Neospora caninum in cattle: Experimental infection with oocysts can result in exogenous transplacental infection, but not endogenous transplacental infection in the subsequent pregnancy

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International Journal for Parasitology 37 (2007) 1631–1639 www.elsevier.com/locate/ijpara

Neospora caninum in cattle: Experimental infection with oocysts can result in exogenous transplacental infection, but not endogenous transplacental infection in the subsequent pregnancy C.M. McCann a, M.M. McAllister b, L.F.P. Gondim b, R.F. Smith c, P.J. Cripps c, A. Kipar d, D.J.L. Williams a, A.J. Trees a,* a

c

Veterinary Parasitology, Liverpool School of Tropical Medicine and Faculty of Veterinary Science, University of Liverpool, Pembroke Place, Liverpool L3 5QA, UK b Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL, USA Department of Veterinary Clinical Science and Animal Husbandry, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 3BX, UK d Department of Veterinary Pathology, Faculty of Veterinary Science, University of Liverpool, Crown Street, Liverpool L69 7ZJ, UK Received 27 March 2007; received in revised form 29 May 2007; accepted 31 May 2007

Abstract Whilst it is presumed that infection of pregnant cattle with Neospora caninum oocysts can provoke abortion and is the likely cause of epidemic abortion outbreaks, only two previous experiments have involved inoculation of pregnant cows with oocysts (and only one abortion was provoked in 22 pregnancies). Here, we describe the oral oocyst challenge of 18 cows synchronously bred and inoculated precisely at 70 (n = 6), 120 (n = 6) and 210 (n = 6) days in pregnancy with a nominal dose of 40,000 oocysts. Only one abortion occurred (at the 120 days challenge) which could be definitively ascribed to N. caninum and no transplacental infection (TPI) was detected in any of the other 11 calves born in the 70 and 120 day challenge groups. In contrast, 4/5 live calves born to cattle challenged at 210 days were transplacentally infected. When cows which had transplacentally infected their calves in the first pregnancy were rebred, no TPI occurred. The results show that the timing of challenge influences clinical and parasitological outcomes and that cattle in late pregnancy are exquisitely sensitive to oocyst challenge leading to exogenous TPI and congenitally infected calves. However, cattle which were indisputably systemically infected in their first pregnancy did not induce endogenous TPI in their subsequent pregnancy. This confirms previous results with experimental tachyzoite challenge and suggests that post-natal infection does not lead to persisting infections which can recrudesce in pregnancy.  2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Neospora caninum; Transplacental infection; Oocysts; Cattle

1. Introduction The intracellular protozoan parasite Neospora caninum was first recognised in 1984 in dogs and in 1989 in cattle and is an important cause of bovine abortion worldwide (Dubey, 2003). Dogs and coyotes are definitive hosts and have been shown to shed unsporulated N. caninum oocysts *

Corresponding author. Tel.: +44 0151 705 3235; fax: +44 0151 705 3373. E-mail address: [email protected] (A.J. Trees).

following the ingestion of infective tissue from intermediate hosts (McAllister et al., 1998; Lindsay et al., 1999; Schares et al., 2001; Dijkstra et al., 2001a; Gondim et al., 2004b). Transplacental infection (TPI) is a major route of transmission of N. caninum in cattle and may result in foetopathy or the birth of congenitally infected calves. Recently, two types of TPI have been defined, namely endogenous and exogenous TPI (Trees and Williams, 2005). Endogenous TPI refers to foetal infection occurring as a result of the recrudescence of a pre-existing persistent maternal infection during pregnancy; exogenous TPI occurs as a result

