Observations on the migration and development of Toxocara vitulorum in natural and experimental hosts

International Journal for Parasitology, 1971, VoL 1, pp. 85-99. Pergamon Press. Printed in Great Britain

OBSERVATIONS ON THE MIGRATION AND DEVELOPMENT OF TOXOCARA VITULORUM IN NATURAL AND EXPERIMENTAL HOSTS E. G. W A R R E N Department of Parasitology, University of Queensland, St. Lucia, Brisbane, Queensland 4067, Australia (Received 13 July 1970) Abstract

WARRENE. G., 1970. Observations on the migration and development of Toxocara vitulorum in natural and experimental hosts. International Jot~nalfor Parasitology, 1: 85-99. Experimental infection of pregnant cows with eggs of T. vitulorum resulted in mature infections. Larvae possessing an oesophageal ventriculus were found in the milk of infected cows; these were considered to be 3rd-stage larvae of T. vitulorum. Some foster calves drinking milk from experimentally infected cows acquired T. vitulorum infections. All calves withdrawn from infected cows within 24 h of birth failed to develop T. vitulorum infections. The migration and fate of T. vitulorum in an experimental host (the mouse) were studied. Measurements and figures are given of lst-stage and 2nd-stage larvae from the egg, 2nd-stage larvae from the mouse, 3rd-stage larvae from bovine milk and 3rd-, 4th- and adult stages from the small intestine contents of calves. The writer concludes that T. vitulorum larvae undertake a transmarrmaarymigration in cattle and that calves acquire infection from their mothers after birth. The writer also concludes that T. vitulorum larvae undertake a somatic migration in mice. INDEX KEY WORDS: Toxocara vitulorum; transmammary; migration; cattle; mouse; development. INTRODUCTION THE TAXONOMYof Toxocara (syn. Neoascaris) vitulorum has recently been revised (Warren, 1970a). This large nematode is found in the small intestine contents of calves of various Bos and Bubalus spp. (Baylis, 1936) and has also been recorded from goats (Shanmugalingam, 1956) and sheep (Matoff & Wassileff, 1959). The life-cycle of T. vitulorum in cattle was assumed by Mayhew (I953), Morgan & Hawkins (1953) and others to be similar to that of Ascaris lumbricoides. Griitiths (1922) and Boulenger (1922) both recorded findings of mature T. vitulorum in calves about 2-weeksold and both authors suggested that either the prepatent period for T. vitulorum was very much shorter than that for Ascaris lumbricoides, which they both took to be approximately 2~ months, or that prenatal infection must have taken place. Prenatal infection was also suggested by Macfie (1922), Schwartz (1922), Sprent (1946), Vaidyanathan (1949) and Keith (1951). Herlich & Porter (1953) first demonstrated the development of a mature infection of T. vitulorum in calves following the experimental oral infection of the pregnant cow. Similar experimental infections were successfully carried out on pregnant cows by Srivastava & Mehra (1955), Matov & Vasilev (1958) and Cvetkovi6 & Neveni6 (1960), on pregnant sheep by Matoff & Wassileff (1959) and on a pregnant goat by Vassilev (1959). In all these investigations it was concluded that the foetus acquired the infection from its dam in utero. An indication of natural postnatal infection and development to adults was given by Refuerzo & Albis-Jiminez (1954), who found a light infection in adult cattle in the Philippines. Davtian (cited by Mozgovoi, 1953) produced a patent infection by feeding eggs to a calf within 2 h of birth. Experimental oral infection of adult cattle and calves (Herlich & Porter, 1953; Refuerzo & Albis-Jiminez, 1954; Matov & Vasilev, 1958), goats and kids 85

