Herbert & Lean, 1970). In some instances a slight rise in egg counts of short duration was noted up to 60 days after infection. Reinfection with 20,000 larvae at 45 kg liveweight in Trial 3 animals showed no rise in egg production after the second infection.
H. RUBIDUS INFECTION IN THE PIG
The mean values recorded for nitrogen retention, apparent digestibility, and feed conversion efficiency in trial 4 animals are plotted in Fig. I. The records are typical of those for all the trials (see Lean, 1969). The data on liveweight gain of all the trials over varying time periods are summarized in Table II. Statistical analysis of these data (by variance ratio
18
2 c co
g"
16 14 12
Z 10
• ___ e
A.
•
Point of Infection
~. ./
8
%
95 90
B.
~.--
85
2
4
6
8
10
12
14
16
18
20
22
24
Days
Fig.
1.
Trial 4 animals. Nitrogen retention (A), apparent digestibility (B) and feed conversion efficiency (C). Each point is the mean of three values. •
• Infected groups, 0 Non-infected groups,
o
TABLE 11 MEAN LIVEWEIGHT GAIN (KG/DAY) OF INFECTED AND NON-INFECTED ANIMALS USED IN TRIALS 1 -5 (SEE TABLE 1 FOR EXPERIMENTAL REGIME)
Trial number and weight range
Infected animals ±S,D, kg/day
Non-infected animals ±S,D, kg/day
I,
From infection for (18-45 kg)
24
days
0'20 0'40
0'10 0'20
0'20 0'40
0'12 0'23
2,
From infection for (18-45 kg)
35
days
0'10 0'40
0'05 0'20
0'10 0'40
0'05 0'20
3,
From infection for ~18-45 kg) 45-90 kg)
35
days
0'20 0'30 0'50
0'07 0'11 0'18
0'20 0'40 0'50
0'10 0'20 0'25
4, From infection for (18-45 kg)
35
days
0'30 0'40
0'17 0'23
0 '3 0 0'40
0'17 0'23
5, From infection for (18-45 kg)
35
days
0'50 0 '40
0'35 0'28
0'50 0'40
0'35 0'28
BRITISH VETERINARY JOURNAL, 128, 3
analyses) shows no consistent differences between infected and control animals in each trial. Analysis of haematological data for each trial also fails to show differences between infected and control animals (see Lean, Herbert & Castelino, 1972). Variation in the nitrogen retention data tends to be high, some of this being attributed to losses of ammonia through evaporation, and difficulties in separating faeces and urine when female animals were used. Examination of the stomachs of all the animals at slaughter revealed a very small residual population of worms, with relatively few immature stages. The stomach surface, especially in the fundic to pyloric region, was usually covered with healed crater type ulcers of a few mm diameter. Table III demonstrates the type of recovery from a single infection of 150,000 larvae (Trial 5). TABLE III WORM RECOVERY DATA
Number of animals
4
ON ANIMALS INFECTED
Days postinfection
21 35 56
WITH
Adults
282 3 53 20 54 2
150,000
LARVAE
(TRIAL
5)
Larvae
Percentage recovery
3461 190- 18 52 196
20'9 3,6-6,8 0'5
These results were collected from Trial 5 animals with others of the same litter which were infected at the same age and slaughtered at varying times from patency onwards. The results show that approximately 2 I per cent of the larvae fed to the pig developed to patency but their numbers subsequently declined rapidly, so that by 56 days post-infection the residual population was no higher than that recorded from groups which had been infected at a much lower level. The egg counts in this group infected with 150,000 larvae, expressed as eggs/g faeces (Castelino et at., 1970), reached a higher peak than that recorded for infective doses of 20,000 larvae given to conventionally reared, as opposed to colostrum deprived, animals. The stomach of the animal slaughtered 2 I days post-infection was inflamed but not haemorrhagic, worms were thickly aggregated in the characteristic clear mucus on the stomach surface. The stomach of the animal slaughtered 56 days post-infection had, as Table III shows, very few worms, the presence of crater shaped ulcers being the only indication of gross pathological change. In no infection recorded here were worms detached in the ingesta, nor were they ever found as Larsen (1967) described in the intestine as far down as the colon. DISCUSSION
