Charles Focant

Fetal infection with Neospora caninum in dairy and beef cattle in Belgium

Neospora caninum is a protozoan parasite, which causes fetal and neonatal mortality in livestock and companion animals. In 224 abortions in Belgian cattle, different diagnostic methods were used to demonstrate infection, and the presence of N. caninum. An indirect fluorescent antibody test (IFAT) was used to analyze fetal and maternal sera and immunohistochemistry (IHC) was performed when lesions consistent with neosporosis were observed in the brain, heart or liver. Twenty dairy cattle sera out of 70 (29%) and 13 beef cattle sera out of 93 (14%) were positive by IFAT. A positive titer to N. caninum was found in seven and three fetuses born to beef and dairy cows, respectively. Lesions consistent with N. caninum infection were observed in 17 fetuses. Of nine positive beef fetuses, five were confirmed by IHC while, all but one dairy fetus were confirmed using the same technique. Age had no influence on the serological status of the mother (P = 0.486) whereas husbandry system had a borderline influence (P = 0.082). However, a strong association (P = 0.004) between the level of antibodies in the dam and the occurrence of lesions in the fetus was observed and lesions were more prominent in dairy than in beef fetuses. Additionally, the distribution of intra-cerebral lesions was more extensive in dairy than in beef fetuses (P < 0.0001). Age and serological status of the fetus were found to influence the occurrence of lesions in beef fetuses (both P < 0.001) but no such significant relationships could be demonstrated in dairy fetuses. The study indicated that N. caninum must be considered as an important cause of bovine abortion in Belgium.

The vertical transmission following the reactivation of a Neospora caninum chronic infection does not seem to be due to an alteration of the systemic immune response in pregnant CBA/Ca mice

The factors responsible for the reactivation of a Neospora caninum latent infection are unknown, but it is postulated that the maternal immune response could be altered during pregnancy. The immune response was investigated in N. caninum chronically infected mice during successive pregnancies as well as in non-pregnant infected mice and mice infected when pregnant. Vertical transmission was demonstrated in chronically infected mice after the first pregnancy but the rate of foetal infection fell after further pregnancies. Non-pregnant chronically infected mice showed a marked specific proliferative response and an IgG2a isotype preferential secretion. During the course of the first pregnancy, no significant modification of the immune response was recorded. After 2 successive pregnancies, the specific cellular response showed a significant fall whereas Th2 cytokine mRNA expression was noted. At the same time, IgG1 secretion increased to reach the IgG2a level. At the third delivery, a partial restoration of the proliferative response was observed. The reactivation of N. caninum chronic infection during pregnancy does not seem to be consecutive to an immunodepression. Nevertheless, pregnancy could favour parasite multiplication in utero after an occasional spontaneous release of bradyzoites.

Clinical, pathological and diagnostic aspects of congenital neosporosis in a series of naturally infected calves

tive analysis of family trees and a high rate of vertical transmission of the parasite (90 per cent) was found in both husbandry systems (F. De Meerschman, N. Speybroeck, D. Berkvens, C. Focant, J. Detry, C. Rettigner, R. Boreux, G. Boseret, D. Cassart, B. Losson, unpublished observations). To gather information on calves showing neurological signs during the follow-up study, and on the performance of the available diagnostic methods, all calves exhibiting neurological signs, such as limb dysfunction (flexion or hyperextension), loss of conscious proprioception or ataxia, were examined clinically, then euthanased and examined postmortem. The age and breed of each affected animal was recorded. In addition, a sample of blood was collected from each newborn calf before it received colostrum, and a sample of blood was also taken from the dam. The postmortem examinations were performed using a standard protocol, and seven sections of the brain and one each of the heart and liver were taken. The tissue specimens were fixed in 10 per cent neutral-buffered formalin, embedded in paraffin and processed for routine histopathology and immunohistochemistry (IHC) using avidin-biotin labelling as previously described by De Meerschman and others (2002). Additionally, samples of approximately 200 μl were randomly taken from five different sites in the brain and pooled. A final 200 μl sample was used in a PCR assay as described by Müller and others (1996). An in-house indirect fluorescent antibody test (IFAT) was used to detect antibodies to N caninum in bovine sera, as described by De Meerschman and others (2000). N caninum tachyzoites were grown in Vero cells in a medium supplemented with horse serum (Life Technologies Gibco BLR). The sera were diluted 1:200 for the assay. Sera were considered positive if the entire surface of the tachyzoites was fluorescent. On microscopic examination, the signal for each sample was classified by a simple grading system (no signal, doubtful, positive, strongly positive, highly positive). Each batch of slides included a known positive and a known negative bovine control serum. For the preparation of DNA, a High Pure PCR Template Preparation kit (Roche Diagnostics) was used according to the manufacturer’s recommendations. The resulting samples were examined by PCR for N caninum DNA using the primers Np6+ and Np21+ (Müller and others 1996) (Invitrogen Life Technologies). Putative false negative results were excluded by performing a parallel inhibition control reaction using bovine primers (F. De Meerschman, N. Speybroeck, D. Berkvens, C. Focant, J. Detry, C. Rettigner, R. Boreux, G. Boseret, D. Cassart, B. Losson, unpublished observations). As well as DNA extraction from tissues being tested, a negative control tissue, from a serologically negative adult cow, was used as an additional negative control. Finally, a sample of water was used together with the test sample in order to monitor possible carry-over contamination. A total of seven calves (two Belgian blue and five Holstein) were examined during the 25-month period of the study. All of them were euthanased and examined postmortem when they were between three and 25 days old. Calf 1 was moribund from birth and had marked neurological signs, consisting of ataxia. Its eyes were deviated ventromedially. Calf 2 was able to stand with assistance but showed severe neurological signs from birth. It was able to rise from lateral recumbency by itself and had a normal suckling reflex. The calf was underweight. Its forelimbs were held flexed but could be extended manually (Fig 1). Its eyes were deviated ventrally. Calf 3 was unable to stand. Its pelvic limbs were held extended and had lateral rotation deformities that could not be corrected by manipulation. Calf 4 was recumbent. Hair loss was observed around the eyes and in the ventral part of the muzzle. Calf 5 was able to stand, but spent most of the time lying down. It was weak and ataxic. Calf 6 had a marked neurological deficit with loss Clinical, pathological and diagnostic aspects of congenital neosporosis in a series of naturally infected calves