0020-7519/$30.00  2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.05.012

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of a de novo infection of a pregnant dam and has been shown experimentally following the administration of N. caninum tachyzoites (Barr et al., 1994; Williams et al., 2000; Innes et al., 2001) and N. caninum oocysts (Gondim et al., 2004a). Natural shedding of N. caninum oocysts by dogs has been described but the prevalence and intensity is low (Basso et al., 2001; McGarry et al., 2003; Schares et al., 2005). Nevertheless, oocysts are the most likely parasite stage to provide the point source of infection which has been associated with epidemic abortions (McAllister et al., 1996, 2000; Dijkstra et al., 2001b, 2002; Crawshaw and Brocklehurst, 2003). However, whilst there is epidemiological evidence that infection with oocysts during pregnancy may result in abortion, the experimental data are more circumspect – to date, in 22 pregnant cows inoculated with oocysts, there has only been a single N. caninum confirmed abortion (Trees et al., 2002; Gondim et al., 2004a). Whilst the ability of N. caninum oocysts to provoke abortion by exogenous TPI requires further investigation, another significant question is whether post-natal infections by oocysts can establish persistency and result in endogenous TPI in subsequent pregnancies. Cattle infected with tachyzoites before pregnancy do not infect their foetuses in a subsequent pregnancy (Williams et al., 2000; Innes et al., 2001), but none of the cattle that have been experimentally inoculated with oocysts during pregnancy have been rebred to determine if a persistent infection was established that could recrudesce during a subsequent pregnancy. Thus this study had two aims: firstly, using precisely timed challenges in synchronously bred cattle to determine if the stage of pregnancy at oocyst challenge affected the parasitological and clinical outcomes; and secondly, to determine if cattle infected with oocysts in one pregnancy in which TPI or abortion had occurred could infect their foetus in a subsequent pregnancy. 2. Materials and methods Infections with N. caninum were carried out in cattle and gerbils in accordance with the Animals (Scientific Procedures) Act, 1986, under licence from the UK Home Office. 2.1. Neospora caninum oocyst production Neospora caninum oocysts were produced at the University of Illinois. A 12–16 weeks old mixed breed female hound pup was fed approximately 3 kg of mixed tissue (brain, spinal cord, tongue, skeletal muscle and kidney) from four calves that have been inoculated i.v. with tachyzoites of the Nc-Liverpool strain (Barber et al., 1995) of N. caninum in March 2003. Oocyst shedding in the pup was observed 13 days later. All the faeces shed during the period of maximum oocyst shedding (15–23 days after the puppy consumed the calf tissue) were collected and oocyst purification was performed as described previously (Gondim et al., 2002). Sporulation of the oocysts was

achieved by suspending them in 2% sulphuric acid and shaking them at room temperature for 6 days, in the dark. After this time 792,000 (71% of detectable oocysts) were sporulated. The oocysts were suspended in 2 L of 2% sulphuric acid, stored in four 500 ml containers and kept refrigerated at 4 C prior to being shipped to Liverpool in October 2003 under refrigerated conditions. The species identity of the oocysts was confirmed as N. caninum by PCR (Gondim et al., 2002). 2.2. Oocyst infection of cattle Twenty-one Friesian–Holstein heifers were purchased from farms in the North-West of England and housed at the University of Liverpool’s Large Animal Experimental Facility in dog- and fox-proof accommodation. Serological tests for evidence of infection with the common abortifacient agents (Bovine Viral Diarrhoea virus, Infectious Bovine Rhinotracheitis virus, N. caninum and Leptospira hardjo) were performed as described previously with negative results (Williams et al., 2000). The cattle were loosehoused on straw bedding and fed straw and concentrates with a mineral block available throughout the experiment. Oestrous was synchronised by means of a progesterone releasing device (PRID, Ceva Animal Health Ltd., Chesham, Buckinghamshire, England) and the heifers were artificially inseminated (AI). Pregnancy was confirmed by transrectal ultrasonography 35 days later. The oocysts were stored at 4 C upon arrival in Liverpool. The four batches of oocysts were mixed and all doses drawn from a single pool. Nominal doses of 40,000 oocysts, based on the estimated concentration of 396 oocysts/ml, were prepared shortly before they were administered to the heifers. The appropriate volume (approximately 100 ml) of oocyst suspension in 2% sulphuric acid was reduced to 20 ml by centrifugation at 1,000g for 20 min, and made up to 1 L with water. The heifers were dosed orally with the 1 L of oocyst suspension and immediately afterwards were given a further 1 L of water. Eighteen animals were challenged with the oocysts between January and April 2004 in three groups of six animals. Group 1 was challenged on day 70 of pregnancy, Group 2 on day 120 and Group 3 on day 210. At the time when each group of animals was challenged, one control animal was dosed with 1 L of water containing 20 ml of 2% sulphuric acid. For 48 h after dosing, the infected animals were isolated from the control animal and kept on sawdust which was later collected and incinerated. The control animal was then kept with the challenged cattle and acted as a sentinel for adventitious infection. Foetal viability was assessed three times a week by transrectal ultrasonography for the first month post-challenge and thereafter weekly. If a foetus was found to be non-viable as indicated by the absence of a heartbeat, it was checked again after 24 h to confirm foetal death. One heifer in Group 2 was found to have a non-viable foetus 33 days after the challenge and was injected with a progesterone receptor antagonist,

