Paraaitology (1977), 74, 235-242 With 2 figures in the text
235
The development of Haemonchus contortus, a nematode parasite of the ovine abomasum, in the laboratory rabbit C. J. MAPES and G. J. GALLIE* Department of Zoology, University of Bristol, and Department of Parasitology, Animal Diseases Research Association, Edinburgh (Received 30 November 1976) SUMMARY
The development and growth of Haemonchus contortus was studied in the stomach of the laboratory rabbit. Less than 1 % of the ensheathed infective larvae given to rabbits became established, whereas after exsheathment in vitro under physiological conditions, 20-30% of the larvae administered developed to the 4th stage. Following doses of up to 10000 larvae, nearly all worms were lost from the 16th to the 21st day after infection, but after doses of 50000 larvae considerable numbers of 4th-stage larvae were recovered 25 days after infection. No adult worms were recovered and no eggs were found in the faeces. Infection was accompanied by inflammation, haemorrhage, disruption and flattening of the mucus secreting cells and by mononuclear infiltration. No cellular reactions in the immediate vicinity of the nematodes in the mucosa or changes in the pH of the stomach contents were observed. INTRODUCTION
Haemonchus contortus, a nematode found in the abomasa of ruminant hosts, has been occasionally reported from other groups of mammals, including the rabbit. Wood & Hansen (1960) recovered small numbers of 5th-stage and adult H. contortus from New Zealand White rabbits infected with 100000 exsheathed larvae. As there are no laboratory hosts available for the study of this nematode, the suitability of the rabbit for this purpose was investigated. This paper describes the effect of exsheathment on infectivity and the development of the nematode in the rabbit following infection with different numbers of larvae.
MATERIALS AND METHODS
Eggs of H. contortus were obtained from experimentally-infected sheep at the Moredun Institute and the larvae were grown and harvested using a modified version of the method of Christie & Patterson (1963). Dutch and New Zealand * Present address: Centre for Tropical Veterinary Medicine, Easter Bush, Roslin, Midlothian, Scotland.
236
C. J. MAPES AND G. J. GALLIE
White rabbits were reared under worm-free conditions and infected by mouth in groups designated A-H, at 7-9 weeks of age with either ensheathed or artificially exsheathed 3rd-stage larvae. Exsheathment was carried out in either 0-1 % (w/v) sodium hypochlorite or in half strength mammalian Ringer or in a 1 % (w/v) aqueous solution of sodium bicarbonate gassed for 10 min with 40-100% carbon dioxide with a balance of nitrogen where necessary. In Expts A-E the Dutch rabbits used were killed by stunning and exsanguination, and the contents of the stomach were iemoved. The stomach was then washed in physiological saline and digested in 1 % (w/v) pepsin and 1 % (v/v) HC1 for 2 h at 39 °C. The contents and digest were then preserved in 5 % (v/v) formaldehyde and 10 and 20 % samples were searched for worms except when few worms were found, when 50% was examined. The worms were classified into larval stages and measured. In Expts F-H New Zealand White rabbits were killed by intravenous injection of sodium pentabarbitone and the stomach and the small intestine were immediately removed. The stomach was opened and the pH of its contents measured. Small portions of the wall were removed, fixed in formol saline and subsequently sectioned and stained in Haematoxylin and Eosin. The stomach was then washed and gently rubbed in warm saline. The washings were pooled together with the stomach contents to form the 'contents fraction'. This contained worms from the contents and those lying superficially on the surface of the mucosa. The washed stomach was placed in 3 % HC1 for 1 h at 39 °C. The mucosa was then scraped off and collected together with the surrounding fluid to form the 'mucosal fraction'. This contained worms closely associated with the mucosa, in the gastric pits and glands. The small intestine was treated in a similar way. All fractions were searched for worms, which were measured and differentiated by sex and developmental stage. Fourth-stage larvae were characterized by the presence of a functional buccal capsule (Mapes, 1969) and 5th-stage larvae by the presence of a buccal tooth and a developed reproductive system. In Expts F and G, male and female 4th-stage larvae were divided into 6 arbitrary phases according to the development of the reproductive structures. These were similar to those described by Mapes & Coop (1972) for Nematodirus battus except that 6 rather than 7 phases of female development were used. Rabbit faeces were examined for nematode eggs by the McMaster method from the 15th day of infection. Four uninfected New Zealand White rabbits were killed using the same method as in Expts F-H. The pH of the stomach contents was measured and mucosal samples were taken for histological examination. RESULTS
Small numbers of 4th-stage H. contortus larvae were recovered 7 days after infection with 20000 ensheathed infective larvae (Table 1). No exsheathed 3rdstage- or 4th-stage larvae were found in rabbits which received 10000 ensheathed
H. contortus in the laboratory rabbit
237
Table 1. Numbers (mean ±S.D.) of Haemonchus contortus in rabbits 7 days after infection with 20 000 ensheathed or artificially exsheathed infective larvae Status of infective larvae Exsheathed Worm population Expt. A 3rd- and 4th-stage larvae 4th-stage larvae
Ensheathed Group 1*
Ringer+ CO2 Group 2
Hypochlorite Group 3
92 + 77 92 ±77
5067 ± 2921 4822 ± 2783
543 ± 493 478+431
6 rabbits in each group.