The effects of nematodes upon liveweight gain, feed conversion efficiency, etc., in the growing pig have tended to take second place to studies involving pathological changes in parasitized tissues, and to the efficiency of anthelmintics on
H. RUBIDUS INFECTION IN THE PIG
143
worm removal. Field reports of prevalence are numerous-Crocker & Biester (1920); Skrjabin & Bekensky (1925); Drezenic (1964); and Gerber (1968) have all found H. rubidus in growing pigs- but experimental work on infections of H. rubidus in the absence of other parasites and its effect on the growing pig seem to be confined to the work of Porter (1940) and Davenport (1967). Porter (1940) failed to demonstrate a difference in the liveweight gains of infected and non-infected animals; Davenport (1967) did, however, show a significant different in the liveweight gains of the animals he used. The results of our trials agree closely with those of Porter (1940) who recorded liveweight gains over periods varying between 34 and 44 days, and who gave small doses of larvae over much of this period. Porter's highest single infection was 4800 larvae to a 35-day-old pig; our single dose to animals of a similar age has been 20,000. Davenport (1968, personal communication) has given a maximum dose of 15,000 larvae to an IS·5-lb animal. He analysed liveweight gain on a percentage increase basis, and demonstrated a significant difference between infected and control animals during the larval development period. This method of analysis was not adopted in the present study. Differences in interpretation of results could be due to this factor and also possibly to the feeding regimes used. Davenport obtained his animals from differing sources and they were housed together. Numerous descriptions have been made of the changes brought about in the stomach wall by the action of larval and adult H. rubidus, one of the most recent being that of Kendall, Thurley & Peirce (1969). Most do not deal with experimentally produced infections but with an examination of material collected after emergency slaughter or in the course of routine veterinary work. Some authors (Connan, 1967; Jacobs, 1967) have suggested that the gastritis due to H. rubidus might cause the same sort of protein digestive upset as occurs in bovine ostertagiasis. Larsen (1967) points out that although gross morphological similarities between gastric and abomasal hyperplasia are seen to exist, important differences in pathogenicity do occur. A primary symptom of ostertagiasis is chronic diarrhoea. Very few authors have found scouring in hyostrongylosis, although Dodd (1960) does mention it as a symptom of infection in very young animals. Shanks (1965) considers the occurrence of this symptom to be due to some other agent. Nielsen (1966) failed to demonstrate a gastro-intestinal protein loss in sows with a history of H. rubidus infection although he has shown this to occur in cattle with ostertagiasis and other diarrhoeic conditions. Larsen (1967) suggests that the emaciation seen in adult swine with H. rubidus infection under field conditions is mainly due to a deficient gastric digestion though he presented no evidence to show that this was so. Although the post-mortem results recorded in the infections noted in this study (Lean, 1969) indicate periods of intense worm activity in the early period of infection, the data in liveweight gain, nitrogen retention and apparent digestibility have failed to show a significant difference between animal groups or individuals on each trial. The results graphed for the infection period of Trial 4 are typical of those for any of the levels of infection so far considered.