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(Vassilev, 1959), and one ewe and two lambs (Matov & Vasilev, t958) failed to produce patent infections. Experimental oral infection of guinea pigs with T. vitulorum eggs (Refuerzo, AlbisJiminez & de la Cerna, 1952; Casarosa, 1953 ; and Irfam & Sarwar, 1954) produced invasion of the liver, lungs and possibly kidneys by 2nd-stage larvae. Herlich (1953) fed T. vitulorum eggs to mice and larvae were found in the liver and lungs up to 12 and 21 days after infection respectively. Larvae were also recovered from the kidneys, intestinal contents and intestinal wall until 8 days after infection. No larvae were recovered from the carcass and no larvae were found in any tissue after 3 weeks of infection. No larvae were recovered from the litters of four mice which had been infected during pregnancy. Herlich suggested that T. vitulorum may undertake a tracheal migration in mice and further suggested that infection of the bovine may follow direct ingestion of embryonated T. vitulorum eggs. The present study was undertaken in an attempt to confirm and to further define infection and development in ruminants and the migration and the fate of larvae in an experimental host the mouse. A preliminary report on the work has been previously published (Warren, 1969). M A T E R I A L S AND METHODS Source o f eggs Professor J. F. A. Sprent, who was visiting the London School of Hygiene and Tropical Medicine in 1965, was sent several cultures of T. vitulorum eggs from cattle in Vom, Nigeria. These eggs were held in London to comply with Australian quarantine regulations and were then forwarded to the author. Salt flotation Diagnosis of mature infections of T. vitulorum was made by salt flotation o f fresh faeces using a McMaster slide (Gordon & Whitlock, 1939). Culture of eggs to the infective stage The methods used were identical to those described for the culture of T. mackerrasae eggs (Warren, 1970b). Mice The mice used were all virgin females, approximately 1 month old and from a mixed strain. Mice were infected with eggs suspended in 0'3 ml of physiological saline by using a stomach tube approximately 1.5 cm long. All mice were fed commercial food pellets. Ruminants Pregnant female cows of mixed Guernsey and Jersey breeds, pregnant female Merino sheep and one pregnant female Saanen goat were purchased locally and fed on pasture and lucerne hay. Suspensions of T. vitulorum eggs were fed to all ruminants by means of a drenching gun. All pregnancies were the product of natural mating and no hormone therapy was used during this study. Some cows were used in more than one experiment. The tissues of mice and ruminants were examined for larvae using the technique described by Sprent (1952) unless otherwise stated.

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87

INFECTIONS

Infection of pregnant sheep T w o p r e g n a n t ewes were each fed a p p r o x i m a t e l y 100,000 T. vitulorum eggs 2½ m o n t h s before each gave b i r t h to one lamb. One l a m b was killed at 36 h after birth. T h e liver a n d lungs were c o m m i n u t e d a n d s e d i m e n t e d a n d contents a n d washings f r o m the trachea, oesophagus, s t o m a c h a n d small intestine were e x a m i n e d for larvae. T h e faeces o f the second l a m b were e x a m i n e d for eggs every 2-3 days between 22 a n d 44 d a y s after birth a n d at 45 days after b i r t h the l a m b was killed a n d the small intestine contents examined. Results. N o T. vitulorum eggs, larvae o r a d u l t w o r m s were f o u n d in either l a m b .

Infection of one pregnant goat One p r e g n a n t g o a t was fed a p p r o x i m a t e l y 25,000 T. vitulorum eggs 23 d a y s before giving b i r t h to one male a n d one female kid. A further 25,000 T. vitulorum eggs were fed to the male k i d within 24 h o f birth. F a e c a l e x a m i n a t i o n s were carried o u t on b o t h kids every 2 - 3 d a y s between 3 a n d 30 days after birth. T h e female k i d was a u t o p s i e d at 33 d a y s after b i r t h a n d the male k i d was a u t o p s i e d 36 d a y s after b i r t h ; the s t o m a c h a n d small intestine contents o f b o t h animals were e x a m i n e d for a d u l t T. vitulorurn. Results. N o eggs were f o u n d in the faeces a n d no a d u l t s were f o u n d in the kids at a u t o p s y . TABLE I ~ E X P E R I M E N T A L INFECTION OF PREGNANT COWS AND CALVES WITH

Pregnancy No.

No. of eggs per cow ( × 103)

Time of infection

Egg production prepartum in calves days

Toxocara vitulorum

Autopsy of calf

postpartum

Recovery of adult T. vitulorum at autopsy

postpartum 1"

I00

1 month

27-29

29 days

I adult female (10 adults found in faeces at 27 days

postpartum) 2*

150

3~ months

26--57

3½ months

None

3*

150 + 50

5 months 3 months

28-39

1½ months

None

4*

100

2 days

None

1½ months

None

5*

100

2 days

None

2 months

None

6**

100

4 months

26--29

29 days

5 adult females + 10 adult males

7**

120

4 months

None

48 days

None

8**

120

5 months

None

30 days

None

50

5½ months

32-35

35 days

8 adult females

9****

* First pregnancy and first infection. ** Second pregnancy and second infection. **** Fourth pregnancy and fourth infection.