Table 2. Haemonchus contortus recovered from rabbits up to 25 days after infection (The results from Expts F and G are pooled and given together with the standard deviation.) No. of larvae adMean no. Age % ministered of infection of at the L4 (days) Experiment Exsheathed worms stage B(2)* B(2) B(l) B(l) B(l) C(l) C(l) C(l) C(l) D(4) E(4)
F & G (6) F & G (5) F & G (8) G(2) H(2) H(2)
50000 50000 50000 50000 50000 1000 1000 1000 1000 5000 100
10000 10000 10000 10000 Ensheathed 10000 10000
5 7 14 21 25 5 7 9 17 16-17 16-17
14825 8635 7500 3530 1775 196 108 178 0 5 0
3 7 14 21
1967 ±836 470 + 527 188 ±238 0
3 7
200 3
100 100 96-5 99-9 100 100 100 96-6t — 100
100 100 54 + 30 — — —
* Number of rabbits in parentheses. •f 29 % of the worm population in the 4th moult.
of all worms in mucosal fraction 1O0 90+20 43 ± 1 9 — — —
238
C. J. MAPES AND G. J. GALLIE
Table 3. The development of Haemonchus contortus in rabbits through the Uh-larval stage following infections with 10000 exsheathed larvae % of the 4th stage in developmental phases Early
larvae
1
2
3
4
5
3
•to
7
S 9 (J
14
•to
Late
Sex of
84 60 11 10 2 2
16 40 30 38 11 36
— 40 20 9 8
— 6 32 22 17
— 4 — 20 25
Day after infection
6 — — 9 — 36 12
Table 4. The mean lengths and range of lengths of Haemonchus contortus recovered from rabbits No.
of larvae administered 1000
Days after infection 5 7 7 9 9
10000
50000
9 9 3 3 7 7 14 14 14 14 5 7 14 14
Mean lengths
235
The development of Haemonchus contortus, a nematode parasite of the ovine abomasum, in the laboratory rabbit C. J. MAPES and G. J. GALLIE* Department of Zoology, University of Bristol, and Department of Parasitology, Animal Diseases Research Association, Edinburgh (Received 30 November 1976) SUMMARY
The development and growth of Haemonchus contortus was studied in the stomach of the laboratory rabbit. Less than 1 % of the ensheathed infective larvae given to rabbits became established, whereas after exsheathment in vitro under physiological conditions, 20-30% of the larvae administered developed to the 4th stage. Following doses of up to 10000 larvae, nearly all worms were lost from the 16th to the 21st day after infection, but after doses of 50000 larvae considerable numbers of 4th-stage larvae were recovered 25 days after infection. No adult worms were recovered and no eggs were found in the faeces. Infection was accompanied by inflammation, haemorrhage, disruption and flattening of the mucus secreting cells and by mononuclear infiltration. No cellular reactions in the immediate vicinity of the nematodes in the mucosa or changes in the pH of the stomach contents were observed. INTRODUCTION
Haemonchus contortus, a nematode found in the abomasa of ruminant hosts, has been occasionally reported from other groups of mammals, including the rabbit. Wood & Hansen (1960) recovered small numbers of 5th-stage and adult H. contortus from New Zealand White rabbits infected with 100000 exsheathed larvae. As there are no laboratory hosts available for the study of this nematode, the suitability of the rabbit for this purpose was investigated. This paper describes the effect of exsheathment on infectivity and the development of the nematode in the rabbit following infection with different numbers of larvae.
MATERIALS AND METHODS
Eggs of H. contortus were obtained from experimentally-infected sheep at the Moredun Institute and the larvae were grown and harvested using a modified version of the method of Christie & Patterson (1963). Dutch and New Zealand * Present address: Centre for Tropical Veterinary Medicine, Easter Bush, Roslin, Midlothian, Scotland.