BRITISH VETERINARY JOURNAL, 128, 3
The values showed no significant deviation from those found in uninfected controls. None of these values were in any way altered when animals received larval doses of 20,000 or up to 150,000. This suggests that neither the larvae nor the adult worms were in sufficiently great numbers in the stomach mucosa, nor present for a sufficiently long time to produce a level of irritation which would disturb gastric activity to an extent which affected the digestive system of the host. It may be postulated that a ration normally given to the growing animal contains an excess of protein such that any effect of the parasite would be masked, whereas a lower crude protein level might increase the effect and duration of H. rubidus infection, since competition for available material would be greater and the animal would be less capable of withstanding the demands of the parasite. Trial 4 animals were therefore fed a diet containing 13 per cent crude protein. The protein level in this diet, although not particularly low, was below that normally fed to growing pigs. Although these animals had a reduced weight gain when compared to other trial groups, there was no significant differences between the infected and non-infected animals on the diet. Animals from each trial were usually slaughtered together, although those in Trial 3 were slaughtered over a three-week period which was a reflection of the differing starting weights of the animals. Crocker & Biester (1920), Castle (1932) and Clay (1938) all considered that H. rubidus was only of importance on those occasions when the vitality of the host animal was lowered as a result of other disease processes or stresses. In the present study, although the animals were exposed to considerably higher infections than they would normally meet on any single occasion in the field, they were not, so far as we are aware, subjected to any stress factors other than those caused by experimentation. There is reason to believe from reinfection experiments not recorded here that the intake of small numbers of larvae over a longer period produces no measureable effect upon the stomach. Nutrition was adequate, and housing conditions good. Animals were not exposed to draughts or adverse temperature conditions. It is consequently suggested that the worms did not reach a position of dominance in the host parasite relationship, and were unable to produce changes of a demonstrable magnitude in our experiments. It is pointed out that the statistical validity of such experiments as those recorded here would be improved by the use of more animals, when marginal differences between infected and non-infected animals might be detectable at the infection levels used. However, such high rates of infection rarely appear to occur naturally in the United Kingdom, and it seems reason able to conclude that H. rubidus is not likely to be commercially important in growing animals under good husbandry conditions and in the absence of other disease-causing agents. ACKNOWLEDGEMENTS
This work was supported by a grant from the Meat and Livestock Commission to whom we offer our thanks.
H. RUBIDUS INFECTION IN THE PIG
145
I. J. Lean was a Sir William Roberts scholar and J. B. Castelino a Commonwealth Scholar. REFER ENCES
CAsTELINO, J . B., HERBERT,!. V. & LEAN, I.J. (1970). R es. Vet. Sci. U,7. CAsTLE, A. (1932) . Vet. J. 88, 474. CLAY, A. L. (1938). Aust. vet. J. 14. 194. CONNAN, R. M. (1967). Vet. Rec. 80, 424. CROCKER, W. T. & BIESTER, H. E. (1920). J. Aust. vet. med. Ass. 57, 427. DAVENPORT, P. G. (1967). Vet. Rec. 81, 390. DODD, D. C. (1960) . N.;:;. vet. J. 8, 100. DREZENIC, 1. (1964). Veter. Arhiv. 34. 283. GERBER, H. C . (1968) . Thesis, Free University, Berlin. HASSALL, A. & STILES, C. W. (1892) . J. camp. Med. vet. Arch. 13,207. JACOBS, D. E. (1967) . Vet. Rec. 81,198. KENDALL, S. B., THURLEY, D. C. & PEIRCE, M. A. (1969). J. camp. Path. 79, 87. LARSEN, S. (1967). Acta vet. scand. 8, 347. LEAN,!. J. (1969). Ph.D. Thesis, University of Wales, Bangor. LEAN, I.J., HERBERT,!. V. & CASTEUNO,J. B. (1972). Br. vet. ]. 128,000. MAIurnAM, R. (1942). Biochem. J. 36, 790. NIELSEN, K. (1966). Acta vet. scand. 7, 321. PORTER, D. A. (1940). Proc. helminth. Soc. Wash. 7, 20. REINICKE, R. K. (1967). Onderstepoort J. vet. Res. 34. 547. SHANKS, P. L. (1965). N;:;. vet.], 13,38. SIRIWARDENE, J. A. DA S. (1966). J. Sci. Fd Agric. 17,456. SKR]ABIN, K . I. & BEKENSKY, P. W. (1925). B erl. tieriir;;tl. Wschr. 41, 52. (Receivedfor publication 20 October 1971)