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Infection o f pregnant cows Experiment 1. On nine occasions pregnant cows were fed T. vitulorum eggs during their first, second or fourth pregnancy under experimental conditions (Table 1). All dams gave birth naturally and their calves were allowed to suckle. Faecal samples f r o m calves were examined for T. vitulorum eggs every 2-3 days f r o m 10 days postpartum. At autopsy the digestive tract contents were examined. Results (Table 1). Five calves first exhibited T. vitulorum eggs in their faeces between 26 and 32 days after birth and 14 adult female and 10 adult male T. vitulorum were recovered f r o m the small intestine contents o f three o f the calves at autopsy. Experiment 2. O n six occasions T. vitulorum eggs were fed to pregnant cows during gestation as shown in Table 2. One calf was removed by Caesarean section, one was aborted dead and four calves were born naturally and allowed to suckle, but were killed between 2 and 11 days after birth (Table 2). Livers and lungs as well as contents and washings f r o m the trachea, oesophagus, stomach and small intestine were examined at autopsy. TABLE 2--EXAMINATION OF FOETAL AND NEWBORN CALVES FOR T. vitulorum

Pregnancy No.

No. of eggs per cow

Time of infection

Autopsy of calf

Recovery of T. vitulorum

( X 10 3)

10"*

100

4 months before Caesarean

2-6 weeks prepartum

None

11'**

20

5 days before abortion

2-6 weeks prepartum

None

12'*

50 q- 200

4~- months prepartum 4 months prepartum

2 days postpartum

None

13'*

120

4 months prepartum

4 days postpartum

None

14"*

120

4 months prepartum

9 days postpartum

1 3rd-stage larva

50

4,} months prepartum

11 days postpartum

None

15'***

** Second pregnancy and second infection. *** Third pregnancy and third infection. **** Fourth pregnancy and fourth infection.

Results (Table 2). One 3rd-stage T. vitulorum moulting to the 4th-stage was f o u n d in the small intestine contents at 9 days postpartum. Measurements o f this larva are given in Table 5. Experiment 3. Five cows were fed T. vitulorum eggs during pregnancy (Table 3). Quantities o f colostrum were collected f r o m 3 cows before they gave birth and, following sedimentation, examined for T. vitulorum. All cows were allowed to give birth naturally. At termination

I

2

3

3

17'**

~18"**

~19"**

t20***

0

0

1"25

0"5

2"5

Volume (1.)

Total output Total output

--

Total output

300 each alternate day

300 each alternate day

Volume (nil)

8

4

13

1

1

No. of larvae

2-9

4-9

4-11

5

18

None

None

None

None

33-39

Egg production in calf days postpartum

7". vitulorum

Period of recovery days postpartum

Colostrum and milk collected up to 30 days postpartum

--

0

0

0

No. of larvae

Colostrum collected prepartum

*** Third pregnancy and third infection with 20,000 T. vitulorum eggs per cow. I" Calves removed at birth and fed on commercial milk.

1

(month)

Infection prepartum

16"**

Pregnancy No.

TABLE 3--EXAMINATION OF COLOSTRUM AND MILK FOR

16

48

54

84

39

Autopsy of calf days postpartum

None

None

None

None

8 adult female + 4 adult male

Recovery of adult T. vitulorum at autopsy

OO ~O

<

7-day-old foster calf

100

10-day-old foster calf

90

4-day-old foster calf

90

3-day-old foster calf

50

2-week-old foster calf

50

4-day-old foster calf

2

3½

3

3½

2~

3

50

(X 10 3)

Time of infection prepartum (months)

N o . eggs per cow

* First pregnancy a n d first infection. **** F o u r t h pregnancy a n d fourth infection.

26*

25****

24****

23****

22*

21'

Pregnancy No.

-0 -0

92 96 95 99

4 --

106

--

102 105

0

101

-

0

91

-

--

94

100

4

Withdrawl hours postpartum

93

Calf No.