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aglepristone (Alizin, Virbac Ltd. England) to induce expulsion of the foetus. Pre-colostral blood samples were collected from all calves after birth; blood was collected for serum at post mortem from one calf that was stillborn.

each titration group as shown by seroconversion. It was assumed that one or more viable oocysts would always cause infection and that no oocysts were lost in washing the inoculum dose.

2.3. Immunological responses in cattle

2.5. Necropsy of aborted foetuses and neonatal calves

Blood samples were collected for serum 1 week prior to oocyst challenge and thereafter weekly until calving, and tested for N. caninum-specific antibody using a commercial ELISA (Mastazyme, Mast Diagnostics, Bootle,UK) as previously described (Williams et al., 1999). The results were calculated as the O.D. expressed as a percentage of a high positive control (percent positivity; PP) (Williams et al., 1999). Pre-colostral serum samples from the calves were also tested using this ELISA. A cut off of PP P20 was used to classify cows and calves as seropositive. From the day of oocyst challenge and weekly post-challenge (pc), blood samples were collected into heparinised vacutainers for the isolation of peripheral blood mononuclear cells (PBMC) which were examined in vitro for antigen-specific proliferation and IFN-c secretion as described (Williams et al., 2000). Animals were considered to have positive proliferation and IFN-c responses if the values were greater than the respective means of the weekly values for 8 and 7 weeks pc of the sentinel controls + 2 SD. These were for PBMC proliferation, expressed as stimulation index (SI), 3.9 and for IFN-c 2.3 ng/ml.

Calves were euthanased within 14 days of birth by i.v. injection of 20% (w/v) pentobarbitone sodium (Euthatal, Merial Animal Health, UK). Samples of brain, heart, kidney, lung, skeletal muscle (from front and hind limbs – deltoideus and quadriceps femoris, respectively) and, when available, placental tissue, were taken from each aborted foetus and calf and stored at 20 C for analysis by PCR or fixed in 10% neutral buffered formalin (NBF). For each calf and foetus, six 50 mg samples from different regions of the brain were pooled and homogenised in liquid nitrogen. DNA was prepared using a DNeasy kit (Qiagen, Crawley, UK). DNA preparations were also made from 50 mg samples of heart tissue from each calf and foetus and from other tissues of aborted foetuses. Replicate PCRs were performed on DNA from the tissues as described above. Pericardial fluid from aborted foetuses was examined by IFAT as for gerbil sera but using anti-bovine IgG FITC as conjugate.