Etudes sur la pathogenicite de I'infection per Hyostrongylus rubidus (neDlatode). Effets des tall" d'infection allant jusqu':ii 150.000 larves en phase infectieuse chez Ie cochon en voie de croissance I. Etudes de nutrition (Lean et al.) Resulne. On a infecte experimentalement avec des larves infectieuses de H . rubidus des cochons en voie de croissance prives de colostrum, sans pathogene specifique (S.P.F.) et eleves conventionellement avec Ie minimum de maladies. L es doses d'infection s'etalient de 3.000 11 150.000 larves administrees en une seule dose; des animaux infectes auparavant ont egalement ete reinfectes. On a maintenu les animaux dans des cages metaboliques avec des regimes contenant 16 pour cent et 13 pour cent de proteine brute et on a enregistre les indices suivants; augmentation du poids du foie, retention d'azote, efficacites de conversion de la nourriture et digestibilite apparente. Les analyses statistiques de ces donnees ne montrent aucune difference consistante entre les animaux infectes et les temoins au cours d e chaque essai. Studien iiber die Pathogenese der InIektion Dlit Hyostrongylus ruhidus (NeIDatoda) Der Effekt von Infektionen Dlit bis zu 150000 Larven iDl infektiosen StadiUln auf heranwachsende Schweine I. Erniihrungsstudien (Lean et al.) ZusaD1Dleruassung. Schweine, die kein Colostrum (S.P.F.) erhalten hat ten und in der ublichen Weise als "minimal disease" Schweine aufgezogen worden waren, wurden experimentell mit H. rubUlus infektioser Larven infiziert. Die Dosis erstreckte sich von 3000 bis zu 150000 Larven, die als Einzeldosis gegeben wurden. Auch vorher infizierte Tiere wurden erneut infiziert. Sie wurden in StoffWechselkafigen mit einer Diat, die 16 Prozent und 13 Prozent Protein enthielt, gehalten, und die folgenden Daten wurden registriert : Gewichts-
BRITISH VETERINARY JOURNAL, 128, ::5 anstieg, Stickstoffretention, Leistungsfahigkeit hinsichtlich der Futterverwertung und augenscheinliche Verdaulichkeit der Nahrung. Estudios sobre la patogenesis de infecci6n con Hyostrongylus rubidus (nelDatodes). Efectos de los niveles de infecci6n de hasta 150.000 larvas en fase infectiva sobre el cerdo en crecimiento I. Estudios nutritivos
(Lean et al.) Res1UDen. Cerdos en crecimiento privados de calostrum, libres de pat6genos especificos y criados con metodos convencionales fueron infectados experimentalmente con larvas infecciosas de H. Tubidus. Las dosis de infecci6n oscilaron entre 3000 y 150.000 larvas adrninistradas en una sola dosis; los animales infectados previamente tam bien fueron reinfectados. Los animales se mantuvieron cajas de metabolismo en dietas que contenian 16 por ciento y 13 por ciento de proteinas crudas, registnindose a continuaci6n los siguientes indices; ganancia en peso en vivo, retenci6n de nitr6geno, eficiencias de conversi6n de los alimentos y digestibilidad aparente. Los analisis estadisticos de estos datos no muestran diferencias importantes entre los ani males infectados y los con troles en cad a una de las pruebas.
ANNOUNCEMENT In October, 1971, a Society of Pharmacological and Environmental Pathologists was incorporated in the state of New Jersey, U.S.A. The objectives of this society are to foster the discipline of pharmacological and environmental p athology, to contribute to the development of criteria and standards for the evaluation of pathological changes induced by drugs, chemicals, or environmental agents, to promote the training of pharmacological and environmental pathologists, and to establish a registry of naturally occurring and induced diseases in laboratory animals. For additional information and application forms for membership please contact the Secretary-Treasurer (P.O. Box 276, Florham Park, New Jersey, 07932, U.S.A.).