-

--

None

--

None

-

None

--

None

28-30

None

--

None

Egg production in calf days after birth by infected dam

TABLE 4~EXPERIMENTAL INFECTION OF FOSTER CALVES WITH Toxocara vitulorum

1 adult None None None None None 35 larvae

40 12 34 16 28 12

None

47 adults

None

30 adults

None

Recovery o f adult T. vitulorum at autopsy

21

33

30

10

15

33

A u t o p s y days after birth by infected dam

i.J.P. VOL.1. 1 9 7 1

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91

of pregnancies 16 and 17 the calves were allowed to suckle normally and in pregnancies 18, 19 and 20 the calves were withdrawn within 24 h of birth and fed on commercial milk only. Samples o f milk were collected following pregnancies 16 and 17 whereas total milk output was collected following pregnancies 18, 19 and 20 up to 30 days after birth. All milk collected was sedimented and examined for larvae of T. vitulorum. Faecal samples from the calves were collected every day from 20 days after birth and examined for eggs. At autopsy the small intestine contents of all calves were examined. Results (Table 3). No T. vitulorum larvae were recovered from colostrum collected before birth. A total of 27 3rd-stage larvae were recovered from all milk collected between 2 and 18 days after birth. One of the two calves left to suckle developed mature T. vitulorum in the small intestine contents but no infection was found in calves withdrawn at birth. Measurements of 3rd-stage larvae from milk are given in Table 5. Experiment 4. Six cows were fed T. vitulorum eggs during pregnancy (Table 4). All six calves were born naturally. Four calves were removed from their dams immediately following birth, two were withdrawn within 4 h of birth. All six calves were rubbed dry, placed in individual pens and fed only on a commercial brand of powdered milk. 'Trespen' longacting penicillin was given intramuscularly to each calf at 2--4 days after birth. Each calf thus withdrawn was immediately replaced with a foster calf, which had been purchased from the same locality as the cows. Each foster calf was kept in an individual pen except while feeding from its foster mother. All calves killed before 20 days after birth, or before 20 days of fosterage, were starved for 36 h before slaughter when they were examined for the presence of T. vitulorum in the liver, lungs, stomach wall and small intestine wall as well as in contents and washings of the trachea, oesophagus, stomach and small intestine. All calves kept alive for more than 20 days after birth, or more than 20 days of fosterage, were examined daily for eggs in their faeces and at autopsy the small intestine contents were examined. Results (Table 4). The six calves withdrawn at birth failed to develop mature infections whereas four of the foster calves were found to possess adult and larval T. vitulorum in the small intestine contents. Measurements of specimens recovered are given in Table 5. TABLE5--MEASUREMENTSIN MMOF LARVALANDADULTT. vitulorum FIXEDIN HOT70 ~ ALCOHOL Site

Autopsy days after birth by infected dam

Milk Foster calf small intestine contents Calf small intestine contents Foster calf small intestine contents Foster calf small intestine contents Foster calf small intestine contents Foster calf small intestine contents Foster calf small intestine contents Foster calf small intestine contents Foster calf small intestine contents Foster calf small intestine contents

* Moulting.

2-18 12 9 12 12 12 15 15 21 30 30

Stage

No. measured Length

3rd 3rd 3rd* 3rd* 4th 4th* adult male adult female adult male adult male oviparous female

11 0.760- 1 . 4 7 0 3 2.850--4-0 1 6"250 11 3.370-5"470 2 5.260-8 . 0 6 0 I 10"030 14 23"0 - 5 9 " 5 0 14 22"30-6 2 " 5 0 1 66.80 23 101"50--154.0 8 71"0- 2 1 7 . 0

Width at level of ventriculus 0.037--0.045 0.060-0.070 0"970 0.060-0.100 0.100-0.1120 0-110 0.250-0.610 0-290-0.730 0.670 0.960-1 "380 0-810-1-670

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Infection of a calf with larvae from milk The twelve 3rd-stage larvae recovered from milk following pregnancies 19 and 20 (Table 3) were fed, immediately following recovery, to a 3-week-old calf purchased in Brisbane. This calf was fed powdered milk only. Faecal samples were examined for eggs each day from 20 days after the first administration of larvae. At 40 days following the first administration of larvae the calf was autopsied and the small intestine contents examined. Results. No eggs were found in the faeces and no T. vitulorum were recovered at autopsy.