2.4. Gerbil bioassay and estimation of the dose of viable oocysts Oocyst viability was assessed using a gerbil bioassay in December 2003 after the oocysts arrived in UK and at the time of each challenge. Nominal doses of 100, 101, 102 and 103 oocysts were prepared from the appropriate volumes of the stock oocyst suspension in 2% sulphuric acid and centrifuged at 1,500g for 15 min. The supernatant was removed and the oocysts washed twice in PBS (centrifuged 1,500g, 15 min each time) before each dose was finally prepared in 300 ll PBS and fed to the gerbils by gavage. Two control animals in each group were dosed with PBS alone. Blood samples were taken prior to infection and at weekly intervals from the tail vein and tested for an antibody to N. caninum using an inhibition ELISA (McGarry et al., 2000). Samples that were positive were also tested by an immunofluorescence antibody test (IFAT) as previously described but using an anti-murine IgG fluorescein isothiocyanate (FITC) conjugate (Trees et al., 1993). The gerbils were euthanased after 28–44 days and samples collected at post mortem for PCR. DNA was extracted and purified from brain and heart tissue with a DNeasy kit (Qiagen, Crawley, UK). PCRs for N. caninum-specific sequences were performed as described by Williams et al. (2000) using the nested PCR of Uggla et al. (1998). The proportion of oocysts that was viable was estimated from the proportion of gerbils infected in

2.6. Histopathological examination Three brain sections taken from all neonatal calves, aborted foetuses and the stillborn calf were examined histologically. A first cross-section was taken in the frontal cortex at the level of the caudate nucleus and putamen, a second section at the level of the hippocampus (Gyrus ectosilvius posterior), Colliculus centralis and Substantia grisea centralis of the mesencephalon and a third cross-section through the cerebellum and brain stem at the level of the proximal part of the cerebellar nucleus. Other tissue sections, including skeletal muscle and heart, from the aborted foetuses were also examined. Tissue sections were fixed in 10% NBF and embedded in paraffin wax. Sections (3–5lm) were cut and stained with H&E or used for immunohistochemistry. 2.7. Immunohistochemistry Immunohistochemistry was performed on brain sections of neonatal calves and on sections from all tissues collected from the aborted foetuses and the stillborn calf using a peroxidase anti-peroxidase (PAP) method modified from that previously described (Kipar et al., 1998). Briefly, after deparaffination and re-hydration, endogenous peroxidise was inactivated by incubation with 0.3% hydrogen peroxide in methanol at room temperature for 30 min. Sections were incubated for 15–18 h at 4 C with a polyclonal rabbit anti-N. caninum antibody (Barber et al., 1995) and incubated with swine anti-rabbit IgG (DakoCytomation, UK) and rabbit PAP (DakoCytomation, UK) complex. Sections

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were washed in Tris-buffered saline (TBS; 0.1 M Tris–HCl with 0.9% NaCl, pH 7.2) between each incubation step. Sections were then incubated (10 min with stirring) in 0.05% 3,3 0 -diaminobenzidine tetrahydrochloride (DAB, Fluka, Buchs, Switzerland) with 0.1% hydrogen peroxide in 0.1 M imidazole buffer, pH 7.1. Slides were counterstaind for 30 s in Papanicolaou’s haematoxylin and rinsed in running tap water before de-hydrating, clearing and mounting. As negative controls, consecutive slides were incubated with normal rabbit serum. A fixed, paraffinembedded cell pellet of a tachyzoite-infected Vero cell culture served as a positive control. 2.8. Rebreeding of cows Seven cows were selected for rebreeding based on their immunological results and the outcome of their first pregnancy. Five of the cows selected had had an N. caninuminfected calf or foetus, one cow had had an undiagnosed abortion and one cow had had a persistently high antibody response from 3 weeks pc. Animals were rebred by AI following oestrous synchronisation as before. Three uninfected control animals were co-housed but not rebred. Foetal viability and N. caninum-specific antibody responses were monitored as before. Pre-colostral blood samples were collected from all the calves at birth and tested for N. caninum-specific antibodies as before. Calves were euthanased and subjected to post mortem as described above. Neospora caninum-specific PCRs were performed on DNA extracted from calf brain and heart tissue. The cows were euthanased within 2 weeks of calving, and brains removed for analysis by PCR. For each cow, 40 · 50 mg samples from different areas of the brain were pooled and homogenised. Ten samples of 50 mg were removed from each pool and DNA extraction, purification and PCRs were performed as described above. 3. Results 3.1. Effect of oocyst infection on pregnancy The results are summarised in Table 1. All six heifers in Group 1, challenged with oocysts at 70 days of pregnancy, gave birth to live calves at term. All six heifers showed