236
C. J. MAPES AND G. J. GALLIE
White rabbits were reared under worm-free conditions and infected by mouth in groups designated A-H, at 7-9 weeks of age with either ensheathed or artificially exsheathed 3rd-stage larvae. Exsheathment was carried out in either 0-1 % (w/v) sodium hypochlorite or in half strength mammalian Ringer or in a 1 % (w/v) aqueous solution of sodium bicarbonate gassed for 10 min with 40-100% carbon dioxide with a balance of nitrogen where necessary. In Expts A-E the Dutch rabbits used were killed by stunning and exsanguination, and the contents of the stomach were iemoved. The stomach was then washed in physiological saline and digested in 1 % (w/v) pepsin and 1 % (v/v) HC1 for 2 h at 39 °C. The contents and digest were then preserved in 5 % (v/v) formaldehyde and 10 and 20 % samples were searched for worms except when few worms were found, when 50% was examined. The worms were classified into larval stages and measured. In Expts F-H New Zealand White rabbits were killed by intravenous injection of sodium pentabarbitone and the stomach and the small intestine were immediately removed. The stomach was opened and the pH of its contents measured. Small portions of the wall were removed, fixed in formol saline and subsequently sectioned and stained in Haematoxylin and Eosin. The stomach was then washed and gently rubbed in warm saline. The washings were pooled together with the stomach contents to form the 'contents fraction'. This contained worms from the contents and those lying superficially on the surface of the mucosa. The washed stomach was placed in 3 % HC1 for 1 h at 39 °C. The mucosa was then scraped off and collected together with the surrounding fluid to form the 'mucosal fraction'. This contained worms closely associated with the mucosa, in the gastric pits and glands. The small intestine was treated in a similar way. All fractions were searched for worms, which were measured and differentiated by sex and developmental stage. Fourth-stage larvae were characterized by the presence of a functional buccal capsule (Mapes, 1969) and 5th-stage larvae by the presence of a buccal tooth and a developed reproductive system. In Expts F and G, male and female 4th-stage larvae were divided into 6 arbitrary phases according to the development of the reproductive structures. These were similar to those described by Mapes & Coop (1972) for Nematodirus battus except that 6 rather than 7 phases of female development were used. Rabbit faeces were examined for nematode eggs by the McMaster method from the 15th day of infection. Four uninfected New Zealand White rabbits were killed using the same method as in Expts F-H. The pH of the stomach contents was measured and mucosal samples were taken for histological examination. RESULTS
Small numbers of 4th-stage H. contortus larvae were recovered 7 days after infection with 20000 ensheathed infective larvae (Table 1). No exsheathed 3rdstage- or 4th-stage larvae were found in rabbits which received 10000 ensheathed
H. contortus in the laboratory rabbit
237
Table 1. Numbers (mean ±S.D.) of Haemonchus contortus in rabbits 7 days after infection with 20 000 ensheathed or artificially exsheathed infective larvae Status of infective larvae Exsheathed Worm population Expt. A 3rd- and 4th-stage larvae 4th-stage larvae
Ensheathed Group 1*
Ringer+ CO2 Group 2
Hypochlorite Group 3
92 + 77 92 ±77
5067 ± 2921 4822 ± 2783
543 ± 493 478+431
6 rabbits in each group.
Table 2. Haemonchus contortus recovered from rabbits up to 25 days after infection (The results from Expts F and G are pooled and given together with the standard deviation.) No. of larvae adMean no. Age % ministered of infection of at the L4 (days) Experiment Exsheathed worms stage B(2)* B(2) B(l) B(l) B(l) C(l) C(l) C(l) C(l) D(4) E(4)
F & G (6) F & G (5) F & G (8) G(2) H(2) H(2)
50000 50000 50000 50000 50000 1000 1000 1000 1000 5000 100
10000 10000 10000 10000 Ensheathed 10000 10000
5 7 14 21 25 5 7 9 17 16-17 16-17
14825 8635 7500 3530 1775 196 108 178 0 5 0
3 7 14 21
1967 ±836 470 + 527 188 ±238 0
3 7
200 3
100 100 96-5 99-9 100 100 100 96-6t — 100
100 100 54 + 30 — — —
* Number of rabbits in parentheses. •f 29 % of the worm population in the 4th moult.
of all worms in mucosal fraction 1O0 90+20 43 ± 1 9 — — —
238
C. J. MAPES AND G. J. GALLIE
Table 3. The development of Haemonchus contortus in rabbits through the Uh-larval stage following infections with 10000 exsheathed larvae % of the 4th stage in developmental phases Early
larvae
1
2
3
4
5
3
•to
7
S 9 (J
14
•to
Late
Sex of
84 60 11 10 2 2
16 40 30 38 11 36
— 40 20 9 8
— 6 32 22 17
— 4 — 20 25
Day after infection
6 — — 9 — 36 12
Table 4. The mean lengths and range of lengths of Haemonchus contortus recovered from rabbits No.
of larvae administered 1000
Days after infection 5 7 7 9 9
10000
50000
9 9 3 3 7 7 14 14 14 14 5 7 14 14
Mean lengths