Infection of mice Experiment 1. Twenty-four mice were fed 1000 or 2000 T. vitulorum eggs each and were sacrificed between ½ and 98 days after infection. All tissues except for the feet, tail and pelt were examined for larvae. The total faecal outputs of l l mice killed at ½, 1, 2, 4, 7, 11, 14, 18, 20, 26 and 33 days after infection were examined for eggs and larvae. Results. No larvae were found in the thoracic cavity washings, stomach, stomach contents, spleen, diaphragm, heart, trachea, oesophagus or eyes. Forty-seven larvae were recovered from the digestive tract and digestive tract contents between ½ and 2 days after infection and one larva was recovered from the wall of the caecum at 14 days after infection. One larva was found in the peritoneal cavity washings at 22 days after infection and one larva was found in the brain at 52 days after infection. Three larvae were recovered from the genital organs between 28 and 34 days after infection. The recovery of larvae from the intestinal mesentery, kidneys, liver, lungs and carcass is shown in Table 6. All larvae recovered measured 0-328--0-630 m m long and 0.014-0.029 m m wide at the level of the ventriculus. Nine larvae recovered from the liver at 18 days after infection and measuring 0.4054).500 m m long and 0.0174).029 m m wide exhibited signs of moulting. Large numbers of eggs and larvae were recovered from the faeces examined up to 48 h after infection but none were found subsequently. TABLE

6----T. vitulorurn

LARVAE RECOVERED FROM VIRGIN FEMALE MICE AT VARIOUS TIMES AFTER INFECTION WITH

1000-2000 EGGS Days after infection No. of mice examined Intestinal mesentery Kidneys Liver Lungs Carcass

1-7

8-14

15-28

5 38 0 143 11 0

3 0 4 36 24 8

5 1 0 38 7 13

29-98 6 0 0 6 1 21

Experiment 2. Seven mice were each fed 2000 T. vitulorum eggs. Individual mice were killed between 3 and 15 days after infection. At autopsy one half of the liver and one lung were fixed in Zenker solution for histological examination. The remaining lung and half of the liver, after the gall bladder had been dissected out and examined for larvae, were squashed separately between two glass plates and examined microscopically for the presence of larvae and lesions. When the larvae and lesions had been counted the squashed liver and lung tissues were comminuted, sedimented and examined for live larvae. Results. The larvae recovered and the lesions counted are recorded in Table 7. By 7 days after infection all larvae seen in the squashed liver were surrounded by a tissue reaction and by 10 days after infection 70 lesions could be counted in the liver tissue but no recognizable

7

10

15

6

5 6

5

4

1

2 3

4

Autopsy days after infection

Mouse No.

--

?

45

6O 51

61

(Larvae)

Squash

--

70

45

0 2

0

(Lesions)

Half Liver

--

0

2

24 11

27

Comminution (Larvae)

TABLE 7--RESULTS OF EXAMINATION OF TISSUES OF MICE FOR

?

16

5

0 0

0

(Larvae)

Squash

T. oitulorum LARVAE

15

16

0

0 0

0

(Lesions)

Lung

0

21

8

0 2

0

Comminution (Larvae)

o~ .r,

94

E . G . WARREN

l.J.e. VOL. 1. 1971

larvae could be seen. The number of live larvae recovered following comminution fell to zero by 10 days after infection. All the larvae seen in the squashed lung at 10 days after infection were surrounded by a tissue reaction and by 15 days after infection only lesions containing no recognizable larvae were found in the lung tissue. Histological examination of livers and lungs of infected mice revealed that at 3 days after infection the larvae in the liver appeared quite normal and were not surrounded by eosinophilic tissue reactions. From 6 days after infection progressively fewer recognizable larvae were found in the liver although there were persistent eosinophilic lesions with associated lymphocytes and macrophages. Larvae in the lung were surrounded by eosinophilic lesions containing some lymphocytes and peripheral macrophages by 10 days after infection. Fifteen days after infection the lesions in the lungs contained macrophages, lymphocytes and eosinophils but no recognizable larvae remained. No larvae were found in the gall bladder.

i\

mouth

2

papil ae E1

--s~.o~e

is, . . . . ~, ° 1 J-

\~'~ i~:, -'11

':.i :i:

oesophagus

I 0025mm I --nerve ring pore ~,"

nucleus

!