evidence of exposure to N. caninum since all had PBMC proliferation responses above threshold within 1 or 2 weeks pc, although the timing and magnitude of these responses varied (Fig. 1). After falling below threshold from 3 to 5 weeks pc in all animals, SIs rose above threshold later in pregnancy in all animals up to or at calving. IFN-c responses were detected in five heifers from 1 or 2 weeks pc with maximum responses at 2 weeks pc (Fig. 2). Four heifers developed a serum antibody response with a PP value of at least 20 (Fig. 3) but in two heifers the PP values remained below 20 PP. In the calves, no evidence of N. caninum infection was found; the pre-colostral serum samples were negative and no parasite DNA was detected by PCR. No evidence of Neospora-associated histological changes were observed in any of the brain sections from the calves and all were negative by immunohistochemistry. In Group 2, inoculated at 120 days, gestation foetal death was detected in one heifer by ultrasound 33 days p.i. and the foetus was expelled 6 days later. One heifer gave birth to a stillborn calf at term and the remaining four animals calved normally (Table 1). Five of the six heifers had a positive antibody response; all six had positive proliferation and IFN-c responses (Figs. 1–3). In the aborted foetus, parasite DNA was detected by PCR in the brain and cotyledons. Histologically, multifocal necrosis with a mild mixed cellular inflammatory infiltration was seen in the skeletal muscle and parasites were detected by immunohistochemistry in skeletal muscle. No histological lesions were detected in brain, lung, kidney, heart or cotyledons and all these tissues were negative in immunohistochemsitry for N. caninum. IFAT on pericardial fluid was negative for N. caninum. In the five calves born at full term, no histological changes were observed and no evidence of N. caninum infection was found by PCR, immunohistochemistry or ELISA in pre-colostral serum samples. In Group 3, inoculated at 210 days of gestation, one heifer aborted 22 days pc; foetal death was not detected prior to the abortion (Table 1). The cause of the abortion was not established and no evidence of N. caninum was found in the foetus by PCR, IFAT on pericardial fluid or immunohistochemistry for N. caninum on brain, heart, liver and skeletal muscle. No histological changes were observed in those same organs. The dam developed SIs and IFN-c responses above threshold values 1 week following

Table 1 Results of oocyst infection in cows at different stages of pregnancy Group

1 2 3

Time in gestation (days)

70 120 210

Cows

Calves

Nc-specific antibodies (PP P20)

IFN-c response (>2.3 ng/ml)

PBMC proliferation (SI > 3.9)

Abortion

4/6 5/6 5/6

5/6 6/6 6/6

6/6 6/6 6/6

0/6 1/6 1/6

Parasite DNA (foetus)

1/1 0/1

Nc, Neospora caninum; PP, percent positivity; PBMC, periferable blood monuclear cells; SI, stimulation index. a Includes one stillborn calf.

Pre-colostral Nc-specific antibodies

Parasite DNA

0/6 0/5a 4/5

0/6 0/5a 1/5

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300

250

Stimulation Index

200

150

100

50

0 0

1

2

3

4

5

6

7

8

Weeks after infection Group 1 - Infected at 70d (n=6)

Group 2 - Infected at 120d (n=6)

Group 3 - Infected at 210d (n=6)

Uninfected controls (n=3)

Fig. 1. Neospora caninum antigen-stimulated proliferation of peripheral blood mononuclear cells of cattle infected with oocysts at different stages of pregnancy (expressed as mean stimulation index + SD).

Interferon-gamma (ng/ml)

100

10

1

0.1 0

1

2

3

4

5

6

7

Weeks after infection Group 1 - Infected at 70d (n=6)

Group 2 - Infected at 120d (n=6)

Group 3 - Infected at 210d (n=6)

Uninfected controls (n=3)

Fig. 2. c-Interferon levels in antigen-stimulated culture supernatants from peripheral blood mononuclear cells of cattle infected with Neospora caninum oocysts at different stages of pregnancy (group mean + SD).

challenge, maintained for at least 13 and 7 weeks, respectively, whilst antibody levels increased above 20 PP 5 weeks pc and remained above threshold until at least 13 weeks pc. The other five heifers in this group calved normally at term; four of the calves showed evidence of N. caninum infection

as shown by high pre-colostral N. caninum ELISA PP values (95, 105, 108 and 110), but the fifth did not. All the heifers in this group, except the one which gave birth to a seronegative calf, seroconverted 4–5 weeks pc with PP levels remaining positive for at least 20 weeks (Fig. 3). All

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70

60

PP

50

40

30

20

10

0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

Weeks of pregnancy 70 days infected (n=6) 210 days infected (n=6)

70 days control (n=1) 210 days control (n=1)

120 days infected (n=6)

120 days control (n=1)

Fig. 3. Neospora caninum-specific IgG responses as ELISA percent positivity (PP) in serum of cattle infected with oocysts at different stages of pregnancy (group mean PP + SD).

heifers in this group developed N. caninum antigen-specific proliferation above threshold for at least 6 weeks duration (Fig. 1) and IFN-c responses from between two to at least 6 weeks duration (Fig. 2). Neospora caninum DNA was detected in the brain of one of the four infected calves but there was no evidence of typical histopathological changes or N. caninum in these calves using immunohistochemistry. No evidence of N. caninum infection was found in the three sentinel heifers or their calves (one heifer failed to become pregnant) and their ELISA PP levels remained consistently less than 15 PP. 3.2. Estimation of the dose of viable oocysts by bioassay in gerbils

eight of the gerbils that were positive by serology. All four gerbils dosed with 1,000 oocysts were infected, which suggests that more than one oocyst was present in each dose, so these findings were not used for further estimation. The results from 30 gerbils given doses between 1 and 100 oocysts were pooled: they showed that from 1,892 oocysts there were six infections. From this, we calculated that the proportion of viable oocysts was 1/315.3 = 0.003171, with 95% exact confidence limits being 0.001165, 0.006890. When multiplied by 40,000 this meant that each heifer received an estimated 127 viable oocysts (95% confidence limits, 47–276). There was no evidence that the viability of the oocysts reduced over the 5 month period that the bioassays were carried out. 3.3. Evidence of TPI during a subsequent pregnancy

Based on seroconversion, N. caninum infection was confirmed in a total of 10 gerbils dosed with oocysts (Table 2). The minimum dose that resulted in infection was 100 oocysts. Neospora caninum DNA was detected by PCR in Table 2 Results of bioassay of Neospora caninum oocysts given to gerbils (number seropositive/number infected) Date infected

December 2003 January 2004 February 2004 April 2004

Nominal oocyst dosea 0

100

101

102

103

0/2 0/2 0/2 0/2

0/2 0/2 nd nd

0/2 0/2 0/2 0/2

1/2 0/2 1/2 4/12

nd nd 2/2 2/2

nd – not done. a Based on dilution of stock oocyst suspension – see Section 2.

Seven cows were selected for rebreeding. The outcomes of the first pregnancy and immune responses are summarised in Table 3. All animals, except number 139, showed persistent antibody levels in excess of 20 PP from 3 to 5 weeks post oocyst challenge to AI for the second pregnancy (i.e were seropositive for at least 10 weeks prior to AI). Individual antibody levels from AI in the second pregnancy are shown in Fig. 4. In the second pregnancy, all seven animals gave birth to live calves at term. Brain and heart samples from the calves were negative for N. caninum by PCR and N. caninum-specific antibodies were not detected in any pre-colostral serum samples. Brain tissues from the cows were negative for N. caninum by PCR and there was no sudden or substantial increase in N. caninum-specific IgG during pregnancy which might have indicated recrudescence