:(~

oesophageaIglands

" :,'"

;"::!?.::: ~ventriculus ~:::: iV][ i

intestin

~ . /~"::~II ~--'"~'1 ] 1"~ :-::,':

phageol. .gl. .a,nd fd

:31

~)

0025 mm

nucl . . . . ....

-. },, '1

~

~ 5 I 0-02:5 mm

~:'i!ii I intestine

t

FIGS. 1-13. Camera lucida drawings of T. vitulorum larvae and adults. FIG. 1. Head of 2ndostage larva 0.352 mm long. FIG. 2. Tail of 2nd-stage larva 0-352 mm long. FXG. 3. Genital rudiment of 2nd-stage larva 0.352 mm long. FIG. 4. Head of 3rd-stage larva 4-00 mm long. FxG. 5. Ventriculus of 3rd-stage larva 4.00 mm long.

I..LP. VOL.

1. 1971

MIGRATION

AND

DEVELOPMENT

OF

Toxocara vitulorum

95

OBSERVATIONS ON DEVELOPMENT The lst-stage larvae of T. vitulorum are fragile and difficult to express from the egg but a single larva was measured and found to be 0.525 mm long and 0.021 mm wide at the level of the ventriculus. Development to the 2nd-stage within the egg takes approximately 50 days at 22°C. Ten 2nd-stage larvae expressed from eggs measured 0"345-0.398 mm long and 0.015-0.023 mm wide at the level of the ventriculus. Camera lucida drawings of the head, tail and genital rudiment of a 2hal-stage larva, expressed from the egg, are shown in Figs. 1, 2 and 3. One 3rd-stage larva recovered from the milk of an infected cow is shown in

ltral rectal II

!

rectum

.-"

:.:~.

.:

,..

ai'lu s

E

{..":i..'.:

tO

"" ::." "

0

OO5

'i,.,..~.;~

mm

~ n u c l e i of • ~' ~ subventreJ ' l .~. ~ d:;~ oesophageat ~

triculua

i

~

aorsat

intestine

FIo. 6. Tail of 3rd-stage larva 4.00 mm long. FIo. 7. Genital rudiment of 3rd-stage larva 3"58mm long. FIG. 8. Tail of 4th-stage larva 8.06 mm long. l:~o. 9. Ventriculus of 4tla-stage larva 8.06 mm long. Fig. 14; an enlargement o f the ventricular region of the same larva is given in Fig. 15. Figures 4-13 show camera lucida drawings of 3rd-, 4th- and adult stages recovered from the small intestine contents o f experimentally infected foster calves. All larval and adult stages described in this study were considered to be Toxocara vitulorum because each specimen possessed an oesophageal ventriculus and because this is

96

E.G. WARREN

LJ.P. VOL.1. 1971

omphid

single papi!la dou ble papilla

undivi uterm

dorsal lip

veginc /

~

oesophagus

intestir 0.1 mm sheath from 4th moult

.;:

• ~: ?-t~:

3:i¢:. ~12-.}:'

!l,

single papillae

OO25mm

FIG. 10. Genital rudiment (1-49 mm long) of 4th-stage larva 10.03 mm long. FIG. 11. Head of 4th-stage larva 8"06mm long. Fro. 12. Head of adult male 66.8 mm long. FI~. 13. Tail of adult male 66.8 mm long. the only Toxocara sp. which has been described as developing to maturity in ruminants. Ascaris lumbricoides, another member of the family Ascaridoidea, is also capable of maturing in cattle (Schwartz, 1925; Dromey, 1953) but may be differentiated from T. vitulorum by the absence of an oesophageal ventriculus. NATURAL INFECTIONS

Incidence of T. vitulorum in Australia Warren & Needham 0969) reviewed published findings of T. vitulorum in New South Wales and reported three new cases. N o infections have been reported from other Australian States. However, in order to determine whether any natural infection occurred in in cattle from the Brisbane area 255 calves 3-13 weeks old from properties and sale yards within 100 km of the city were examined for nematode eggs in the faeces, but no T. vitulorum eggs were found.