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Table 3 Details of cows selected for rebreeding: immunological parameters and clinical outcomes of their first pregnancies Cow identification

Day of pregnancy when challenged with oocysts

Outcome of pregnancy

Calf/foetus infected with Neospora caninum

Maximum PP value by ELISA

Maximum antigen-specific PBMC proliferation (SI)

Maximum IFN-c response (ng/ml)

165 142 96 187 84 139 95

210 210 210 210 120 210 70

Live calf Live calf Live calf Live calf Abortion Abortion Live calf

Yes Yes Yes Yes Yes No No

63 40 44 68 78 24 65

235 15 283 178 58 115 67

14.0 21.8 3.3 5.0 3.6 11.5 1.4

PP, percent positivity; PBMC, periferal blood mononuclear cells; SI, stimulation index.

70

60

COW # 165(I) 142(I) 96(I) 187(I) 84(I) 139(I) 95(I) 199(NI) 160(NI) 154(NI)

50

PP

40

30

20

10

0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

Weeks of pregnancy

Fig. 4. Neospora caninum-specific IgG responses as ELISA percent positivity (PP) in serum during the second gestation of cows infected (I) with N. caninum oocysts during their first gestation. IgG responses of non-infected (NI) cows are also shown.

of infection (Fig. 4) (Guy et al., 2001). Only two cows had PP values greater than 20 at calving and five cows had a PP value at calving that was less than the PP value at the start of pregnancy (Table 4). The PP values of the control cows stayed at about 10 PP throughout the experiment (Fig. 4).

4. Discussion Here, we report what to our knowledge is the third attempt to infect pregnant cattle with N. caninum oocysts. The immunological responses indicated that all animals were exposed to infection since they all developed

Table 4 Rebred cows: Neospora caninum-specific antibody responses during second pregnancy and pregnancy outcome Cow identification

Interval (weeks) between oocyst challenge and AI for rebreeding

PP values at AI

PP values at 20 weeks in gestation

PP values at calving

Outcome of pregnancy

Calf infected with N. caninum (pre-colostral serology and PCR)

N. caninum PCR on cow brain tissue

165 142 96 187 84 139 95

15 15 15 15 29 18 15

60 48 61 29 32 5 18

25 36 17 26 26 10 21

19 31 16 21 17 8 18

Live Live Live Live Live Live Live

No No No No No No No

Negative Negative Negative Negative Negative Negative Negative

AI, artifical insemination; PP, percent positivity.

calf calf calf calf calf calf calf

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antigen-specific responses in at least two of the three assays employed (PBMC proliferation, IFN-c and antibody). Nonetheless, and fully in accordance with previous results (Trees et al., 2002; Gondim et al., 2004a), the incidence of abortion was low. Only one abortion in 18 pregnancies could be definitively associated with N. caninum; in a second abortion occurring 22 days after infection, no evidence of foetal infection was found. Similarly, challenging 19 cattle at various stages of gestation from 70 days, Gondim et al. (2004a) observed only one proven N. caninum-associated abortion and that followed challenge at 120 days in gestation, as here. Inoculation early in pregnancy, at 70 days gestation, did not result in foetal infection; conversely, inoculation later in pregnancy resulted in non-lethal foetal infection and the birth of congenitally infected, otherwise healthy calves. Thus, these results involving synchronised breeding and precisely timed challenges confirm the conclusions of Gondim et al. (2004a), that the time of infection in gestation is an important determinant of parasitological and clinical outcome. This in turn is broadly consistent with the results of experimental tachyzoite challenge at different stages of pregnancy (Williams et al., 2000) with the exception that tachyzoite challenge at 70 days gestation invariably causes foetal infection and death (Williams et al., 2000; Macaldowie et al., 2004). This is likely to be due to the substantial doses (usually 107 tachyzoites) and route of administration (i.v.) employed in the tachyzoite challenge model. These and the earlier results of Gondim et al. (2004a) demonstrate that oocyst challenge can lead to bovine abortion if animals are challenged at a critical period. This suggests that in epidemic abortion outbreaks which, epidemiologically, have been ascribed to oocyst exposure (McAllister et al., 1996, 2000; Dijkstra et al., 2001b, 2002; Crawshaw and Brocklehurst, 2003), challenge has coincided with this susceptible period. Clearly, oocyst dose may also be a key factor in influencing clinical outcome. There is evidence from experimental uterine challenge with tachyzoites that the ability to infect is dose related, with a threshold of 50 · 103 tachyzoites being required to cause persistent seroconversion (Serrano-Martı´nez et al., 2007). The oocyst dose used in this experiment is not inconsistent with likely levels of environmental contamination. The intensity of N. caninum oocysts in faeces from either naturally or experimentally infected dogs is generally remarkably low compared with comparable coccidians such as Toxoplasma gondii, and total oocyst output over 5–10 days rarely exceeds 1 · 106 (McAllister et al., 1998; and others) although exceptions have been noted (McAllister et al., unpublished observations) and more research is needed on the nature of oocyst shedding in natural infections. With many canine infections the density of oocysts in faeces is frequently too low to count. Because of this we deliberately did not centrifuge and wash the sporulated oocyst suspension to remove the sulphuric acid for cattle infections, but simply diluted the appropriate aliquot of stock suspension with excess water. This was not possible for

the low volume gerbil inocula. The gerbil bioassay results suggested that cattle received a minimum viable dose of 127 oocysts but this is undoubtedly an underestimate because it presumes: (i) there was no loss of oocysts when the stock suspension was centrifuged and washed – which there undoubtedly would have been; and (ii) that one oocyst will infect a gerbil with 100% certainty, which is unlikely, although gerbils are sensitive to oocyst infection (Dubey and Lindsay, 2000). Thus the dose of oocyst received by cattle lay between 127 and 40,000. Whilst there was likely to be some loss of viability of oocysts over time from original collection, bioassays over the 5 months between receipt of oocysts in Liverpool and the final challenge did not indicate any reduction in viability. Notwithstanding, uncertainty over the true dose of oocysts, at 210 days of gestation it caused systemic infection of the dam leading to infection in four of five foetuses. It is clear that late in pregnancy, cattle are highly sensitive to exogenous transplacental infection. To determine if this oocyst infection would lead to chronic infection and endogenous transplacental infection, seven cows were rebred. They were selected from the 18 primiparous animals, on the basis that five had indisputably been systemically infected in that their foetuses were infected, and the other two had substantial immunological responses. In spite of this, there was no evidence that their infections persisted, recrudesced and infected the foetus in the second pregnancy. This suggests that whilst post-natal oocyst infection can cause exogenous TPI, it seems unlikely to cause endogenous TPI. However, the congenitally infected calves which are the result of exogenous TPI may themselves mature, carry a persistent infection and induce endogenous TPI. This important point has yet to be proven, but may be crucial in maintaining the basic reproductive rate at greater than one to sustain N. caninum infection in cattle populations (French et al., 1999). Acknowledgements We are grateful to the Department for Food, the Environment and Rural Affairs (DEFRA) for funding for (Project # OZ0404). Support for Milton McAllister and Pita Gondim was provided by United States Department of Agriculture – National Research Initiative Competitive Grants Program 2000-01997). We thank the farm staff of the University of Liverpool for their dedicated care of the animals in the study, and the technicians in the Histology Service, Faculty of Veterinary Science, University of Liverpool, for excellent technical assistance. References Barber, J.S., Holmdahl, J.O.M., Owen, M.R., Guy, F., Uggla, A., Trees, A.J., 1995. Characterisation of the first European isolate of Neospora caninum (Dubey et al., 1988). Parasitology 111, 563–568. Barr, B.C., Rowe, J.D., Sverlow, K.W., BonDurant, R.H., Ardans, A.A., Oliver, M.N., Conrad, P.A., 1994. Experimental reproduction of

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