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MIGRATION AND DEVELOP~,~NT OF Toxocara vitulorum

97

Recovery of T. vitulorum from milk from Iran

In response to a request from the writer Dr. A. A. Shahlapour sent sediments of milk from two cows known to have given birth to calves which developed mature T. vitulorum infections. These cows (A and B) were the property of Mr. Mohammad of Mossein-Abad village near Tehran, Iran. The milk was collected between I and 7 days after birth during December 1968 and January 1969 and the sediments fixed in 6 ~ formalin for posting to Brisbane where they were examined. Results. One 3rd-stage larva 1.13 mm long was found in the sediment of milk collected from Cow A at 2 days after birth. Two 3rd-stage larvae 1.2 and 1.4 mm long and 0.040 mm and 0-043 mm wide at the level of the ventriculus respectively were found in the sediment of milk collected from Cow B at 5-6 days postpartum. These larvae were identical to those found in milk in experimental T. vitulorum infections. DISCUSSION

No natural infection of cattle with T. vitulorum has ever been found in S.E. Queensland. Therefore it was assumed that all cows and calves purchased in this area for this study were free from natural infection. When pregnant cows were fed T. vitulorum eggs some of their calves developed patent infections but examinations of foetal and new born calves failed to demonstrate any evidence of prenatal infection. A search of milk produced by infected cows did, however, reveal the presence of ascaridoid larvae which possessed an oesophageal ventrieulus and it was found that when foster calves ingested milk from infected cows some calves developed patent T. vitulorum infection. In nine cases calves were withdrawn from infected dams and maintained on commercial milk or powdered milk but none of them developed an infection. This study, therefore, failed to produce any evidence of prenatal infection of calves by T. vitulorum. A possible criticism is that the artificial maintenance of these calves may have been detrimental to the development of any infection acquired in utero. This study demonstrates that T. vitulorum larvae in cattle may utilize milk as a means of transfer from one host to another in both experimental and natural infections. This facility for transmammary migration is also possessed by Uncinaria lucasi in seals (Olsen & Lyons, 1965), Strongyloides ransomi in the pig (Moncol & Batte, 1966), Toxocara canis in the dog (Stone & Girardeau, 1967) and by Ancylostoma canium in the dog (Stone & Girardeau, 1966 and 1968). Limited attempts to produce T. vitulorum infections in lambs and kids were unsuccessful. Infection of calves evidently takes place after birth through suckling and larvae develop to maturity entirely within the digestive tract. The migration and development of larvae in the pregnant cow remain unknown and confirmation is required to determine whether or not there can be migration and development to adults following ingestion of infective ova. Although only a few examples of certain developmental stages of T. vitulorum were recovered during this study it appears that the morphological development of this species is similar to that described for Toxocara cati (Sprent, 1956), Toxocara canis (Sprent, 1958) and Toxocara mackerrasae (Warren, 1970b). This study failed to produce any evidence of tracheal migration by T. vitulorum in mice. Evidence was found of encapsulation of larvae by inflammatory reactions in the liver and lungs where many were killed. However, a small proportion of the larvae was capable of undertaking a somatic migration to the skeletal musculature. Nevertheless it was evident that there was a gradual disappearance of larvae from mouse tissues either by being killed or by taking some other route out of the host. The possibility that some of these larvae were I.P. I/l---O

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l o s t v i a t h e gall b l a d d e r w a s i n v e s t i g a t e d b u t w i t h n e g a t i v e r e s u l t s . I t s e e m s p r o b a b l e , t h e r e f o r e , t h a t m o s t T. vitulorum l a r v a e in m i c e a r e d e s t r o y e d b y t h e t i s s u e r e a c t i o n s o f t h e host.

Acknowledgements--This work was financed by Research Grants from the University of Queensland, the National Health and Medical Research Council of Australia, the Australian Research Grants Committee and the United States Department of Health, Education and Welfare No. AI-07023. I wish to thank Dr. A. A. Shahlapour, Institut D'Etat Razi, B.P. 656, Tehran,/.ran for his most generous help in providing the milk samples from Iran. I also wish to thank Professor J. F. A. Sprent, Head of the Department of Parasitology, University of Queensland for stimulating and facilitating this study and Mr. G. Orr, Mr. R. Leutton and Mr. J. Leonard for their care of the cattle and for giving up their own time to help the writer to withdraw calves at birth. I also wish to thank Dr. G. Carlson, Department of Veterinary Clinical Studies, University of Queensland for conducting the Caesarean section of a foetal calf.

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